Microarray-Based Comparative Genomic Hybridization Using Sex-Matched Reference DNA Provides Greater Sensitivity for Detection of Sex Chromosome Imbalances than Array-Comparative Genomic Hybridization with Sex-Mismatched Reference DNA

PubMed Central

Yatsenko, Svetlana A.; Shaw, Chad A.; Ou, Zhishuo; Pursley, Amber N.; Patel, Ankita; Bi, Weimin; Cheung, Sau Wai; Lupski, James R.; Chinault, A. Craig; Beaudet, Arthur L.

2009-01-01

In array-comparative genomic hybridization (array-CGH) experiments, the measurement of DNA copy number of sex chromosomal regions depends on the sex of the patient and the reference DNAs used. We evaluated the ability of bacterial artificial chromosomes/P1-derived artificial and oligonucleotide array-CGH analyses to detect constitutional sex chromosome imbalances using sex-mismatched reference DNAs. Twenty-two samples with imbalances involving either the X or Y chromosome, including deletions, duplications, triplications, derivative or isodicentric chromosomes, and aneuploidy, were analyzed. Although concordant results were obtained for approximately one-half of the samples when using sex-mismatched and sex-matched reference DNAs, array-CGH analyses with sex-mismatched reference DNAs did not detect genomic imbalances that were detected using sex-matched reference DNAs in 6 of 22 patients. Small duplications and deletions of the X chromosome were most difficult to detect in female and male patients, respectively, when sex-mismatched reference DNAs were used. Sex-matched reference DNAs in array-CGH analyses provides optimal sensitivity and enables an automated statistical evaluation for the detection of sex chromosome imbalances when compared with an experimental design using sex-mismatched reference DNAs. Using sex-mismatched reference DNAs in array-CGH analyses may generate false-negative, false-positive, and ambiguous results for sex chromosome-specific probes, thus masking potential pathogenic genomic imbalances. Therefore, to optimize both detection of clinically relevant sex chromosome imbalances and ensure proper experimental performance, we suggest that alternative internal controls be developed and used instead of using sex-mismatched reference DNAs. PMID:19324990

Application of Nexus copy number software for CNV detection and analysis.

PubMed

Darvishi, Katayoon

2010-04-01

Among human structural genomic variation, copy number variants (CNVs) are the most frequently known component, comprised of gains/losses of DNA segments that are generally 1 kb in length or longer. Array-based comparative genomic hybridization (aCGH) has emerged as a powerful tool for detecting genomic copy number variants (CNVs). With the rapid increase in the density of array technology and with the adaptation of new high-throughput technology, a reliable and computationally scalable method for accurate mapping of recurring DNA copy number aberrations has become a main focus in research. Here we introduce Nexus Copy Number software, a platform-independent tool, to analyze the output files of all types of commercial and custom-made comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays, such as those manufactured by Affymetrix, Agilent Technologies, Illumina, and Roche NimbleGen. It also supports data generated by various array image-analysis software tools such as GenePix, ImaGene, and BlueFuse. (c) 2010 by John Wiley & Sons, Inc.

Evaluation of Genomic Instability in the Abnormal Prostate

DTIC Science & Technology

2006-12-01

array CGH maps copy number aberrations relative to the genome sequence by using arrays of BAC or cDNA clones as the hybridization target instead of...data produced from these analyses complicate the interpretation of results . For these reasons, and as outlined by Davies et al., 22 it is desirable...There have been numerous studies of these abnormalities and several techniques, including 9 chromosome painting, array CGH and SNP arrays , have

SeeGH--a software tool for visualization of whole genome array comparative genomic hybridization data.

PubMed

Chi, Bryan; DeLeeuw, Ronald J; Coe, Bradley P; MacAulay, Calum; Lam, Wan L

2004-02-09

Array comparative genomic hybridization (CGH) is a technique which detects copy number differences in DNA segments. Complete sequencing of the human genome and the development of an array representing a tiling set of tens of thousands of DNA segments spanning the entire human genome has made high resolution copy number analysis throughout the genome possible. Since array CGH provides signal ratio for each DNA segment, visualization would require the reassembly of individual data points into chromosome profiles. We have developed a visualization tool for displaying whole genome array CGH data in the context of chromosomal location. SeeGH is an application that translates spot signal ratio data from array CGH experiments to displays of high resolution chromosome profiles. Data is imported from a simple tab delimited text file obtained from standard microarray image analysis software. SeeGH processes the signal ratio data and graphically displays it in a conventional CGH karyotype diagram with the added features of magnification and DNA segment annotation. In this process, SeeGH imports the data into a database, calculates the average ratio and standard deviation for each replicate spot, and links them to chromosome regions for graphical display. Once the data is displayed, users have the option of hiding or flagging DNA segments based on user defined criteria, and retrieve annotation information such as clone name, NCBI sequence accession number, ratio, base pair position on the chromosome, and standard deviation. SeeGH represents a novel software tool used to view and analyze array CGH data. The software gives users the ability to view the data in an overall genomic view as well as magnify specific chromosomal regions facilitating the precise localization of genetic alterations. SeeGH is easily installed and runs on Microsoft Windows 2000 or later environments.

A Rapid Method of Genomic Array Analysis of Scaffold/Matrix Attachment Regions (S/MARs) Identifies a 2.5-Mb Region of Enhanced Scaffold/Matrix Attachment at a Human Neocentromere

PubMed Central

Sumer, Huseyin; Craig, Jeffrey M.; Sibson, Mandy; Choo, K.H. Andy

2003-01-01

Human neocentromeres are fully functional centromeres that arise at previously noncentromeric regions of the genome. We have tested a rapid procedure of genomic array analysis of chromosome scaffold/matrix attachment regions (S/MARs), involving the isolation of S/MAR DNA and hybridization of this DNA to a genomic BAC/PAC array. Using this procedure, we have defined a 2.5-Mb domain of S/MAR-enriched chromatin that fully encompasses a previously mapped centromere protein-A (CENP-A)-associated domain at a human neocentromere. We have independently verified this procedure using a previously established fluorescence in situ hybridization method on salt-treated metaphase chromosomes. In silico sequence analysis of the S/MAR-enriched and surrounding regions has revealed no outstanding sequence-related predisposition. This study defines the S/MAR-enriched domain of a higher eukaryotic centromere and provides a method that has broad application for the mapping of S/MAR attachment sites over large genomic regions or throughout a genome. PMID:12840048

HIA: a genome mapper using hybrid index-based sequence alignment.

PubMed

Choi, Jongpill; Park, Kiejung; Cho, Seong Beom; Chung, Myungguen

2015-01-01

A number of alignment tools have been developed to align sequencing reads to the human reference genome. The scale of information from next-generation sequencing (NGS) experiments, however, is increasing rapidly. Recent studies based on NGS technology have routinely produced exome or whole-genome sequences from several hundreds or thousands of samples. To accommodate the increasing need of analyzing very large NGS data sets, it is necessary to develop faster, more sensitive and accurate mapping tools. HIA uses two indices, a hash table index and a suffix array index. The hash table performs direct lookup of a q-gram, and the suffix array performs very fast lookup of variable-length strings by exploiting binary search. We observed that combining hash table and suffix array (hybrid index) is much faster than the suffix array method for finding a substring in the reference sequence. Here, we defined the matching region (MR) is a longest common substring between a reference and a read. And, we also defined the candidate alignment regions (CARs) as a list of MRs that is close to each other. The hybrid index is used to find candidate alignment regions (CARs) between a reference and a read. We found that aligning only the unmatched regions in the CAR is much faster than aligning the whole CAR. In benchmark analysis, HIA outperformed in mapping speed compared with the other aligners, without significant loss of mapping accuracy. Our experiments show that the hybrid of hash table and suffix array is useful in terms of speed for mapping NGS sequencing reads to the human reference genome sequence. In conclusion, our tool is appropriate for aligning massive data sets generated by NGS sequencing.

Analysis of Chinese women with primary ovarian insufficiency by high resolution array-comparative genomic hybridization.

PubMed

Liao, Can; Fu, Fang; Yang, Xin; Sun, Yi-Min; Li, Dong-Zhi

2011-06-01

Primary ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 years. The etiology of primary ovarian insufficiency in human female patients is still unclear. The purpose of this study is to investigate the potential genetic causes in primary amenorrhea patients by high resolution array based comparative genomic hybridization (array-CGH) analysis. Following the standard karyotyping analysis, genomic DNA from whole blood of 15 primary amenorrhea patients and 15 normal control women was hybridized with Affymetrix cytogenetic 2.7M arrays following the standard protocol. Copy number variations identified by array-CGH were confirmed by real time polymerase chain reaction. All the 30 samples were negative by conventional karyotyping analysis. Microdeletions on chromosome 17q21.31-q21.32 with approximately 1.3 Mb were identified in four patients by high resolution array-CGH analysis. This included the female reproductive secretory pathway related factor N-ethylmaleimide-sensitive factor (NSF) gene. The results of the present study suggest that there may be critical regions regulating primary ovarian insufficiency in women with a 17q21.31-q21.32 microdeletion. This effect might be due to the loss of function of the NSF gene/genes within the deleted region or to effects on contiguous genes.

Single-cell copy number variation detection

PubMed Central

2011-01-01

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data. PMID:21854607

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Application of Array Comparative Genomic Hybridization in Newborns with Multiple Congenital Anomalies.

PubMed

Szczałuba, Krzysztof; Nowakowska, Beata; Sobecka, Katarzyna; Smyk, Marta; Castaneda, Jennifer; Klapecki, Jakub; Kutkowska-Kaźmierczak, Anna; Śmigiel, Robert; Bocian, Ewa; Radkowski, Marek; Demkow, Urszula

2016-01-01

Major congenital anomalies are detectable in 2-3 % of the newborn population. Some of their genetic causes are attributable to copy number variations identified by array comparative genomic hybridization (aCGH). The value of aCGH screening as a first-tier test in children with multiple congenital anomalies has been studied and consensus adopted. However, array resolution has not been agreed upon, specifically in the newborn or infant population. Moreover, most array studies have been focused on mixed populations of intellectual disability/developmental delay with or without multiple congenital anomalies, making it difficult to assess the value of microarrays in newborns. The aim of the study was to determine the optimal quality and clinical sensitivity of high-resolution array comparative genomic hybridization in neonates with multiple congenital anomalies. We investigated a group of 54 newborns with multiple congenital anomalies defined as two or more birth defects from more than one organ system. Cytogenetic studies were performed using OGT CytoSure 8 × 60 K microarray. We found ten rearrangements in ten newborns. Of these, one recurrent syndromic microduplication was observed, whereas all other changes were unique. Six rearrangements were definitely pathogenic, including one submicroscopic and five that could be seen on routine karyotype analysis. Four other copy number variants were likely pathogenic. The candidate genes that may explain the phenotype were discussed. In conclusion, high-resolution array comparative hybridization can be applied successfully in newborns with multiple congenital anomalies as the method detects a significant number of pathogenic changes, resulting in early diagnoses. We hypothesize that small changes previously considered benign or even inherited rearrangements should be classified as potentially pathogenic at least until a subsequent clinical assessment would exclude a developmental delay or dysmorphism.

Array-Based Comparative Genomic Hybridization for the Genomewide Detection of Submicroscopic Chromosomal Abnormalities

PubMed Central

Vissers, Lisenka E. L. M. ; de Vries, Bert B. A. ; Osoegawa, Kazutoyo ; Janssen, Irene M. ; Feuth, Ton ; Choy, Chik On ; Straatman, Huub ; van der Vliet, Walter ; Huys, Erik H. L. P. G. ; van Rijk, Anke ; Smeets, Dominique ; van Ravenswaaij-Arts, Conny M. A. ; Knoers, Nine V. ; van der Burgt, Ineke ; de Jong, Pieter J. ; Brunner, Han G. ; van Kessel, Ad Geurts ; Schoenmakers, Eric F. P. M. ; Veltman, Joris A. 

2003-01-01

Microdeletions and microduplications, not visible by routine chromosome analysis, are a major cause of human malformation and mental retardation. Novel high-resolution, whole-genome technologies can improve the diagnostic detection rate of these small chromosomal abnormalities. Array-based comparative genomic hybridization allows such a high-resolution screening by hybridizing differentially labeled test and reference DNAs to arrays consisting of thousands of genomic clones. In this study, we tested the diagnostic capacity of this technology using ∼3,500 flourescent in situ hybridization–verified clones selected to cover the genome with an average of 1 clone per megabase (Mb). The sensitivity and specificity of the technology were tested in normal-versus-normal control experiments and through the screening of patients with known microdeletion syndromes. Subsequently, a series of 20 cytogenetically normal patients with mental retardation and dysmorphisms suggestive of a chromosomal abnormality were analyzed. In this series, three microdeletions and two microduplications were identified and validated. Two of these genomic changes were identified also in one of the parents, indicating that these are large-scale genomic polymorphisms. Deletions and duplications as small as 1 Mb could be reliably detected by our approach. The percentage of false-positive results was reduced to a minimum by use of a dye-swap-replicate analysis, all but eliminating the need for laborious validation experiments and facilitating implementation in a routine diagnostic setting. This high-resolution assay will facilitate the identification of novel genes involved in human mental retardation and/or malformation syndromes and will provide insight into the flexibility and plasticity of the human genome. PMID:14628292

A comparative genomic hybridization approach to study gene copy number variations among Chinese hamster cell lines.

PubMed

Vishwanathan, Nandita; Bandyopadhyay, Arpan; Fu, Hsu-Yuan; Johnson, Kathryn C; Springer, Nathan M; Hu, Wei-Shou

2017-08-01

Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Array comparative genomic hybridization and computational genome annotation in constitutional cytogenetics: suggesting candidate genes for novel submicroscopic chromosomal imbalance syndromes.

PubMed

Van Vooren, Steven; Coessens, Bert; De Moor, Bart; Moreau, Yves; Vermeesch, Joris R

2007-09-01

Genome-wide array comparative genomic hybridization screening is uncovering pathogenic submicroscopic chromosomal imbalances in patients with developmental disorders. In those patients, imbalances appear now to be scattered across the whole genome, and most patients carry different chromosomal anomalies. Screening patients with developmental disorders can be considered a forward functional genome screen. The imbalances pinpoint the location of genes that are involved in human development. Because most imbalances encompass regions harboring multiple genes, the challenge is to (1) identify those genes responsible for the specific phenotype and (2) disentangle the role of the different genes located in an imbalanced region. In this review, we discuss novel tools and relevant databases that have recently been developed to aid this gene discovery process. Identification of the functional relevance of genes will not only deepen our understanding of human development but will, in addition, aid in the data interpretation and improve genetic counseling.

Design and coverage of high throughput genotyping arrays optimized for individuals of East Asian, African American, and Latino race/ethnicity using imputation and a novel hybrid SNP selection algorithm.

PubMed

Hoffmann, Thomas J; Zhan, Yiping; Kvale, Mark N; Hesselson, Stephanie E; Gollub, Jeremy; Iribarren, Carlos; Lu, Yontao; Mei, Gangwu; Purdy, Matthew M; Quesenberry, Charles; Rowell, Sarah; Shapero, Michael H; Smethurst, David; Somkin, Carol P; Van den Eeden, Stephen K; Walter, Larry; Webster, Teresa; Whitmer, Rachel A; Finn, Andrea; Schaefer, Catherine; Kwok, Pui-Yan; Risch, Neil

2011-12-01

Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Genome-Wide Mapping of Copy Number Variation in Humans: Comparative Analysis of High Resolution Array Platforms

PubMed Central

Haraksingh, Rajini R.; Abyzov, Alexej; Gerstein, Mark; Urban, Alexander E.; Snyder, Michael

2011-01-01

Accurate and efficient genome-wide detection of copy number variants (CNVs) is essential for understanding human genomic variation, genome-wide CNV association type studies, cytogenetics research and diagnostics, and independent validation of CNVs identified from sequencing based technologies. Numerous, array-based platforms for CNV detection exist utilizing array Comparative Genome Hybridization (aCGH), Single Nucleotide Polymorphism (SNP) genotyping or both. We have quantitatively assessed the abilities of twelve leading genome-wide CNV detection platforms to accurately detect Gold Standard sets of CNVs in the genome of HapMap CEU sample NA12878, and found significant differences in performance. The technologies analyzed were the NimbleGen 4.2 M, 2.1 M and 3×720 K Whole Genome and CNV focused arrays, the Agilent 1×1 M CGH and High Resolution and 2×400 K CNV and SNP+CGH arrays, the Illumina Human Omni1Quad array and the Affymetrix SNP 6.0 array. The Gold Standards used were a 1000 Genomes Project sequencing-based set of 3997 validated CNVs and an ultra high-resolution aCGH-based set of 756 validated CNVs. We found that sensitivity, total number, size range and breakpoint resolution of CNV calls were highest for CNV focused arrays. Our results are important for cost effective CNV detection and validation for both basic and clinical applications. PMID:22140474

Comparative Genomic Hybridization–Array Analysis Enhances the Detection of Aneuploidies and Submicroscopic Imbalances in Spontaneous Miscarriages

PubMed Central

Schaeffer, Anthony J. ; Chung, June ; Heretis, Konstantina ; Wong, Andrew ; Ledbetter, David H. ; Lese Martin, Christa 

2004-01-01

Miscarriage is a condition that affects 10%–15% of all clinically recognized pregnancies, most of which occur in the first trimester. Approximately 50% of first-trimester miscarriages result from fetal chromosome abnormalities. Currently, G-banded chromosome analysis is used to determine if large-scale genetic imbalances are the cause of these pregnancy losses. This technique relies on the culture of cells derived from the fetus, a technique that has many limitations, including a high rate of culture failure, maternal overgrowth of fetal cells, and poor chromosome morphology. Comparative genomic hybridization (CGH)–array analysis is a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number. By hybridizing patient DNA and normal reference DNA to arrays of genomic clones, unbalanced gains or losses of genetic material across the genome can be detected. In this study, 41 product-of-conception (POC) samples, which were previously analyzed by G-banding, were tested using CGH arrays to determine not only if the array could identify all reported abnormalities, but also whether any previously undetected genomic imbalances would be discovered. The array methodology detected all abnormalities as reported by G-banding analysis and revealed new abnormalities in 4/41 (9.8%) cases. Of those, one trisomy 21 POC was also mosaic for trisomy 20, one had a duplication of the 10q telomere region, one had an interstitial deletion of chromosome 9p, and the fourth had an interstitial duplication of the Prader-Willi/Angelman syndrome region on chromosome 15q, which, if maternally inherited, has been implicated in autism. This retrospective study demonstrates that the DNA-based CGH-array technology overcomes many of the limitations of routine cytogenetic analysis of POC samples while enhancing the detection of fetal chromosome aberrations. PMID:15127362

Customized Oligonucleotide Array-Based Comparative Genomic Hybridization as a Clinical Assay for Genomic Profiling of Chronic Lymphocytic Leukemia

PubMed Central

Sargent, Rachel; Jones, Dan; Abruzzo, Lynne V.; Yao, Hui; Bonderover, Jaime; Cisneros, Marissa; Wierda, William G.; Keating, Michael J.; Luthra, Rajyalakshmi

2009-01-01

Chromosome gains and losses used for risk stratification in chronic lymphocytic leukemia (CLL) are commonly assessed by multiprobe fluorescence in situ hybridization (FISH) studies. We designed and validated a customized array-comparative genomic hybridization (aCGH) platform as a clinical assay for CLL genomic profiling. A 60-mer, 44,000-probe oligonucleotide array with a 50-kb average spatial resolution was augmented with high-density probe tiling at loci that are frequently aberrant in CLL. Aberrations identified by aCGH were compared with those identified by a FISH panel, including locus-specific probes to ATM (11q22.3), the centromeric region of chromosome 12 (12p11.1–q11), D13S319 (13q14.3), LAMP1 (13q34), and TP53 (17p13.1). In 100 CLL samples, aCGH/FISH concordance was seen for 89% of FISH-called aberrations at the ATM (n = 18), D13S319 (n = 42), LAMP (n = 12), and TP53 (n = 22) loci and for chromosome 12 (n = 14). Eighty-four percentage of FISH/aCGH discordant calls were in samples either at or below the limit of aCGH sensitivity (10% to 25% FISH aberration-containing cells). Therefore, aCGH profiling is a feasible routine clinical test with comparable results to multiprobe FISH studies; however, it may be less sensitive than FISH in cases with low-level aberrations. Further, a customized array design can provide comprehensive genomic profiling with additional accuracy in both identifying and defining the extent of small aberrations at target loci. PMID:19074592

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas

PubMed Central

Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

2010-01-01

Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

An oligonucleotide array for the identification and differentiation of bacteria pathogenic on potato.

PubMed

Fessehaie, Anania; De Boer, Solke H; Lévesque, C André

2003-03-01

ABSTRACT Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.

The Diagnostic Yield of Array Comparative Genomic Hybridization Is High Regardless of Severity of Intellectual Disability/Developmental Delay in Children.

PubMed

D'Arrigo, Stefano; Gavazzi, Francesco; Alfei, Enrico; Zuffardi, Orsetta; Montomoli, Cristina; Corso, Barbara; Buzzi, Erika; Sciacca, Francesca L; Bulgheroni, Sara; Riva, Daria; Pantaleoni, Chiara

2016-05-01

Microarray-based comparative genomic hybridization is a method of molecular analysis that identifies chromosomal anomalies (or copy number variants) that correlate with clinical phenotypes. The aim of the present study was to apply a clinical score previously designated by de Vries to 329 patients with intellectual disability/developmental disorder (intellectual disability/developmental delay) referred to our tertiary center and to see whether the clinical factors are associated with a positive outcome of aCGH analyses. Another goal was to test the association between a positive microarray-based comparative genomic hybridization result and the severity of intellectual disability/developmental delay. Microarray-based comparative genomic hybridization identified structural chromosomal alterations responsible for the intellectual disability/developmental delay phenotype in 16% of our sample. Our study showed that causative copy number variants are frequently found even in cases of mild intellectual disability (30.77%). We want to emphasize the need to conduct microarray-based comparative genomic hybridization on all individuals with intellectual disability/developmental delay, regardless of the severity, because the degree of intellectual disability/developmental delay does not predict the diagnostic yield of microarray-based comparative genomic hybridization. © The Author(s) 2015.

A new normalizing algorithm for BAC CGH arrays with quality control metrics.

PubMed

Miecznikowski, Jeffrey C; Gaile, Daniel P; Liu, Song; Shepherd, Lori; Nowak, Norma

2011-01-01

The main focus in pin-tip (or print-tip) microarray analysis is determining which probes, genes, or oligonucleotides are differentially expressed. Specifically in array comparative genomic hybridization (aCGH) experiments, researchers search for chromosomal imbalances in the genome. To model this data, scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise. In this paper we propose "SmoothArray", a new method to preprocess comparative genomic hybridization (CGH) bacterial artificial chromosome (BAC) arrays and we show the effects on a cancer dataset. As part of our R software package "aCGHplus," this freely available algorithm removes the variation due to the intensity effects, pin/print-tip, the spatial location on the microarray chip, and the relative location from the well plate. removal of this variation improves the downstream analysis and subsequent inferences made on the data. Further, we present measures to evaluate the quality of the dataset according to the arrayer pins, 384-well plates, plate rows, and plate columns. We compare our method against competing methods using several metrics to measure the biological signal. With this novel normalization algorithm and quality control measures, the user can improve their inferences on datasets and pinpoint problems that may arise in their BAC aCGH technology.

Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization

PubMed Central

Yang, Wen-Xu; Pan, Hong; Li, Lin; Wu, Hai-Rong; Wang, Song-Tao; Bao, Xin-Hua; Jiang, Yu-Wu; Qi, Yu

2016-01-01

Background: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH). Methods: Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results: All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions: The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype. PMID:26960370

Characterization of Deletions of the HBA and HBB Loci by Array Comparative Genomic Hybridization

PubMed Central

Sabath, Daniel E.; Bender, Michael A.; Sankaran, Vijay G.; Vamos, Esther; Kentsis, Alex; Yi, Hye-Son; Greisman, Harvey A.

2017-01-01

Thalassemia is among the most common genetic diseases worldwide. α-Thalassemia is usually caused by deletion of one or more of the duplicated HBA genes on chromosome 16. In contrast, most β-thalassemia results from point mutations that decrease or eliminate expression of the HBB gene on chromosome 11. Deletions within the HBB locus result in thalassemia or hereditary persistence of fetal Hb. Although routine diagnostic testing cannot distinguish thalassemia deletions from point mutations, deletional hereditary persistence of fetal Hb is notable for having an elevated HbF level with a normal mean corpuscular volume. A small number of deletions accounts for most α-thalassemias; in contrast, there are no predominant HBB deletions causing β-thalassemia. To facilitate the identification and characterization of deletions of the HBA and HBB globin loci, we performed array-based comparative genomic hybridization using a custom oligonucleotide microarray. We accurately mapped the breakpoints of known and previously uncharacterized HBB deletions defining previously uncharacterized deletion breakpoints by PCR amplification and sequencing. The array also successfully identified the common HBA deletions --SEA and --FIL. In summary, comparative genomic hybridization can be used to characterize deletions of the HBA and HBB loci, allowing high-resolution characterization of novel deletions that are not readily detected by PCR-based methods. PMID:26612711

Array-CGH Analysis in a Cohort of Phenotypically Well-Characterized Individuals with "Essential" Autism Spectrum Disorders

ERIC Educational Resources Information Center

Napoli, Eleonora; Russo, Serena; Casula, Laura; Alesi, Viola; Amendola, Filomena Alessandra; Angioni, Adriano; Novelli, Antonio; Valeri, Giovanni; Menghini, Deny; Vicari, Stefano

2018-01-01

Copy-number variants (CNVs) are associated with susceptibility to autism spectrum disorder (ASD). To detect the presence of CNVs, we conducted an array-comparative genomic hybridization (array-CGH) analysis in 133 children with "essential" ASD phenotype. Genetic analyses documented that 12 children had causative CNVs (C-CNVs), 29…

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

PubMed

Hartmann, Luise; Stephenson, Christine F; Verkamp, Stephanie R; Johnson, Krystal R; Burnworth, Bettina; Hammock, Kelle; Brodersen, Lisa Eidenschink; de Baca, Monica E; Wells, Denise A; Loken, Michael R; Zehentner, Barbara K

2014-12-01

Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated. © 2014 American Association for Clinical Chemistry.

The Utility of Chromosomal Microarray Analysis in Developmental and Behavioral Pediatrics

ERIC Educational Resources Information Center

Beaudet, Arthur L.

2013-01-01

Chromosomal microarray analysis (CMA) has emerged as a powerful new tool to identify genomic abnormalities associated with a wide range of developmental disabilities including congenital malformations, cognitive impairment, and behavioral abnormalities. CMA includes array comparative genomic hybridization (CGH) and single nucleotide polymorphism…

LS-CAP: an algorithm for identifying cytogenetic aberrations in hepatocellular carcinoma using microarray data.

PubMed

He, Xianmin; Wei, Qing; Sun, Meiqian; Fu, Xuping; Fan, Sichang; Li, Yao

2006-05-01

Biological techniques such as Array-Comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH) and affymetrix single nucleotide pleomorphism (SNP) array have been used to detect cytogenetic aberrations. However, on genomic scale, these techniques are labor intensive and time consuming. Comparative genomic microarray analysis (CGMA) has been used to identify cytogenetic changes in hepatocellular carcinoma (HCC) using gene expression microarray data. However, CGMA algorithm can not give precise localization of aberrations, fails to identify small cytogenetic changes, and exhibits false negatives and positives. Locally un-weighted smoothing cytogenetic aberrations prediction (LS-CAP) based on local smoothing and binomial distribution can be expected to address these problems. LS-CAP algorithm was built and used on HCC microarray profiles. Eighteen cytogenetic abnormalities were identified, among them 5 were reported previously, and 12 were proven by CGH studies. LS-CAP effectively reduced the false negatives and positives, and precisely located small fragments with cytogenetic aberrations.

Whole genome amplification of DNA extracted from FFPE tissues.

PubMed

Bosso, Mira; Al-Mulla, Fahd

2011-01-01

Whole genome amplification systems were developed to meet the increasing research demands on DNA resources and to avoid DNA shortage. The technology enables amplification of nanogram amounts of DNA into microgram quantities and is increasingly used in the amplification of DNA from multiple origins such as blood, fresh frozen tissue, formalin-fixed paraffin-embedded tissues, saliva, buccal swabs, bacteria, and plant and animal sources. This chapter focuses on the use of GenomePlex(®) tissue Whole Genome Amplification Kit, to amplify DNA directly from archived tissue. In addition, this chapter documents our unique experience with the utilization of GenomePlex(®) amplified DNA using several molecular techniques including metaphase Comparative Genomic Hybridization, array Comparative Genomic Hybridization, and real-time quantitative polymerase chain reaction assays. GenomePlex(®) is a registered trademark of Rubicon Genomics Incorporation.

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

PubMed Central

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian; Ulrich, Eldon L.; Zhao, Qin; Wrobel, Russell L.; Newman, Craig S.; Fox, Brian G.; Phillips, George N.; Markley, John L.; Sussman, Michael R.

2005-01-01

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron–exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions “antisense” to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage. PMID:15755812

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian;

Genome profiling of ovarian adenocarcinomas using pangenomic BACs microarray comparative genomic hybridization

PubMed Central

Caserta, Donatella; Benkhalifa, Moncef; Baldi, Marina; Fiorentino, Francesco; Qumsiyeh, Mazin; Moscarini, Massimo

2008-01-01

Background Routine cytogenetic investigations for ovarian cancers are limited by culture failure and poor growth of cancer cells compared to normal cells. Fluorescence in situ Hybridization (FISH) application or classical comparative genome hybridization techniques are also have their own limitations in detecting genome imbalance especially for small changes that are not known ahead of time and for which FISH probes could not be thus designed. Methods We applied microarray comparative genomic hybridization (A-CGH) using one mega base BAC arrays to investigate chromosomal disorders in ovarian adenocarcinoma in patients with familial history. Results Our data on 10 cases of ovarian cancer revealed losses of 6q (4 cases mainly mosaic loss), 9p (4 cases), 10q (3 cases), 21q (3 cases), 22q (4 cases) with association to a monosomy X and gains of 8q and 9q (occurring together in 8 cases) and gain of 12p. There were other abnormalities such as loss of 17p that were noted in two profiles of the studied cases. Total or mosaic segmental gain of 2p, 3q, 4q, 7q and 13q were also observed. Seven of 10 patients were investigated by FISH to control array CGH results. The FISH data showed a concordance between the 2 methods. Conclusion The data suggest that A-CGH detects unique and common abnormalities with certain exceptions such as tetraploidy and balanced translocation, which may lead to understanding progression of genetic changes as well as aid in early diagnosis and have an impact on therapy and prognosis. PMID:18492273

Comprehensive performance comparison of high-resolution array platforms for genome-wide Copy Number Variation (CNV) analysis in humans.

PubMed

Haraksingh, Rajini R; Abyzov, Alexej; Urban, Alexander Eckehart

2017-04-24

High-resolution microarray technology is routinely used in basic research and clinical practice to efficiently detect copy number variants (CNVs) across the entire human genome. A new generation of arrays combining high probe densities with optimized designs will comprise essential tools for genome analysis in the coming years. We systematically compared the genome-wide CNV detection power of all 17 available array designs from the Affymetrix, Agilent, and Illumina platforms by hybridizing the well-characterized genome of 1000 Genomes Project subject NA12878 to all arrays, and performing data analysis using both manufacturer-recommended and platform-independent software. We benchmarked the resulting CNV call sets from each array using a gold standard set of CNVs for this genome derived from 1000 Genomes Project whole genome sequencing data. The arrays tested comprise both SNP and aCGH platforms with varying designs and contain between ~0.5 to ~4.6 million probes. Across the arrays CNV detection varied widely in number of CNV calls (4-489), CNV size range (~40 bp to ~8 Mbp), and percentage of non-validated CNVs (0-86%). We discovered strikingly strong effects of specific array design principles on performance. For example, some SNP array designs with the largest numbers of probes and extensive exonic coverage produced a considerable number of CNV calls that could not be validated, compared to designs with probe numbers that are sometimes an order of magnitude smaller. This effect was only partially ameliorated using different analysis software and optimizing data analysis parameters. High-resolution microarrays will continue to be used as reliable, cost- and time-efficient tools for CNV analysis. However, different applications tolerate different limitations in CNV detection. Our study quantified how these arrays differ in total number and size range of detected CNVs as well as sensitivity, and determined how each array balances these attributes. This analysis will inform appropriate array selection for future CNV studies, and allow better assessment of the CNV-analytical power of both published and ongoing array-based genomics studies. Furthermore, our findings emphasize the importance of concurrent use of multiple analysis algorithms and independent experimental validation in array-based CNV detection studies.

[Dandy-walker syndrome and microdeletions on chromosome 7].

PubMed

Liao, Can; Fu, Fang; Li, Ru; Pan, Min; Yang, Xin; Yi, Cui-xing; Li, Jian; Li, Dong-zhi

2012-02-01

To investigate genetic etiology of Dandy-Walker syndrome with array-based comparative genomic hybridization (array-CGH). Eight fetuses with Dandy-Walker malformations but normal karyotypes by conventional cytogenetic technique were selected. DNA samples were extracted and hybridized with Affymetrix cytogenetic 2.7 M arrays by following the manufacturer's standard protocol. The data were analyzed by special software packages. By using array-CGH technique, common deletions and duplication on chromosome 7p21.3 were identified in three cases, within which were central nervous system disease associated genes NDUFA4 and PHF14. Copy number variations (CNVs) of chromosome 7p21.3 region are associated with Dandy-Walker malformations which may be due to haploinsufficiency or overexpression of NDUFA4 and PHF14 genes.

CGI: Java Software for Mapping and Visualizing Data from Array-based Comparative Genomic Hybridization and Expression Profiling

PubMed Central

Gu, Joyce Xiuweu-Xu; Wei, Michael Yang; Rao, Pulivarthi H.; Lau, Ching C.; Behl, Sanjiv; Man, Tsz-Kwong

2007-01-01

With the increasing application of various genomic technologies in biomedical research, there is a need to integrate these data to correlate candidate genes/regions that are identified by different genomic platforms. Although there are tools that can analyze data from individual platforms, essential software for integration of genomic data is still lacking. Here, we present a novel Java-based program called CGI (Cytogenetics-Genomics Integrator) that matches the BAC clones from array-based comparative genomic hybridization (aCGH) to genes from RNA expression profiling datasets. The matching is computed via a fast, backend MySQL database containing UCSC Genome Browser annotations. This program also provides an easy-to-use graphical user interface for visualizing and summarizing the correlation of DNA copy number changes and RNA expression patterns from a set of experiments. In addition, CGI uses a Java applet to display the copy number values of a specific BAC clone in aCGH experiments side by side with the expression levels of genes that are mapped back to that BAC clone from the microarray experiments. The CGI program is built on top of extensible, reusable graphic components specifically designed for biologists. It is cross-platform compatible and the source code is freely available under the General Public License. PMID:19936083

CGI: Java software for mapping and visualizing data from array-based comparative genomic hybridization and expression profiling.

PubMed

Gu, Joyce Xiuweu-Xu; Wei, Michael Yang; Rao, Pulivarthi H; Lau, Ching C; Behl, Sanjiv; Man, Tsz-Kwong

2007-10-06

With the increasing application of various genomic technologies in biomedical research, there is a need to integrate these data to correlate candidate genes/regions that are identified by different genomic platforms. Although there are tools that can analyze data from individual platforms, essential software for integration of genomic data is still lacking. Here, we present a novel Java-based program called CGI (Cytogenetics-Genomics Integrator) that matches the BAC clones from array-based comparative genomic hybridization (aCGH) to genes from RNA expression profiling datasets. The matching is computed via a fast, backend MySQL database containing UCSC Genome Browser annotations. This program also provides an easy-to-use graphical user interface for visualizing and summarizing the correlation of DNA copy number changes and RNA expression patterns from a set of experiments. In addition, CGI uses a Java applet to display the copy number values of a specific BAC clone in aCGH experiments side by side with the expression levels of genes that are mapped back to that BAC clone from the microarray experiments. The CGI program is built on top of extensible, reusable graphic components specifically designed for biologists. It is cross-platform compatible and the source code is freely available under the General Public License.

Micro-Scale Genomic DNA Copy Number Aberrations as Another Means of Mutagenesis in Breast Cancer

PubMed Central

Chao, Hann-Hsiang; He, Xiaping; Parker, Joel S.; Zhao, Wei; Perou, Charles M.

2012-01-01

Introduction In breast cancer, the basal-like subtype has high levels of genomic instability relative to other breast cancer subtypes with many basal-like-specific regions of aberration. There is evidence that this genomic instability extends to smaller scale genomic aberrations, as shown by a previously described micro-deletion event in the PTEN gene in the Basal-like SUM149 breast cancer cell line. Methods We sought to identify if small regions of genomic DNA copy number changes exist by using a high density, gene-centric Comparative Genomic Hybridizations (CGH) array on cell lines and primary tumors. A custom tiling array for CGH (244,000 probes, 200 bp tiling resolution) was created to identify small regions of genomic change, which was focused on previously identified basal-like-specific, and general cancer genes. Tumor genomic DNA from 94 patients and 2 breast cancer cell lines was labeled and hybridized to these arrays. Aberrations were called using SWITCHdna and the smallest 25% of SWITCHdna-defined genomic segments were called micro-aberrations (<64 contiguous probes, ∼ 15 kb). Results Our data showed that primary tumor breast cancer genomes frequently contained many small-scale copy number gains and losses, termed micro-aberrations, most of which are undetectable using typical-density genome-wide aCGH arrays. The basal-like subtype exhibited the highest incidence of these events. These micro-aberrations sometimes altered expression of the involved gene. We confirmed the presence of the PTEN micro-amplification in SUM149 and by mRNA-seq showed that this resulted in loss of expression of all exons downstream of this event. Micro-aberrations disproportionately affected the 5′ regions of the affected genes, including the promoter region, and high frequency of micro-aberrations was associated with poor survival. Conclusion Using a high-probe-density, gene-centric aCGH microarray, we present evidence of small-scale genomic aberrations that can contribute to gene inactivation. These events may contribute to tumor formation through mechanisms not detected using conventional DNA copy number analyses. PMID:23284754

Comparative genomic hybridization.

PubMed

Pinkel, Daniel; Albertson, Donna G

2005-01-01

Altering DNA copy number is one of the many ways that gene expression and function may be modified. Some variations are found among normal individuals ( 14, 35, 103 ), others occur in the course of normal processes in some species ( 33 ), and still others participate in causing various disease states. For example, many defects in human development are due to gains and losses of chromosomes and chromosomal segments that occur prior to or shortly after fertilization, whereas DNA dosage alterations that occur in somatic cells are frequent contributors to cancer. Detecting these aberrations, and interpreting them within the context of broader knowledge, facilitates identification of critical genes and pathways involved in biological processes and diseases, and provides clinically relevant information. Over the past several years array comparative genomic hybridization (array CGH) has demonstrated its value for analyzing DNA copy number variations. In this review we discuss the state of the art of array CGH and its applications in medical genetics and cancer, emphasizing general concepts rather than specific results.

Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias

PubMed Central

2012-01-01

Background High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs) in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. Results We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO) probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno) that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. Conclusion The problems of ascertainment bias and missing information due to genotyping errors are widely recognized as limiting factors in genetic studies. We have conducted the first formal analysis of the effect of novel variants on genotyping arrays, and we have shown that these variants account for a large portion of miscalled and uncalled genotypes. Genetic studies will benefit from substantial improvements in the accuracy of their results by incorporating VINOs in their analyses. PMID:22260749

Phylogenomic evidence for ancient hybridization in the genomes of living cats (Felidae)

PubMed Central

Li, Gang; Davis, Brian W.; Eizirik, Eduardo; Murphy, William J.

2016-01-01

Inter-species hybridization has been recently recognized as potentially common in wild animals, but the extent to which it shapes modern genomes is still poorly understood. Distinguishing historical hybridization events from other processes leading to phylogenetic discordance among different markers requires a well-resolved species tree that considers all modes of inheritance and overcomes systematic problems due to rapid lineage diversification by sampling large genomic character sets. Here, we assessed genome-wide phylogenetic variation across a diverse mammalian family, Felidae (cats). We combined genotypes from a genome-wide SNP array with additional autosomal, X- and Y-linked variants to sample ∼150 kb of nuclear sequence, in addition to complete mitochondrial genomes generated using light-coverage Illumina sequencing. We present the first robust felid time tree that accounts for unique maternal, paternal, and biparental evolutionary histories. Signatures of phylogenetic discordance were abundant in the genomes of modern cats, in many cases indicating hybridization as the most likely cause. Comparison of big cat whole-genome sequences revealed a substantial reduction of X-linked divergence times across several large recombination cold spots, which were highly enriched for signatures of selection-driven post-divergence hybridization between the ancestors of the snow leopard and lion lineages. These results highlight the mosaic origin of modern felid genomes and the influence of sex chromosomes and sex-biased dispersal in post-speciation gene flow. A complete resolution of the tree of life will require comprehensive genomic sampling of biparental and sex-limited genetic variation to identify and control for phylogenetic conflict caused by ancient admixture and sex-biased differences in genomic transmission. PMID:26518481

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

PubMed Central

Wiszniewska, Joanna; Bi, Weimin; Shaw, Chad; Stankiewicz, Pawel; Kang, Sung-Hae L; Pursley, Amber N; Lalani, Seema; Hixson, Patricia; Gambin, Tomasz; Tsai, Chun-hui; Bock, Hans-Georg; Descartes, Maria; Probst, Frank J; Scaglia, Fernando; Beaudet, Arthur L; Lupski, James R; Eng, Christine; Wai Cheung, Sau; Bacino, Carlos; Patel, Ankita

2014-01-01

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner. PMID:23695279

A case of female epispadias.

PubMed

Tantibhedhyangkul, Julierut; Copland, Susannah D; Haqq, Andrea M; Price, Thomas M

2008-11-01

To present a case of unrecognized female epispadias. Case report. University-based reproductive endocrinology and fertility clinic. A 16-year-old girl with epispadias, history of mild urinary incontinence, auditory neuropathy, and functional hyperandrogenism. None. Peripheral blood array-based comparative genomic hybridization. The patient was referred for evaluation of excessive weight gain, secondary amenorrhea, and abnormal external genitalia. Examination under anesthesia revealed bilateral labia minora hypertrophy, bifid clitoris, and a patulous urethra, consistent with female epispadias. Hormonal evaluation showed functional hyperandrogenism, and peripheral blood array-based comparative genomic hybridization showed no chromosomal deletions or duplications. Female epispadias is a rare abnormality, not commonly recognized by most practitioners. The diagnosis is supported by a history of urinary incontinence and physical findings of bifid clitoris and patulous urethra. The condition can have serious physical and psychological consequences leading to a gross disruption of social function.

Genomic Alteration in Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Inferred from Karyotyping, Molecular Cytogenetics, and Array Comparative Genomic Hybridization

PubMed Central

Rerkarmnuaychoke, Budsaba; Suntronpong, Aorarat; Fu, Beiyuan; Bodhisuwan, Winai; Peyachoknagul, Surin; Yang, Fengtang; Koontongkaew, Sittichai; Srikulnath, Kornsorn

2016-01-01

Genomic alteration in head and neck squamous cell carcinoma (HNSCC) was studied in two cell line pairs (HN30-HN31 and HN4-HN12) using conventional C-banding, multiplex fluorescence in situ hybridization (M-FISH), and array comparative genomic hybridization (array CGH). HN30 and HN4 were derived from primary lesions in the pharynx and base of tongue, respectively, and HN31 and HN12 were derived from lymph-node metastatic lesions belonging to the same patients. Gain of chromosome 1, 7, and 11 were shared in almost all cell lines. Hierarchical clustering revealed that HN31 was closely related to HN4, which shared eight chromosome alteration cases. Large C-positive heterochromatins were found in the centromeric region of chromosome 9 in HN31 and HN4, which suggests complex structural amplification of the repetitive sequence. Array CGH revealed amplification of 7p22.3p11.2, 8q11.23q12.1, and 14q32.33 in all cell lines involved with tumorigenesis and inflammation genes. The amplification of 2p21 (SIX3), 11p15.5 (H19), and 11q21q22.3 (MAML2, PGR, TRPC6, and MMP family) regions, and deletion of 9p23 (PTPRD) and 16q23.1 (WWOX) regions were identified in HN31 and HN12. Interestingly, partial loss of PTPRD (9p23) and WWOX (16q23.1) genes was identified in HN31 and HN12, and the level of gene expression tended to be the down-regulation of PTPRD, with no detectable expression of the WWOX gene. This suggests that the scarcity of PTPRD and WWOX genes might have played an important role in progression of HNSCC, and could be considered as a target for cancer therapy or a biomarker in molecular pathology. PMID:27501229

Analysis of genomic alterations in neuroblastoma by multiplex ligation-dependent probe amplification and array comparative genomic hybridization: a comparison of results.

PubMed

Combaret, Valérie; Iacono, Isabelle; Bréjon, Stéphanie; Schleiermacher, Gudrun; Pierron, Gäelle; Couturier, Jérôme; Bergeron, Christophe; Blay, Jean-Yves

2012-12-01

In cases of neuroblastoma, recurring genetic alterations--losses of the 1p, 3p, 4p, and 11q and/or gains of 1q, 2p, and 17q chromosome arms--are currently used to define the therapeutic strategy in therapeutic protocols for low- and intermediate-risk patients. Different genome-wide analysis techniques, such as array comparative genomic hybridization (aCGH) or multiplex ligation-dependent probe amplification (MLPA), have been suggested for detecting chromosome segmental abnormalities. In this study, we compared the results of the two technologies in the analyses of the DNA of tumor samples from 91 neuroblastoma patients. Similar results were obtained with the two techniques for 75 samples (82%). In five cases (5.5%), the MLPA results were not interpretable. Discrepancies between the aCGH and MLPA results were observed in 11 cases (12%). Among the discrepancies, a 18q21.2-qter gain and 16p11.2 and 11q14.1-q14.3 losses were detected only by aCGH. The MLPA results showed that the 7p, 7q, and 14q chromosome arms were affected in six cases, while in two cases, 2p and 17q gains were observed; these results were confirmed by neither aCGH nor fluorescence in situ hybridization (FISH) analysis. Because of the higher sensitivity and specificity of genome-wide information, reasonable cost, and shorter time of aCGH analysis, we recommend the aCGH procedure for the analysis of genomic alterations in neuroblastoma. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular Breeding of Rice Restorer Lines and Hybrids for Brown Planthopper (BPH) Resistance Using the Bph14 and Bph15 Genes.

PubMed

Wang, Hongbo; Ye, Shengtuo; Mou, Tongmin

2016-12-01

The development of hybrid rice is a practical approach for increasing rice production. However, the brown planthopper (BPH), Nilaparvata lugens Stål, causes severe yield loss of rice (Oryza sativa L.) and can threaten food security. Therefore, breeding hybrid rice resistant to BPH is the most effective and economical strategy to maintain high and stable production. Fortunately, numerous BPH resistance genes have been identified, and abundant linkage markers are available for molecular marker-assisted selection (MAS) in breeding programs. Hence, we pyramided two BPH resistance genes, Bph14 and Bph15, into a susceptive CMS restorer line Huahui938 and its derived hybrids using MAS to improve the BPH resistance of hybrid rice. Three near-isogenic lines (NILs) with pyramided Bph14 and Bph15 were obtained by molecular marker-assisted backcross (MAB) and phenotypic selection. The genomic components of these NILs were detected using the whole-genome SNP (Single nucleotide polymorphism) array, RICE6K, suggesting that the recurrent parent genome (RPG) recovery of the NILs was 87.88, 87.70 and 86.62 %, respectively. BPH bioassays showed that the improved NILs and their derived hybrids carrying homozygous Bph14 and Bph15 were resistant to BPH. However, the hybrids with heterozygous Bph14 and Bph15 remained susceptible to BPH. The developed NILs showed no significant differences in major agronomic traits and rice qualities compared with the recurrent parent. Moreover, the improved hybrids derived from the NILs exhibited better agronomic performance and rice quality compared with the controls under natural field conditions. This study demonstrates that it is essential to stack Bph14 and Bph15 into both the maternal and paternal parents for developing BPH-resistant hybrid rice varieties. The SNP array with abundant DNA markers is an efficient tool for analyzing the RPG recovery of progenies and can be used to monitor the donor segments in NILs, thus being extremely important for rice molecular breeding.

Mild Intellectual Disability Associated with a Progeny of Father-Daughter Incest: Genetic and Environmental Considerations

ERIC Educational Resources Information Center

Ansermet, Francois; Lespinasse, James; Gimelli, Stefania; Bena, Frederique; Paoloni-Giacobino, Ariane

2010-01-01

We report the case of a 34-year-old female resulting from a father-daughter sexual abuse and presenting a phenotype of mild intellectual disability with minor dysmorphic features. Karyotyping showed a normal 46, XX constitution. Array-based comparative genomic hybridization (array-CGH) revealed a heterozygote 320kb 6p22.3 microdeletion in the…

A Novel 6.14 Mb Duplication of Chromosome 8p21 in a Patient with Autism and Self Mutilation

ERIC Educational Resources Information Center

Ozgen, Heval M.; Staal, Wouter G.; Barber, John C.; de Jonge, Maretha V.; Eleveld, Marc J.; Beemer, Frits A.; Hochstenbach, Ron; Poot, Martin

2009-01-01

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with a strong genetic etiology. Cytogenetic abnormalities have been detected in 5-10% of the patients with autism. In this study, we present the clinical, cytogenetic and array-comparative genomic hybridization (array-CGH) evaluation of a 13-year-old male with severe…

Caryoscope: An Open Source Java application for viewing microarray data in a genomic context

PubMed Central

Awad, Ihab AB; Rees, Christian A; Hernandez-Boussard, Tina; Ball, Catherine A; Sherlock, Gavin

2004-01-01

Background Microarray-based comparative genome hybridization experiments generate data that can be mapped onto the genome. These data are interpreted more easily when represented graphically in a genomic context. Results We have developed Caryoscope, which is an open source Java application for visualizing microarray data from array comparative genome hybridization experiments in a genomic context. Caryoscope can read General Feature Format files (GFF files), as well as comma- and tab-delimited files, that define the genomic positions of the microarray reporters for which data are obtained. The microarray data can be browsed using an interactive, zoomable interface, which helps users identify regions of chromosomal deletion or amplification. The graphical representation of the data can be exported in a number of graphic formats, including publication-quality formats such as PostScript. Conclusion Caryoscope is a useful tool that can aid in the visualization, exploration and interpretation of microarray data in a genomic context. PMID:15488149

Enteropathy-associated T-cell lymphoma--a clinicopathologic and array comparative genomic hybridization study.

PubMed

Ko, Young Hyeh; Karnan, Sivasundaram; Kim, Kyeong Mee; Park, Cheol Keun; Kang, Eun Suk; Kim, Young Ho; Kang, Won Ki; Kim, Seok Jin; Kim, Won Seog; Lee, Woo Yong; Chun, Ho Kyung; Seto, Masao

2010-09-01

According to the new World Health Organization classification system, there are 2 types of enteropathy-associated T-cell lymphoma. Type 1 is associated with celiac disease and accounts for the majority of cases in Western countries, whereas type 2 is not associated with celiac disease. To characterize enteropathy-associated T-cell lymphoma types in Korea, we carried out clinicopathologic and immunophenotypic analyses of 8 Koreans with enteropathy-associated T-cell lymphoma and investigated genomic profile using array comparative genomic hybridization. The tumors involved the small intestine in 5 patients and the colorectum in 3 patients. Two patients carried an HLA DQB10302 allele that corresponds to HLA DQ8. None of the patients had gluten-sensitive malabsorption syndrome. Intraepithelial lymphocytosis was observed in all patients. The sizes of the tumor cells were small or small-to-medium in 7 cases and medium-to-large in 1 case. The immunophenotypes of the tumor cells were CD4-CD8+CD56+ in 4 cases, CD4-CD8+CD56- in 1 case, CD4-CD8-CD56+ in 2 cases, and CD4-CD8-CD56- in 1 case. Array comparative genomic hybridization analysis showed that chromosome 9q33-q34.1 gain was present in 4 (80%) of the 5 cases examined. Other recurrent genomic alterations were gain of 6p21.1-21.31 (3/5, 60%), gain of 19q (2/5), and the loss of 3p12.1-p12.2 (2/5) and 3q26.31 (2/5). These results suggest that the most prevalent type of enteropathy-associated T-cell lymphoma in this geographic region is type 2, and the genetic changes associated with it are similar to those in Western countries. Copyright 2010 Elsevier Inc. All rights reserved.

Predicting Hybrid Performances for Quality Traits through Genomic-Assisted Approaches in Central European Wheat

PubMed Central

Liu, Guozheng; Zhao, Yusheng; Gowda, Manje; Longin, C. Friedrich H.; Reif, Jochen C.; Mette, Michael F.

2016-01-01

Bread-making quality traits are central targets for wheat breeding. The objectives of our study were to (1) examine the presence of major effect QTLs for quality traits in a Central European elite wheat population, (2) explore the optimal strategy for predicting the hybrid performance for wheat quality traits, and (3) investigate the effects of marker density and the composition and size of the training population on the accuracy of prediction of hybrid performance. In total 135 inbred lines of Central European bread wheat (Triticum aestivum L.) and 1,604 hybrids derived from them were evaluated for seven quality traits in up to six environments. The 135 parental lines were genotyped using a 90k single-nucleotide polymorphism array. Genome-wide association mapping initially suggested presence of several quantitative trait loci (QTLs), but cross-validation rather indicated the absence of major effect QTLs for all quality traits except of 1000-kernel weight. Genomic selection substantially outperformed marker-assisted selection in predicting hybrid performance. A resampling study revealed that increasing the effective population size in the estimation set of hybrids is relevant to boost the accuracy of prediction for an unrelated test population. PMID:27383841

Progressive but Previously Untreated CLL Patients with Greater Array CGH Complexity Exhibit a Less Durable Response to Chemoimmunotherapy

PubMed Central

Kay, Neil E.; Eckel-Passow, Jeanette E.; Braggio, Esteban; VanWier, Scott; Shanafelt, Tait D.; Van Dyke, Daniel L.; Jelinek, Diane F.; Tschumper, Renee C.; Kipps, Thomas; Byrd, John C.; Fonseca, Rafael

2010-01-01

To better understand the implications of genomic instability and outcome in B-cell CLL, we sought to address genomic complexity as a predictor of chemosensitivity and ultimately clinical outcome in this disease. We employed array-based comparative genomic hybridization (aCGH), using a one-million probe array and identified gains and losses of genetic material in 48 patients treated on a chemoimmunotherapy (CIT) clinical trial. We identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. Higher genomic complexity, as a mechanism favoring clonal selection, was associated with shorter progression-free survival and predicted a poor response to treatment. Of interest, CLL cases with loss of p53 surveillance showed more complex genomic features and were found both in patients with a 17p13.1 deletion and in the more favorable genetic subtype characterized by the presence of 13q14.1 deletion. This aCGH study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. PMID:21156228

Copy number variants analysis in a cohort of isolated and syndromic developmental delay/intellectual disability reveals novel genomic disorders, position effects and candidate disease genes.

PubMed

Di Gregorio, E; Riberi, E; Belligni, E F; Biamino, E; Spielmann, M; Ala, U; Calcia, A; Bagnasco, I; Carli, D; Gai, G; Giordano, M; Guala, A; Keller, R; Mandrile, G; Arduino, C; Maffè, A; Naretto, V G; Sirchia, F; Sorasio, L; Ungari, S; Zonta, A; Zacchetti, G; Talarico, F; Pappi, P; Cavalieri, S; Giorgio, E; Mancini, C; Ferrero, M; Brussino, A; Savin, E; Gandione, M; Pelle, A; Giachino, D F; De Marchi, M; Restagno, G; Provero, P; Cirillo Silengo, M; Grosso, E; Buxbaum, J D; Pasini, B; De Rubeis, S; Brusco, A; Ferrero, G B

2017-10-01

Array-comparative genomic hybridization (array-CGH) is a widely used technique to detect copy number variants (CNVs) associated with developmental delay/intellectual disability (DD/ID). Identification of genomic disorders in DD/ID. We performed a comprehensive array-CGH investigation of 1,015 consecutive cases with DD/ID and combined literature mining, genetic evidence, evolutionary constraint scores, and functional information in order to assess the pathogenicity of the CNVs. We identified non-benign CNVs in 29% of patients. Amongst the pathogenic variants (11%), detected with a yield consistent with the literature, we found rare genomic disorders and CNVs spanning known disease genes. We further identified and discussed 51 cases with likely pathogenic CNVs spanning novel candidate genes, including genes encoding synaptic components and/or proteins involved in corticogenesis. Additionally, we identified two deletions spanning potential Topological Associated Domain (TAD) boundaries probably affecting the regulatory landscape. We show how phenotypic and genetic analyses of array-CGH data allow unraveling complex cases, identifying rare disease genes, and revealing unexpected position effects. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH

PubMed Central

2009-01-01

Background Comparative genomic hybridization microarrays for the detection of constitutional chromosomal aberrations is the application of microarray technology coming fastest into routine clinical application. Through genotype-phenotype association, it is also an important technique towards the discovery of disease causing genes and genomewide functional annotation in human. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing a patient against a normal reference sample. Two major disadvantages of this setup are (1) the use of half of the resources to measure a (little informative) reference sample and (2) the possibility that deviating signals are caused by benign copy number variation in the "normal" reference instead of a patient aberration. Instead, we apply an experimental loop design that compares three patients in three hybridizations. Results We develop and compare two statistical methods (linear models of log ratios and mixed models of absolute measurements). In an analysis of 27 patients seen at our genetics center, we observed that the linear models of the log ratios are advantageous over the mixed models of the absolute intensities. Conclusion The loop design and the performance of the statistical analysis contribute to the quick adoption of array CGH as a routine diagnostic tool. They lower the detection limit of mosaicisms and improve the assignment of copy number variation for genetic association studies. PMID:19925645

An experimental loop design for the detection of constitutional chromosomal aberrations by array CGH.

PubMed

Allemeersch, Joke; Van Vooren, Steven; Hannes, Femke; De Moor, Bart; Vermeesch, Joris Robert; Moreau, Yves

2009-11-19

Comparative genomic hybridization microarrays for the detection of constitutional chromosomal aberrations is the application of microarray technology coming fastest into routine clinical application. Through genotype-phenotype association, it is also an important technique towards the discovery of disease causing genes and genomewide functional annotation in human. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing a patient against a normal reference sample. Two major disadvantages of this setup are (1) the use of half of the resources to measure a (little informative) reference sample and (2) the possibility that deviating signals are caused by benign copy number variation in the "normal" reference instead of a patient aberration. Instead, we apply an experimental loop design that compares three patients in three hybridizations. We develop and compare two statistical methods (linear models of log ratios and mixed models of absolute measurements). In an analysis of 27 patients seen at our genetics center, we observed that the linear models of the log ratios are advantageous over the mixed models of the absolute intensities. The loop design and the performance of the statistical analysis contribute to the quick adoption of array CGH as a routine diagnostic tool. They lower the detection limit of mosaicisms and improve the assignment of copy number variation for genetic association studies.

[Application of array-based comparative genomic hybridization technique in genetic analysis of patients with spontaneous abortion].

PubMed

Chu, Y; Wu, D; Hou, Q F; Huo, X D; Gao, Y; Wang, T; Wang, H D; Yang, Y L; Liao, S X

2016-08-25

To investigate the value of array-based comparative genomic hybridization (array-CGH) technique for the detection of chromosomal analysis of miscarried embryo, and to provide genetic counseling for couples with spontaneous abortion. Totally 382 patients who underwent miscarriage were enrolled in this study. All aborted tissues were analyzed with conventional cytogenetic karyotyping and array-CGH, respectively. Through genetic analysis, all of the 382 specimens were successfully analyzed by array-CGH (100.0%, 382/382), and the detection rate of chromosomal aberrations was 46.6% (178/382). However, conventional karyotype analysis was successfully performed in 281 cases (73.6%, 281/382), and 113 (40.2%, 113/281) were found with chromosomal aberrations. Of these 178 samples identified by array-CGH, 163 samples (91.6%, 163/178) were aneuploidy, 15 samples (8.4%, 15/178) were segmental deletion and (or) duplication cases. Four of 10 cases with small segmental deletion and duplication were validated to be transferred from their fathers or mathers who were carriers of submicroscopic reciprocal translocation. Of these 113 abnormal karyotypes founded by conventional karyotyping, 108 cases (95.6%, 108/113) were aneuploidy and 5 cases (4.4%, 5/113) had chromosome structural aberrations. Most array-CGH results were consistent with conventional karyotyping but with 3 cases of discrepancy, which included 2 cases of triploids, 1 case of low-level mosaicism that undetcted by array-CGH. Compared with conventional karyotyping, there is an increased detection rate of chromosomal abnormalities when array-CGH is used to analyse the products of conception, primarilly because of its sucess with nonviable tissues. It could be a first-line method to determine the reason of miscarrage with higher accuracy and sensitivity.

DASH-2: Flexible, Low-Cost, and High-Throughput SNP Genotyping by Dynamic Allele-Specific Hybridization on Membrane Arrays

PubMed Central

Jobs, Magnus; Howell, W. Mathias; Strömqvist, Linda; Mayr, Torsten; Brookes, Anthony J.

2003-01-01

Genotyping technologies need to be continually improved in terms of their flexibility, cost-efficiency, and throughput, to push forward genome variation analysis. To this end, we have leveraged the inherent simplicity of dynamic allele-specific hybridization (DASH) and coupled it to recent innovations of centrifugal arrays and iFRET. We have thereby created a new genotyping platform we term DASH-2, which we demonstrate and evaluate in this report. The system is highly flexible in many ways (any plate format, PCR multiplexing, serial and parallel array processing, spectral-multiplexing of hybridization probes), thus supporting a wide range of application scales and objectives. Precision is demonstrated to be in the range 99.8–100%, and assay costs are 0.05 USD or less per genotype assignment. DASH-2 thus provides a powerful new alternative for genotyping practice, which can be used without the need for expensive robotics support. PMID:12727908

High-resolution array comparative genomic hybridization (aCGH) identifies copy number alterations in diffuse large B-cell lymphoma that predict response to immuno-chemotherapy

PubMed Central

Kreisel, F.; Kulkarni, S.; Kerns, R. T.; Hassan, A.; Deshmukh, H.; Nagarajan, R.; Frater, J. L.; Cashen, A.

2013-01-01

Despite recent attempts at sub-categorization, including gene expression profiling into prognostically different groups of “germinal center B-cell type” and “activated B-cell type”, diffuse large B-cell lymphoma (DLBCL) remains a biologically heterogenous tumor with no clear prognostic biomarkers to guide therapy. Whole genome, high resolution array comparative genomic hybridization (aCGH) was performed on 4 cases of chemoresistant DLBCL and 4 cases of chemo-responsive DLBCL to identify genetic differences which may correlate with response to R-CHOP therapy. Array CGH analysis identified 7 DNA copy number alteration (CNA) regions exclusive to the chemoresistant group, consisting of amplifications at 1p36.13, 1q42.3, 3p21.31, 7q11.23, and 16p13.3, and loss at 9p21.3, and 14p21.31. Copy number loss of the tumor suppressor genes CDKN2A (p16, p14) and CDKN2B (p15) at 9p21.3 was validated by fluorescence in situ hybridization and immunohistochemistry as independent techniques. In the chemo-sensitive group, 12 CNAs were detected consisting of segment gains on 1p36.11, 1p36.22, 2q11.2, 8q24.3, 12p13.33, and 22q13.2 and segment loss on 6p21.32. RUNX3, a tumor suppressor gene located on 1p36.11 and MTHFR, which encodes for the enzyme methylenetetrahydrofolate reductase, located on 1p36.22 are the only known genes in this group associated with lymphoma. Whole genome aCGH analysis has detected copy number alterations exclusive to either chemoresistant or chemo-responsive DLBCL that may represent consistent clonal changes predictive for prognosis and outcome of chemotherapy. PMID:21504712

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives.

PubMed

Zhao, Min; Wang, Qingguo; Wang, Quan; Jia, Peilin; Zhao, Zhongming

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development.

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives

PubMed Central

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development. PMID:24564169

Comparative cytogenetic characterization of primary canine melanocytic lesions using array CGH and fluorescence in situ hybridization

PubMed Central

Poorman, Kelsey; Borst, Luke; Moroff, Scott; Roy, Siddharth; Labelle, Philippe; Motsinger-Reif, Alison

2017-01-01

Melanocytic lesions originating from the oral mucosa or cutaneous epithelium are common in the general dog population, with up to 100,000 diagnoses each year in the USA. Oral melanoma is the most frequent canine neoplasm of the oral cavity, exhibiting a highly aggressive course. Cutaneous melanocytomas occur frequently, but rarely develop into a malignant form. Despite the differential prognosis, it has been assumed that subtypes of melanocytic lesions represent the same disease. To address the relative paucity of information about their genomic status, molecular cytogenetic analysis was performed on the three recognized subtypes of canine melanocytic lesions. Using array comparative genomic hybridization (aCGH) analysis, highly aberrant distinct copy number status across the tumor genome for both of the malignant melanoma subtypes was revealed. The most frequent aberrations included gain of dog chromosome (CFA) 13 and 17 and loss of CFA 22. Melanocytomas possessed fewer genome wide aberrations, yet showed a recurrent gain of CFA 20q15.3–17. A distinctive copy number profile, evident only in oral melanomas, displayed a sigmoidal pattern of copy number loss followed immediately by a gain, around CFA 30q14. Moreover, when assessed by fluorescence in situ hybridization (FISH), copy number aberrations of targeted genes, such as gain of c-MYC (80 % of cases) and loss of CDKN2A (68 % of cases), were observed. This study suggests that in concordance with what is known for human melanomas, canine melanomas of the oral mucosa and cutaneous epithelium are discrete and initiated by different molecular pathways. PMID:25511566

Population sequencing reveals breed and sub-species specific CNVs in cattle

USDA-ARS?s Scientific Manuscript database

Individualized copy number variation (CNV) maps have highlighted the need for population surveys of cattle to detect rare and common variants. While SNP and comparative genomic hybridization (CGH) arrays have provided preliminary data, next-generation sequence (NGS) data analysis offers an increased...

Early-onset seizures due to mosaic exonic deletions of CDKL5 in a male and two females.

PubMed

Bartnik, Magdalena; Derwińska, Katarzyna; Gos, Monika; Obersztyn, Ewa; Kołodziejska, Katarzyna E; Erez, Ayelet; Szpecht-Potocka, Agnieszka; Fang, Ping; Terczyńska, Iwona; Mierzewska, Hanna; Lohr, Naomi J; Bellus, Gary A; Reimschisel, Tyler; Bocian, Ewa; Mazurczak, Tadeusz; Cheung, Sau Wai; Stankiewicz, Paweł

2011-05-01

Mutations in the CDKL5 gene have been associated with an X-linked dominant early infantile epileptic encephalopathy-2. The clinical presentation is usually of severe encephalopathy with refractory seizures and Rett syndrome (RTT)-like phenotype. We attempted to assess the role of mosaic intragenic copy number variation in CDKL5. We have used comparative genomic hybridization with a custom-designed clinical oligonucleotide array targeting exons of selected disease and candidate genes, including CDKL5. We have identified mosaic exonic deletions of CDKL5 in one male and two females with developmental delay and medically intractable seizures. These three mosaic changes represent 60% of all deletions detected in 12,000 patients analyzed by array comparative genomic hybridization and involving the exonic portion of CDKL5. We report the first case of an exonic deletion of CDKL5 in a male and emphasize the importance of underappreciated mosaic exonic copy number variation in patients with early-onset seizures and RTT-like features of both genders.

Characterization of canine osteosarcoma by array comparative genomic hybridization and RT-qPCR: signatures of genomic imbalance in canine osteosarcoma parallel the human counterpart.

PubMed

Angstadt, Andrea Y; Motsinger-Reif, Alison; Thomas, Rachael; Kisseberth, William C; Guillermo Couto, C; Duval, Dawn L; Nielsen, Dahlia M; Modiano, Jaime F; Breen, Matthew

2011-11-01

Osteosarcoma (OS) is the most commonly diagnosed malignant bone tumor in humans and dogs, characterized in both species by extremely complex karyotypes exhibiting high frequencies of genomic imbalance. Evaluation of genomic signatures in human OS using array comparative genomic hybridization (aCGH) has assisted in uncovering genetic mechanisms that result in disease phenotype. Previous low-resolution (10-20 Mb) aCGH analysis of canine OS identified a wide range of recurrent DNA copy number aberrations, indicating extensive genomic instability. In this study, we profiled 123 canine OS tumors by 1 Mb-resolution aCGH to generate a dataset for direct comparison with current data for human OS, concluding that several high frequency aberrations in canine and human OS are orthologous. To ensure complete coverage of gene annotation, we identified the human refseq genes that map to these orthologous aberrant dog regions and found several candidate genes warranting evaluation for OS involvement. Specifically, subsequenct FISH and qRT-PCR analysis of RUNX2, TUSC3, and PTEN indicated that expression levels correlated with genomic copy number status, showcasing RUNX2 as an OS associated gene and TUSC3 as a possible tumor suppressor candidate. Together these data demonstrate the ability of genomic comparative oncology to identify genetic abberations which may be important for OS progression. Large scale screening of genomic imbalance in canine OS further validates the use of the dog as a suitable model for human cancers, supporting the idea that dysregulation discovered in canine cancers will provide an avenue for complementary study in human counterparts. Copyright © 2011 Wiley-Liss, Inc.

Automated array-based genomic profiling in chronic lymphocytic leukemia: Development of a clinical tool and discovery of recurrent genomic alterations

PubMed Central

Schwaenen, Carsten; Nessling, Michelle; Wessendorf, Swen; Salvi, Tatjana; Wrobel, Gunnar; Radlwimmer, Bernhard; Kestler, Hans A.; Haslinger, Christian; Stilgenbauer, Stephan; Döhner, Hartmut; Bentz, Martin; Lichter, Peter

2004-01-01

B cell chronic lymphocytic leukemia (B-CLL) is characterized by a highly variable clinical course. Recurrent chromosomal imbalances provide significant prognostic markers. Risk-adapted therapy based on genomic alterations has become an option that is currently being tested in clinical trials. To supply a robust tool for such large scale studies, we developed a comprehensive DNA microarray dedicated to the automated analysis of recurrent genomic imbalances in B-CLL by array-based comparative genomic hybridization (matrix–CGH). Validation of this chip in a series of 106 B-CLL cases revealed a high specificity and sensitivity that fulfils the criteria for application in clinical oncology. This chip is immediately applicable within clinical B-CLL treatment trials that evaluate whether B-CLL cases with distinct chromosomal abnormalities should be treated with chemotherapy of different intensities and/or stem cell transplantation. Through the control set of DNA fragments equally distributed over the genome, recurrent genomic imbalances were discovered: trisomy of chromosome 19 and gain of the MYCN oncogene correlating with an elevation of MYCN mRNA expression. PMID:14730057

Characterization of hemizygous deletions in Citrus using array-Comparative Genomic Hybridization and microsynteny comparisons with the poplar genome

PubMed Central

Ríos, Gabino; Naranjo, Miguel A; Iglesias, Domingo J; Ruiz-Rivero, Omar; Geraud, Marion; Usach, Antonio; Talón, Manuel

2008-01-01

Background Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background. Results Two independent fast neutron mutants of self-incompatible clementine (Citrus clementina Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a Citrus cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available Citrus BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues. Conclusion In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana. This work corroborates the potential of Citrus genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement. PMID:18691431

MobilomeFINDER: web-based tools for in silico and experimental discovery of bacterial genomic islands

PubMed Central

Ou, Hong-Yu; He, Xinyi; Harrison, Ewan M.; Kulasekara, Bridget R.; Thani, Ali Bin; Kadioglu, Aras; Lory, Stephen; Hinton, Jay C. D.; Barer, Michael R.; Rajakumar, Kumar

2007-01-01

MobilomeFINDER (http://mml.sjtu.edu.cn/MobilomeFINDER) is an interactive online tool that facilitates bacterial genomic island or ‘mobile genome’ (mobilome) discovery; it integrates the ArrayOme and tRNAcc software packages. ArrayOme utilizes a microarray-derived comparative genomic hybridization input data set to generate ‘inferred contigs’ produced by merging adjacent genes classified as ‘present’. Collectively these ‘fragments’ represent a hypothetical ‘microarray-visualized genome (MVG)’. ArrayOme permits recognition of discordances between physical genome and MVG sizes, thereby enabling identification of strains rich in microarray-elusive novel genes. Individual tRNAcc tools facilitate automated identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites and other integration hotspots in closely related sequenced genomes. Accessory tools facilitate design of hotspot-flanking primers for in silico and/or wet-science-based interrogation of cognate loci in unsequenced strains and analysis of islands for features suggestive of foreign origins; island-specific and genome-contextual features are tabulated and represented in schematic and graphical forms. To date we have used MobilomeFINDER to analyse several Enterobacteriaceae, Pseudomonas aeruginosa and Streptococcus suis genomes. MobilomeFINDER enables high-throughput island identification and characterization through increased exploitation of emerging sequence data and PCR-based profiling of unsequenced test strains; subsequent targeted yeast recombination-based capture permits full-length sequencing and detailed functional studies of novel genomic islands. PMID:17537813

Population sequencing reveals breed and sub-species specific CNVs in cattle

USDA-ARS?s Scientific Manuscript database

Individualized copy number variation (CNV) maps have highlighted the need for population surveys of cattle to detect the rare and common variants. While SNP and comparative genomic hybridization (CGH) arrays have provided preliminary data, next-generation sequence (NGS) data analysis offers an incre...

Comparison of Comparative Genomic Hybridization Technologies across Microarray Platforms

EPA Science Inventory

In the 2007 Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) project, we analyzed HL-60 DNA with five platforms: Agilent, Affymetrix 500K, Affymetrix U133 Plus 2.0, Illumina, and RPCI 19K BAC arrays. Copy number variation (CNV) was analyzed ...

Human minisatellite alleles detectable only after PCR amplification.

PubMed

Armour, J A; Crosier, M; Jeffreys, A J

1992-01-01

We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.

Identification of DNA copy number aberrations by array comparative genomic hybridization in patients with ruptured intracranial aneurysms.

PubMed

Choi, Jin Soo; Kim, Seong-Rim; Jeon, Yang-Whan; Lee, Kweon-Haeng; Rha, Hyoung Kyun

2009-02-01

We aimed to use array comparative genomic hybridization (CGH) to identify chromosomal loci that contribute to the pathogenesis of ruptured intracranial aneurysms (IAs) in a Korean population and to confirm the results using real-time polymerase chain reaction (PCR). Twenty-three patients with ruptured IAs were enrolled in this study. Array CGH revealed copy number aberrations in 19 chromosomal regions. Chromosomal gains were identified at a high frequency in regions 1p12, 4q24, 5p15.31, 5p15.33, 6p12.2, 6q22.33, 7p21.1, 9q22.1, 10q24.32, 10q26.3, 12q13.13, 17p12, 18q12.3, 18q23, 19p13.3, 20q13.33, 21q11.2, and 21q22.3, whereas chromosomal losses were identified at 15q11.2 and 22q11.21. Real-time PCR confirmed the results of the array CGH studies of the COL6A2, GRIN3B, MUC17, and PRODH genes. This is the first study to identify candidate regions by array CGH in patients with IAs. The identification of genes that may predispose an individual to the development of IAs may lead to a better understanding of the mechanism of IA formation. Multicenter studies comparing cohorts of patients of different ethnicities are needed to better understand the mechanism of IA formation.

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community.

PubMed

Yokoi, Takahide; Kaku, Yoshiko; Suzuki, Hiroyuki; Ohta, Masayuki; Ikuta, Hajime; Isaka, Kazuichi; Sumino, Tatsuo; Wagatsuma, Masako

2007-08-01

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.

Array-based comparative genomic hybridization in 190 Korean patients with developmental delay and/or intellectual disability: a single tertiary care university center study.

PubMed

Lee, Cha Gon; Park, Sang-Jin; Yun, Jun-No; Ko, Jung Min; Kim, Hyon-Ju; Yim, Shin-Young; Sohn, Young Bae

2013-11-01

This study analyzed and evaluated the demographic, clinical, and cytogenetic data [G-banded karyotyping and array-based comparative genomic hybridization (array CGH)] of patients with unexplained developmental delay or intellectual disability at a single Korean institution. We collected clinical and cytogenetic data based on retrospective charts at Ajou University Medical Center, Suwon, Korea from April 2008 to March 2012. A total of 190 patients were identified. Mean age was 5.1±1.87 years. Array CGH yielded abnormal results in 26 of 190 patients (13.7%). Copy number losses were about two-fold more frequent than gains. A total of 61.5% of all patients had copy number losses. The most common deletion disorders included 22q11.2 deletion syndrome, 15q11.2q12 deletion and 18q deletion syndrome. Copy number gains were identified in 34.6% of patients, and common diseases among these included Potocki-Lupski syndrome, 15q11-13 duplication syndrome and duplication 22q. Abnormal karyotype with normal array CGH results was exhibited in 2.6% of patients; theses included balanced translocation (n=2), inversion (n=2) and low-level mosaicism (n=1). Facial abnormalities (p<0.001) and failure to thrive were (p<0.001) also more frequent in the group of patients with abnormal CGH findings. Array CGH is a useful diagnostic tool in clinical settings in patients with developmental delay or intellectual disability combined with facial abnormalities or failure to thrive.

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers.

PubMed

Martins, C; Galetti, P M

2001-10-01

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.

Systematic validation and atomic force microscopy of non-covalent short oligonucleotide barcode microarrays.

PubMed

Cook, Michael A; Chan, Chi-Kin; Jorgensen, Paul; Ketela, Troy; So, Daniel; Tyers, Mike; Ho, Chi-Yip

2008-02-06

Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20-60 base) unique sequence tags, or "barcodes", associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5'-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.

The use of population-scale sequencing to identify CNVs impacting productive traits in different cattle breeds

USDA-ARS?s Scientific Manuscript database

Individualized copy number variation (CNV) maps have highlighted the need for population surveys of cattle to detect rare and common variants. While SNP and comparative genomic hybridization (CGH) arrays have provided preliminary data, next-generation sequence (NGS) data analysis offers an increased...

Syntenic conservation of HSP70 genes in cattle and humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grosz, M.D.; Womack, J.E.; Skow, L.C.

1992-12-01

A phage library of bovine genomic DNA was screened for hybridization with a human HSP70 cDNA probe, and 21 positive plaques were identified and isolated. Restriction mapping and blot hybridization analysis of DNA from the recombinant plaques demonstrated that the cloned DNAs were derived from three different regions of the bovine genome. Ore region contains two tandemly arrayed HSP70 sequences, designated HSP70-1 and HSP70-2, separated by approximately 8 kb of DNA. Single HSP70 sequences, designated HSP70-3 and HSP70-4, were found in two other genomic regions. Locus-specific probes of unique flanking sequences from representative HSP70 clones were hybridized to restriction endonuclease-digestedmore » DNA from bovine-hamster and bovine-mouse somatic cell hybrid panels to determine the chromosomal location of the HSP70 sequences. The probe for the tandemly arrayed HSP70-1 and HSP70-2 sequences mapped to bovine chromosome 23, syntenic with glyoxalase 1, 21 steroid hydroxylase, and major histocompatibility class I loci. HSP70-3 sequences mapped to bovine chromosome 10, syntenic with nucleoside phosphorylase and murine osteosarcoma viral oncogene (v-fos), and HSP70-4 mapped to bovine syntenic group U6, syntenic with amylase 1 and phosphoglucomutase 1. On the basis of these data, the authors propose that bovine HSP70-1,2 are homologous to human HSPA1 and HSPA1L on chromosome 6p21.3, bovine HSP70-3 is the homolog of an unnamed human HSP70 gene on chromosome 14q22-q24, and bovine HSP70-4 is homologous to one of the human HSPA-6,-7 genes on chromosome 1. 34 refs., 2 figs., 1 tab.« less

Improved analytical methods for microarray-based genome-composition analysis

PubMed Central

Kim, Charles C; Joyce, Elizabeth A; Chan, Kaman; Falkow, Stanley

2002-01-01

Background Whereas genome sequencing has given us high-resolution pictures of many different species of bacteria, microarrays provide a means of obtaining information on genome composition for many strains of a given species. Genome-composition analysis using microarrays, or 'genomotyping', can be used to categorize genes into 'present' and 'divergent' categories based on the level of hybridization signal. This typically involves selecting a signal value that is used as a cutoff to discriminate present (high signal) and divergent (low signal) genes. Current methodology uses empirical determination of cutoffs for classification into these categories, but this methodology is subject to several problems that can result in the misclassification of many genes. Results We describe a method that depends on the shape of the signal-ratio distribution and does not require empirical determination of a cutoff. Moreover, the cutoff is determined on an array-to-array basis, accounting for variation in strain composition and hybridization quality. The algorithm also provides an estimate of the probability that any given gene is present, which provides a measure of confidence in the categorical assignments. Conclusions Many genes previously classified as present using static methods are in fact divergent on the basis of microarray signal; this is corrected by our algorithm. We have reassigned hundreds of genes from previous genomotyping studies of Helicobacter pylori and Campylobacter jejuni strains, and expect that the algorithm should be widely applicable to genomotyping data. PMID:12429064

Array comparative genome hybridization in patients with developmental delay: two example cases.

PubMed

Hancarova, Miroslava; Drabova, Jana; Zmitkova, Zuzana; Vlckova, Marketa; Hedvicakova, Petra; Novotna, Drahuse; Vlckova, Zdenka; Vejvalkova, Sarka; Marikova, Tatana; Sedlacek, Zdenek

2012-02-15

Developmental delay is often a predictor of mental retardation (MR) or autism, two relatively frequent developmental disorders severely affecting intellectual and social functioning. The causes of these conditions remain unknown in most patients. They have a strong genetic component, but the specific genetic defects can only be identified in a fraction of patients. Recent developments in genomics supported the establishment of the causal link between copy number variants in the genomes of some patients and their affection. One of the techniques suitable for this analysis is array comparative genome hybridization, which can be used both for detailed mapping of chromosome rearrangements identified by classical cytogenetics and for the identification of novel submicroscopic gains or losses of genetic material. We illustrate the power of this approach in two patients. Patient 1 had a cytogenetically visible deletion of chromosome X and the molecular analysis was used to specify the gene content of the deletion and the prognosis of the child. Patient 2 had a seemingly normal karyotype and the analysis revealed a small recurrent deletion of chromosome 1 likely to be responsible for his phenotype. However, the genetic dissection of MR and autism is complicated by high heterogeneity of the genetic aberrations among patients and by broad variability of phenotypic effects of individual genetic defects. Copyright © 2010 Elsevier B.V. All rights reserved.

Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array.

PubMed

Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina; Guldberg, Per; Dufva, Martin; Wang, Shan X; Hansen, Mikkel F

2017-09-26

Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors

PubMed Central

Brunner, Clemens; Brunner-Herglotz, Bettina; Ziegler, Andrea; Frech, Christian; Amann, Gabriele; Ladenstein, Ruth; Ambros, Inge M.; Ambros, Peter F.

2016-01-01

Introduction Tumor touch imprints (TTIs) are routinely used for the molecular diagnosis of neuroblastomas by interphase fluorescence in-situ hybridization (I-FISH). However, in order to facilitate a comprehensive, up-to-date molecular diagnosis of neuroblastomas and to identify new markers to refine risk and therapy stratification methods, whole genome approaches are needed. We examined the applicability of an ultra-high density SNP array platform that identifies copy number changes of varying sizes down to a few exons for the detection of genomic changes in tumor DNA extracted from TTIs. Material and Methods DNAs were extracted from TTIs of 46 neuroblastoma and 4 other pediatric tumors. The DNAs were analyzed on the Cytoscan HD SNP array platform to evaluate numerical and structural genomic aberrations. The quality of the data obtained from TTIs was compared to that from randomly chosen fresh or fresh frozen solid tumors (n = 212) and I-FISH validation was performed. Results SNP array profiles were obtained from 48 (out of 50) TTI DNAs of which 47 showed genomic aberrations. The high marker density allowed for single gene analysis, e.g. loss of nine exons in the ATRX gene and the visualization of chromothripsis. Data quality was comparable to fresh or fresh frozen tumor SNP profiles. SNP array results were confirmed by I-FISH. Conclusion TTIs are an excellent source for SNP array processing with the advantage of simple handling, distribution and storage of tumor tissue on glass slides. The minimal amount of tumor tissue needed to analyze whole genomes makes TTIs an economic surrogate source in the molecular diagnostic work up of tumor samples. PMID:27560999

Chromosomal Distribution of Endogenous Jaagsiekte Sheep Retrovirus Proviral Sequences in the Sheep Genome

PubMed Central

Carlson, Jonathan; Lyon, Monique; Bishop, Jeanette; Vaiman, Anne; Cribiu, Edmond; Mornex, Jean-François; Brown, Susan; Knudson, Dennis; DeMartini, James; Leroux, Caroline

2003-01-01

A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known. PMID:12915578

Cryptic insertion of 3'FOXO1 into inverted chromosome arm 2q in the presence of two normal chromosome 13s and 13 small interstitial duplications in a patient with alveolar rhabdomyosarcoma.

PubMed

Hackman, Sarah; Calvey, Laura; Bernreuter, Kristen; Mark, Mengya Wang; Starnes, Sarah; Batanian, Jacqueline R

2015-09-01

Alveolar rhabdomyosarcoma (ARMS) is a pediatric soft tissue neoplasm with a characteristic translocation, t(2;13)(q35;q14), which is detected in 70-80% of cases. This well-described translocation produces the gene fusion product PAX3-FOXO1. Cryptic rearrangements of this fusion have never before been reported in ARMS. Here we describe a patient with ARMS that showed, by fluorescence in situ hybridization and G-banded chromosomes, a cryptic insertion of 3'FOXO1 into inverted chromosome 2q. The inversion breakpoints were depicted by array comparative genomic hybridization as two small interstitial duplications, one of which involved the PAX3 gene. In addition, the array comparative genomic hybridization results revealed 1q gain, 16q loss, and 11 more small duplications, with one of them involving the FOXO1 gene. Although the pathogenesis in classic ARMS cases is thought to be driven by the 5'PAX3-3'FOXO1 fusion on derivative chromosome 13, here we report a novel cryptic insertion of 3'FOXO1 resulting in a pathogenic fusion with 5'PAX3 on inverted chromosome 2q. Copyright © 2015 Elsevier Inc. All rights reserved.

Array-CGH analysis in Rwandan patients presenting development delay/intellectual disability with multiple congenital anomalies.

PubMed

Uwineza, Annette; Caberg, Jean-Hubert; Hitayezu, Janvier; Hellin, Anne Cecile; Jamar, Mauricette; Dideberg, Vinciane; Rusingiza, Emmanuel K; Bours, Vincent; Mutesa, Leon

2014-07-12

Array-CGH is considered as the first-tier investigation used to identify copy number variations. Right now, there is no available data about the genetic etiology of patients with development delay/intellectual disability and congenital malformation in East Africa. Array comparative genomic hybridization was performed in 50 Rwandan patients with development delay/intellectual disability and multiple congenital abnormalities, using the Agilent's 180 K microarray platform. Fourteen patients (28%) had a global development delay whereas 36 (72%) patients presented intellectual disability. All patients presented multiple congenital abnormalities. Clinically significant copy number variations were found in 13 patients (26%). Size of CNVs ranged from 0,9 Mb to 34 Mb. Six patients had CNVs associated with known syndromes, whereas 7 patients presented rare genomic imbalances. This study showed that CNVs are present in African population and show the importance to implement genetic testing in East-African countries.

Custom Array Comparative Genomic Hybridization: the Importance of DNA Quality, an Expert Eye, and Variant Validation

PubMed Central

Lantieri, Francesca; Malacarne, Michela; Gimelli, Stefania; Santamaria, Giuseppe; Coviello, Domenico; Ceccherini, Isabella

2017-01-01

The presence of false positive and false negative results in the Array Comparative Genomic Hybridization (aCGH) design is poorly addressed in literature reports. We took advantage of a custom aCGH recently carried out to analyze its design performance, the use of several Agilent aberrations detection algorithms, and the presence of false results. Our study provides a confirmation that the high density design does not generate more noise than standard designs and, might reach a good resolution. We noticed a not negligible presence of false negative and false positive results in the imbalances call performed by the Agilent software. The Aberration Detection Method 2 (ADM-2) algorithm with a threshold of 6 performed quite well, and the array design proved to be reliable, provided that some additional filters are applied, such as considering only intervals with average absolute log2ratio above 0.3. We also propose an additional filter that takes into account the proportion of probes with log2ratio exceeding suggestive values for gain or loss. In addition, the quality of samples was confirmed to be a crucial parameter. Finally, this work raises the importance of evaluating the samples profiles by eye and the necessity of validating the imbalances detected. PMID:28287439

Construction, database integration, and application of an Oenothera EST library.

PubMed

Mrácek, Jaroslav; Greiner, Stephan; Cho, Won Kyong; Rauwolf, Uwe; Braun, Martha; Umate, Pavan; Altstätter, Johannes; Stoppel, Rhea; Mlcochová, Lada; Silber, Martina V; Volz, Stefanie M; White, Sarah; Selmeier, Renate; Rudd, Stephen; Herrmann, Reinhold G; Meurer, Jörg

2006-09-01

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.

Microarray slide hybridization using fluorescently labeled cDNA.

PubMed

Ares, Manuel

2014-01-01

Microarray hybridization is used to determine the amount and genomic origins of RNA molecules in an experimental sample. Unlabeled probe sequences for each gene or gene region are printed in an array on the surface of a slide, and fluorescently labeled cDNA derived from the RNA target is hybridized to it. This protocol describes a blocking and hybridization protocol for microarray slides. The blocking step is particular to the chemistry of "CodeLink" slides, but it serves to remind us that almost every kind of microarray has a treatment step that occurs after printing but before hybridization. We recommend making sure of the precise treatment necessary for the particular chemistry used in the slides to be hybridized because the attachment chemistries differ significantly. Hybridization is similar to northern or Southern blots, but on a much smaller scale.

A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis

PubMed Central

Marquis-Nicholson, Renate; Lai, Daniel; Love, Jennifer M.; Love, Donald R.

2013-01-01

Purpose. The aim of this study was to develop a streamlined mutation screening protocol for the DMD gene in order to confirm a clinical diagnosis of Duchenne or Becker muscular dystrophy in affected males and to clarify the carrier status of female family members. Methods. Sequence analysis and array comparative genomic hybridization (aCGH) were used to identify mutations in the dystrophin DMD gene. We analysed genomic DNA from six individuals with a range of previously characterised mutations and from eight individuals who had not previously undergone any form of molecular analysis. Results. We successfully identified the known mutations in all six patients. A molecular diagnosis was also made in three of the four patients with a clinical diagnosis who had not undergone prior genetic screening, and testing for familial mutations was successfully completed for the remaining four patients. Conclusion. The mutation screening protocol described here meets best practice guidelines for molecular testing of the DMD gene in a diagnostic laboratory. The aCGH method is a superior alternative to more conventional assays such as multiplex ligation-dependent probe amplification (MLPA). The combination of aCGH and sequence analysis will detect mutations in 98% of patients with the Duchenne or Becker muscular dystrophy. PMID:23476807

Ecological Success of a Group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii Hybrids in the Northern European Wine-Making Environment

PubMed Central

Erny, C.; Raoult, P.; Alais, A.; Butterlin, G.; Delobel, P.; Matei-Radoi, F.; Casaregola, S.

2012-01-01

The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24 Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) profile of the Eg8 genome revealed a typical chimeric profile. Measurement of hybrids DNA content per cell by flow cytometry revealed multiple ploidy levels (2n, 3n, or 4n), and restriction fragment length polymorphism analysis of 22 genes indicated variable amounts of S. kudriavzevii genetic content in three representative strains. We developed microsatellite markers for S. kudriavzevii and used them to analyze the diversity of a population isolated from oaks in Ardèche (France). This analysis revealed new insights into the diversity of this species. We then analyzed the diversity of the wine hybrids for 12 S. cerevisiae and 7 S. kudriavzevii microsatellite loci and found that these strains are the products of multiple hybridization events between several S. cerevisiae wine yeast isolates and various S. kudriavzevii strains. The Eg8 lineage appeared remarkable, since it harbors strains found over a wide geographic area, and the interstrain divergence measured with a (δμ)2 genetic distance indicates an ancient origin. These findings reflect the specific adaptations made by S. cerevisiae/S. kudriavzevii cryophilic hybrids to winery environments in cool climates. PMID:22344648

Mutations in X-linked PORCN, a putative regulator of Wnt signaling, cause focal dermal hypoplasia

USDA-ARS?s Scientific Manuscript database

Focal dermal hypoplasia is an X-linked dominant disorder characterized by patchy hypoplastic skin and digital, ocular, and dental malformations. We used array comparative genomic hybridization to identify a 219-kb deletion in Xp11.23 in two affected females. We sequenced genes in this region and fou...

Two Siblings with Alternate Unbalanced Recombinants Derived from a Large Cryptic Maternal Pericentric Inversion of Chromosome 20

PubMed Central

DeScipio, Cheryl; Morrissette, Jennifer J.D.; Conlin, Laura K.; Clark, Dinah; Kaur, Maninder; Coplan, James; Riethman, Harold; Spinner, Nancy B.; Krantz, Ian D.

2009-01-01

Two brothers, with dissimilar clinical features, were each found to have different abnormalities of chromosome 20 by subtelomere fluorescence in situ hybridization (FISH). The proband had deletion of 20p subtelomere and duplication of 20q subtelomere, while his brother was found to have a duplication of 20p subtelomere and deletion of 20q subtelomere. Parental cytogenetic studies were initially thought to be normal, both by G-banding and by subtelomere FISH analysis. Since chromosome 20 is a metacentric chromosome and an inversion was suspected, we used anchored FISH to assist in identifying a possible inversion. This approach employed concomitant hybridization of a FISH probe to the short (p) arm of chromosome 20 with the 20q subtelomere probe. We identified a cytogenetically non-visible, mosaic pericentric inversion of one of the maternal chromosome 20 homologues, providing a mechanistic explanation for the chromosomal abnormalities present in these brothers. Array comparative genomic hybridization (CGH) with both a custom-made BAC and cosmid-based subtelomere specific array (TEL array) and a commercially-available SNP-based array confirmed and further characterized these rearrangements, identifying this as the largest pericentric inversion of chromosome 20 described to date. TEL array data indicate that the 20p breakpoint is defined by BAC RP11-978M13, ~900 kb from the pter; SNP array data reveal this breakpoint to occur within BAC RP11-978M13. The 20q breakpoint is defined by BAC RP11-93B14, ~1.7 Mb from the qter, by TEL array; SNP array data refine this breakpoint to within a gap between BACs on the TEL array (i.e. between RP11-93B14 and proximal BAC RP11-765G16). PMID:20101690

Two siblings with alternate unbalanced recombinants derived from a large cryptic maternal pericentric inversion of chromosome 20.

PubMed

Descipio, Cheryl; Morrissette, Jennifer D; Conlin, Laura K; Clark, Dinah; Kaur, Maninder; Coplan, James; Riethman, Harold; Spinner, Nancy B; Krantz, Ian D

2010-02-01

Two brothers, with dissimilar clinical features, were each found to have different abnormalities of chromosome 20 by subtelomere fluorescence in situ hybridization (FISH). The proband had deletion of 20p subtelomere and duplication of 20q subtelomere, while his brother was found to have a duplication of 20p subtelomere and deletion of 20q subtelomere. Parental cytogenetic studies were initially thought to be normal, both by G-banding and by subtelomere FISH analysis. Since chromosome 20 is a metacentric chromosome and an inversion was suspected, we used anchored FISH to assist in identifying a possible inversion. This approach employed concomitant hybridization of a FISH probe to the short (p) arm of chromosome 20 with the 20q subtelomere probe. We identified a cytogenetically non-visible, mosaic pericentric inversion of one of the maternal chromosome 20 homologs, providing a mechanistic explanation for the chromosomal abnormalities present in these brothers. Array comparative genomic hybridization (CGH) with both a custom-made BAC and cosmid-based subtelomere specific array (TEL array) and a commercially available SNP-based array confirmed and further characterized these rearrangements, identifying this as the largest pericentric inversion of chromosome 20 described to date. TEL array data indicate that the 20p breakpoint is defined by BAC RP11-978M13, approximately 900 kb from the pter; SNP array data reveal this breakpoint to occur within BAC RP11-978M13. The 20q breakpoint is defined by BAC RP11-93B14, approximately 1.7 Mb from the qter, by TEL array; SNP array data refine this breakpoint to within a gap between BACs on the TEL array (i.e., between RP11-93B14 and proximal BAC RP11-765G16). Copyright 2010 Wiley-Liss, Inc.

A Complex 6p25 Rearrangement in a Child With Multiple Epiphyseal Dysplasia

PubMed Central

Bedoyan, Jirair K.; Lesperance, Marci M.; Ackley, Todd; Iyer, Ramaswamy K.; Innis, Jeffrey W.; Misra, Vinod K.

2015-01-01

Genomic rearrangements are increasingly recognized as important contributors to human disease. Here we report on an 11½-year-old child with myopia, Duane retraction syndrome, bilateral mixed hearing loss, skeletal anomalies including multiple epiphyseal dysplasia, and global developmental delay, and a complex 6p25 genomic rearrangement. We have employed oligonucleotide-based comparative genomic hybridization arrays (aCGH) of different resolutions (44 and 244K) as well as a 1 M single nucleotide polymorphism (SNP) array to analyze this complex rearrangement. Our analyses reveal a complex rearrangement involving a ~2.21 Mb interstitial deletion, a ~240 kb terminal deletion, and a 70–80 kb region in between these two deletions that shows maintenance of genomic copy number. The interstitial deletion contains eight known genes, including three Forkhead box containing (FOX) transcription factors (FOXQ1, FOXF2, and FOXC1). The region maintaining genomic copy number partly overlaps the dual specificity protein phosphatase 22 (DUSP22) gene. Array analyses suggest a homozygous loss of genomic material at the 5′ end of DUSP22, which was corroborated using TaqMan® copy number analysis. It is possible that this homozygous genomic loss may render both copies of DUSP22 or its products non-functional. Our analysis suggests a rearrangement mechanism distinct from a previously reported replication-based error-prone mechanism without template switching for a specific 6p25 rearrangement with a 1.22 Mb interstitial deletion. Our study demonstrates the utility and limitations of using oligonucleotide-based aCGH and SNP array technologies of increasing resolutions in order to identify complex DNA rearrangements and gene disruptions. PMID:21204225

Systematic Validation and Atomic Force Microscopy of Non-Covalent Short Oligonucleotide Barcode Microarrays

PubMed Central

Cook, Michael A.; Chan, Chi-Kin; Jorgensen, Paul; Ketela, Troy; So, Daniel; Tyers, Mike; Ho, Chi-Yip

2008-01-01

Background Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20–60 base) unique sequence tags, or “barcodes”, associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. Methodology/Principal Findings Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5′-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. Conclusions/Significance These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis. PMID:18253494

GEAR: genomic enrichment analysis of regional DNA copy number changes.

PubMed

Kim, Tae-Min; Jung, Yu-Chae; Rhyu, Mun-Gan; Jung, Myeong Ho; Chung, Yeun-Jun

2008-02-01

We developed an algorithm named GEAR (genomic enrichment analysis of regional DNA copy number changes) for functional interpretation of genome-wide DNA copy number changes identified by array-based comparative genomic hybridization. GEAR selects two types of chromosomal alterations with potential biological relevance, i.e. recurrent and phenotype-specific alterations. Then it performs functional enrichment analysis using a priori selected functional gene sets to identify primary and clinical genomic signatures. The genomic signatures identified by GEAR represent functionally coordinated genomic changes, which can provide clues on the underlying molecular mechanisms related to the phenotypes of interest. GEAR can help the identification of key molecular functions that are activated or repressed in the tumor genomes leading to the improved understanding on the tumor biology. GEAR software is available with online manual in the website, http://www.systemsbiology.co.kr/GEAR/.

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

PubMed Central

2013-01-01

Background There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. Results A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. Conclusions We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes. PMID:23758725

Integrated analysis of copy number alteration and RNA expression profiles of cancer using a high-resolution whole-genome oligonucleotide array.

PubMed

Jung, Seung-Hyun; Shin, Seung-Hun; Yim, Seon-Hee; Choi, Hye-Sun; Lee, Sug-Hyung; Chung, Yeun-Jun

2009-07-31

Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.

Microarray-based comparative genomic hybridization analysis in neonates with congenital anomalies: detection of chromosomal imbalances.

PubMed

Emy Dorfman, Luiza; Leite, Júlio César L; Giugliani, Roberto; Riegel, Mariluce

2015-01-01

To identify chromosomal imbalances by whole-genome microarray-based comparative genomic hybridization (array-CGH) in DNA samples of neonates with congenital anomalies of unknown cause from a birth defects monitoring program at a public maternity hospital. A blind genomic analysis was performed retrospectively in 35 stored DNA samples of neonates born between July of 2011 and December of 2012. All potential DNA copy number variations detected (CNVs) were matched with those reported in public genomic databases, and their clinical significance was evaluated. Out of a total of 35 samples tested, 13 genomic imbalances were detected in 12/35 cases (34.3%). In 4/35 cases (11.4%), chromosomal imbalances could be defined as pathogenic; in 5/35 (14.3%) cases, DNA CNVs of uncertain clinical significance were identified; and in 4/35 cases (11.4%), normal variants were detected. Among the four cases with results considered causally related to the clinical findings, two of the four (50%) showed causative alterations already associated with well-defined microdeletion syndromes. In two of the four samples (50%), the chromosomal imbalances found, although predicted as pathogenic, had not been previously associated with recognized clinical entities. Array-CGH analysis allowed for a higher rate of detection of chromosomal anomalies, and this determination is especially valuable in neonates with congenital anomalies of unknown etiology, or in cases in which karyotype results cannot be obtained. Moreover, although the interpretation of the results must be refined, this method is a robust and precise tool that can be used in the first-line investigation of congenital anomalies, and should be considered for prospective/retrospective analyses of DNA samples by birth defect monitoring programs. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Gene conversion events and variable degree of homogenization of rDNA loci in cultivars of Brassica napus

PubMed Central

Sochorová, Jana; Coriton, Olivier; Kuderová, Alena; Lunerová, Jana; Chèvre, Anne-Marie; Kovařík, Aleš

2017-01-01

Background and aims Brassica napus (AACC, 2n = 38, oilseed rape) is a relatively recent allotetraploid species derived from the putative progenitor diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). To determine the influence of intensive breeding conditions on the evolution of its genome, we analysed structure and copy number of rDNA in 21 cultivars of B. napus, representative of genetic diversity. Methods We used next-generation sequencing genomic approaches, Southern blot hybridization, expression analysis and fluorescence in situ hybridization (FISH). Subgenome-specific sequences derived from rDNA intergenic spacers (IGS) were used as probes for identification of loci composition on chromosomes. Key Results Most B. napus cultivars (18/21, 86 %) had more A-genome than C-genome rDNA copies. Three cultivars analysed by FISH (‘Darmor’, ‘Yudal’ and ‘Asparagus kale’) harboured the same number (12 per diploid set) of loci. In B. napus ‘Darmor’, the A-genome-specific rDNA probe hybridized to all 12 rDNA loci (eight on the A-genome and four on the C-genome) while the C-genome-specific probe showed weak signals on the C-genome loci only. Deep sequencing revealed high homogeneity of arrays suggesting that the C-genome genes were largely overwritten by the A-genome variants in B. napus ‘Darmor’. In contrast, B. napus ‘Yudal’ showed a lack of gene conversion evidenced by additive inheritance of progenitor rDNA variants and highly localized hybridization signals of subgenome-specific probes on chromosomes. Brassica napus ‘Asparagus kale’ showed an intermediate pattern to ‘Darmor’ and ‘Yudal’. At the expression level, most cultivars (95 %) exhibited stable A-genome nucleolar dominance while one cultivar (‘Norin 9’) showed co-dominance. Conclusions The B. napus cultivars differ in the degree and direction of rDNA homogenization. The prevalent direction of gene conversion (towards the A-genome) correlates with the direction of expression dominance indicating that gene activity may be needed for interlocus gene conversion. PMID:27707747

Identification of IL11RA and MELK amplification in gastric cancer by comprehensive genomic profiling of gastric cancer cell lines

PubMed Central

Calcagno, Danielle Queiroz; Takeno, Sylvia Santomi; Gigek, Carolina Oliveira; Leal, Mariana Ferreira; Wisnieski, Fernanda; Chen, Elizabeth Suchi; Araújo, Taíssa Maíra Thomaz; Lima, Eleonidas Moura; Melaragno, Maria Isabel; Demachki, Samia; Assumpção, Paulo Pimentel; Burbano, Rommel Rodriguez; Smith, Marília Cardoso

2016-01-01

AIM To identify common copy number alterations on gastric cancer cell lines. METHODS Four gastric cancer cell lines (ACP02, ACP03, AGP01 and PG100) underwent chromosomal comparative genome hybridization and array comparative genome hybridization. We also confirmed the results by fluorescence in situ hybridization analysis using the bacterial artificial chromosome clone and quantitative real time PCR analysis. RESULTS The amplification of 9p13.3 was detected in all cell lines by both methodologies. An increase in the copy number of 9p13.3 was also confirmed by fluorescence in situ hybridization analysis. Moreover, the interleukin 11 receptor alpha (IL11RA) and maternal embryonic leucine zipper kinase (MELK) genes, which are present in the 9p13.3 amplicon, revealed gains of the MELK gene in all the cell lines studied. Additionally, a gain in the copy number of IL11RA and MELK was observed in 19.1% (13/68) and 55.9% (38/68) of primary gastric adenocarcinoma samples, respectively. CONCLUSION The characterization of a small gain region at 9p13.3 in gastric cancer cell lines and primary gastric adenocarcinoma samples has revealed MELK as a candidate target gene that is possibly related to the development of gastric cancer. PMID:27920471

Global assessment of genomic variation in cattle by genome resequencing and high-throughput genotyping

PubMed Central

2011-01-01

Background Integration of genomic variation with phenotypic information is an effective approach for uncovering genotype-phenotype associations. This requires an accurate identification of the different types of variation in individual genomes. Results We report the integration of the whole genome sequence of a single Holstein Friesian bull with data from single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) array technologies to determine a comprehensive spectrum of genomic variation. The performance of resequencing SNP detection was assessed by combining SNPs that were identified to be either in identity by descent (IBD) or in copy number variation (CNV) with results from SNP array genotyping. Coding insertions and deletions (indels) were found to be enriched for size in multiples of 3 and were located near the N- and C-termini of proteins. For larger indels, a combination of split-read and read-pair approaches proved to be complementary in finding different signatures. CNVs were identified on the basis of the depth of sequenced reads, and by using SNP and CGH arrays. Conclusions Our results provide high resolution mapping of diverse classes of genomic variation in an individual bovine genome and demonstrate that structural variation surpasses sequence variation as the main component of genomic variability. Better accuracy of SNP detection was achieved with little loss of sensitivity when algorithms that implemented mapping quality were used. IBD regions were found to be instrumental for calculating resequencing SNP accuracy, while SNP detection within CNVs tended to be less reliable. CNV discovery was affected dramatically by platform resolution and coverage biases. The combined data for this study showed that at a moderate level of sequencing coverage, an ensemble of platforms and tools can be applied together to maximize the accurate detection of sequence and structural variants. PMID:22082336

A functional gene array for detection of bacterial virulence elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaing, C

2007-11-01

We report our development of the first of a series of microarrays designed to detect pathogens with known mechanisms of virulence and antibiotic resistance. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. To validate our approach, we developed a first generation array targeting genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for microorganism detection and discrimination, measured the required target concentration, and assessedmore » tolerance for mismatches between probe and target sequences. Mismatch tolerance is a priority for this application, due to DNA sequence variability among members of gene families. Arrays were created using the NimbleGen Maskless Array Synthesizer at Lawrence Livermore National Laboratory. Purified genomic DNA from combinations of one or more of the four target organisms, pure cultures of four related organisms, and environmental aerosol samples with spiked-in genomic DNA were hybridized to the arrays. Based on the success of this prototype, we plan to design further arrays in this series, with the goal of detecting all known virulence and antibiotic resistance gene families in a greatly expanded set of organisms.« less

Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort.

PubMed

Retterer, Kyle; Scuffins, Julie; Schmidt, Daniel; Lewis, Rachel; Pineda-Alvarez, Daniel; Stafford, Amanda; Schmidt, Lindsay; Warren, Stephanie; Gibellini, Federica; Kondakova, Anastasia; Blair, Amanda; Bale, Sherri; Matyakhina, Ludmila; Meck, Jeanne; Aradhya, Swaroop; Haverfield, Eden

2015-08-01

Detection of copy-number variation (CNV) is important for investigating many genetic disorders. Testing a large clinical cohort by array comparative genomic hybridization provides a deep perspective on the spectrum of pathogenic CNV. In this context, we describe a bioinformatics approach to extract CNV information from whole-exome sequencing and demonstrate its utility in clinical testing. Exon-focused arrays and whole-genome chromosomal microarray analysis were used to test 14,228 and 14,000 individuals, respectively. Based on these results, we developed an algorithm to detect deletions/duplications in whole-exome sequencing data and a novel whole-exome array. In the exon array cohort, we observed a positive detection rate of 2.4% (25 duplications, 318 deletions), of which 39% involved one or two exons. Chromosomal microarray analysis identified 3,345 CNVs affecting single genes (18%). We demonstrate that our whole-exome sequencing algorithm resolves CNVs of three or more exons. These results demonstrate the clinical utility of single-exon resolution in CNV assays. Our whole-exome sequencing algorithm approaches this resolution but is complemented by a whole-exome array to unambiguously identify intragenic CNVs and single-exon changes. These data illustrate the next advancements in CNV analysis through whole-exome sequencing and whole-exome array.Genet Med 17 8, 623-629.

Uprobe: a genome-wide universal probe resource for comparative physical mapping in vertebrates.

PubMed

Kellner, Wendy A; Sullivan, Robert T; Carlson, Brian H; Thomas, James W

2005-01-01

Interspecies comparisons are important for deciphering the functional content and evolution of genomes. The expansive array of >70 public vertebrate genomic bacterial artificial chromosome (BAC) libraries can provide a means of comparative mapping, sequencing, and functional analysis of targeted chromosomal segments that is independent and complementary to whole-genome sequencing. However, at the present time, no complementary resource exists for the efficient targeted physical mapping of the majority of these BAC libraries. Universal overgo-hybridization probes, designed from regions of sequenced genomes that are highly conserved between species, have been demonstrated to be an effective resource for the isolation of orthologous regions from multiple BAC libraries in parallel. Here we report the application of the universal probe design principal across entire genomes, and the subsequent creation of a complementary probe resource, Uprobe, for screening vertebrate BAC libraries. Uprobe currently consists of whole-genome sets of universal overgo-hybridization probes designed for screening mammalian or avian/reptilian libraries. Retrospective analysis, experimental validation of the probe design process on a panel of representative BAC libraries, and estimates of probe coverage across the genome indicate that the majority of all eutherian and avian/reptilian genes or regions of interest can be isolated using Uprobe. Future implementation of the universal probe design strategy will be used to create an expanded number of whole-genome probe sets that will encompass all vertebrate genomes.

A ddRAD Based Linkage Map of the Cultivated Strawberry, Fragaria xananassa

PubMed Central

Davik, Jahn; Sargent, Daniel James; Brurberg, May Bente; Lien, Sigbjørn; Kent, Matthew; Alsheikh, Muath

2015-01-01

The cultivated strawberry (Fragaria ×ananassa Duch.) is an allo-octoploid considered difficult to disentangle genetically due to its four relatively similar sub-genomic chromosome sets. This has been alleviated by the recent release of the strawberry IStraw90 whole genome genotyping array. However, array resolution relies on the genotypes used in the array construction and may be of limited general use. SNP detection based on reduced genomic sequencing approaches has the potential of providing better coverage in cases where the studied genotypes are only distantly related from the SNP array’s construction foundation. Here we have used double digest restriction-associated DNA sequencing (ddRAD) to identify SNPs in a 145 seedling F1 hybrid population raised from the cross between the cultivars Sonata (♀) and Babette (♂). A linkage map containing 907 markers which spanned 1,581.5 cM across 31 linkage groups representing the 28 chromosomes of the species. Comparing the physical span of the SNP markers with the F. vesca genome sequence, the linkage groups resolved covered 79% of the estimated 830 Mb of the F. ×ananassa genome. Here, we have developed the first linkage map for F. ×ananassa using ddRAD and show that this technique and other related techniques are useful tools for linkage map development and downstream genetic studies in the octoploid strawberry. PMID:26398886

Multiplex PCR-based DNA array for simultaneous detection of three human herpesviruses, EVB, CMV and KSHV.

PubMed

Fujimuro, Masahiro; Nakaso, Kazuhiro; Nakashima, Kenji; Sadanari, Hidetaka; Hisanori, Inoue; Teishikata, Yasuhiro; Hayward, S Diane; Yokosawa, Hideyoshi

2006-04-01

Human lymphotropic herpesviruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are responsible for a wide variety of human diseases. Due to an increase in diseased states associated with immunosuppression, more instances of co-morbid infections with these herpesviruses have resulted in viral reactivations that have caused numerous fatalities. Therefore, the development of rapid and accurate method to detect these viruses in immunocompromised patients is vital for immediate treatment with antiviral prophylactic drugs. In this study, we developed a new multiplex PCR method coupled to DNA array hybridization, which can simultaneously detect all three human herpesviruses in one single cell sample. Multiplex PCR primers were designed to amplify specific regions of the EBV (EBER1), CMV (IE) and KSHV (LANA) viral genomes. Pre-clinical application of this method revealed that this approach is capable of detecting as few as 1 copy of the viral genomes for KSHV and CMV and 100 copies of the genome for EBV. Furthermore, this highly sensitive test showed no cross-reactivity among the three viruses and is capable of detecting both KSHV and EBV viral genomes simultaneously in the lymphoblastoid cells that have been double infected with both viruses. Thus, this array-based approach serves as a rapid and reliable diagnostic tool for clinical applications.

[Analysis of genetics mechanism for the phenotypic diversity in a patient carrying a rare ring chromosome 9].

PubMed

Qin, Shengfang; Wang, Xueyan; Li, Yunxing; Wei, Ping; Chen, Chun; Zeng, Lan

2016-02-01

To explore the genetics mechanism for the phenotypic variability in a patient carrying a rare ring chromosome 9. The karyotype of the patient was analyzed with cytogenetics method. Presence of sex chromosome was confirmed with fluorescence in situ hybridization. The SRY gene was subjected to PCR amplification and direct sequencing. Potential deletion and duplication were detected with array-based comparative genomic hybridization (array-CGH). The karyotype of the patient has comprised 6 types of cell lines containing a ring chromosome 9. The SRY gene sequence was normal. By array-CGH, the patient has carried a hemizygous deletion at 9p24.3-p23 (174 201-9 721 761) encompassing 30 genes from Online Mendelian Inheritance in Man. The phenotypic variability of the 9p deletion syndrome in conjunct with ring chromosome 9 may be attributable to multiple factors including loss of chromosomal material, insufficient dosage of genes, instability of ring chromosome, and pattern of inheritance.

Development of a High-Throughput Resequencing Array for the Detection of Pathogenic Mutations in Osteogenesis Imperfecta

PubMed Central

Wang, Yao; Cui, Yazhou; Zhou, Xiaoyan; Han, Jinxiang

2015-01-01

Objective Osteogenesis imperfecta (OI) is a rare inherited skeletal disease, characterized by bone fragility and low bone density. The mutations in this disorder have been widely reported to be on various exonal hotspots of the candidate genes, including COL1A1, COL1A2, CRTAP, LEPRE1, and FKBP10, thus creating a great demand for precise genetic tests. However, large genome sizes make the process daunting and the analyses, inefficient and expensive. Therefore, we aimed at developing a fast, accurate, efficient, and cheaper sequencing platform for OI diagnosis; and to this end, use of an advanced array-based technique was proposed. Method A CustomSeq Affymetrix Resequencing Array was established for high-throughput sequencing of five genes simultaneously. Genomic DNA extraction from 13 OI patients and 85 normal controls and amplification using long-range PCR (LR-PCR) were followed by DNA fragmentation and chip hybridization, according to standard Affymetrix protocols. Hybridization signals were determined using GeneChip Sequence Analysis Software (GSEQ). To examine the feasibility, the outcome from new resequencing approach was validated by conventional capillary sequencing method. Result Overall call rates using resequencing array was 96–98% and the agreement between microarray and capillary sequencing was 99.99%. 11 out of 13 OI patients with pathogenic mutations were successfully detected by the chip analysis without adjustment, and one mutation could also be identified using manual visual inspection. Conclusion A high-throughput resequencing array was developed that detects the disease-associated mutations in OI, providing a potential tool to facilitate large-scale genetic screening for OI patients. Through this method, a novel mutation was also found. PMID:25742658

Comprehensive identification of mutations induced by heavy-ion beam irradiation in Arabidopsis thaliana.

PubMed

Hirano, Tomonari; Kazama, Yusuke; Ishii, Kotaro; Ohbu, Sumie; Shirakawa, Yuki; Abe, Tomoko

2015-04-01

Heavy-ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy-ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole-genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array-CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy-ion beams. Array-CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar-ion and Fe-ion irradiation, respectively, with deletion sizes ranging from 149 to 602,180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar-ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy-ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy-ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Molecular karyotyping by array CGH in a Russian cohort of children with intellectual disability, autism, epilepsy and congenital anomalies

PubMed Central

2012-01-01

Background Array comparative genomic hybridization (CGH) has been repeatedly shown to be a successful tool for the identification of genomic variations in a clinical population. During the last decade, the implementation of array CGH has resulted in the identification of new causative submicroscopic chromosome imbalances and copy number variations (CNVs) in neuropsychiatric (neurobehavioral) diseases. Currently, array-CGH-based technologies have become an integral part of molecular diagnosis and research in individuals with neuropsychiatric disorders and children with intellectual disability (mental retardation) and congenital anomalies. Here, we introduce the Russian cohort of children with intellectual disability, autism, epilepsy and congenital anomalies analyzed by BAC array CGH and a novel bioinformatic strategy. Results Among 54 individuals highly selected according to clinical criteria and molecular and cytogenetic data (from 2426 patients evaluated cytogenetically and molecularly between November 2007 and May 2012), chromosomal imbalances were detected in 26 individuals (48%). In two patients (4%), a previously undescribed condition was observed. The latter has been designated as meiotic (constitutional) genomic instability resulted in multiple submicroscopic rearrangements (including CNVs). Using bioinformatic strategy, we were able to identify clinically relevant CNVs in 15 individuals (28%). Selected cases were confirmed by molecular cytogenetic and molecular genetic methods. Eight out of 26 chromosomal imbalances (31%) have not been previously reported. Among them, three cases were co-occurrence of subtle chromosome 9 and 21 deletions. Conclusions We conducted an array CGH study of Russian patients suffering from intellectual disability, autism, epilepsy and congenital anomalies. In total, phenotypic manifestations of clinically relevant genomic variations were found to result from genomic rearrangements affecting 1247 disease-causing and pathway-involved genes. Obviously, a significantly lesser part of them are true candidates for intellectual disability, autism or epilepsy. The success of our preliminary array CGH and bioinformatic study allows us to expand the cohort. According to the available literature, this is the first comprehensive array CGH evaluation of a Russian cohort of children with neuropsychiatric disorders and congenital anomalies. PMID:23272938

X-chromosome tiling path array detection of copy number variants in patients with chromosome X-linked mental retardation

PubMed Central

Madrigal, I; Rodríguez-Revenga, L; Armengol, L; González, E; Rodriguez, B; Badenas, C; Sánchez, A; Martínez, F; Guitart, M; Fernández, I; Arranz, JA; Tejada, MI; Pérez-Jurado, LA; Estivill, X; Milà, M

2007-01-01

Background Aproximately 5–10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects. Results Known genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%). Conclusion This tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients. PMID:18047645

Development and mapping of DArT markers within the Festuca - Lolium complex

PubMed Central

Kopecký, David; Bartoš, Jan; Lukaszewski, Adam J; Baird, James H; Černoch, Vladimír; Kölliker, Roland; Rognli, Odd Arne; Blois, Helene; Caig, Vanessa; Lübberstedt, Thomas; Studer, Bruno; Shaw, Paul; Doležel, Jaroslav; Kilian, Andrzej

2009-01-01

Background Grasses are among the most important and widely cultivated plants on Earth. They provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Among cultivated grasses, species within the Festuca-Lolium complex predominate, especially in temperate regions. To facilitate high-throughput genome profiling and genetic mapping within the complex, we have developed a Diversity Arrays Technology (DArT) array for five grass species: F. pratensis, F. arundinacea, F. glaucescens, L. perenne and L. multiflorum. Results The DArTFest array contains 7680 probes derived from methyl-filtered genomic representations. In a first marker discovery experiment performed on 40 genotypes from each species (with the exception of F. glaucescens for which only 7 genotypes were used), we identified 3884 polymorphic markers. The number of DArT markers identified in every single genotype varied from 821 to 1852. To test the usefulness of DArTFest array for physical mapping, DArT markers were assigned to each of the seven chromosomes of F. pratensis using single chromosome substitution lines while recombinants of F. pratensis chromosome 3 were used to allocate the markers to seven chromosome bins. Conclusion The resources developed in this project will facilitate the development of genetic maps in Festuca and Lolium, the analysis on genetic diversity, and the monitoring of the genomic constitution of the Festuca × Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions. PMID:19832973

Bridging the gap from prenatal karyotyping to whole-genome array comparative genomic hybridization in Hong Kong: survey on knowledge and acceptance of health-care providers and pregnant women.

PubMed

Cheng, Hiu Yee Heidi; Kan, Anita Sik-Yau; Hui, Pui Wah; Lee, Chin Peng; Tang, Mary Hoi Yin

2017-12-01

The use of array comparative genomic hybridization (aCGH) has been increasingly widespread. The challenge of integration of this technology into prenatal diagnosis was the interpretation of results and communicating findings of unclear clinical significance. This study assesses the knowledge and acceptance of prenatal aCGH in Hong Kong obstetricians and pregnant women. The aim is to identify the needs and gaps before implementing the replacement of karyotyping with aCGH. Questionnaires with aCGH information in the form of pamphlets were sent by post to obstetrics and gynecology doctors. For the pregnant women group, a video presentation, pamphlets on aCGH and a self-administered questionnaire were provided at the antenatal clinic. The perception of aCGH between doctors and pregnant women was similar. Doctors not choosing aCGH were more concerned about the difficulty in counseling of variants of unknown significance and adult-onset disease in pregnant women, whereas pregnant women not choosing aCGH were more concerned about the increased waiting time leading to increased anxiety. Prenatal aCGH is perceived as a better test by both doctors and patients. Counseling support, training, and better understanding and communication of findings of unclear clinical significance are necessary to improve doctor-patient experience.

Assessment of the role of copy-number variants in 150 patients with congenital heart defects.

PubMed

Derwińska, Katarzyna; Bartnik, Magdalena; Wiśniowiecka-Kowalnik, Barbara; Jagła, Mateusz; Rudziński, Andrzej; Pietrzyk, Jacek J; Kawalec, Wanda; Ziółkowska, Lidia; Kutkowska-Kaźmierczak, Anna; Gambin, Tomasz; Sykulski, Maciej; Shaw, Chad A; Gambin, Anna; Mazurczak, Tadeusz; Obersztyn, Ewa; Bocian, Ewa; Stankiewicz, Paweł

2012-01-01

Congenital heart defects are the most common group of major birth anomalies and one of the leading causes of infant deaths. Mendelian and chromosomal syndromes account for about 20% of congenital heart defects and in some cases are associated with other malformations, intellectual disability, and/or dysmorphic features. The remarkable conservation of genetic pathways regulating heart development in animals suggests that genetic factors can be responsible for a significantly higher percentage of cases. Assessment of the role of CNVs in the etiology of congenital heart defects using microarray studies. Genome-wide array comparative genomic hybridization, targeting genes known to play an important role in heart development or responsible for abnormal cardiac phenotype was used in the study on 150 patients. In addition, we have used multiplex ligation-dependent probe amplification specific for chromosome 22q11.2 region. We have identified 21 copy-number variants, including 13 known causative recurrent rearrangements (12 deletions 22q11.2 and one deletion 7q11.23), three potentially pathogenic duplications (5q14.2, 15q13.3, and 22q11.2), and five variants likely benign for cardiac anomalies. We suggest that abnormal copy-number of the ARRDC3 and KLF13 genes can be responsible for heart defects. Our study demonstrates that array comparative genomic hybridization enables detection of clinically significant chromosomal imbalances in patients with congenital heart defects.

Detection limit of intragenic deletions with targeted array comparative genomic hybridization

PubMed Central

2013-01-01

Background Pathogenic mutations range from single nucleotide changes to deletions or duplications that encompass a single exon to several genes. The use of gene-centric high-density array comparative genomic hybridization (aCGH) has revolutionized the detection of intragenic copy number variations. We implemented an exon-centric design of high-resolution aCGH to detect single- and multi-exon deletions and duplications in a large set of genes using the OGT 60 K and 180 K arrays. Here we describe the molecular characterization and breakpoint mapping of deletions at the smaller end of the detectable range in several genes using aCGH. Results The method initially implemented to detect single to multiple exon deletions, was able to detect deletions much smaller than anticipated. The selected deletions we describe vary in size, ranging from over 2 kb to as small as 12 base pairs. The smallest of these deletions are only detectable after careful manual review during data analysis. Suspected deletions smaller than the detection size for which the method was optimized, were rigorously followed up and confirmed with PCR-based investigations to uncover the true detection size limit of intragenic deletions with this technology. False-positive deletion calls often demonstrated single nucleotide changes or an insertion causing lower hybridization of probes demonstrating the sensitivity of aCGH. Conclusions With optimizing aCGH design and careful review process, aCGH can uncover intragenic deletions as small as dozen bases. These data provide insight that will help optimize probe coverage in array design and illustrate the true assay sensitivity. Mapping of the breakpoints confirms smaller deletions and contributes to the understanding of the mechanism behind these events. Our knowledge of the mutation spectra of several genes can be expected to change as previously unrecognized intragenic deletions are uncovered. PMID:24304607

Transcript Profiling of Common Bean (Phaseolus vulgaris) Using the GeneChip Soybean Genome Array: Optimizing Analysis by Masking Biased Probes

USDA-ARS?s Scientific Manuscript database

Common bean (Phaseolus vulgaris) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common b...

Programmable Self-Assembly of DNA-Dendrimer and DNA-Fullerene Nanostructures

DTIC Science & Technology

2004-10-01

separated by high pressure liquid chromatography ( HPLC ). The resulting material was analytically pure (99%) and monodisperse. Hybridization...bacterial and viral recognition, and gene expression analysis . These major accomplishments have been disseminated by various applications including 16...designing DNA strands with specific structural properties. The direct analysis of genomic DNA and RNA in an array format without labeling or

Cytogenic and molecular analyses of 46,XX male syndrome with clinical comparison to other groups with testicular azoospermia of genetic origin.

PubMed

Chiang, Han-Sun; Wu, Yi-No; Wu, Chien-Chih; Hwang, Jiann-Loung

2013-02-01

XX male is a rare sex chromosomal disorder in infertile men. The purpose of this study was to distinguish the clinical and genetic features of the 46,XX male syndrome from other more frequent, testicular-origin azoospermic causes of male infertility. To study 46,XX male syndrome, we compared clinical and endocrinological parameters to other groups with testicular-origin azoospermia, and to an age-matched group of healthy males and females as normal control. Fluorescent in situ hybridization for detection and localization of the sex-determining region of the Y gene (SRY), array-based comparative genomic hybridization screening, and real-time qualitative polymerase chain reaction of FGF9, WT1, NR5A1, and SPRY2 genes were performed in this genetic investigation. Our three patients with 46,XX male syndrome had a much higher follicular-stimulating hormone level, lower body height, lower testosterone level, and ambiguous external genitalia. One of the three patients with 46,XX male syndrome was SRY-negative. A further genetic study, including a comparative genomic hybridization array and real-time polymerase chain reaction, showed a gain of FGF9 copy numbers only in the SRY-negative 46,XX male. The genetic copy number of the FGF9 gene was duplicated in that case compared to the normal female control and was significantly lower than that of the normal male control. No such genomic gain was observed in the case of the two SRY-positive 46,XX males. Similar to clinical manifestations of 46,XX male syndrome, genetic evidence in this study suggests that FGF9 may contribute to sex reversal, but additional confirmation with more cases is still needed. Copyright © 2012. Published by Elsevier B.V.

Array Comparative Genomic Hybridization as the First-line Investigation for Neonates with Congenital Heart Disease: Experience in a Single Tertiary Center.

PubMed

Choi, Bo Geum; Hwang, Su Kyung; Kwon, Jung Eun; Kim, Yeo Hyang

2018-03-01

The purpose of the present study was to investigate the advantages and disadvantages of verifying genetic abnormalities using array comparative genomic hybridization (a-CGH) immediately after diagnosis of congenital heart disease (CHD). Among neonates under the age of 28 days who underwent echocardiography from January 1, 2014 to April 30, 2016, neonates whose chromosomal and genomic abnormalities were tested using a-CGH in cases of an abnormal finding on echocardiography were enrolled. Of the 166 patients diagnosed with CHD, 81 underwent a-CGH and 11 patients (11/81, 13.5%) had abnormal findings on a-CGH. 22q11.2 deletion syndrome was the most common (4/11, 36.4%). On the first a-CGH, 4 patients were negative (4/81, 5%). Three of them were finally diagnosed with Williams syndrome using fluorescent in situ hybridization (FISH), 1 patient was diagnosed with Noonan syndrome through exome sequencing. All of them exhibited diffuse pulmonary artery branch hypoplasia, as well as increased velocity of blood flow, on repeated echocardiography. Five patients started rehabilitation therapy at mean 6 months old age in outpatient clinics and epilepsy was diagnosed in 2 patients. Parents of 2 patients (22q11.2 deletion syndrome and Patau syndrome) refused treatment due to the anticipated prognosis. Screening tests for genetic abnormalities using a-CGH in neonates with CHD has the advantage of early diagnosis of genetic abnormality during the neonatal period in which there is no obvious symptom of genetic abnormality. However, there are disadvantages that some genetic abnormalities cannot be identified on a-CGH. Copyright © 2018. The Korean Society of Cardiology.

Array Comparative Genomic Hybridization as the First-line Investigation for Neonates with Congenital Heart Disease: Experience in a Single Tertiary Center

PubMed Central

2018-01-01

Background and Objectives The purpose of the present study was to investigate the advantages and disadvantages of verifying genetic abnormalities using array comparative genomic hybridization (a-CGH) immediately after diagnosis of congenital heart disease (CHD). Methods Among neonates under the age of 28 days who underwent echocardiography from January 1, 2014 to April 30, 2016, neonates whose chromosomal and genomic abnormalities were tested using a-CGH in cases of an abnormal finding on echocardiography were enrolled. Results Of the 166 patients diagnosed with CHD, 81 underwent a-CGH and 11 patients (11/81, 13.5%) had abnormal findings on a-CGH. 22q11.2 deletion syndrome was the most common (4/11, 36.4%). On the first a-CGH, 4 patients were negative (4/81, 5%). Three of them were finally diagnosed with Williams syndrome using fluorescent in situ hybridization (FISH), 1 patient was diagnosed with Noonan syndrome through exome sequencing. All of them exhibited diffuse pulmonary artery branch hypoplasia, as well as increased velocity of blood flow, on repeated echocardiography. Five patients started rehabilitation therapy at mean 6 months old age in outpatient clinics and epilepsy was diagnosed in 2 patients. Parents of 2 patients (22q11.2 deletion syndrome and Patau syndrome) refused treatment due to the anticipated prognosis. Conclusions Screening tests for genetic abnormalities using a-CGH in neonates with CHD has the advantage of early diagnosis of genetic abnormality during the neonatal period in which there is no obvious symptom of genetic abnormality. However, there are disadvantages that some genetic abnormalities cannot be identified on a-CGH. PMID:29557107

High-resolution array comparative genomic hybridization analysis of human bronchial and salivary adenoid cystic carcinoma.

PubMed

Bernheim, Alain; Toujani, Saloua; Saulnier, Patrick; Robert, Thomas; Casiraghi, Odile; Validire, Pierre; Temam, Stéphane; Menard, Philippe; Dessen, Philippe; Fouret, Pierre

2008-05-01

Adenoid cystic carcinoma (ACC) is a rare but distinctive tumor. Oligonucleotide array comparative genomic hybridization has been applied for cataloging genomic copy number alterations (CNAs) in 17 frozen salivary or bronchial tumors. Only four whole chromosome CNAs were found, and most cases had 2-4 segmental CNAs. No high level amplification was observed. There were recurrent gains at 7p15.2, 17q21-25, and 22q11-13, and recurrent losses at 1p35, 6q22-25, 8q12-13, 9p21, 12q12-13, and 17p11-13. The minimal region of gain at 7p15.2 contained the HOXA cluster. The minimal common regions of deletions contained the CDKN2A/CDKN2B, TP53, and LIMA1 tumor suppressor genes. The recurrent deletion at 8q12.3-13.1 contained no straightforward tumor suppressor gene, but the MIRN124A2 microRNA gene, whose product regulates MMP2 and CDK6. Among unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, MDM2, and JAK2. The CNAs involving CCND1, MDM2, KIT, CDKN2A/2B, and TP53 were validated by FISH and/or multiplex ligation-dependent probe amplification. Although most tumors overexpressed cyclin D1 compared with surrounding glands, the only case to overexpress MDM2 had the corresponding CNA. In conclusion, our report suggests that ACC is characterized by a relatively low level of structural complexity. Array CGH and immunohistochemical data implicate MDM2 as the oncogene targeted at 12q15. The gain at 4q12 warrants further exploration as it contains a cluster of receptor kinase genes (KIT/PDGFRA/KDR), whose products can be responsive to specific therapies.

Sunflower Hybrid Breeding: From Markers to Genomic Selection

PubMed Central

Dimitrijevic, Aleksandra; Horn, Renate

2018-01-01

In sunflower, molecular markers for simple traits as, e.g., fertility restoration, high oleic acid content, herbicide tolerance or resistances to Plasmopara halstedii, Puccinia helianthi, or Orobanche cumana have been successfully used in marker-assisted breeding programs for years. However, agronomically important complex quantitative traits like yield, heterosis, drought tolerance, oil content or selection for disease resistance, e.g., against Sclerotinia sclerotiorum have been challenging and will require genome-wide approaches. Plant genetic resources for sunflower are being collected and conserved worldwide that represent valuable resources to study complex traits. Sunflower association panels provide the basis for genome-wide association studies, overcoming disadvantages of biparental populations. Advances in technologies and the availability of the sunflower genome sequence made novel approaches on the whole genome level possible. Genotype-by-sequencing, and whole genome sequencing based on next generation sequencing technologies facilitated the production of large amounts of SNP markers for high density maps as well as SNP arrays and allowed genome-wide association studies and genomic selection in sunflower. Genome wide or candidate gene based association studies have been performed for traits like branching, flowering time, resistance to Sclerotinia head and stalk rot. First steps in genomic selection with regard to hybrid performance and hybrid oil content have shown that genomic selection can successfully address complex quantitative traits in sunflower and will help to speed up sunflower breeding programs in the future. To make sunflower more competitive toward other oil crops higher levels of resistance against pathogens and better yield performance are required. In addition, optimizing plant architecture toward a more complex growth type for higher plant densities has the potential to considerably increase yields per hectare. Integrative approaches combining omic technologies (genomics, transcriptomics, proteomics, metabolomics and phenomics) using bioinformatic tools will facilitate the identification of target genes and markers for complex traits and will give a better insight into the mechanisms behind the traits. PMID:29387071

Characterization of Capsicum annuum Genetic Diversity and Population Structure Based on Parallel Polymorphism Discovery with a 30K Unigene Pepper GeneChip

PubMed Central

Hill, Theresa A.; Ashrafi, Hamid; Reyes-Chin-Wo, Sebastian; Yao, JiQiang; Stoffel, Kevin; Truco, Maria-Jose; Kozik, Alexander; Michelmore, Richard W.; Van Deynze, Allen

2013-01-01

The widely cultivated pepper, Capsicum spp., important as a vegetable and spice crop world-wide, is one of the most diverse crops. To enhance breeding programs, a detailed characterization of Capsicum diversity including morphological, geographical and molecular data is required. Currently, molecular data characterizing Capsicum genetic diversity is limited. The development and application of high-throughput genome-wide markers in Capsicum will facilitate more detailed molecular characterization of germplasm collections, genetic relationships, and the generation of ultra-high density maps. We have developed the Pepper GeneChip® array from Affymetrix for polymorphism detection and expression analysis in Capsicum. Probes on the array were designed from 30,815 unigenes assembled from expressed sequence tags (ESTs). Our array design provides a maximum redundancy of 13 probes per base pair position allowing integration of multiple hybridization values per position to detect single position polymorphism (SPP). Hybridization of genomic DNA from 40 diverse C. annuum lines, used in breeding and research programs, and a representative from three additional cultivated species (C. frutescens, C. chinense and C. pubescens) detected 33,401 SPP markers within 13,323 unigenes. Among the C. annuum lines, 6,426 SPPs covering 3,818 unigenes were identified. An estimated three-fold reduction in diversity was detected in non-pungent compared with pungent lines, however, we were able to detect 251 highly informative markers across these C. annuum lines. In addition, an 8.7 cM region without polymorphism was detected around Pun1 in non-pungent C. annuum. An analysis of genetic relatedness and diversity using the software Structure revealed clustering of the germplasm which was confirmed with statistical support by principle components analysis (PCA) and phylogenetic analysis. This research demonstrates the effectiveness of parallel high-throughput discovery and application of genome-wide transcript-based markers to assess genetic and genomic features among Capsicum annuum. PMID:23409153

Characterization of Capsicum annuum genetic diversity and population structure based on parallel polymorphism discovery with a 30K unigene Pepper GeneChip.

PubMed

Hill, Theresa A; Ashrafi, Hamid; Reyes-Chin-Wo, Sebastian; Yao, JiQiang; Stoffel, Kevin; Truco, Maria-Jose; Kozik, Alexander; Michelmore, Richard W; Van Deynze, Allen

2013-01-01

The widely cultivated pepper, Capsicum spp., important as a vegetable and spice crop world-wide, is one of the most diverse crops. To enhance breeding programs, a detailed characterization of Capsicum diversity including morphological, geographical and molecular data is required. Currently, molecular data characterizing Capsicum genetic diversity is limited. The development and application of high-throughput genome-wide markers in Capsicum will facilitate more detailed molecular characterization of germplasm collections, genetic relationships, and the generation of ultra-high density maps. We have developed the Pepper GeneChip® array from Affymetrix for polymorphism detection and expression analysis in Capsicum. Probes on the array were designed from 30,815 unigenes assembled from expressed sequence tags (ESTs). Our array design provides a maximum redundancy of 13 probes per base pair position allowing integration of multiple hybridization values per position to detect single position polymorphism (SPP). Hybridization of genomic DNA from 40 diverse C. annuum lines, used in breeding and research programs, and a representative from three additional cultivated species (C. frutescens, C. chinense and C. pubescens) detected 33,401 SPP markers within 13,323 unigenes. Among the C. annuum lines, 6,426 SPPs covering 3,818 unigenes were identified. An estimated three-fold reduction in diversity was detected in non-pungent compared with pungent lines, however, we were able to detect 251 highly informative markers across these C. annuum lines. In addition, an 8.7 cM region without polymorphism was detected around Pun1 in non-pungent C. annuum. An analysis of genetic relatedness and diversity using the software Structure revealed clustering of the germplasm which was confirmed with statistical support by principle components analysis (PCA) and phylogenetic analysis. This research demonstrates the effectiveness of parallel high-throughput discovery and application of genome-wide transcript-based markers to assess genetic and genomic features among Capsicum annuum.

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

PubMed Central

2011-01-01

Background With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. Results RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. Conclusions An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment. PMID:21235785

Identification of De Novo and Rare Inherited Copy Number Variants in Children with Syndromic Congenital Heart Defects.

PubMed

Hussein, Ibtessam R; Bader, Rima S; Chaudhary, Adeel G; Bassiouni, Randa; Alquaiti, Maha; Ashgan, Fai; Schulten, Hans-Juergen; Al Qahtani, Mohammad H

2018-06-01

Congenital heart defects (CHDs) are the most common birth defects in neonatal life. CHDs could be presented as isolated defects or associated with developmental delay (DD) and/or other congenital malformations. A small proportion of cardiac defects are caused by chromosomal abnormalities or single gene defects; however, in a large proportion of cases no genetic diagnosis could be achieved by clinical examination and conventional genetic analysis. The development of genome wide array-Comparative Genomic Hybridization technique (array-CGH) allowed for the detection of cryptic chromosomal imbalances and pathogenic copy number variants (CNVs) not detected by conventional techniques. We investigated 94 patients having CHDs associated with other malformations and/or DD. Clinical examination and Echocardiography was done to all patients to evaluate the type of CHD. To investigate for genome defects we applied high-density array-CGH 2 × 400K (41 patients) and CGH/SNP microarray 2 × 400K (Agilent) for 53 patients. Confirmation of results was done using Fluorescent in situ hybridization (FISH) or qPCR techniques in certain cases. Chromosomal abnormalities such as trisomy 18, 13, 21, microdeletions: del22q11.2, del7q11.23, del18 (p11.32; p11.21), tetrasomy 18p, trisomy 9p, del11q24-q25, add 15p, add(18)(q21.3), and der 9, 15 (q34.2; q11.2) were detected in 21/94 patients (22%) using both conventional cytogenetics methods and array-CGH technique. Cryptic chromosomal anomalies and pathogenic variants were detected in 15/73 (20.5%) cases. CNVs were observed in a large proportion of the studied samples (27/56) (48%). Clustering of variants was observed in chromosome 1p36, 1p21.1, 2q37, 3q29, 5p15, 7p22.3, 8p23, 11p15.5, 14q11.2, 15q11.2, 16p13.3, 16p11.2, 18p11, 21q22, and 22q11.2. CGH/SNP array could detect loss of heterozygosity (LOH) in different chromosomal loci in 10/25 patients. Array-CGH technique allowed for detection of cryptic chromosomal imbalances that could not be detected by conventional cytogenetics methods. CHDs associated with DD/congenital malformations presented with a relatively high rate of cryptic chromosomal abnormalities. Clustering of CNVs in certain genome loci needs further analysis to identify candidate genes that may provide clues for understanding the molecular pathway of cardiac development.

Enhancing Results of Microarray Hybridizations Through Microagitation

PubMed Central

Toegl, Andreas; Kirchner, Roland; Gauer, Christoph; Wixforth, Achim

2003-01-01

Protein and DNA microarrays have become a standard tool in proteomics/genomics research. In order to guarantee fast and reproducible hybridization results, the diffusion limit must be overcome. Surface acoustic wave (SAW) micro-agitation chips efficiently agitate the smallest sample volumes (down to 10 μL and below) without introducing any dead volume. The advantages are reduced reaction time, increased signal-to-noise ratio, improved homogeneity across the microarray, and better slide-to-slide reproducibility. The SAW micromixer chips are the heart of the Advalytix ArrayBooster, which is compatible with all microarrays based on the microscope slide format. PMID:13678150

[Middle ear salivary gland choristoma related to branchio-oto-renal syndrome diagnosed by array-CGH].

PubMed

Amrhein, P; Sittel, C; Spaich, C; Kohlhase, J; Boppert, R; Kohlhof, P; Koitschev, A

2014-05-01

Branchio-oto-renal (BOR) syndrome is characterized by ear malformations associated with sensorineural or mixed hearing loss. In addition, preauricular tags, preauricular pits, branchial cleft fistulas and cysts, as well as renal dysplasia are seen. A genetic mutation on chromosome 8, either autosomal dominantly inherited or occuring as a spontaneous mutation, is the cause in the majority of cases. Using array-based comparative genomic hybridization (CGH), it is possible to detect even the smallest genetic changes. Salivary gland choristoma in the middle ear is very rare. Surgical removal and histological clarification are required.

Application of bacterial artificial chromosome array-based comparative genomic hybridization and spectral karyotyping to the analysis of glioblastoma multiforme.

PubMed

Cowell, John K; Matsui, Sei-Ichi; Wang, Yong D; LaDuca, Jeffrey; Conroy, Jeffrey; McQuaid, Devin; Nowak, Norma J

2004-05-01

Identification of genetic losses and gains is valuable in analysis of brain tumors. Locus-by-locus analyses have revealed correlations between prognosis and response to chemotherapy and loss or gain of specific genes and loci. These approaches are labor intensive and do not provide a global view of the genetic changes within the tumor cells. Bacterial artificial chromosome (BAC) arrays, which cover the genome with an average resolution of less than 1 MbP, allow defining the sum total of these genetic changes in a single comparative genomic hybridization (CGH) experiment. These changes are directly overlaid on the human genome sequence, thus providing the extent of the amplification or deletion, reflected by a megabase position, and gene content of the abnormal region. Although this array-based CGH approach (CGHa) seems to detect the extent of the genetic changes in tumors reliably, it has not been robustly tested. We compared genetic changes in four newly derived, early-passage glioma cell lines, using spectral karyotyping (SKY) and CGHa. Chromosome changes seen in cell lines under SKY analysis were also detected with CGHa. In addition, CGHa detected cryptic genetic gains and losses and resolved the nature of subtle marker chromosomes that could not be resolved with SKY, thus providing distinct advantages over previous technologies. There was remarkable general concordance between the CGHa results comparing the cell lines to the original tumor, except that the magnitude of the changes seen in the tumor sample was generally suppressed compared with the cell lines, a consequence of normal cells contaminating the tumor sample. CGHa revealed changes in cell lines that were not present in the original tumors and vice versa, even when analyzed at the earliest passage possible, which highlights the adaptation of the cells to in vitro culture. CGHa proved to be highly accurate and efficient for identifying genetic changes in tumor cells. This approach can accurately identify subtle, novel genetic abnormalities in tumors directly linked to the human genome sequence. CGHa far surpasses the resolution and information provided by conventional metaphase CGH, without relying on in vitro culture of tumors for metaphase spreads.

Chromosome catastrophes involve replication mechanisms generating complex genomic rearrangements

PubMed Central

Liu, Pengfei; Erez, Ayelet; Sreenath Nagamani, Sandesh C.; Dhar, Shweta U.; Kołodziejska, Katarzyna E.; Dharmadhikari, Avinash V.; Cooper, M. Lance; Wiszniewska, Joanna; Zhang, Feng; Withers, Marjorie A.; Bacino, Carlos A.; Campos-Acevedo, Luis Daniel; Delgado, Mauricio R.; Freedenberg, Debra; Garnica, Adolfo; Grebe, Theresa A.; Hernández-Almaguer, Dolores; Immken, LaDonna; Lalani, Seema R.; McLean, Scott D.; Northrup, Hope; Scaglia, Fernando; Strathearn, Lane; Trapane, Pamela; Kang, Sung-Hae L.; Patel, Ankita; Cheung, Sau Wai; Hastings, P. J.; Stankiewicz, Paweł; Lupski, James R.; Bi, Weimin

2011-01-01

SUMMARY Complex genomic rearrangements (CGR) consisting of two or more breakpoint junctions have been observed in genomic disorders. Recently, a chromosome catastrophe phenomenon termed chromothripsis, in which numerous genomic rearrangements are apparently acquired in one single catastrophic event, was described in multiple cancers. Here we show that constitutionally acquired CGRs share similarities with cancer chromothripsis. In the 17 CGR cases investigated we observed localization and multiple copy number changes including deletions, duplications and/or triplications, as well as extensive translocations and inversions. Genomic rearrangements involved varied in size and complexities; in one case, array comparative genomic hybridization revealed 18 copy number changes. Breakpoint sequencing identified characteristic features, including small templated insertions at breakpoints and microhomology at breakpoint junctions, which have been attributed to replicative processes. The resemblance between CGR and chromothripsis suggests similar mechanistic underpinnings. Such chromosome catastrophic events appear to reflect basic DNA metabolism operative throughout an organism’s life cycle. PMID:21925314

The role of genetic and epigenetic alterations in neuroblastoma disease pathogenesis

PubMed Central

Domingo-Fernandez, Raquel; Watters, Karen; Piskareva, Olga; Bray, Isabella

2013-01-01

Neuroblastoma is a highly heterogeneous tumor accounting for 15 % of all pediatric cancer deaths. Clinical behavior ranges from the spontaneous regression of localized, asymptomatic tumors, as well as metastasized tumors in infants, to rapid progression and resistance to therapy. Genomic amplification of the MYCN oncogene has been used to predict outcome in neuroblastoma for over 30 years, however, recent methodological advances including miR-NA and mRNA profiling, comparative genomic hybridization (array-CGH), and whole-genome sequencing have enabled the detailed analysis of the neuroblastoma genome, leading to the identification of new prognostic markers and better patient stratification. In this review, we will describe the main genetic factors responsible for these diverse clinical phenotypes in neuroblastoma, the chronology of their discovery, and the impact on patient prognosis. PMID:23274701

Conventional and array-based comparative genomic hybridization analysis of nasopharyngeal carcinomas from the Mediterranean area.

PubMed

Rodriguez, S; Khabir, A; Keryer, C; Perrot, C; Drira, M; Ghorbel, A; Jlidi, R; Bernheim, A; Valent, A; Busson, P

2005-03-01

Nasopharyngeal carcinoma (NPC) occurs with a high incidence in Southeast Asia and to a lesser extent in the Mediterranean area, especially in Tunisia, Algeria, and Morocco. Cellular gene alterations combined with latent Epstein-Barr virus infection are thought to be essential for NPC oncogenesis. To date, chromosome analysis with comparative genomic hybridization (CGH) has been reported exclusively for NPCs from Southeast Asia. Although NPCs from the Mediterranean area have several distinct clinical and epidemiological features, CGH investigations have been lacking. Chromosome analysis was therefore undertaken on a series of NPC xenografts and biopsies derived from patients of Mediterranean origin. Four xenografts were investigated with a combination of conventional CGH, array-based CGH, and comparative expressed sequence hybridization. In addition, 23 fresh NPC biopsies were analyzed with conventional CGH. Data obtained from xenografts and fresh biopsies were consistent, except that amplification of genes at 18p was observed only in xenografts derived from metastatic tissues. Frequent gains associated with gene overexpression were detected at 1q25 approximately qter (64%) and 12p13 (50%). Losses were noticed mainly at 11q14 approximately q23 (50%), 13q12 approximately q31 (50%), 14q24 approximately q31 (43%), and 3p13 approximately p23 (43%). Comparison with previous reports suggests that Mediterranean NPCs have higher frequencies of gains at 1q and losses at 13q than their Asian counterparts.

BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci.

PubMed

Milbredt, Sarah; Waldminghaus, Torsten

2017-06-07

Fluorescence-based methods are widely used to analyze elementary cell processes such as DNA replication or chromosomal folding and segregation. Labeling DNA with a fluorescent protein allows the visualization of its temporal and spatial organization. One popular approach is FROS (fluorescence repressor operator system). This method specifically labels DNA in vivo through binding of a fusion of a fluorescent protein and a repressor protein to an operator array, which contains numerous copies of the repressor binding site integrated into the genomic site of interest. Bound fluorescent proteins are then visible as foci in microscopic analyses and can be distinguished from the background fluorescence caused by unbound fusion proteins. Even though this method is widely used, no attempt has been made so far to decrease the background fluorescence to facilitate analysis of the actual signal of interest. Here, we present a new method that greatly reduces the background signal of FROS. BiFCROS (Bimolecular Fluorescence Complementation and Repressor Operator System) is based on fusions of repressor proteins to halves of a split fluorescent protein. Binding to a hybrid FROS array results in fluorescence signals due to bimolecular fluorescence complementation. Only proteins bound to the hybrid FROS array fluoresce, greatly improving the signal to noise ratio compared to conventional FROS. We present the development of BiFCROS and discuss its potential to be used as a fast and single-cell readout for copy numbers of genetic loci. Copyright © 2017 Milbredt and Waldminghaus.

BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci

PubMed Central

Milbredt, Sarah; Waldminghaus, Torsten

2017-01-01

Fluorescence-based methods are widely used to analyze elementary cell processes such as DNA replication or chromosomal folding and segregation. Labeling DNA with a fluorescent protein allows the visualization of its temporal and spatial organization. One popular approach is FROS (fluorescence repressor operator system). This method specifically labels DNA in vivo through binding of a fusion of a fluorescent protein and a repressor protein to an operator array, which contains numerous copies of the repressor binding site integrated into the genomic site of interest. Bound fluorescent proteins are then visible as foci in microscopic analyses and can be distinguished from the background fluorescence caused by unbound fusion proteins. Even though this method is widely used, no attempt has been made so far to decrease the background fluorescence to facilitate analysis of the actual signal of interest. Here, we present a new method that greatly reduces the background signal of FROS. BiFCROS (Bimolecular Fluorescence Complementation and Repressor Operator System) is based on fusions of repressor proteins to halves of a split fluorescent protein. Binding to a hybrid FROS array results in fluorescence signals due to bimolecular fluorescence complementation. Only proteins bound to the hybrid FROS array fluoresce, greatly improving the signal to noise ratio compared to conventional FROS. We present the development of BiFCROS and discuss its potential to be used as a fast and single-cell readout for copy numbers of genetic loci. PMID:28450375

Detection of recurrent transmission of 17q12 microdeletion by array comparative genomic hybridization in a fetus with prenatally diagnosed hydronephrosis, hydroureter, and multicystic kidney, and variable clinical spectrum in the family.

PubMed

Chen, Chih-Ping; Chang, Shuenn-Dyh; Wang, Tzu-Hao; Wang, Liang-Kai; Tsai, Jeng-Daw; Liu, Yu-Peng; Chern, Schu-Rern; Wu, Peih-Shan; Su, Jun-Wei; Chen, Yu-Ting; Wang, Wayseen

2013-12-01

This study was aimed at detection of recurrent transmission of the 17q12 microdeletion in a fetus with congenital anomalies of the kidney and urinary tract. A 35-year-old woman was referred to the hospital at 20 weeks' gestation because of hydronephrosis in the fetus. The mother was normal and healthy. Her second child was a girl who had bilateral dysplastic kidneys that required hemodialysis, and died at the age of 5 years. During this pregnancy, the woman underwent amniocentesis at 18 weeks' gestation because of advanced maternal age. Cytogenetic analysis revealed a karyotype of 46,XY. Prenatal ultrasound showed left hydronephrosis with a tortuous ureter, right hydronephrosis, and increased echogenicity of the kidneys. Fetal magnetic resonance imaging showed right dilated renal calyces, left hydronephrosis, hydroureter, and multicystic kidney. The pregnancy was subsequently terminated. Array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization were applied for genetic analysis using umbilical cord, maternal blood, and cultured amniocytes. aCGH analysis on umbilical cord detected a 1.75-Mb deletion at 17q12 including haploinsufficiency of LHX1 and HNF1B. aCGH analysis on maternal blood detected a 1.54-Mb deletion at 17q12 including haploinsufficiency of LHX1 and HNF1B. Metaphase fluorescence in situ hybridization analysis on cultured amniocytes and maternal blood lymphocytes using 17q12-specific bacterial artificial chromosome probe showed 17q12 microdeletion in the fetus and the mother. Prenatal diagnosis of recurrent renal and urinary tract abnormalities in the fetus should include a differential diagnosis of familial 17q12 microdeletion. Copyright © 2013. Published by Elsevier B.V.

Dynamic evolution at pericentromeres.

PubMed

Hall, Anne E; Kettler, Gregory C; Preuss, Daphne

2006-03-01

Pericentromeres are exceptional genomic regions: in animals they contain extensive segmental duplications implicated in gene creation, and in plants they sustain rearrangements and insertions uncommon in euchromatin. To examine the mechanisms and patterns of plant pericentromere evolution, we compared pericentromere sequence from four Brassicaceae species separated by <15 million years (Myr). This flowering plant family is ideal for studying relationships between genome reorganization and pericentromere evolution-its members have undergone recent polyploidization and hybridization, with close relatives changing in genome size and chromosome number. Through sequence and hybridization analyses, we examined regions from Arabidopsis arenosa, Capsella rubella, and Olimarabidopsis pumila that are homologous to Arabidopsis thaliana pericentromeres (peri-CENs) III and V, and used FISH to demonstrate they have been maintained near centromere satellite arrays in each species. Sequence analysis revealed a set of highly conserved genes, yet we discovered substantial differences in intergenic length and species-specific changes in sequence content and gene density. We discovered that A. thaliana has undergone recent, significant expansions within its pericentromeres, in some cases measuring hundreds of kilobases; these findings are in marked contrast to euchromatic segments in these species that exhibit only minor length changes. While plant pericentromeres do contain some duplications, we did not find evidence of extensive segmental duplications, as has been documented in primates. Our data support a model in which plant pericentromeres may experience selective pressures distinct from euchromatin, tolerating rapid, dynamic changes in structure and sequence content, including large insertions of mobile elements, 5S rDNA arrays and pseudogenes.

Preimplantation genetic screening for all 24 chromosomes by microarray comparative genomic hybridization significantly increases implantation rates and clinical pregnancy rates in patients undergoing in vitro fertilization with poor prognosis

PubMed Central

Majumdar, Gaurav; Majumdar, Abha; Lall, Meena; Verma, Ishwar C.; Upadhyaya, Kailash C.

2016-01-01

CONTEXT: A majority of human embryos produced in vitro are aneuploid, especially in couples undergoing in vitro fertilization (IVF) with poor prognosis. Preimplantation genetic screening (PGS) for all 24 chromosomes has the potential to select the most euploid embryos for transfer in such cases. AIM: To study the efficacy of PGS for all 24 chromosomes by microarray comparative genomic hybridization (array CGH) in Indian couples undergoing IVF cycles with poor prognosis. SETTINGS AND DESIGN: A retrospective, case–control study was undertaken in an institution-based tertiary care IVF center to compare the clinical outcomes of twenty patients, who underwent 21 PGS cycles with poor prognosis, with 128 non-PGS patients in the control group, with the same inclusion criterion as for the PGS group. MATERIALS AND METHODS: Single cells were obtained by laser-assisted embryo biopsy from day 3 embryos and subsequently analyzed by array CGH for all 24 chromosomes. Once the array CGH results were available on the morning of day 5, only chromosomally normal embryos that had progressed to blastocyst stage were transferred. RESULTS: The implantation rate and clinical pregnancy rate (PR) per transfer were found to be significantly higher in the PGS group than in the control group (63.2% vs. 26.2%, P = 0.001 and 73.3% vs. 36.7%, P = 0.006, respectively), while the multiple PRs sharply declined from 31.9% to 9.1% in the PGS group. CONCLUSIONS: In this pilot study, we have shown that PGS by array CGH can improve the clinical outcome in patients undergoing IVF with poor prognosis. PMID:27382234

Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies.

PubMed

Capalbo, Antonio; Treff, Nathan R; Cimadomo, Danilo; Tao, Xin; Upham, Kathleen; Ubaldi, Filippo Maria; Rienzi, Laura; Scott, Richard T

2015-07-01

Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.

Array CGH analysis of a cohort of Russian patients with intellectual disability.

PubMed

Kashevarova, Anna A; Nazarenko, Lyudmila P; Skryabin, Nikolay A; Salyukova, Olga A; Chechetkina, Nataliya N; Tolmacheva, Ekaterina N; Sazhenova, Elena A; Magini, Pamela; Graziano, Claudio; Romeo, Giovanni; Kučinskas, Vaidutis; Lebedev, Igor N

2014-02-15

The use of array comparative genomic hybridization (array CGH) as a diagnostic tool in molecular genetics has facilitated the identification of many new microdeletion/microduplication syndromes (MMSs). Furthermore, this method has allowed for the identification of copy number variations (CNVs) whose pathogenic role has yet to be uncovered. Here, we report on our application of array CGH for the identification of pathogenic CNVs in 79 Russian children with intellectual disability (ID). Twenty-six pathogenic or likely pathogenic changes in copy number were detected in 22 patients (28%): 8 CNVs corresponded to known MMSs, and 17 were not associated with previously described syndromes. In this report, we describe our findings and comment on genes potentially associated with ID that are located within the CNV regions. Copyright © 2013 Elsevier B.V. All rights reserved.

Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana.

PubMed

Wittenberg, Alexander H J; van der Lee, Theo; Cayla, Cyril; Kilian, Andrzej; Visser, Richard G F; Schouten, Henk J

2005-08-01

Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. This paper demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana. Restriction fragments from a genomic representation of the ecotype Landsberg erecta (Ler) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypes Columbia (Col) and Ler and of individuals from an F(2) population obtained from a Col x Ler cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the Ler markers and alignment with the available Col genome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms.

Analysis of the Saccharomyces cerevisiae pan-genome reveals a pool of copy number variants distributed in diverse yeast strains from differing industrial environments.

PubMed

Dunn, Barbara; Richter, Chandra; Kvitek, Daniel J; Pugh, Tom; Sherlock, Gavin

2012-05-01

Although the budding yeast Saccharomyces cerevisiae is arguably one of the most well-studied organisms on earth, the genome-wide variation within this species--i.e., its "pan-genome"--has been less explored. We created a multispecies microarray platform containing probes covering the genomes of several Saccharomyces species: S. cerevisiae, including regions not found in the standard laboratory S288c strain, as well as the mitochondrial and 2-μm circle genomes-plus S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum, S. kluyveri, and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) on 83 different S. cerevisiae strains collected across a wide range of habitats; of these, 69 were commercial wine strains, while the remaining 14 were from a diverse set of other industrial and natural environments. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole-genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae.

Integrative Genomics Reveals Mechanisms of Copy Number Alterations Responsible for Transcriptional Deregulation in Colorectal Cancer

PubMed Central

Camps, Jordi; Nguyen, Quang Tri; Padilla-Nash, Hesed M.; Knutsen, Turid; McNeil, Nicole E.; Wangsa, Danny; Hummon, Amanda B.; Grade, Marian; Ried, Thomas; Difilippantonio, Michael J.

2016-01-01

To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations. PMID:19691111

A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

PubMed

Comeron, Josep M; Reed, Jordan; Christie, Matthew; Jacobs, Julia S; Dierdorff, Jason; Eberl, Daniel F; Manak, J Robert

2016-04-05

Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

Evaluation of the X-Linked High-Grade Myopia Locus (MYP1) with Cone Dysfunction and Color Vision Deficiencies

PubMed Central

Metlapally, Ravikanth; Michaelides, Michel; Bulusu, Anuradha; Li, Yi-Ju; Schwartz, Marianne; Rosenberg, Thomas; Hunt, David M.; Moore, Anthony T.; Züchner, Stephan; Rickman, Catherine Bowes; Young, Terri L.

2014-01-01

Purpose X-linked high myopia with mild cone dysfunction and color vision defects has been mapped to chromosome Xq28 (MYP1 locus). CXorf2/TEX28 is a nested, intercalated gene within the red-green opsin cone pigment gene tandem array on Xq28. The authors investigated whether TEX28 gene alterations were associated with the Xq28-linked myopia phenotype. Genomic DNA from five pedigrees (with high myopia and either protanopia or deuteranopia) that mapped to Xq28 were screened for TEX28 copy number variations (CNVs) and sequence variants. Methods To examine for CNVs, ultra-high resolution array-comparative genomic hybridization (array-CGH) assays were performed comparing the subject genomic DNA with control samples (two pairs from two pedigrees). Opsin or TEX28 gene-targeted quantitative real-time gene expression assays (comparative CT method) were performed to validate the array-CGH findings. All exons of TEX28, including intron/exon boundaries, were amplified and sequenced using standard techniques. Results Array-CGH findings revealed predicted duplications in affected patient samples. Although only three copies of TEX28 were previously reported within the opsin array, quantitative real-time analysis of the TEX28 targeted assay of affected male or carrier female individuals in these pedigrees revealed either fewer (one) or more (four or five) copies than did related and control unaffected individuals. Sequence analysis of TEX28 did not reveal any variants associated with the disease status. Conclusions CNVs have been proposed to play a role in disease inheritance and susceptibility as they affect gene dosage. TEX28 gene CNVs appear to be associated with the MYP1 X-linked myopia phenotypes. PMID:19098318

Genetic traits of avascular necrosis of the femoral head analyzed by array comparative genomic hybridization and real-time polymerase chain reaction.

PubMed

Hwang, Jung-Taek; Baik, Seung-Ho; Choi, Jin-Soo; Lee, Kweon-Haeng; Rhee, Seung-Koo

2011-01-03

In an attempt to observe the genetic traits of avascular necrosis of the femoral head, we analyzed the genomic alterations in blood samples of 18 patients with avascular necrosis of the femoral head (9 idiopathic and 9 alcoholic cases) using the array comparative genomic hybridization method and real-time polymerase chain reaction. Several candidate genes were identified that may induce avascular necrosis of the femoral head, and we investigated their role in the pathomechanism of osteonecrosis of bone. The frequency of each candidate gene over all the categories of avascular necrosis of the femoral head was also calculated by real-time polymerase chain reaction. The highest frequency specific genes in each category were FLJ40296, CYP27C1, and CTDP1. FLJ40296 and CYP27C1 had the highest frequency (55.6%) in the idiopathic category. FLJ40296 had a high frequency (44.4%) in the alcoholic category, but CYP27C1 had a relatively low frequency (33.3%) in the alcoholic category. However, CTDP1 showed a significantly high frequency (55.6%) in the alcoholic category and a low frequency (22.2%) in the idiopathic category. Although we statistically analyzed the frequency of each gene with Fisher's exact test, we could not prove statistical significance due to the small number of samples. Further studies are needed with larger sample numbers. If the causal genes of avascular necrosis of the femoral head are found, they may be used for early detection, prognosis prediction, and genomic treatment of avascular necrosis of the femoral head in the future. Copyright 2011, SLACK Incorporated.

Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.

PubMed

Chin, Chen-Shan; Alexander, David H; Marks, Patrick; Klammer, Aaron A; Drake, James; Heiner, Cheryl; Clum, Alicia; Copeland, Alex; Huddleston, John; Eichler, Evan E; Turner, Stephen W; Korlach, Jonas

2013-06-01

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

Avoiding pitfalls in molecular genetic testing: case studies of high-resolution array comparative genomic hybridization testing in the definitive diagnosis of Mowat-Wilson syndrome.

PubMed

Kluk, Michael Joseph; An, Yu; James, Philip; Coulter, David; Harris, David; Wu, Bai-Lin; Shen, Yiping

2011-05-01

The molecular testing options available for the diagnosis of genetic disorders are numerous and include a variety of different assay platforms. The consultative input of molecular pathologists and cytogeneticists, working closely with the ordering clinicians, is often important for definitive diagnosis. Herein, we describe two patients who had long histories of unexplained signs and symptoms with a high clinical suspicion of an underlying genetic etiology. Initial molecular testing in both cases was negative, but the application of high-resolution array comparative genomic hybridization technology lead to definitive diagnosis in both cases. We summarize the clinical findings and molecular testing in each case, discuss the differential diagnoses, and review the clinical and pathological findings of Mowat-Wilson syndrome. This report highlights the importance for those involved in molecular testing to know the nature of the underlying genetic abnormalities associated with the suspected diagnosis, to recognize the limitations of each testing platform, and to persistently pursue repeat testing using high-resolution technologies when indicated. This concept is applicable to both germline and somatic molecular genetic testing. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study.

PubMed

Feichtinger, Michael; Vaccari, Enrico; Carli, Luca; Wallner, Elisabeth; Mädel, Ulrike; Figl, Katharina; Palini, Simone; Feichtinger, Wilfried

2017-06-01

The aim of this pilot study was to assess if array comparative genomic hybridization (aCGH), non-invasive preimplantation genetic screening (PGS) on blastocyst culture media is feasible. Therefore, aCGH analysis was carried out on 22 spent blastocyst culture media samples after polar body PGS because of advanced maternal age. All oocytes were fertilized by intracytoplasmic sperm injection and all embryos underwent assisted hatching. Concordance of polar body analysis and culture media genetic results was assessed. Thirteen out of 18 samples (72.2%) revealed general concordance of ploidy status (euploid or aneuploid). At least one chromosomal aberration was found concordant in 10 out of 15 embryos found to be aneuploid by both polar body and culture media analysis. Overall, 17 out of 35 (48.6%) single chromosomal aneuploidies were concordant between the culture media and polar body analysis. By analysing negative controls (oocytes with fertilization failure), notable maternal contamination was observed. Therefore, non-invasive PGS could serve as a second matrix after polar body or cleavage stage PGS; however, in euploid results, maternal contamination needs to be considered and results interpreted with caution. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

A copy number variation genotyping method for aneuploidy detection in spontaneous abortion specimens.

PubMed

Chen, Songchang; Liu, Deyuan; Zhang, Junyu; Li, Shuyuan; Zhang, Lanlan; Fan, Jianxia; Luo, Yuqin; Qian, Yeqing; Huang, Hefeng; Liu, Chao; Zhu, Huanhuan; Jiang, Zhengwen; Xu, Chenming

2017-02-01

Chromosomal abnormalities such as aneuploidy have been shown to be responsible for causing spontaneous abortion. Genetic evaluation of abortions is currently underperformed. Screening for aneuploidy in the products of conception can help determine the etiology. We designed a high-throughput ligation-dependent probe amplification (HLPA) assay to examine aneuploidy of 24 chromosomes in miscarriage tissues and aimed to validate the performance of this technique. We carried out aneuploidy screening in 98 fetal tissue samples collected from female subjects with singleton pregnancies who experienced spontaneous abortion. The mean maternal age was 31.6 years (range: 24-43), and the mean gestational age was 10.2 weeks (range: 4.6-14.1). HLPA was performed in parallel with array comparative genomic hybridization, which is the gold standard for aneuploidy detection in clinical practices. The results from the two platforms were compared. Forty-nine out of ninety-eight samples were found to be aneuploid. HLPA showed concordance with array comparative genomic hybridization in diagnosing aneuploidy. High-throughput ligation-dependent probe amplification is a rapid and accurate method for aneuploidy detection. It can be used as a cost-effective screening procedure in clinical spontaneous abortions. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Molecular cytogenetics: an indispensable tool for cancer diagnosis.

PubMed

Wan, Thomas Sk; Ma, Edmond Sk

2012-01-01

Cytogenetic aberrations may escape detection or recognition in traditional karyotyping. The past decade has seen an explosion of methodological advances in molecular cytogenetics technology. These cytogenetics techniques add color to the black and white world of conventional banding. Fluorescence in-situ hybridization (FISH) study has emerged as an indispensable tool for both basic and clinical research, as well as diagnostics, in leukemia and cancers. FISH can be used to identify chromosomal abnormalities through fluorescent labeled DNA probes that target specific DNA sequences. Subsequently, FISH-based tests such as multicolor karyotyping, comparative genomic hybridization (CGH) and array CGH have been used in emerging clinical applications as they enable resolution of complex karyotypic aberrations and whole global scanning of genomic imbalances. More recently, crossspecies array CGH analysis has also been employed in cancer gene identification. The clinical impact of FISH is pivotal, especially in the diagnosis, prognosis and treatment decisions for hematological diseases, all of which facilitate the practice of personalized medicine. This review summarizes the methodology and current utilization of these FISH techniques in unraveling chromosomal changes and highlights how the field is moving away from conventional methods towards molecular cytogenetics approaches. In addition, the potential of the more recently developed FISH tests in contributing information to genetic abnormalities is illustrated.

New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

PubMed Central

Cobo, Ana Cristina; Milán, Miguel; Al-Asmar, Nasser; García-Herrero, Sandra; Mir, Pere; Simón, Carlos

2014-01-01

The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS) using array comparative genomic hybridization (aCGH). The study included 1420 CCS cycles for recurrent miscarriage (n = 203); repetitive implantation failure (n = 188); severe male factor (n = 116); previous trisomic pregnancy (n = 33); and advanced maternal age (n = 880). CCS was performed in cycles with fresh oocytes and embryos (n = 774); mixed cycles with fresh and vitrified oocytes (n = 320); mixed cycles with fresh and vitrified day-2 embryos (n = 235); and mixed cycles with fresh and vitrified day-3 embryos (n = 91). Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2%) and pregnancy rates per transfer (range: 46.0–62.9%) were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1%) due to the higher percentage of aneuploid embryos (85.3%) and lower number of cycles with at least one euploid embryo available per transfer (40.3%). We concluded that aneuploidy is one of the major factors which affect embryo implantation. PMID:24877108

A novel deletion in the thyrotropin Beta-subunit gene identified by array comparative genomic hybridization analysis causes central congenital hypothyroidism in a boy originating from Turkey.

PubMed

Hermanns, Pia; Couch, Robert; Leonard, Norma; Klotz, Cherise; Pohlenz, Joachim

2014-01-01

Isolated central congenital hypothyroidism (ICCH) is rare but important. Most ICCH patients are diagnosed later, which results in severe growth failure and intellectual disability. We describe a boy with ICCH due to a large homozygous TSHβ gene deletion. A 51-day-old male Turkish infant, whose parents were first cousins, was admitted for evaluation of prolonged jaundice. His clinical appearance was compatible with hypothyroidism. Venous thyrotropin (TSH) was undetectably low, with a subsequent low free T4 and a low free T3, suggestive of central hypothyroidism. Using different PCR protocols, we could not amplify both coding exons of the boy's TSHβ gene, which suggested a deletion. An array comparative genomic hybridization (aCGH) using specific probes around the TSHβ gene locus showed him to be homozygous for a 6-kb deletion spanning all exons and parts of the 5' untranslated region of the gene. Infants who are clinically suspected of having hypothyroidism should be evaluated thoroughly, even if their TSH-based screening result is normal. In cases with ICCH and undetectably low TSH serum concentrations, a TSHβ gene deletion should be considered; aCGH should be performed when gene deletions are suspected. In such cases, PCR-based sequencing techniques give negative results.

Chromosomal imbalances are associated with outcome of Helicobacter pylori eradication in t(11;18)(q21;q21) negative gastric mucosa-associated lymphoid tissue lymphomas.

PubMed

Fukuhara, Noriko; Nakamura, Tsuneya; Nakagawa, Masao; Tagawa, Hiroyuki; Takeuchi, Ichiro; Yatabe, Yasushi; Morishima, Yasuo; Nakamura, Shigeo; Seto, Masao

2007-08-01

Approximately 70% of gastric mucosa-associated lymphoid tissue (MALT) lymphomas can be successfully treated with H. pylori eradication. The translocation t(11;18)(q21;q21) characteristic of MALT lymphoma is recognized as a marker for H. pylori independency, but this marker is found in only a half of the MALT lymphomas resistant to H. pylori eradication. Detailed analyses of the genomic features of eradication resistant as well as responsive groups are important for understanding their molecular basis. We performed array-based comparative genomic hybridization (array-CGH) for 29 gastric MALT lymphomas treated with H. pylori eradication. These comprised ten cases of t(11;18) positive MALT, nine cases of t(11;18) negative MALT with H. pylori dependency, and ten cases of t(11;18) negative MALT with H. pylori independency. Array-CGH analysis demonstrated that no significant genetic alterations were found in t(11;18) positive MALT lymphomas, but numerous genomic alterations were detected in t(11;18) negative MALT lymphomas. Many of these alterations were similar to those found in diffuse large B-cell lymphoma with trisomy 3 being the most recurrent alteration. Within the t(11;18) negative MALT lymphoma without large cell components group, genomic imbalances occurred more frequently in the H. pylori independent than in the H. pylori dependent group (P = 0.02). Genomic imbalances are associated with H. pylori independency in t(11;18) negative gastric MALT lymphomas. They may thus play an important role in the development of H. pylori independency.

Prenatal diagnosis and molecular cytogenetic characterization of rec(10)dup(10p)inv(10)(p11.2q26.3) in a fetus associated with paternal pericentric inversion.

PubMed

Chen, Chih-Ping; Ko, Tsang-Ming; Su, Yi-Ning; Wang, Liang-Kai; Chern, Schu-Rern; Wu, Peih-Shan; Chen, Yen-Ni; Chen, Shin-Wen; Ko, Kevin; Lee, Chen-Chi; Chen, Li-Feng; Yang, Chien-Wen; Wang, Wayseen

2016-10-01

We present prenatal diagnosis and molecular cytogenetic characterization of a recombinant chromosome 10 in a fetus associated with a paternal pericentric inversion. A 35-year-old woman underwent amniocentesis at 18 weeks of gestation because of an advanced maternal age. Amniocentesis revealed a karyotype of 46,XY,der(10)del(10) (q26.3)dup(10)(p11.2p15). She underwent repeat amniocentesis at 21 weeks of gestation and array comparative genomic hybridization revealed a 31.65-Mb duplication of chromosome 10p15.3-p11.22 and a 3.07-Mb deletion of chromosome 10q26.3. Prenatal ultrasound findings were unremarkable. She was referred for genetic counseling and cytogenetic analysis revealed a karyotype of 46,XY,inv(10)(p11.2q26.3) in the father and a karyotype of 46,XX in the mother. The pregnancy was subsequently terminated, and a fetus was delivered with prominent facial dysmorphism. Postnatal cytogenetic analysis of the placenta revealed a karyotype of 46,XY, rec(10)dup(10p)inv(10)(p11.2q26.3). Fluorescence in situ hybridization analysis revealed a duplication of terminal 10p and a deletion of terminal 10q in the recombinant chromosome 10. Array comparative genomic hybridization analysis of the cord blood and umbilical cord confirmed the prenatal diagnosis. Prenatal diagnosis of a recombinant chromosome because of an advanced maternal age should alert the possibility of a paternal pericentric inversion. Copyright © 2016. Published by Elsevier B.V.

Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae).

PubMed

Mahelka, Václav; Kopecky, David; Baum, Bernard R

2013-09-01

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

Genomic alterations identified by array comparative genomic hybridization as prognostic markers in tamoxifen-treated estrogen receptor-positive breast cancer

PubMed Central

Han, Wonshik; Han, Mi-Ryung; Kang, Jason Jongho; Bae, Ji-Yeon; Lee, Ji Hyun; Bae, Young Ju; Lee, Jeong Eon; Shin, Hyuk-Jae; Hwang, Ki-Tae; Hwang, Sung-Eun; Kim, Sung-Won; Noh, Dong-Young

2006-01-01

Background A considerable proportion of estrogen receptor (ER)-positive breast cancer recurs despite tamoxifen treatment, which is a serious problem commonly encountered in clinical practice. We tried to find novel prognostic markers in this subtype of breast cancer. Methods We performed array comparative genomic hybridization (CGH) with 1,440 human bacterial artificial chromosome (BAC) clones to assess copy number changes in 28 fresh-frozen ER-positive breast cancer tissues. All of the patients included had received at least 1 year of tamoxifen treatment. Nine patients had distant recurrence within 5 years (Recurrence group) of diagnosis and 19 patients were alive without disease at least 5 years after diagnosis (Non-recurrence group). Results Potential prognostic variables were comparable between the two groups. In an unsupervised clustering analysis, samples from each group were well separated. The most common regions of gain in all samples were 1q32.1, 17q23.3, 8q24.11, 17q12-q21.1, and 8p11.21, and the most common regions of loss were 6q14.1-q16.3, 11q21-q24.3, and 13q13.2-q14.3, as called by CGH-Explorer software. The average frequency of copy number changes was similar between the two groups. The most significant chromosomal alterations found more often in the Recurrence group using two different statistical methods were loss of 11p15.5-p15.4, 1p36.33, 11q13.1, and 11p11.2 (adjusted p values <0.001). In subgroup analysis according to lymph node status, loss of 11p15 and 1p36 were found more often in Recurrence group with borderline significance within the lymph node positive patients (adjusted p = 0.052). Conclusion Our array CGH analysis with BAC clones could detect various genomic alterations in ER-positive breast cancers, and Recurrence group samples showed a significantly different pattern of DNA copy number changes than did Non-recurrence group samples. PMID:16608533

A Novel Microdeletion in 1(p34.2p34.3), Involving the "SLC2A1" ("GLUT1") Gene, and Severe Delayed Development

ERIC Educational Resources Information Center

Vermeer, Sascha; Koolen, David A; Visser, Gepke; Brackel, Hein J. L.; van der Burgt, Ineke; de Leeuw, Nicole; Willemsen, Michel A. A. P.; Sistermans, Erik A.; Pfundt, Rolph; de Vries, Bert B. A.

2007-01-01

A "de novo" 4.1-megabase microdeletion of chromosome 1p34.2p34.3 has been identified by array-based comparative genomic hybridization in a young male with severely delayed development, microcephaly, pronounced hypotonia, and facial dysmorphism. The deleted region encompasses 48 genes, among them the glucose transporter 1 ("SLC2A1" or "GLUT1")…

Array-based comparative genomic hybridization analysis reveals recurrent chromosomal alterations and prognostic parameters in primary cutaneous large B-cell lymphoma.

PubMed

Dijkman, Remco; Tensen, Cornelis P; Jordanova, Ekaterina S; Knijnenburg, Jeroen; Hoefnagel, Juliette J; Mulder, Aat A; Rosenberg, Carla; Raap, Anton K; Willemze, Rein; Szuhai, Károly; Vermeer, Maarten H

2006-01-10

To evaluate the clinical relevance of genomic aberrations in primary cutaneous large B-cell lymphoma (PCLBCL). Skin biopsy samples of 31 patients with a PCLBCL classified as either primary cutaneous follicle center lymphoma (PCFCL; n = 19) or PCLBCL, leg type (n = 12), according to the WHO-European Organisation for Research and Treatment of Cancer (EORTC) classification, were investigated using array-based comparative genomic hybridization, fluorescence in situ hybridization (FISH), and examination of promoter hypermethylation. The most recurrent alterations in PCFCL were high-level DNA amplifications at 2p16.1 (63%) and deletion of chromosome 14q32.33 (68%). FISH analysis confirmed c-REL amplification in patients with gains at 2p16.1. In PCLBCL, leg type, most prominent aberrations were a high-level DNA amplification of 18q21.31-q21.33 (67%), including the BCL-2 and MALT1 genes as confirmed by FISH, and deletions of a small region within 9p21.3 containing the CDKN2A, CDKN2B, and NSG-x genes. Homozygous deletion of 9p21.3 was detected in five of 12 patients with PCLBCL, leg type, but in zero of 19 patients with PCFCL. Complete methylation of the promoter region of the CDKN2A gene was demonstrated in one PCLBCL, leg type, patient with hemizygous deletion, in one patient without deletion, but in zero of 19 patients with PCFCL. Seven of seven PCLBCL, leg type, patients with deletion of 9p21.3 and/or complete methylation of CDKN2A died as a result of their lymphoma. Our results demonstrate prominent differences in chromosomal alterations between PCFCL and PCLBCL, leg type, that support their classification as separate entities within the WHO-EORTC scheme. Inactivation of CDKN2A by either deletion or methylation of its promoter could be an important prognostic parameter for the group of PCLBCL, leg type.

A powerful tool for genome analysis in maize: development and evaluation of the high density 600 k SNP genotyping array.

PubMed

Unterseer, Sandra; Bauer, Eva; Haberer, Georg; Seidel, Michael; Knaak, Carsten; Ouzunova, Milena; Meitinger, Thomas; Strom, Tim M; Fries, Ruedi; Pausch, Hubert; Bertani, Christofer; Davassi, Alessandro; Mayer, Klaus Fx; Schön, Chris-Carolin

2014-09-29

High density genotyping data are indispensable for genomic analyses of complex traits in animal and crop species. Maize is one of the most important crop plants worldwide, however a high density SNP genotyping array for analysis of its large and highly dynamic genome was not available so far. We developed a high density maize SNP array composed of 616,201 variants (SNPs and small indels). Initially, 57 M variants were discovered by sequencing 30 representative temperate maize lines and then stringently filtered for sequence quality scores and predicted conversion performance on the array resulting in the selection of 1.2 M polymorphic variants assayed on two screening arrays. To identify high-confidence variants, 285 DNA samples from a broad genetic diversity panel of worldwide maize lines including the samples used for sequencing, important founder lines for European maize breeding, hybrids, and proprietary samples with European, US, semi-tropical, and tropical origin were used for experimental validation. We selected 616 k variants according to their performance during validation, support of genotype calls through sequencing data, and physical distribution for further analysis and for the design of the commercially available Affymetrix® Axiom® Maize Genotyping Array. This array is composed of 609,442 SNPs and 6,759 indels. Among these are 116,224 variants in coding regions and 45,655 SNPs of the Illumina® MaizeSNP50 BeadChip for study comparison. In a subset of 45,974 variants, apart from the target SNP additional off-target variants are detected, which show only a minor bias towards intermediate allele frequencies. We performed principal coordinate and admixture analyses to determine the ability of the array to detect and resolve population structure and investigated the extent of LD within a worldwide validation panel. The high density Affymetrix® Axiom® Maize Genotyping Array is optimized for European and American temperate maize and was developed based on a diverse sample panel by applying stringent quality filter criteria to ensure its suitability for a broad range of applications. With 600 k variants it is the largest currently publically available genotyping array in crop species.

Genetic Alterations in Primary Gastric Carcinomas Correlated with Clinicopathological Variables by Array Comparative Genomic Hybridization

PubMed Central

Kang, Ji Un; Kang, Jason Jongho; Kwon, Kye Chul; Park, Jong Woo; Jeong, Tae Eun; Noh, Seung Mu

2006-01-01

Genetic alterations have been recognized as an important event in the carcinogenesis of gastric cancer (GC). We conducted high resolution bacterial artificial chromosome array-comparative genomic hybridization, to elucidate in more detail the genomic alterations, and to establish a pattern of DNA copy number changes with distinct clinical variables in GC. Our results showed some correlations between novel amplified or deleted regions and clinical status. Copy-number gains were frequently detected at 1p, 5p, 7q, 8q, 11p, 16p, 20p and 20q, and losses at 1p, 2q, 4q, 5q, 7q, 9p, 14q, and 18q. Losses at 4q23, 9p23, 14q31.1, or 18q21.1 as well as a gain at 20q12 were correlated with tumor-node-metastasis tumor stage. Losses at 9p23 or 14q31.1 were associated with lymph node status. Metastasis was determined to be related to losses at 4q23 or 4q28.2, as well as losses at 4q15.2, 4q21.21, 4q 28.2, or 14q31.1, with differentiation. One of the notable aspects of this study was that the losses at 4q or 14q could be employed in the evaluation of the metastatic status of GC. Our results should provide a potential resource for the molecular cytogenetic events in GC, and should also provide clues in the hunt for genes associated with GC. PMID:16891809

Detection of chromothripsis‐like patterns with a custom array platform for chronic lymphocytic leukemia

PubMed Central

Salaverria, Itziar; Martín‐Garcia, David; López, Cristina; Clot, Guillem; García‐Aragonés, Manel; Navarro, Alba; Delgado, Julio; Baumann, Tycho; Pinyol, Magda; Martin‐Guerrero, Idoia; Carrió, Ana; Costa, Dolors; Queirós, Ana C.; Jayne, Sandrine; Aymerich, Marta; Villamor, Neus; Colomer, Dolors; González, Marcos; López‐Guillermo, Armando; Campo, Elías; Dyer, Martin J. S.; Siebert, Reiner; Armengol, Lluís

2015-01-01

Chronic lymphocytic leukemia (CLL) is a common disease with highly variable clinical course. Several recurrent chromosomal alterations are associated with prognosis and may guide risk‐adapted therapy. We have developed a targeted genome‐wide array to provide a robust tool for ascertaining abnormalities in CLL and to overcome limitations of the 4‐marker fluorescence in situ hybridization (FISH). DNA from 180 CLL patients were hybridized to the qChip®Hemo array with a high density of probes covering commonly altered loci in CLL (11q22‐q23, 13q14, and 17p13), nine focal regions (2p15‐p16.1, 2p24.3, 2q13, 2q36.3‐q37.1, 3p21.31, 8q24.21, 9p21.3, 10q24.32, and 18q21.32‐q21.33) and two larger regions (6q14.1‐q22.31 and 7q31.33‐q33). Overall, 86% of the cases presented copy number alterations (CNA) by array. There was a high concordance of array findings with FISH (84% sensitivity, 100% specificity); all discrepancies corresponded to subclonal alterations detected only by FISH. A chromothripsis‐like pattern was detected in eight cases. Three showed concomitant shattered 5p with gain of TERT along with isochromosome 17q. Presence of 11q loss was associated with shorter time to first treatment (P = 0.003), whereas 17p loss, increased genomic complexity, and chromothripsis were associated with shorter overall survival (P < 0.001, P = 0.001, and P = 0.02, respectively). In conclusion, we have validated a targeted array for the diagnosis of CLL that accurately detects, in a single experiment, all relevant CNAs, genomic complexity, chromothripsis, copy number neutral loss of heterozygosity, and CNAs not covered by the FISH panel. This test may be used as a practical tool to stratify CLL patients for routine diagnostics or clinical trials. © 2015 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc. PMID:26305789

Whole-genome multiple displacement amplification from single cells.

PubMed

Spits, Claudia; Le Caignec, Cédric; De Rycke, Martine; Van Haute, Lindsey; Van Steirteghem, André; Liebaers, Inge; Sermon, Karen

2006-01-01

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.

Genotype–phenotype correlations in Down syndrome identified by array CGH in 30 cases of partial trisomy and partial monosomy chromosome 21

PubMed Central

Lyle, Robert; Béna, Frédérique; Gagos, Sarantis; Gehrig, Corinne; Lopez, Gipsy; Schinzel, Albert; Lespinasse, James; Bottani, Armand; Dahoun, Sophie; Taine, Laurence; Doco-Fenzy, Martine; Cornillet-Lefèbvre, Pascale; Pelet, Anna; Lyonnet, Stanislas; Toutain, Annick; Colleaux, Laurence; Horst, Jürgen; Kennerknecht, Ingo; Wakamatsu, Nobuaki; Descartes, Maria; Franklin, Judy C; Florentin-Arar, Lina; Kitsiou, Sophia; Aït Yahya-Graison, Emilie; Costantine, Maher; Sinet, Pierre-Marie; Delabar, Jean M; Antonarakis, Stylianos E

2009-01-01

Down syndrome (DS) is one of the most frequent congenital birth defects, and the most common genetic cause of mental retardation. In most cases, DS results from the presence of an extra copy of chromosome 21. DS has a complex phenotype, and a major goal of DS research is to identify genotype–phenotype correlations. Cases of partial trisomy 21 and other HSA21 rearrangements associated with DS features could identify genomic regions associated with specific phenotypes. We have developed a BAC array spanning HSA21q and used array comparative genome hybridization (aCGH) to enable high-resolution mapping of pathogenic partial aneuploidies and unbalanced translocations involving HSA21. We report the identification and mapping of 30 pathogenic chromosomal aberrations of HSA21 consisting of 19 partial trisomies and 11 partial monosomies for different segments of HSA21. The breakpoints have been mapped to within ∼85 kb. The majority of the breakpoints (26 of 30) for the partial aneuploidies map within a 10-Mb region. Our data argue against a single DS critical region. We identify susceptibility regions for 25 phenotypes for DS and 27 regions for monosomy 21. However, most of these regions are still broad, and more cases are needed to narrow down the phenotypic maps to a reasonable number of candidate genomic elements per phenotype. PMID:19002211

Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

PubMed

Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej

2016-09-01

The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement.

Application of Genomic In Situ Hybridization in Horticultural Science

PubMed Central

Ramzan, Fahad; Lim, Ki-Byung

2017-01-01

Molecular cytogenetic techniques, such as in situ hybridization methods, are admirable tools to analyze the genomic structure and function, chromosome constituents, recombination patterns, alien gene introgression, genome evolution, aneuploidy, and polyploidy and also genome constitution visualization and chromosome discrimination from different genomes in allopolyploids of various horticultural crops. Using GISH advancement as multicolor detection is a significant approach to analyze the small and numerous chromosomes in fruit species, for example, Diospyros hybrids. This analytical technique has proved to be the most exact and effective way for hybrid status confirmation and helps remarkably to distinguish donor parental genomes in hybrids such as Clivia, Rhododendron, and Lycoris ornamental hybrids. The genome characterization facilitates in hybrid selection having potential desirable characteristics during the early hybridization breeding, as this technique expedites to detect introgressed sequence chromosomes. This review study epitomizes applications and advancements of genomic in situ hybridization (GISH) techniques in horticultural plants. PMID:28459054

Integrated high-resolution array CGH and SKY analysis of homozygous deletions and other genomic alterations present in malignant mesothelioma cell lines.

PubMed

Klorin, Geula; Rozenblum, Ester; Glebov, Oleg; Walker, Robert L; Park, Yoonsoo; Meltzer, Paul S; Kirsch, Ilan R; Kaye, Frederic J; Roschke, Anna V

2013-05-01

High-resolution oligonucleotide array comparative genomic hybridization (aCGH) and spectral karyotyping (SKY) were applied to a panel of malignant mesothelioma (MMt) cell lines. SKY has not been applied to MMt before, and complete karyotypes are reported based on the integration of SKY and aCGH results. A whole genome search for homozygous deletions (HDs) produced the largest set of recurrent and non-recurrent HDs for MMt (52 recurrent HDs in 10 genomic regions; 36 non-recurrent HDs). For the first time, LINGO2, RBFOX1/A2BP1, RPL29, DUSP7, and CCSER1/FAM190A were found to be homozygously deleted in MMt, and some of these genes could be new tumor suppressor genes for MMt. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements that led to the formation of HDs. Our data imply that only with acquisition of structural and/or numerical karyotypic instability can MMt cells attain a complete loss of tumor suppressor genes located in 9p21.3, which is the most frequently homozygously deleted region. Tetraploidization is a late event in the karyotypic progression of MMt cells, after HDs in the 9p21.3 region have already been acquired. Published by Elsevier Inc.

A Trichosporonales genome tree based on 27 haploid and three evolutionarily conserved 'natural' hybrid genomes.

PubMed

Takashima, Masako; Sriswasdi, Sira; Manabe, Ri-Ichiroh; Ohkuma, Moriya; Sugita, Takashi; Iwasaki, Wataru

2018-01-01

To construct a backbone tree consisting of basidiomycetous yeasts, draft genome sequences from 25 species of Trichosporonales (Tremellomycetes, Basidiomycota) were generated. In addition to the hybrid genomes of Trichosporon coremiiforme and Trichosporon ovoides that we described previously, we identified an interspecies hybrid genome in Cutaneotrichosporon mucoides (formerly Trichosporon mucoides). This hybrid genome had a gene retention rate of ~55%, and its closest haploid relative was Cutaneotrichosporon dermatis. After constructing the C. mucoides subgenomes, we generated a phylogenetic tree using genome data from the 27 haploid species and the subgenome data from the three hybrid genome species. It was a high-quality tree with 100% bootstrap support for all of the branches. The genome-based tree provided superior resolution compared with previous multi-gene analyses. Although our backbone tree does not include all Trichosporonales genera (e.g. Cryptotrichosporon), it will be valuable for future analyses of genome data. Interest in interspecies hybrid fungal genomes has recently increased because they may provide a basis for new technologies. The three Trichosporonales hybrid genomes described in this study are different from well-characterized hybrid genomes (e.g. those of Saccharomyces pastorianus and Saccharomyces bayanus) because these hybridization events probably occurred in the distant evolutionary past. Hence, they will be useful for studying genome stability following hybridization and speciation events. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Assessment of copy number variations in 120 patients with Poland syndrome.

PubMed

Vaccari, Carlotta Maria; Tassano, Elisa; Torre, Michele; Gimelli, Stefania; Divizia, Maria Teresa; Romanini, Maria Victoria; Bossi, Simone; Musante, Ilaria; Valle, Maura; Senes, Filippo; Catena, Nunzio; Bedeschi, Maria Francesca; Baban, Anwar; Calevo, Maria Grazia; Acquaviva, Massimo; Lerone, Margherita; Ravazzolo, Roberto; Puliti, Aldamaria

2016-11-25

Poland Syndrome (PS) is a rare congenital disorder presenting with agenesis/hypoplasia of the pectoralis major muscle variably associated with thoracic and/or upper limb anomalies. Most cases are sporadic, but familial recurrence, with different inheritance patterns, has been observed. The genetic etiology of PS remains unknown. Karyotyping and array-comparative genomic hybridization (CGH) analyses can identify genomic imbalances that can clarify the genetic etiology of congenital and neurodevelopmental disorders. We previously reported a chromosome 11 deletion in twin girls with pectoralis muscle hypoplasia and skeletal anomalies, and a chromosome six deletion in a patient presenting a complex phenotype that included pectoralis muscle hypoplasia. However, the contribution of genomic imbalances to PS remains largely unknown. To investigate the prevalence of chromosomal imbalances in PS, standard cytogenetic and array-CGH analyses were performed in 120 PS patients. Following the application of stringent filter criteria, 14 rare copy number variations (CNVs) were identified in 14 PS patients in different regions outside known common copy number variations: seven genomic duplications and seven genomic deletions, enclosing the two previously reported PS associated chromosomal deletions. These CNVs ranged from 0.04 to 4.71 Mb in size. Bioinformatic analysis of array-CGH data indicated gene enrichment in pathways involved in cell-cell adhesion, DNA binding and apoptosis processes. The analysis also provided a number of candidate genes possibly causing the developmental defects observed in PS patients, among others REV3L, a gene coding for an error-prone DNA polymerase previously associated with Möbius Syndrome with variable phenotypes including pectoralis muscle agenesis. A number of rare CNVs were identified in PS patients, and these involve genes that represent candidates for further evaluation. Rare inherited CNVs may contribute to, or represent risk factors of PS in a multifactorial mode of inheritance.

Analysis of the Saccharomyces cerevisiae pan-genome reveals a pool of copy number variants distributed in diverse yeast strains from differing industrial environments

PubMed Central

Dunn, Barbara; Richter, Chandra; Kvitek, Daniel J.; Pugh, Tom; Sherlock, Gavin

2012-01-01

Although the budding yeast Saccharomyces cerevisiae is arguably one of the most well-studied organisms on earth, the genome-wide variation within this species—i.e., its “pan-genome”—has been less explored. We created a multispecies microarray platform containing probes covering the genomes of several Saccharomyces species: S. cerevisiae, including regions not found in the standard laboratory S288c strain, as well as the mitochondrial and 2-μm circle genomes–plus S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum, S. kluyveri, and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) on 83 different S. cerevisiae strains collected across a wide range of habitats; of these, 69 were commercial wine strains, while the remaining 14 were from a diverse set of other industrial and natural environments. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole-genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae. PMID:22369888

A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays

PubMed Central

2012-01-01

Background DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin. Results For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform. Conclusions To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These two factors are sequence dependent and have a large impact on probe intensity. The results presented here provide novel insight into the effect of probe synthesis errors on Affymetrix microarrays; furthermore, the algorithms developed in this work provide useful tools for the analysis of cross-hybridization, probe synthesis efficiency, fragmentation, wash stringency, temperature, and salt concentration on microarray intensities. PMID:23270536

Genetic Dosage Compensation in a Family with Velo-cardio-facial/DiGeorge/22q11.2 Deletion Syndrome

PubMed Central

Alkalay, Avishai A.; Guo, Tingwei; Montagna, Cristina; Digilio, M. Cristina; Marino, Bruno; Dallapiccola, Bruno; Morrow, Bernice

2014-01-01

Cytogenetic studies of a male child carrying the 22q11.2 deletion common in patients with velo-cardio-facial/DiGeorge syndrome revealed an unexpected rearrangement of the 22q11.2 region in his normal appearing mother. The mother carries a 3 Mb deletion on one copy and a reciprocal, similar sized duplication on the other copy of chromosome 22q11.2 as revealed by fluorescence in situ hybridization and array comparative genome hybridization analysis. The most parsimonious mechanism for the rearrangement is a mitotic non-allelic homologous recombination event in a cell in the early embryo soon after fertilization. The normal phenotype of the mother can be explained by the theory of genetic dosage compensation. This is the second documented case of such an event for this or any genomic disorder. This finding helps to reinforce this phenomenon in a human model, and has significant implications for genetic counseling of future children. PMID:21337693

Copy Number Variants and Congenital Anomalies Surveillance: A Suggested Coding Strategy Using the Royal College of Paediatrics and Child Health Version of ICD-10.

PubMed

Bedard, Tanya; Lowry, R Brian; Sibbald, Barbara; Thomas, Mary Ann; Innes, A Micheil

2016-01-01

The use of array-based comparative genomic hybridization to assess DNA copy number is increasing in many jurisdictions. Such technology identifies more genetic causes of congenital anomalies; however, the clinical significance of some results may be challenging to interpret. A coding strategy to address cases with copy number variants has recently been implemented by the Alberta Congenital Anomalies Surveillance System and is described.

Incidental findings on array comparative genomic hybridization: detection of carrier females of dystrophinopathy without any family history.

PubMed

Nguyen, K; Putoux, A; Busa, T; Cordier, M P; Sigaudy, S; Till, M; Chabrol, B; Michel-Calemard, L; Bernard, R; Julia, S; Malzac, P; Labalme, A; Missirian, C; Edery, P; Popovici, C; Philip, N; Sanlaville, D

2015-05-01

Array comparative genomic hybridization (aCGH) has progressively replaced conventional karyotype in the diagnostic strategy of intellectual disability (ID) and congenital malformations. This technique increases not only the diagnostic rate but also the possibility of finding unexpected variants unrelated to the indication of referral, namely incidental findings. The incidental finding of copy number variants (CNVs) located in X-linked genes in girls addresses the crucial question of genetic counseling in the family. We report here five cases of CNVs involving the dystrophin gene detected by aCGH in girls referred for developmental delay, without any family history of dystrophinopathy. The rearrangements included three in-frame deletions; one maternally and two paternally inherited, and two frameshift duplications: one de novo and one from undetermined inheritance. In two cases, the deletion identified in a girl was transmitted by the asymptomatic father. In the case of the maternally inherited deletion, prenatal diagnosis of dystrophinopathy was proposed for an ongoing pregnancy, whereas the cause of developmental delay in the index case remained unknown. Through these cases, we discussed the challenges of genetic counseling in the family, regarding the predictive issues for male individuals at risk for a muscular dystrophy without precise knowledge of the clinical consequences of some CNVs in the DMD gene. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Epidemiology of transmissible diseases: Array hybridization and next generation sequencing as universal nucleic acid-mediated typing tools.

PubMed

Michael Dunne, W; Pouseele, Hannes; Monecke, Stefan; Ehricht, Ralf; van Belkum, Alex

2017-09-21

The magnitude of interest in the epidemiology of transmissible human diseases is reflected in the vast number of tools and methods developed recently with the expressed purpose to characterize and track evolutionary changes that occur in agents of these diseases over time. Within the past decade a new suite of such tools has become available with the emergence of the so-called "omics" technologies. Among these, two are exponents of the ongoing genomic revolution. Firstly, high-density nucleic acid probe arrays have been proposed and developed using various chemical and physical approaches. Via hybridization-mediated detection of entire genes or genetic polymorphisms in such genes and intergenic regions these so called "DNA chips" have been successfully applied for distinguishing very closely related microbial species and strains. Second and even more phenomenal, next generation sequencing (NGS) has facilitated the assessment of the complete nucleotide sequence of entire microbial genomes. This technology currently provides the most detailed level of bacterial genotyping and hence allows for the resolution of microbial spread and short-term evolution in minute detail. We will here review the very recent history of these two technologies, sketch their usefulness in the elucidation of the spread and epidemiology of mostly hospital-acquired infections and discuss future developments. Copyright © 2017 Elsevier B.V. All rights reserved.

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains.

PubMed

Dumas, Laura; Dickens, C Michael; Anderson, Nathan; Davis, Jonathan; Bennett, Beth; Radcliffe, Richard A; Sikela, James M

2014-06-01

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to contribute to the alcohol-related phenotypic differences associated with these strains.

Amplification and chromosomal dispersion of human endogenous retroviral sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steele, P.E.; Martin, M.A.; Rabson, A.B.

1986-09-01

Endogenous retroviral sequences have undergone amplification events involving both viral and flanking cellular sequences. The authors cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification andmore » dispersion events may be linked.« less

Space and power efficient hybrid counters array

DOEpatents

Gara, Alan G [Mount Kisco, NY; Salapura, Valentina [Chappaqua, NY

2009-05-12

A hybrid counter array device for counting events. The hybrid counter array includes a first counter portion comprising N counter devices, each counter device for receiving signals representing occurrences of events from an event source and providing a first count value corresponding to a lower order bits of the hybrid counter array. The hybrid counter array includes a second counter portion comprising a memory array device having N addressable memory locations in correspondence with the N counter devices, each addressable memory location for storing a second count value representing higher order bits of the hybrid counter array. A control device monitors each of the N counter devices of the first counter portion and initiates updating a value of a corresponding second count value stored at the corresponding addressable memory location in the second counter portion. Thus, a combination of the first and second count values provide an instantaneous measure of number of events received.

Space and power efficient hybrid counters array

DOEpatents

Gara, Alan G.; Salapura, Valentina

2010-03-30

A hybrid counter array device for counting events. The hybrid counter array includes a first counter portion comprising N counter devices, each counter device for receiving signals representing occurrences of events from an event source and providing a first count value corresponding to a lower order bits of the hybrid counter array. The hybrid counter array includes a second counter portion comprising a memory array device having N addressable memory locations in correspondence with the N counter devices, each addressable memory location for storing a second count value representing higher order bits of the hybrid counter array. A control device monitors each of the N counter devices of the first counter portion and initiates updating a value of a corresponding second count value stored at the corresponding addressable memory location in the second counter portion. Thus, a combination of the first and second count values provide an instantaneous measure of number of events received.

Genome wide analysis in a discordant monozygotic twin with caudal appendage and multiple congenital anomalies.

PubMed

Cogulu, O; Pariltay, E; Koroglu, O A; Aykut, A; Ozyurek, R; Levent, E; Kultursay, N; Ozkinay, F

2013-01-01

Caudal appendage is a rare dysmorphic feature of which etiologic mechanisms are not well understood. Here we report monozygotic (MZ) twin brothers who are discordant for the caudal appendage and multiple congenital anomalies. Twins were the product of a 33 weeks of gestation, monochorionic-diamniotic pregnancy. On admission the proband had micrognathia, beaked nose, hypospadias, caudal appendage and juxtaductal aorta coarctation. At birth, he was small for gestational age and he had transient hypothyroidism which was detected in the newborn period. Karyotype analysis showed 46,XY. Monozygosity was shown by 15 microsatellite markers plus amelogenin (AmpFlSTR Identifiler PCR Amplification Kit, Applied Biosystems). Genome-wide copy number analysis of the twins by DNA-DNA hybridization of whole genomic DNA (NimbleGen Human CGH 385K WG-T v2.0 array) showed a significant difference at two neighboring probes with Log2 ratio: 0.72088 which are located on chromosome 3p12.3. Further analysis by high resolution of chromosome 3 array (Roche NimbleGen Human HG18 CHR3 FT Median Probe Spacing 475 bp) and quantitative PCR analysis did not confirm the deletion.

Rhabdoid glioblastoma is distinguishable from classical glioblastoma by cytogenetics and molecular genetics.

PubMed

Byeon, Sun-Ju; Cho, Hwa Jin; Baek, Hae Woon; Park, Chul-Kee; Choi, Seung-Hong; Kim, Se-Hoon; Kim, Hee Kyung; Park, Sung-Hye

2014-03-01

The clinicopathologic and molecular genetic features of 5 cases of rhabdoid glioblastoma, an extremely rare variant of glioblastoma that tends to affect patients at a young age, were investigated by immunohistochemical analysis and focused molecular genetic studies including array-based comparative genomic hybridization. All 5 cases had supratentorial tumors that immunohistochemical analysis revealed to be robustly positive for epithelial membrane antigen, vimentin, p53, and PDGFRα (platelet-derived growth factor receptor, alpha polypeptide) but only focally positive for glial fibrillary acidic protein. Although complete retention of SMARCB1 (INI1) was observed in all 5 cases, epidermal growth factor receptor (EGFR) amplification, PTEN (phosphatase and tensin homolog) loss, homozygous deletion of cyclin-dependent kinase inhibitor 2A, 1p/19q codeletion, and isocitrate dehydrogenase 1 R132/IDH2 R172 mutation were not observed in any case, although a high level of EGFR polysomy was detected in 1 recurrent tumor. Although c-MET (MET protein) expression was focal but robustly positive in 3 cases, met proto-oncogene (MET) fluorescence in situ hybridization revealed low polysomy but not MET amplification. MGMT (O-6-methylguanine-DNA methyl-40 transferase) methylation-specific polymerase chain reaction revealed MGMT methylation in only 1 case. Furthermore, array-based comparative genomic hybridization revealed gain of chromosome 7 and loss of 1p, 6, 8p, 11, 13q, and 18q but no deletion of chromosome 22. In contrast to the classical subtype of primary glioblastoma, the cases studied here were characterized by the absence of EGFR amplification, PTEN loss, and 9p homozygous deletion and overexpression of p53, PDGFRα, and c-MET, suggesting that they can be classified as the proneural or mesenchymal subtype of glioblastoma and benefit from intensive therapy that includes temozolomide. Copyright © 2014 Elsevier Inc. All rights reserved.

Diversity Arrays Technology (DArT) for Pan-Genomic Evolutionary Studies of Non-Model Organisms

PubMed Central

James, Karen E.; Schneider, Harald; Ansell, Stephen W.; Evers, Margaret; Robba, Lavinia; Uszynski, Grzegorz; Pedersen, Niklas; Newton, Angela E.; Russell, Stephen J.; Vogel, Johannes C.; Kilian, Andrzej

2008-01-01

Background High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms. Methodology/Principal Findings This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples. Conclusions/Significance These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography. PMID:18301759

COMPARISON OF COMPARATIVE GENOMIC HYBRIDIZATIONS TECHNOLOGIES ACROSS MICROARRAY PLATFORMS

EPA Science Inventory

Comparative Genomic Hybridization (CGH) measures DNA copy number differences between a reference genome and a test genome. The DNA samples are differentially labeled and hybridized to an immobilized substrate. In early CGH experiments, the DNA targets were hybridized to metaphase...

High resolution array CGH and gene expression profiling of alveolar soft part sarcoma

PubMed Central

Selvarajah, Shamini; Pyne, Saumyadipta; Chen, Eleanor; Sompallae, Ramakrishna; Ligon, Azra H.; Nielsen, Gunnlaugur P.; Dranoff, Glenn; Stack, Edward; Loda, Massimo; Flavin, Richard

2014-01-01

Purpose Alveolar soft part sarcoma (ASPS) is a soft tissue sarcoma with poor prognosis, and little molecular evidence for its origin, initiation and progression. The aim of this study was to elucidate candidate molecular pathways involved in tumor pathogenesis. Experimental Design We employed high-throughput array comparative genomic hybridization and cDNA-Mediated Annealing, Selection, Ligation, and Extension Assay to profile the genomic and expression signatures of primary and metastatic ASPS from 17 tumors derived from 11 patients. We used an integrative bioinformatics approach to elucidate the molecular pathways associated with ASPS progression. Fluorescence in situ hybridization was performed to validate the presence of the t(X;17)(p11.2;q25) ASPL-TFE3 fusion and hence confirm the aCGH observations. Results FISH analysis identified the ASPL-TFE3 fusion in all cases. ArrayCGH revealed a higher number of numerical aberrations in metastatic tumors relative to primaries, but failed to identify consistent alterations in either group. Gene expression analysis highlighted 1,063 genes which were differentially expressed between the two groups. Gene set enrichment analysis identified 16 enriched gene sets (p < 0.1) associated with differentially expressed genes. Notable among these were several stem cell gene expression signatures and pathways related to differentiation. In particular, the paired box transcription factor PAX6 was up-regulated in the primary tumors, along with several genes whose mouse orthologs have previously been implicated in Pax6-DNA binding during neural stem cell differentiation. Conclusion In addition to suggesting a tentative neural line of differentiation for ASPS, these results implicate transcriptional deregulation from fusion genes in the pathogenesis of ASPS. PMID:24493828

Array-Based Comparative Genomic Hybridization Analysis Reveals Chromosomal Copy Number Aberrations Associated with Clinical Outcome in Canine Diffuse Large B-Cell Lymphoma

PubMed Central

Bresolin, Silvia; Marconato, Laura; Comazzi, Stefano; Te Kronnie, Geertruy; Aresu, Luca

2014-01-01

Canine Diffuse Large B-cell Lymphoma (cDLBCL) is an aggressive cancer with variable clinical response. Despite recent attempts by gene expression profiling to identify the dog as a potential animal model for human DLBCL, this tumor remains biologically heterogeneous with no prognostic biomarkers to predict prognosis. The aim of this work was to identify copy number aberrations (CNAs) by high-resolution array comparative genomic hybridization (aCGH) in 12 dogs with newly diagnosed DLBCL. In a subset of these dogs, the genetic profiles at the end of therapy and at relapse were also assessed. In primary DLBCLs, 90 different genomic imbalances were counted, consisting of 46 gains and 44 losses. Two gains in chr13 were significantly correlated with clinical stage. In addition, specific regions of gains and losses were significantly associated to duration of remission. In primary DLBCLs, individual variability was found, however 14 recurrent CNAs (>30%) were identified. Losses involving IGK, IGL and IGH were always found, and gains along the length of chr13 and chr31 were often observed (>41%). In these segments, MYC, LDHB, HSF1, KIT and PDGFRα are annotated. At the end of therapy, dogs in remission showed four new CNAs, whereas three new CNAs were observed in dogs at relapse compared with the previous profiles. One ex novo CNA, involving TCR, was present in dogs in remission after therapy, possibly induced by the autologous vaccine. Overall, aCGH identified small CNAs associated with outcome, which, along with future expression studies, may reveal target genes relevant to cDLBCL. PMID:25372838

Indium Hybridization of Large Format TES Bolometer Arrays to Readout Multiplexers for Far-Infrared Astronomy

NASA Technical Reports Server (NTRS)

Miller, Timothy M.; Costen, Nick; Allen, Christine

2007-01-01

This conference poster reviews the Indium hybridization of the large format TES bolometer arrays. We are developing a key technology to enable the next generation of detectors. That is the Hybridization of Large Format Arrays using Indium bonded detector arrays containing 32x40 elements which conforms to the NIST multiplexer readout architecture of 1135 micron pitch. We have fabricated and hybridized mechanical models with the detector chips bonded after being fully back-etched. The mechanical support consists of 30 micron walls between elements Demonstrated electrical continuity for each element. The goal is to hybridize fully functional array of TES detectors to NIST readout.

Centromere Locations in Brassica A and C Genomes Revealed Through Half-Tetrad Analysis

PubMed Central

Mason, Annaliese S.; Rousseau-Gueutin, Mathieu; Morice, Jérôme; Bayer, Philipp E.; Besharat, Naghmeh; Cousin, Anouska; Pradhan, Aneeta; Parkin, Isobel A. P.; Chèvre, Anne-Marie; Batley, Jacqueline; Nelson, Matthew N.

2016-01-01

Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromere-specific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate. PMID:26614742

Centromere Locations in Brassica A and C Genomes Revealed Through Half-Tetrad Analysis.

PubMed

Mason, Annaliese S; Rousseau-Gueutin, Mathieu; Morice, Jérôme; Bayer, Philipp E; Besharat, Naghmeh; Cousin, Anouska; Pradhan, Aneeta; Parkin, Isobel A P; Chèvre, Anne-Marie; Batley, Jacqueline; Nelson, Matthew N

2016-02-01

Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromere-specific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate. Copyright © 2016 by the Genetics Society of America.

Branchio-otic syndrome caused by a genomic rearrangement: clinical findings and molecular cytogenetic studies in a patient with a pericentric inversion of chromosome 8.

PubMed

Schmidt, T; Bierhals, T; Kortüm, F; Bartels, I; Liehr, T; Burfeind, P; Shoukier, M; Frank, V; Bergmann, C; Kutsche, K

2014-01-01

Branchio-oto-renal (BOR) syndrome is an autosomal dominantly inherited developmental disorder, which is characterized by anomalies of the ears, the branchial arches and the kidneys. It is caused by mutations in the genes EYA1,SIX1 and SIX5. Genomic rearrangements of chromosome 8 affecting the EYA1 gene have also been described. Owing to this fact, methods for the identification of abnormal copy numbers such as multiplex ligation-dependent probe amplification (MLPA) have been introduced as routine laboratory techniques for molecular diagnostics of BOR syndrome. The advantages of these techniques are clear compared to standard cytogenetic and array approaches as well as Southern blot. MLPA detects deletions or duplications of a part or the entire gene of interest, but not balanced structural aberrations such as inversions and translocations. Consequently, disruption of a gene by a genomic rearrangement may escape detection by a molecular genetic analysis, although this gene interruption results in haploinsufficiency and, therefore, causes the disease. In a patient with clinical features of BOR syndrome, such as hearing loss, preauricular fistulas and facial dysmorphisms, but no renal anomalies, neither sequencing of the 3 genes linked to BOR syndrome nor array comparative genomic hybridization and MLPA were able to uncover a causative mutation. By routine cytogenetic analysis, we finally identified a pericentric inversion of chromosome 8 in the affected female. High-resolution multicolor banding confirmed the chromosome 8 inversion and narrowed down the karyotype to 46,XX,inv(8)(p22q13). By applying fluorescence in situ hybridization, we narrowed down both breakpoints on chromosome 8 and found the EYA1 gene in q13.3 to be directly disrupted. We conclude that standard karyotyping should not be neglected in the genetic diagnostics of BOR syndrome or other Mendelian disorders, particularly when molecular testing failed to detect any causative alteration in patients with a convincing phenotype. © 2013 S. Karger AG, Basel.

Fine mapping of regulatory loci for mammalian gene expression using radiation hybrids

PubMed Central

Park, Christopher C; Ahn, Sangtae; Bloom, Joshua S; Lin, Andy; Wang, Richard T; Wu, Tongtong; Sekar, Aswin; Khan, Arshad H; Farr, Christine J; Lusis, Aldons J; Leahy, Richard M; Lange, Kenneth; Smith, Desmond J

2010-01-01

We mapped regulatory loci for nearly all protein-coding genes in mammals using comparative genomic hybridization and expression array measurements from a panel of mouse–hamster radiation hybrid cell lines. The large number of breaks in the mouse chromosomes and the dense genotyping of the panel allowed extremely sharp mapping of loci. As the regulatory loci result from extra gene dosage, we call them copy number expression quantitative trait loci, or ceQTLs. The −2log10P support interval for the ceQTLs was <150 kb, containing an average of <2–3 genes. We identified 29,769 trans ceQTLs with −log10P > 4, including 13 hotspots each regulating >100 genes in trans. Further, this work identifies 2,761 trans ceQTLs harboring no known genes, and provides evidence for a mode of gene expression autoregulation specific to the X chromosome. PMID:18362883

The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization

PubMed Central

2012-01-01

Background Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis. Methods Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients. Results We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian. Conclusions Our study provides an invaluable database revealing common and differential imbalance regions at specific chromosomes among Asian and Caucasian lung cancer patients. Four validation methods confirmed our database, which would help in further studies on the mechanism of lung tumorigenesis. PMID:22691236

Colloidal silica films for high-capacity DNA arrays

NASA Astrophysics Data System (ADS)

Glazer, Marc Irving

The human genome project has greatly expanded the amount of genetic information available to researchers, but before this vast new source of data can be fully utilized, techniques for rapid, large-scale analysis of DNA and RNA must continue to develop. DNA arrays have emerged as a powerful new technology for analyzing genomic samples in a highly parallel format. The detection sensitivity of these arrays is dependent on the quantity and density of immobilized probe molecules. We have investigated substrates with a porous, "three-dimensional" surface layer as a means of increasing the surface area available for the synthesis of oligonucleotide probes, thereby increasing the number of available probes and the amount of detectable bound target. Porous colloidal silica films were created by two techniques. In the first approach, films were deposited by spin-coating silica colloid suspensions onto flat glass substrates, with the pores being formed by the natural voids between the solid particles (typically 23nm pores, 35% porosity). In the second approach, latex particles were co-deposited with the silica and then pyrolyzed, creating films with larger pores (36 nm), higher porosity (65%), and higher surface area. For 0.3 mum films, enhancements of eight to ten-fold and 12- to 14-fold were achieved with the pure silica films and the films "templated" with polymer latex, respectively. In gene expression assays for up to 7,000 genes using complex biological samples, the high-capacity films provided enhanced signals and performed equivalently or better than planar glass on all other functional measures, confirming that colloidal silica films are a promising platform for high-capacity DNA arrays. We have also investigated the kinetics of hybridization on planar glass and high-capacity substrates. Adsorption on planar arrays is similar to ideal Langmuir-type adsorption, although with an "overshoot" at high solution concentration. Hybridization on high-capacity films is controlled by traditional adsorption (ka) and desorption (kd) coefficients, as well as morphology factors and transient binding interactions between the target and probes. The strength of the transient probe/target binding interactions are on the order of 5--7 DNA base pairs, which suggests the formation of nucleation or other metastable complexes, rather than fully-zippered duplexes.

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes).

PubMed

Yamada, Kazuhiko; Nishida-Umehara, Chizuko; Matsuda, Yoichi

2004-03-01

We isolated a new family of satellite DNA sequences from HaeIII- and EcoRI-digested genomic DNA of the Blakiston's fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

PubMed

Deng, Wulan; Shi, Xinghua; Tjian, Robert; Lionnet, Timothée; Singer, Robert H

2015-09-22

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

Transposable Element Proliferation and Genome Expansion Are Rare in Contemporary Sunflower Hybrid Populations Despite Widespread Transcriptional Activity of LTR Retrotransposons

PubMed Central

Kawakami, Takeshi; Dhakal, Preeti; Katterhenry, Angela N.; Heatherington, Chelsea A.; Ungerer, Mark C.

2011-01-01

Hybridization is a natural phenomenon that has been linked in several organismal groups to transposable element derepression and copy number amplification. A noteworthy example involves three diploid annual sunflower species from North America that have arisen via ancient hybridization between the same two parental taxa, Helianthus annuus and H. petiolaris. The genomes of the hybrid species have undergone large-scale increases in genome size attributable to long terminal repeat (LTR) retrotransposon proliferation. The parental species that gave rise to the hybrid taxa are widely distributed, often sympatric, and contemporary hybridization between them is common. Natural H. annuus × H. petiolaris hybrid populations likely served as source populations from which the hybrid species arose and, as such, represent excellent natural experiments for examining the potential role of hybridization in transposable element derepression and proliferation in this group. In the current report, we examine multiple H. annuus × H. petiolaris hybrid populations for evidence of genome expansion, LTR retrotransposon copy number increases, and LTR retrotransposon transcriptional activity. We demonstrate that genome expansion and LTR retrotransposon proliferation are rare in contemporary hybrid populations, despite independent proliferation events that took place in the genomes of the ancient hybrid species. Interestingly, LTR retrotransposon lineages that proliferated in the hybrid species genomes remain transcriptionally active in hybrid and nonhybrid genotypes across the entire sampling area. The finding of transcriptional activity but not copy number increases in hybrid genotypes suggests that proliferation and genome expansion in contemporary hybrid populations may be mitigated by posttranscriptional mechanisms of repression. PMID:21282712

Multiple displacement amplification on single cell and possible PGD applications.

PubMed

Hellani, Ali; Coskun, Serdar; Benkhalifa, Moncef; Tbakhi, Abelghani; Sakati, Nadia; Al-Odaib, Ali; Ozand, Pinar

2004-11-01

Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.

Characterization of a chromosome-specific chimpanzee alpha satellite subset: Evolutionary relationship to subsets on human chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warburton, P.E.; Gosden, J.; Lawson, D.

1996-04-15

Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize andmore » spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.« less

A Genome-Wide Survey of Genetic Instability by Transposition in Drosophila Hybrids

PubMed Central

Vela, Doris; Fontdevila, Antonio; Vieira, Cristina; García Guerreiro, María Pilar

2014-01-01

Hybridization between species is a genomic instability factor involved in increasing mutation rate and new chromosomal rearrangements. Evidence of a relationship between interspecific hybridization and transposable element mobilization has been reported in different organisms, but most studies are usually performed with particular TEs and do not discuss the real effect of hybridization on the whole genome. We have therefore studied whole genome instability of Drosophila interspecific hybrids, looking for the presence of new AFLP markers in hybrids. A high percentage (27–90%) of the instability markers detected corresponds to TEs belonging to classes I and II. Moreover, three transposable elements (Osvaldo, Helena and Galileo) representative of different families, showed an overall increase of transposition rate in hybrids compared to parental species. This research confirms the hypothesis that hybridization induces genomic instability by transposition bursts and suggests that genomic stress by transposition could contribute to a relaxation of mechanisms controlling TEs in the Drosophila genome. PMID:24586475

Drosophila Females Undergo Genome Expansion after Interspecific Hybridization

PubMed Central

Romero-Soriano, Valèria; Burlet, Nelly; Vela, Doris; Fontdevila, Antonio; Vieira, Cristina; García Guerreiro, María Pilar

2016-01-01

Genome size (or C-value) can present a wide range of values among eukaryotes. This variation has been attributed to differences in the amplification and deletion of different noncoding repetitive sequences, particularly transposable elements (TEs). TEs can be activated under different stress conditions such as interspecific hybridization events, as described for several species of animals and plants. These massive transposition episodes can lead to considerable genome expansions that could ultimately be involved in hybrid speciation processes. Here, we describe the effects of hybridization and introgression on genome size of Drosophila hybrids. We measured the genome size of two close Drosophila species, Drosophila buzzatii and Drosophila koepferae, their F1 offspring and the offspring from three generations of backcrossed hybrids; where mobilization of up to 28 different TEs was previously detected. We show that hybrid females indeed present a genome expansion, especially in the first backcross, which could likely be explained by transposition events. Hybrid males, which exhibit more variable C-values among individuals of the same generation, do not present an increased genome size. Thus, we demonstrate that the impact of hybridization on genome size can be detected through flow cytometry and is sex-dependent. PMID:26872773

Complex rearranged small supernumerary marker chromosomes (sSMC), three new cases; evidence for an underestimated entity?

PubMed Central

Trifonov, Vladimir; Fluri, Simon; Binkert, Franz; Nandini, Adayapalam; Anderson, Jasen; Rodriguez, Laura; Gross, Madeleine; Kosyakova, Nadezda; Mkrtchyan, Hasmik; Ewers, Elisabeth; Reich, Daniela; Weise, Anja; Liehr, Thomas

2008-01-01

Background Small supernumerary marker chromosomes (sSMC) are present ~2.6 × 106 human worldwide. sSMC are a heterogeneous group of derivative chromosomes concerning their clinical consequences as well as their chromosomal origin and shape. Besides the sSMC present in Emanuel syndrome, i.e. der(22)t(11;22)(q23;q11), only few so-called complex sSMC are reported. Results Here we report three new cases of unique complex sSMC. One was a de novo case with a dic(13 or 21;22) and two were maternally derived: a der(18)t(8;18) and a der(13 or 21)t(13 or 21;18). Thus, in summary, now 22 cases of unique complex sSMC are available in the literature. However, this special kind of sSMC might be under-diagnosed among sSMC-carriers. Conclusion More comprehensive characterization of sSMC and approaches like reverse fluorescence in situ hybridization (FISH) or array based comparative genomic hybridization (array-CGH) might identify them to be more frequent than only ~0.9% among all sSMC. PMID:18471318

The Hybridization Barrier between Herbaceous Medicago sativa and Woody M. arborea Is Weakened by Selection of Seed Parents

PubMed Central

Bingham, Edwin; Armour, David; Irwin, John

2013-01-01

Medicago sativa, alfalfa or lucerne, and M. arborea were considered reproductively isolated until recently. Then, in 2003, an alfalfa genotype was identified that produced a few seeds and progeny with hybrid traits after a large number of pollinations by M. arborea. A derivative of this alfalfa genotype also produced a low frequency of progeny with hybrid traits. Thus, the hybridization barrier was weakened by selection of seed parents. Hybrids from both events expressed traits from M. arborea and M. arborea-specific DNA bands, although more of the M. sativa genome was retained, based on the DNA results. Thus, there was chromatin elimination during embryogenesis, resulting in partial hybrids (hereafter hybrids). However, more than 30 hybrids with an array of M. arborea traits have been obtained thus far, and research continues on the nature of the hybrids. Traits have been genetically transmitted in crosses, and selected traits are in use for alfalfa breeding. This paper reviews the first hybrids and then focuses on further weakening of the hybridization barrier with the discovery of a more efficient hybridizer derived from crossing Medicago sativa subspecies, sativa, coerulea and falcata. This genotype was found to have reproductive abnormalities associated with its complex subspecies origin that are best described as hybrid breakdown. In effect, this subspecies derivative is a bridge-cross parent that consistently produces hybrids. Reproductive abnormalities in the bridge-cross parent are reported and discussed. PMID:27137379

Value for money? Array genomic hybridization for diagnostic testing for genetic causes of intellectual disability.

PubMed

Regier, Dean A; Friedman, Jan M; Marra, Carlo A

2010-05-14

Array genomic hybridization (AGH) provides a higher detection rate than does conventional cytogenetic testing when searching for chromosomal imbalance causing intellectual disability (ID). AGH is more costly than conventional cytogenetic testing, and it remains unclear whether AGH provides good value for money. Decision analytic modeling was used to evaluate the trade-off between costs, clinical effectiveness, and benefit of an AGH testing strategy compared to a conventional testing strategy. The trade-off between cost and effectiveness was expressed via the incremental cost-effectiveness ratio. Probabilistic sensitivity analysis was performed via Monte Carlo simulation. The baseline AGH testing strategy led to an average cost increase of $217 (95% CI $172-$261) per patient and an additional 8.2 diagnoses in every 100 tested (0.082; 95% CI 0.044-0.119). The mean incremental cost per additional diagnosis was $2646 (95% CI $1619-$5296). Probabilistic sensitivity analysis demonstrated that there was a 95% probability that AGH would be cost effective if decision makers were willing to pay $4550 for an additional diagnosis. Our model suggests that using AGH instead of conventional karyotyping for most ID patients provides good value for money. Deterministic sensitivity analysis found that employing AGH after first-line cytogenetic testing had proven uninformative did not provide good value for money when compared to using AGH as first-line testing. Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Exonic duplication CNV of NDRG1 associated with autosomal-recessive HMSN-Lom/CMT4D.

PubMed

Okamoto, Yuji; Goksungur, Meryem Tuba; Pehlivan, Davut; Beck, Christine R; Gonzaga-Jauregui, Claudia; Muzny, Donna M; Atik, Mehmed M; Carvalho, Claudia M B; Matur, Zeliha; Bayraktar, Serife; Boone, Philip M; Akyuz, Kaya; Gibbs, Richard A; Battaloglu, Esra; Parman, Yesim; Lupski, James R

2014-05-01

Copy-number variations as a mutational mechanism contribute significantly to human disease. Approximately one-half of the patients with Charcot-Marie-Tooth (CMT) disease have a 1.4 Mb duplication copy-number variation as the cause of their neuropathy. However, non-CMT1A neuropathy patients rarely have causative copy-number variations, and to date, autosomal-recessive disease has not been associated with copy-number variation as a mutational mechanism. We performed Agilent 8 × 60 K array comparative genomic hybridization on DNA from 12 recessive Turkish families with CMT disease. Additional molecular studies were conducted to detect breakpoint junctions and to evaluate gene expression levels in a family in which we detected an intragenic duplication copy-number variation. We detected an ~6.25 kb homozygous intragenic duplication in NDRG1, a gene known to be causative for recessive HMSNL/CMT4D, in three individuals from a Turkish family with CMT neuropathy. Further studies showed that this intragenic copy-number variation resulted in a homozygous duplication of exons 6-8 that caused decreased mRNA expression of NDRG1. Exon-focused high-resolution array comparative genomic hybridization enables the detection of copy-number variation carrier states in recessive genes, particularly small copy-number variations encompassing or disrupting single genes. In families for whom a molecular diagnosis has not been elucidated by conventional clinical assays, an assessment for copy-number variations in known CMT genes might be considered.

Array-based comparative genomic hybridization-guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs.

PubMed

Tsai, Pei-Chien; Breen, Matthew

2012-09-01

To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs. Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues. Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs. LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes. Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

Marker chromosome genomic structure and temporal origin implicate a chromoanasynthesis event in a family with pleiotropic psychiatric phenotypes.

PubMed

Grochowski, Christopher M; Gu, Shen; Yuan, Bo; Tcw, Julia; Brennand, Kristen J; Sebat, Jonathan; Malhotra, Dheeraj; McCarthy, Shane; Rudolph, Uwe; Lindstrand, Anna; Chong, Zechen; Levy, Deborah L; Lupski, James R; Carvalho, Claudia M B

2018-04-25

Small supernumerary marker chromosomes (sSMC) are chromosomal fragments difficult to characterize genomically. Here, we detail a proband with schizoaffective disorder and a mother with bipolar disorder with psychotic features who present with a marker chromosome that segregates with disease. We explored the architecture of this marker and investigated its temporal origin. Array comparative genomic hybridization (aCGH) analysis revealed three duplications and three triplications that spanned the short arm of chromosome 9, suggestive of a chromoanasynthesis-like event. Segregation of marker genotypes, phased using sSMC mosaicism in the mother, provided evidence that it was generated during a germline-level event in the proband's maternal grandmother. Whole-genome sequencing (WGS) was performed to resolve the structure and junctions of the chromosomal fragments, revealing further complexities. While structural variations have been previously associated with neuropsychiatric disorders and marker chromosomes, here we detail the precise architecture, human life-cycle genesis, and propose a DNA replicative/repair mechanism underlying formation. © 2018 Wiley Periodicals, Inc.

A Roadmap for Functional Structural Variants in the Soybean Genome

PubMed Central

Anderson, Justin E.; Kantar, Michael B.; Kono, Thomas Y.; Fu, Fengli; Stec, Adrian O.; Song, Qijian; Cregan, Perry B.; Specht, James E.; Diers, Brian W.; Cannon, Steven B.; McHale, Leah K.; Stupar, Robert M.

2014-01-01

Gene structural variation (SV) has recently emerged as a key genetic mechanism underlying several important phenotypic traits in crop species. We screened a panel of 41 soybean (Glycine max) accessions serving as parents in a soybean nested association mapping population for deletions and duplications in more than 53,000 gene models. Array hybridization and whole genome resequencing methods were used as complementary technologies to identify SV in 1528 genes, or approximately 2.8%, of the soybean gene models. Although SV occurs throughout the genome, SV enrichment was noted in families of biotic defense response genes. Among accessions, SV was nearly eightfold less frequent for gene models that have retained paralogs since the last whole genome duplication event, compared with genes that have not retained paralogs. Increases in gene copy number, similar to that described at the Rhg1 resistance locus, account for approximately one-fourth of the genic SV events. This assessment of soybean SV occurrence presents a target list of genes potentially responsible for rapidly evolving and/or adaptive traits. PMID:24855315

Novel fabrication technique of hybrid structure lens array for 3D images

NASA Astrophysics Data System (ADS)

Lee, Junsik; Kim, Junoh; Kim, Cheoljoong; Shin, Dooseub; Koo, Gyohyun; Won, Yong Hyub

2016-03-01

Tunable liquid lens arrays can produce three dimensional images by using electrowetting principle that alters surface tensions by applying voltage. This method has advantages of fast response time and low power consumption. However, it is challenging to fabricate a high fill factor liquid lens array and operate three dimensional images which demand high diopter. This study describes a hybrid structure lens array which has not only a liquid lens array but a solid lens array. A concave-shape lens array is unavoidable when using only the liquid lens array and some voltages are needed to make the lens flat. By placing the solid lens array on the liquid lens array, initial diopter can be positive. To fabricate the hybrid structure lens array, a conventional lithographic process in semiconductor manufacturing is needed. A negative photoresist SU-8 was used as chamber master molds. PDMS and UV adhesive replica molding are done sequentially. Two immiscible liquids, DI water and dodecane, are injected in the fabricated chamber, followed by sealing. The fabricated structure has a 20 by 20 pattern of cylindrical shaped circle array and the aperture size of each lens is 1mm. The thickness of the overall hybrid structure is about 2.8mm. Hybrid structure lens array has many advantages. Solid lens array has almost 100% fill factor and allow high efficiency. Diopter can be increased by more than 200 and negative diopter can be shifted to the positive region. This experiment showed several properties of the hybrid structure and demonstrated its superiority.

Mitochondrial Recombination and Introgression during Speciation by Hybridization.

PubMed

Leducq, Jean-Baptiste; Henault, Mathieu; Charron, Guillaume; Nielly-Thibault, Lou; Terrat, Yves; Fiumera, Heather L; Shapiro, B Jesse; Landry, Christian R

2017-08-01

Genome recombination is a major source of genotypic diversity and contributes to adaptation and speciation following interspecies hybridization. The contribution of recombination in these processes has been thought to be largely limited to the nuclear genome because organelles are mostly uniparentally inherited in animals and plants, which prevents recombination. Unicellular eukaryotes such as budding yeasts do, however, transmit mitochondria biparentally, suggesting that during hybridization, both parents could provide alleles that contribute to mitochondrial functions such as respiration and metabolism in hybrid populations or hybrid species. We examined the dynamics of mitochondrial genome transmission and evolution during speciation by hybridization in the natural budding yeast Saccharomyces paradoxus. Using population-scale mitochondrial genome sequencing in two endemic North American incipient species SpB and SpC and their hybrid species SpC*, we found that both parental species contributed to the hybrid mitochondrial genome through recombination. We support our findings by showing that mitochondrial recombination between parental types is frequent in experimental crosses that recreate the early step of this speciation event. In these artificial hybrids, we observed that mitochondrial genome recombination enhances phenotypic variation among diploid hybrids, suggesting that it could play a role in the phenotypic differentiation of hybrid species. Like the nuclear genome, the mitochondrial genome can, therefore, also play a role in hybrid speciation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chronic arsenic trioxide exposure leads to enhanced aggressiveness via Met oncogene addiction in cancer cells

PubMed Central

Kryeziu, Kushtrim; Pirker, Christine; Englinger, Bernhard; van Schoonhoven, Sushilla; Spitzwieser, Melanie; Mohr, Thomas; Körner, Wilfried; Weinmüllner, Regina; Tav, Koray; Grillari, Johannes; Cichna-Markl, Margit; Berger, Walter; Heffeter, Petra

2016-01-01

As an environmental poison, arsenic is responsible for many cancer deaths. Paradoxically, arsenic trioxide (ATO) presents also a powerful therapy used to treat refractory acute promyelocytic leukemia (APL) and is intensively investigated for treatment of other cancer types. Noteworthy, cancer therapy is frequently hampered by drug resistance, which is also often associated with enhancement of tumor aggressiveness. In this study, we analyzed ATO-selected cancer cells (A2780ATO) for the mechanisms underlying their enhanced tumorigenicity and aggressiveness. These cells were characterized by enhanced proliferation and spheroid growth as well as increased tumorigenicity of xenografts in SCID mice. Noteworthy, subsequent studies revealed that overexpression of Met receptor was the underlying oncogenic driver of these effects, as A2780ATO cells were characterized by collateral sensitivity against Met inhibitors. This finding was also confirmed by array comparative genomic hybridization (array CGH) and whole genome gene expression arrays, which revealed that Met overexpression by chronic ATO exposure was based on the transcriptional regulation via activation of AP-1. Finally, it was shown that treatment with the Met inhibitor crizotinib was also effective against A2780ATO cell xenografts in vivo, indicating that targeting of Met presents a promising strategy for the treatment of Met-overexpressing tumors after either arsenic exposure or failure to ATO treatment. PMID:27036042

Origin of year-long bean (Phaseolus dumosus Macfady, Fabaceae) from reticulated hybridization events between multiple Phaseolus species.

PubMed

Mina-Vargas, Angela M; McKeown, Peter C; Flanagan, Nicola S; Debouck, Daniel G; Kilian, Andrzej; Hodkinson, Trevor R; Spillane, Charles

2016-08-06

Improved understanding of the secondary gene pools of crops is essential for advancing genetic gain in breeding programmes. Common bean, Phaseolus vulgaris, is a staple crop with several wild relatives in its secondary gene pool. The year-long bean, P. dumosus, an important crop in Guatemala, is considered particularly closely related to P. vulgaris and a potential source of novel variation. However, the genetic diversity and relationship to other Phaseolus species of P. dumosus remain unclear. We conducted the first comprehensive investigation of P. dumosus genetic diversity using both nuclear and chloroplast genome markers. Our nuclear marker set included over 700 markers present within the Phaseolus DArT (Diversity Arrays Technology) array, which we applied to P. dumosus and other relatives of P. vulgaris (including every secondary gene pool species: P. acutifolius, P. albescens, P. coccineus and P. costaricensis). Phaseolus dumosus arose from hybridization of P. vulgaris and P. coccineus, followed by at least two later hybridizations with sympatric congener populations. Existing P. dumosus collections have low genetic diversity. The under-utilized crop P. dumosus has a complex hybrid origin. Further sampling in the region in which it arose may uncover additional germplasm for introgressing favourable traits into crops within the P. vulgaris gene pool. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genomic Imbalances Are Confined to Non-Proliferating Cells in Paediatric Patients with Acute Myeloid Leukaemia and a Normal or Incomplete Karyotype

PubMed Central

Ballabio, Erica; Regan, Regina; Garimberti, Elisa; Harbott, Jochen; Bradtke, Jutta; Teigler-Schlegel, Andrea; Biondi, Andrea; Cazzaniga, Giovanni; Giudici, Giovanni; Wainscoat, James S.; Boultwood, Jacqueline; Bridger, Joanna M.; Knight, Samantha J. L.; Tosi, Sabrina

2011-01-01

Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status. PMID:21694761

MYC and MYCN amplification can be reliably assessed by aCGH in medulloblastoma.

PubMed

Bourdeaut, Franck; Grison, Camille; Maurage, Claude-Alain; Laquerriere, Annie; Vasiljevic, Alexandre; Delisle, Marie-Bernadette; Michalak, Sophie; Figarella-Branger, Dominique; Doz, François; Richer, Wilfrid; Pierron, Gaelle; Miquel, Catherine; Delattre, Olivier; Couturier, Jérôme

2013-04-01

As prognostic factors, MYC and MYCN amplifications are routinely assessed in medulloblastomas. Fluorescence in situ hybridization (FISH) is currently considered as the technique of reference. Recently, array comparative genomic hybridization (aCGH) has been developed as an alternative technique to evaluate genomic abnormalities in other tumor types; however, this technique has not been widely adopted as a replacement for FISH in medulloblastoma. In this study, 34 tumors were screened by both FISH and aCGH. In all cases showing amplification by FISH, aCGH also unambiguously revealed the abnormality. The aCGH technique was also performed on tumors showing no amplification by FISH, and the absence of amplification was confirmed in all cases. Interestingly, one tumor showed a subclonal MYC amplification by FISH. This subclonal amplification was observed in approximately 20% of tumor cells and was clearly evident on aCGH. In conclusion, our analysis confirms that aCGH is as safe as FISH for the detection of MYC/MYCN gene amplification. Given its cost efficiency in comparison to two FISH tests and the global genomic information additionally provided by an aCGH experiment, this reproducible technique can be safely retained as an alternative to FISH for routine investigation of medulloblastoma. Copyright © 2013 Elsevier Inc. All rights reserved.

Discovery and mapping of single feature polymorphisms in wheat using Affymetrix arrays

PubMed Central

Bernardo, Amy N; Bradbury, Peter J; Ma, Hongxiang; Hu, Shengwa; Bowden, Robert L; Buckler, Edward S; Bai, Guihua

2009-01-01

Background Wheat (Triticum aestivum L.) is a staple food crop worldwide. The wheat genome has not yet been sequenced due to its huge genome size (~17,000 Mb) and high levels of repetitive sequences; the whole genome sequence may not be expected in the near future. Available linkage maps have low marker density due to limitation in available markers; therefore new technologies that detect genome-wide polymorphisms are still needed to discover a large number of new markers for construction of high-resolution maps. A high-resolution map is a critical tool for gene isolation, molecular breeding and genomic research. Single feature polymorphism (SFP) is a new microarray-based type of marker that is detected by hybridization of DNA or cRNA to oligonucleotide probes. This study was conducted to explore the feasibility of using the Affymetrix GeneChip to discover and map SFPs in the large hexaploid wheat genome. Results Six wheat varieties of diverse origins (Ning 7840, Clark, Jagger, Encruzilhada, Chinese Spring, and Opata 85) were analyzed for significant probe by variety interactions and 396 probe sets with SFPs were identified. A subset of 164 unigenes was sequenced and 54% showed polymorphism within probes. Microarray analysis of 71 recombinant inbred lines from the cross Ning 7840/Clark identified 955 SFPs and 877 of them were mapped together with 269 simple sequence repeat markers. The SFPs were randomly distributed within a chromosome but were unevenly distributed among different genomes. The B genome had the most SFPs, and the D genome had the least. Map positions of a selected set of SFPs were validated by mapping single nucleotide polymorphism using SNaPshot and comparing with expressed sequence tags mapping data. Conclusion The Affymetrix array is a cost-effective platform for SFP discovery and SFP mapping in wheat. The new high-density map constructed in this study will be a useful tool for genetic and genomic research in wheat. PMID:19480702

A segmentation/clustering model for the analysis of array CGH data.

PubMed

Picard, F; Robin, S; Lebarbier, E; Daudin, J-J

2007-09-01

Microarray-CGH (comparative genomic hybridization) experiments are used to detect and map chromosomal imbalances. A CGH profile can be viewed as a succession of segments that represent homogeneous regions in the genome whose representative sequences share the same relative copy number on average. Segmentation methods constitute a natural framework for the analysis, but they do not provide a biological status for the detected segments. We propose a new model for this segmentation/clustering problem, combining a segmentation model with a mixture model. We present a new hybrid algorithm called dynamic programming-expectation maximization (DP-EM) to estimate the parameters of the model by maximum likelihood. This algorithm combines DP and the EM algorithm. We also propose a model selection heuristic to select the number of clusters and the number of segments. An example of our procedure is presented, based on publicly available data sets. We compare our method to segmentation methods and to hidden Markov models, and we show that the new segmentation/clustering model is a promising alternative that can be applied in the more general context of signal processing.

Copy Number Variants in Obesity-Related Syndromes: Review and Perspectives on Novel Molecular Approaches

PubMed Central

Koiffmann, Celia Priszkulnik

2012-01-01

In recent decades, obesity has reached epidemic proportions worldwide and became a major concern in public health. Despite heritability estimates of 40 to 70% and the long-recognized genetic basis of obesity in a number of rare cases, the list of common obesity susceptibility variants by the currently published genome-wide association studies (GWASs) only explain a small proportion of the individual variation in risk of obesity. It was not until very recently that GWASs of copy number variants (CNVs) in individuals with extreme phenotypes reported a number of large and rare CNVs conferring high risk to obesity, and specifically deletions on chromosome 16p11.2. In this paper, we comment on the recent advances in the field of genetics of obesity with an emphasis on the genes and genomic regions implicated in highly penetrant forms of obesity associated with developmental disorders. Array genomic hybridization in this patient population has afforded discovery opportunities for CNVs that have not previously been detectable. This information can be used to generate new diagnostic arrays and sequencing platforms, which will likely enhance detection of known genetic conditions with the potential to elucidate new disease genes and ultimately help in developing a next-generation sequencing protocol relevant to clinical practice. PMID:23316347

A Discovery Resource of Rare Copy Number Variations in Individuals with Autism Spectrum Disorder

PubMed Central

Prasad, Aparna; Merico, Daniele; Thiruvahindrapuram, Bhooma; Wei, John; Lionel, Anath C.; Sato, Daisuke; Rickaby, Jessica; Lu, Chao; Szatmari, Peter; Roberts, Wendy; Fernandez, Bridget A.; Marshall, Christian R.; Hatchwell, Eli; Eis, Peggy S.; Scherer, Stephen W.

2012-01-01

The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection. PMID:23275889

Stress-induced rearrangement of Fusarium retrotransposon sequences.

PubMed

Anaya, N; Roncero, M I

1996-11-27

Rearrangement of fusarium oxysporum retrotransposon skippy was induced by growth in the presence of potassium chlorate. Three fungal strains, one sensitive to chlorate (Co60) and two resistant to chlorate and deficient for nitrate reductase (Co65 and Co94), were studied by Southern analysis of their genomic DNA. Polymorphism was detected in their hybridization banding pattern, relative to the wild type grown in the absence of chlorate, using various enzymes with or without restriction sites within the retrotransposon. Results were consistent with the assumption that three different events had occurred in strain Co60: genomic amplification of skippy yielding tandem arrays of the element, generation of new skippy sequences, and deletion of skippy sequences. Amplification of Co60 genomic DNA using the polymerase chain reaction and divergent primers derived from the retrotransposon generated a new band, corresponding to one long terminal repeat plus flanking sequences, that was not present in the wild-type strain. Molecular analysis of nitrate reductase-deficient mutants showed that generation and deletion of skippy sequences, but not genomic amplification in tandem repeats, had occurred in their genomes.

Mechanisms Underlying Mammalian Hybrid Sterility in Two Feline Interspecies Models

PubMed Central

Davis, Brian W.; Seabury, Christopher M.; Brashear, Wesley A.; Li, Gang; Roelke-Parker, Melody; Murphy, William J.

2015-01-01

The phenomenon of male sterility in interspecies hybrids has been observed for over a century, however, few genes influencing this recurrent phenotype have been identified. Genetic investigations have been primarily limited to a small number of model organisms, thus limiting our understanding of the underlying molecular basis of this well-documented “rule of speciation.” We utilized two interspecies hybrid cat breeds in a genome-wide association study employing the Illumina 63 K single-nucleotide polymorphism array. Collectively, we identified eight autosomal genes/gene regions underlying associations with hybrid male sterility (HMS) involved in the function of the blood-testis barrier, gamete structural development, and transcriptional regulation. We also identified several candidate hybrid sterility regions on the X chromosome, with most residing in close proximity to complex duplicated regions. Differential gene expression analyses revealed significant chromosome-wide upregulation of X chromosome transcripts in testes of sterile hybrids, which were enriched for genes involved in chromatin regulation of gene expression. Our expression results parallel those reported in Mus hybrids, supporting the “Large X-Effect” in mammalian HMS and the potential epigenetic basis for this phenomenon. These results support the value of the interspecies feline model as a powerful tool for comparison to rodent models of HMS, demonstrating unique aspects and potential commonalities that underpin mammalian reproductive isolation. PMID:26006188

A diversity study of Saccharomycopsis fibuligera in rice wine starter nuruk, reveals the evolutionary process associated with its interspecies hybrid.

PubMed

Farh, Mohamed El-Agamy; Cho, Yunjoo; Lim, Jae Yun; Seo, Jeong-Ah

2017-05-01

The amylolytic yeast Saccharomycopsis fibuligera is the predominant yeast in the starter product, nuruk, which is utilized for rice wine production in South Korea. Latest molecular studies explore a recently developed interspecific hybridization among stains of S. fibuligera with a unique genetic feature. However, the origin of the natural hybridization occurrence is still unclear. Thus, to respectively distinguish parental and hybrid strains, specific primer sets were applied on 141 yeast strains isolated from different nuruk samples fermented in different provinces. Sixty-seven strains were defined accordingly as parental species with genome A while 8 strains were defined as hybrid strains. Unexpectedly, another parental species with genome B could not be found among the strain pools yet. Furthermore, it was observed that hybrid strains are phenotypically different from A genome strains; asci containing tetrad ascospores were observed in A genome strains more frequent than in hybrid strains. Nevertheless, hybrid strains were slightly more thermotolerant than A genome strains. Interestingly, all hybrid strains were located only in Jeju province. Based on these sets of data, we speculated that the unique climate of Jeju province might play an evolutionary role in the interspecific hybridization between A genome strains, as well as the unculturable allopatric B genome strains.

Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex

PubMed Central

Chand Dakal, Tikam; Giudici, Paolo; Solieri, Lisa

2016-01-01

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5’ end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding. PMID:27501051

Mild intellectual disability associated with a progeny of father-daughter incest: genetic and environmental considerations.

PubMed

Ansermet, Francois; Lespinasse, James; Gimelli, Stefania; Béna, Frédérique; Paoloni-Giacobino, Ariane

2010-05-01

We report the case of a 34-year-old female resulting from a father-daughter sexual abuse and presenting a phenotype of mild intellectual disability with minor dysmorphic features. Karyotyping showed a normal 46, XX constitution. Array-based comparative genomic hybridization (array-CGH) revealed a heterozygote 320kb 6p22.3 microdeletion in the proband, encompassing only one known gene, and therefore unlikely to be the cause of the phenotype. However, the role of other genetic factors, such as a recessive condition, could not be ruled out as a putative cause for the phenotype. On the other hand, the role played by a heavily detrimental familial situation on the development and outcome, and possibly leading or contributing to a mild intellectual disability, should be taken into account.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

PubMed

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing

PubMed Central

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing. PMID:14519201

Cytoplasmic and Genomic Effects on Meiotic Pairing in Brassica Hybrids and Allotetraploids from Pair Crosses of Three Cultivated Diploids

PubMed Central

Cui, Cheng; Ge, Xianhong; Gautam, Mayank; Kang, Lei; Li, Zaiyun

2012-01-01

Interspecific hybridization and allopolyploidization contribute to the origin of many important crops. Synthetic Brassica is a widely used model for the study of genetic recombination and “fixed heterosis” in allopolyploids. To investigate the effects of the cytoplasm and genome combinations on meiotic recombination, we produced digenomic diploid and triploid hybrids and trigenomic triploid hybrids from the reciprocal crosses of three Brassica diploids (B. rapa, AA; B. nigra, BB; B. oleracea, CC). The chromosomes in the resultant hybrids were doubled to obtain three allotetraploids (B. juncea, AA.BB; B. napus, AA.CC; B. carinata, BB.CC). Intra- and intergenomic chromosome pairings in these hybrids were quantified using genomic in situ hybridization and BAC-FISH. The level of intra- and intergenomic pairings varied significantly, depending on the genome combinations and the cytoplasmic background and/or their interaction. The extent of intragenomic pairing was less than that of intergenomic pairing within each genome. The extent of pairing variations within the B genome was less than that within the A and C genomes, each of which had a similar extent of pairing. Synthetic allotetraploids exhibited nondiploidized meiotic behavior, and their chromosomal instabilities were correlated with the relationship of the genomes and cytoplasmic background. Our results highlight the specific roles of the cytoplasm and genome to the chromosomal behaviors of hybrids and allopolyploids. PMID:22505621

Imputation-Based Genomic Coverage Assessments of Current Human Genotyping Arrays

PubMed Central

Nelson, Sarah C.; Doheny, Kimberly F.; Pugh, Elizabeth W.; Romm, Jane M.; Ling, Hua; Laurie, Cecelia A.; Browning, Sharon R.; Weir, Bruce S.; Laurie, Cathy C.

2013-01-01

Microarray single-nucleotide polymorphism genotyping, combined with imputation of untyped variants, has been widely adopted as an efficient means to interrogate variation across the human genome. “Genomic coverage” is the total proportion of genomic variation captured by an array, either by direct observation or through an indirect means such as linkage disequilibrium or imputation. We have performed imputation-based genomic coverage assessments of eight current genotyping arrays that assay from ~0.3 to ~5 million variants. Coverage was determined separately in each of the four continental ancestry groups in the 1000 Genomes Project phase 1 release. We used the subset of 1000 Genomes variants present on each array to impute the remaining variants and assessed coverage based on correlation between imputed and observed allelic dosages. More than 75% of common variants (minor allele frequency > 0.05) are covered by all arrays in all groups except for African ancestry, and up to ~90% in all ancestries for the highest density arrays. In contrast, less than 40% of less common variants (0.01 < minor allele frequency < 0.05) are covered by low density arrays in all ancestries and 50–80% in high density arrays, depending on ancestry. We also calculated genome-wide power to detect variant-trait association in a case-control design, across varying sample sizes, effect sizes, and minor allele frequency ranges, and compare these array-based power estimates with a hypothetical array that would type all variants in 1000 Genomes. These imputation-based genomic coverage and power analyses are intended as a practical guide to researchers planning genetic studies. PMID:23979933

Gene amplification of the Hps locus in Glycine max

PubMed Central

Gijzen, Mark; Kuflu, Kuflom; Moy, Pat

2006-01-01

Background Hydrophobic protein from soybean (HPS) is an 8 kD cysteine-rich polypeptide that causes asthma in persons allergic to soybean dust. HPS is synthesized in the pod endocarp and deposited on the seed surface during development. Past evidence suggests that the protein may mediate the adherence or dehiscence of endocarp tissues during maturation and affect the lustre, or glossiness of the seed surface. Results A comparison of soybean germplasm by genomic DNA blot hybridization shows that the copy number and structure of the Hps locus is polymorphic among soybean cultivars and related species. Changes in Hps gene copy number were also detected by comparative genomic DNA hybridization using cDNA microarrays. The Hps copy number polymorphisms co-segregated with seed lustre phenotype and HPS surface protein in a cross between dull- and shiny-seeded soybeans. In soybean cultivar Harosoy 63, a minimum of 27 ± 5 copies of the Hps gene were estimated to be present in each haploid genome. The isolation and analysis of genomic clones indicates that the core Hps locus is comprised of a tandem array of reiterated units, with each 8.6 kb unit containing a single HPS open reading frame. Conclusion This study shows that polymorphisms at the Hps locus arise from changes in the gene copy number via gene amplification. We present a model whereby Hps copy number modulates protein expression levels and seed lustre, and we suggest that gene amplification may result from selection pressures imposed on crop plants. PMID:16536872

Expanding probe repertoire and improving reproducibility in human genomic hybridization

PubMed Central

Dorman, Stephanie N.; Shirley, Ben C.; Knoll, Joan H. M.; Rogan, Peter K.

2013-01-01

Diagnostic DNA hybridization relies on probes composed of single copy (sc) genomic sequences. Sc sequences in probe design ensure high specificity and avoid cross-hybridization to other regions of the genome, which could lead to ambiguous results that are difficult to interpret. We examine how the distribution and composition of repetitive sequences in the genome affects sc probe performance. A divide and conquer algorithm was implemented to design sc probes. With this approach, sc probes can include divergent repetitive elements, which hybridize to unique genomic targets under higher stringency experimental conditions. Genome-wide custom probe sets were created for fluorescent in situ hybridization (FISH) and microarray genomic hybridization. The scFISH probes were developed for detection of copy number changes within small tumour suppressor genes and oncogenes. The microarrays demonstrated increased reproducibility by eliminating cross-hybridization to repetitive sequences adjacent to probe targets. The genome-wide microarrays exhibited lower median coefficients of variation (17.8%) for two HapMap family trios. The coefficients of variations of commercial probes within 300 nt of a repetitive element were 48.3% higher than the nearest custom probe. Furthermore, the custom microarray called a chromosome 15q11.2q13 deletion more consistently. This method for sc probe design increases probe coverage for FISH and lowers variability in genomic microarrays. PMID:23376933

Genovar: a detection and visualization tool for genomic variants.

PubMed

Jung, Kwang Su; Moon, Sanghoon; Kim, Young Jin; Kim, Bong-Jo; Park, Kiejung

2012-05-08

Along with single nucleotide polymorphisms (SNPs), copy number variation (CNV) is considered an important source of genetic variation associated with disease susceptibility. Despite the importance of CNV, the tools currently available for its analysis often produce false positive results due to limitations such as low resolution of array platforms, platform specificity, and the type of CNV. To resolve this problem, spurious signals must be separated from true signals by visual inspection. None of the previously reported CNV analysis tools support this function and the simultaneous visualization of comparative genomic hybridization arrays (aCGH) and sequence alignment. The purpose of the present study was to develop a useful program for the efficient detection and visualization of CNV regions that enables the manual exclusion of erroneous signals. A JAVA-based stand-alone program called Genovar was developed. To ascertain whether a detected CNV region is a novel variant, Genovar compares the detected CNV regions with previously reported CNV regions using the Database of Genomic Variants (DGV, http://projects.tcag.ca/variation) and the Single Nucleotide Polymorphism Database (dbSNP). The current version of Genovar is capable of visualizing genomic data from sources such as the aCGH data file and sequence alignment format files. Genovar is freely accessible and provides a user-friendly graphic user interface (GUI) to facilitate the detection of CNV regions. The program also provides comprehensive information to help in the elimination of spurious signals by visual inspection, making Genovar a valuable tool for reducing false positive CNV results. http://genovar.sourceforge.net/.

Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies

PubMed Central

Euskirchen, Ghia M.; Rozowsky, Joel S.; Wei, Chia-Lin; Lee, Wah Heng; Zhang, Zhengdong D.; Hartman, Stephen; Emanuelsson, Olof; Stolc, Viktor; Weissman, Sherman; Gerstein, Mark B.; Ruan, Yijun; Snyder, Michael

2007-01-01

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides ≥50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%–86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies. PMID:17568005

Genomic networks of hybrid sterility.

PubMed

Turner, Leslie M; White, Michael A; Tautz, Diethard; Payseur, Bret A

2014-02-01

Hybrid dysfunction, a common feature of reproductive barriers between species, is often caused by negative epistasis between loci ("Dobzhansky-Muller incompatibilities"). The nature and complexity of hybrid incompatibilities remain poorly understood because identifying interacting loci that affect complex phenotypes is difficult. With subspecies in the early stages of speciation, an array of genetic tools, and detailed knowledge of reproductive biology, house mice (Mus musculus) provide a model system for dissecting hybrid incompatibilities. Male hybrids between M. musculus subspecies often show reduced fertility. Previous studies identified loci and several X chromosome-autosome interactions that contribute to sterility. To characterize the genetic basis of hybrid sterility in detail, we used a systems genetics approach, integrating mapping of gene expression traits with sterility phenotypes and QTL. We measured genome-wide testis expression in 305 male F2s from a cross between wild-derived inbred strains of M. musculus musculus and M. m. domesticus. We identified several thousand cis- and trans-acting QTL contributing to expression variation (eQTL). Many trans eQTL cluster into eleven 'hotspots,' seven of which co-localize with QTL for sterility phenotypes identified in the cross. The number and clustering of trans eQTL-but not cis eQTL-were substantially lower when mapping was restricted to a 'fertile' subset of mice, providing evidence that trans eQTL hotspots are related to sterility. Functional annotation of transcripts with eQTL provides insights into the biological processes disrupted by sterility loci and guides prioritization of candidate genes. Using a conditional mapping approach, we identified eQTL dependent on interactions between loci, revealing a complex system of epistasis. Our results illuminate established patterns, including the role of the X chromosome in hybrid sterility. The integrated mapping approach we employed is applicable in a broad range of organisms and we advocate for widespread adoption of a network-centered approach in speciation genetics.

Genomic Networks of Hybrid Sterility

PubMed Central

Turner, Leslie M.; White, Michael A.; Tautz, Diethard; Payseur, Bret A.

2014-01-01

Hybrid dysfunction, a common feature of reproductive barriers between species, is often caused by negative epistasis between loci (“Dobzhansky-Muller incompatibilities”). The nature and complexity of hybrid incompatibilities remain poorly understood because identifying interacting loci that affect complex phenotypes is difficult. With subspecies in the early stages of speciation, an array of genetic tools, and detailed knowledge of reproductive biology, house mice (Mus musculus) provide a model system for dissecting hybrid incompatibilities. Male hybrids between M. musculus subspecies often show reduced fertility. Previous studies identified loci and several X chromosome-autosome interactions that contribute to sterility. To characterize the genetic basis of hybrid sterility in detail, we used a systems genetics approach, integrating mapping of gene expression traits with sterility phenotypes and QTL. We measured genome-wide testis expression in 305 male F2s from a cross between wild-derived inbred strains of M. musculus musculus and M. m. domesticus. We identified several thousand cis- and trans-acting QTL contributing to expression variation (eQTL). Many trans eQTL cluster into eleven ‘hotspots,’ seven of which co-localize with QTL for sterility phenotypes identified in the cross. The number and clustering of trans eQTL—but not cis eQTL—were substantially lower when mapping was restricted to a ‘fertile’ subset of mice, providing evidence that trans eQTL hotspots are related to sterility. Functional annotation of transcripts with eQTL provides insights into the biological processes disrupted by sterility loci and guides prioritization of candidate genes. Using a conditional mapping approach, we identified eQTL dependent on interactions between loci, revealing a complex system of epistasis. Our results illuminate established patterns, including the role of the X chromosome in hybrid sterility. The integrated mapping approach we employed is applicable in a broad range of organisms and we advocate for widespread adoption of a network-centered approach in speciation genetics. PMID:24586194

Karyological evidence of hybridogenesis in Greenlings (Teleostei: Hexagrammidae).

PubMed

Suzuki, Shota; Arai, Katsutoshi; Munehara, Hiroyuki

2017-01-01

Two types of natural hybrids were discovered in populations of three Hexagrammos species (Teleostei: Hexagrammidae) distributed off the southern coast of Hokkaido in the North Pacific Ocean. Both hybrids reproduce by hybridogenesis, in which the maternal haploid genome is transmitted to offspring without recombination and the paternal haploid genome is eliminated during gametogenesis. While natural hybrids are unisexual and reproduce hemiclonally by backcrossing with the paternal species (BC-P), artificial F1-hybrids between the pure species produce recombinant gametes. Thus, despite having the same genome composition, the natural hybrids and the F1-hybrids are not genetically identical. Here, to clarify the differences between both hybrids, we examined the karyotypes of the three Hexagrammos species, their natural hybrids, the artificial F1-hybrids, and several backcrosses. Artificial F1-hybrids have karyotypes and chromosome numbers that are intermediate between those of the parental species. Conversely, the natural hybrids differed from F1-hybrids by having several large metacentric chromosomes and microchromosomes. Since the entire maternal haploid genome is inherited by the natural hybrids, maternal backcrosses (BC-M) between natural hybrids and males of the maternal species (H. octogrammus; Hoc) have a hemiclonal Hoc genome with large chromosomes from the mother and a normal Hoc genome from the father. However, the large chromosomes disappear in offspring of BC-M, probably due to fissuring during gametogenesis. Similarly, microsatellite DNA analysis revealed that chromosomes of BC-M undergo recombination. These findings suggest that genetic factors associated with hemiclonal reproduction may be located on the large metacentric chromosomes of natural hybrids.

Genomic Hypomethylation in the Human Germline Associates with Selective Structural Mutability in the Human Genome

PubMed Central

Li, Jian; Harris, R. Alan; Cheung, Sau Wai; Coarfa, Cristian; Jeong, Mira; Goodell, Margaret A.; White, Lisa D.; Patel, Ankita; Kang, Sung-Hae; Shaw, Chad; Chinault, A. Craig; Gambin, Tomasz; Gambin, Anna; Lupski, James R.; Milosavljevic, Aleksandar

2012-01-01

The hotspots of structural polymorphisms and structural mutability in the human genome remain to be explained mechanistically. We examine associations of structural mutability with germline DNA methylation and with non-allelic homologous recombination (NAHR) mediated by low-copy repeats (LCRs). Combined evidence from four human sperm methylome maps, human genome evolution, structural polymorphisms in the human population, and previous genomic and disease studies consistently points to a strong association of germline hypomethylation and genomic instability. Specifically, methylation deserts, the ∼1% fraction of the human genome with the lowest methylation in the germline, show a tenfold enrichment for structural rearrangements that occurred in the human genome since the branching of chimpanzee and are highly enriched for fast-evolving loci that regulate tissue-specific gene expression. Analysis of copy number variants (CNVs) from 400 human samples identified using a custom-designed array comparative genomic hybridization (aCGH) chip, combined with publicly available structural variation data, indicates that association of structural mutability with germline hypomethylation is comparable in magnitude to the association of structural mutability with LCR–mediated NAHR. Moreover, rare CNVs occurring in the genomes of individuals diagnosed with schizophrenia, bipolar disorder, and developmental delay and de novo CNVs occurring in those diagnosed with autism are significantly more concentrated within hypomethylated regions. These findings suggest a new connection between the epigenome, selective mutability, evolution, and human disease. PMID:22615578

Non-invasive prenatal screening versus prenatal diagnosis by array comparative genomic hybridization: a comparative retrospective study.

PubMed

Sotiriadis, Alexandros; Papoulidis, Ioannis; Siomou, Elisavet; Papageorgiou, Elena; Eleftheriades, Makarios; Papadopoulos, Vasilios; Alexiou, Maria; Manolakos, Emmanouil; Athanasiadis, Apostolos

2017-06-01

To calculate the proportion of array comparative genomic hybridization (aCGH) pathogenic results, that would not be detectable by non-invasive prenatal screening (NIPS). This is a comparative study using data from 2779 fetuses, which underwent invasive prenatal diagnosis, and the samples were analyzed using aCGH. The simulated NIPS assay would test for trisomies 21, 18, 13, monosomy X, 47, XXX, 47, XYY, and 47, XXY. Indications for invasive testing were grouped into categories and the absolute, relative rates of pathogenic/likely pathogenic results of aCGH analysis that would not be detectable by NIPS were calculated. The expected rate of aCGH-detected abnormalities that would not be detectable by NIPS was 28.0% (95% CI 14.3-47.6) for nuchal translucency (NT) 95 to 99th centile; 14.3% (95% 5.0-34.6) for NT > 99th centile; 34.2% (95% CI 21.1-50.1) for high-risk first-trimester results (regardless of NT); 52.4% (95% CI 32.4-71.7) for second-trimester markers; and 50.0% (95% CI 26.8-73.2) for advanced maternal age. The overall rate of aCGH pathogenic/likely pathogenic results was 5.0% and 44.0% (95% CI 36.0-52.2) of them would not be detected by NIPS. Approximately half of the abnormal aCGH results would not be detectable by standard NIPS assays, highlighting the necessity of pre-test counseling, and illustrating the limitations of NIPS. © 2017 John Wiley & Sons, Ltd. © 2017 John Wiley & Sons, Ltd.

Copy Number Variations of TBK1 in Australian Patients With Primary Open-Angle Glaucoma

PubMed Central

AWADALLA, MONA S.; FINGERT, JOHN H.; ROOS, BENJAMIN E.; CHEN, SIMON; HOLMES, RICHARD; GRAHAM, STUART L.; CHEHADE, MARK; GALANOPOLOUS, ANNA; RIDGE, BRONWYN; SOUZEAU, EMMANUELLE; ZHOU, TIGER; SIGGS, OWEN M.; HEWITT, ALEX W.; MACKEY, DAVID A.; BURDON, KATHRYN P.; CRAIG, JAMIE E.

2015-01-01

PURPOSE To investigate the presence of TBK1 copy number variations in a large, well-characterized Australian cohort of patients with glaucoma comprising both normal-tension glaucoma and high-tension glaucoma cases. DESIGN A retrospective cohort study. METHODS DNA samples from patients with normal-tension glaucoma and high-tension glaucoma and unaffected controls were screened for TBK1 copy number variations using real-time quantitative polymerase chain reaction. Samples with additional copies of the TBK1 gene were further tested using custom comparative genomic hybridization arrays. RESULTS Four out of 334 normal-tension glaucoma cases (1.2%) were found to carry TBK1 copy number variations using quantitative polymerase chain reaction. One extra dose of the TBK1 gene (duplication) was detected in 3 normal-tension glaucoma patients, while 2 extra doses of the gene (triplication) were detected in a fourth normal-tension glaucoma patient. The results were further confirmed by custom comparative genomic hybridization arrays. Further, the TBK1 copy number variation segregated with normal-tension glaucoma in the family members of the probands, showing an autosomal dominant pattern of inheritance. No TBK1 copy number variations were detected in 1045 Australian patients with high-tension glaucoma or in 254 unaffected controls. CONCLUSION We report the presence of TBK1 copy number variations in our Australian normal-tension glaucoma cohort, including the first example of more than 1 extra copy of this gene in glaucoma patients (gene triplication). These results confirm TBK1 to be an important cause of normal-tension glaucoma, but do not suggest common involvement in high-tension glaucoma. PMID:25284765

Molecular cytogenetic characterization of Xp22.32→pter deletion and Xq26.3→qter duplication in a male fetus associated with 46,Y,rec(X)dup(Xq) inv(X)(p22.3q26.3), a hypoplastic left heart, short stature, and maternal X chromosome pericentric inversion.

PubMed

Chen, Chih-Ping; Chen, Chen-Yu; Chern, Schu-Rern; Wu, Peih-Shan; Chen, Yen-Ni; Chen, Shin-Wen; Lee, Chen-Chi; Town, Dai-Dyi; Lee, Meng-Shan; Yang, Chien-Wen; Wang, Wayseen

2016-10-01

We present molecular cytogenetic characterization of an Xp22.32→pter deletion and an Xq26.3→qter duplication in a male fetus with congenital malformations and maternal X chromosome pericentric inversion. A 22-year-old woman underwent amniocentesis at 17 weeks of gestation because of an abnormal maternal serum screening result. Prenatal ultrasound revealed a hypoplastic left heart and short limbs. Amniocentesis revealed a karyotype of 46,Y,der(X) t(X;?)(p22.31;?). The pregnancy was subsequently terminated, and a malformed fetus was delivered with short stature and facial dysmorphism. Repeat amniocentesis was performed before termination of the pregnancy. Array comparative genomic hybridization was performed on uncultured amniocytes and maternal blood. Conventional cytogenetic analysis was performed on cultured amniocytes, cord blood, and blood from both parents. Fluorescence in situ hybridization was performed on cultured amniocytes. The maternal karyotype was 46,X,inv(X)(p22.3q26.3). The fetal karyotype was 46,Y, rec(X)dup(Xq)inv(X)(p22.3q26.3) or 46,Y, rec(X)(qter→q26.3::p22.3→qter). Array comparative genomic hybridization on uncultured amniocytes revealed a 4.56-Mb deletion of Xp22.33-p22.32 encompassing SHOX, CSF2RA, and ARSE, and a 19.22-Mb duplication of Xq26.3-q28 encompassing SOX3, FMR1, MECP2, RAB39B, and CLIC2 in the fetus. The mother did not have X chromosome imbalance. Detection of X chromosome aberration in a male fetus should give suspicion of a recombinant X chromosome derived from maternal X chromosome pericentric inversion. Copyright © 2016. Published by Elsevier B.V.

Genomic predictive model for recurrence and metastasis development in head and neck squamous cell carcinoma patients.

PubMed

Ribeiro, Ilda Patrícia; Caramelo, Francisco; Esteves, Luísa; Menoita, Joana; Marques, Francisco; Barroso, Leonor; Miguéis, Jorge; Melo, Joana Barbosa; Carreira, Isabel Marques

2017-10-24

The head and neck squamous cell carcinoma (HNSCC) population consists mainly of high-risk for recurrence and locally advanced stage patients. Increased knowledge of the HNSCC genomic profile can improve early diagnosis and treatment outcomes. The development of models to identify consistent genomic patterns that distinguish HNSCC patients that will recur and/or develop metastasis after treatment is of utmost importance to decrease mortality and improve survival rates. In this study, we used array comparative genomic hybridization data from HNSCC patients to implement a robust model to predict HNSCC recurrence/metastasis. This predictive model showed a good accuracy (>80%) and was validated in an independent population from TCGA data portal. This predictive genomic model comprises chromosomal regions from 5p, 6p, 8p, 9p, 11q, 12q, 15q and 17p, where several upstream and downstream members of signaling pathways that lead to an increase in cell proliferation and invasion are mapped. The introduction of genomic predictive models in clinical practice might contribute to a more individualized clinical management of the HNSCC patients, reducing recurrences and improving patients' quality of life. The power of this genomic model to predict the recurrence and metastases development should be evaluated in other HNSCC populations.

GAB2 Amplification in Squamous Cell Lung Cancer of Non-Smokers

PubMed Central

2017-01-01

Lung squamous cell cancer (SCC) is typically found in smokers and has a very low incidence in non-smokers, indicating differences in the tumor biology of lung SCC in smokers and non-smokers. However, the specific mutations that drive tumor growth in non-smokers have not been identified. To identify mutations in lung SCC of non-smokers, we performed a genetic analysis using arrays comparative genomic hybridization (ArrayCGH). We analyzed 19 patients with lung SCC who underwent surgical treatment between April 2005 and April 2015. Clinical characteristics were reviewed, and DNA was extracted from fresh frozen lung cancer specimens. All of copy number alterations from ArrayCGH were validated using The Cancer Genome Atlas (TCGA) copy number variation (CNV) data of lung SCC. We examined the frequency of copy number changes according to the smoking status (non-smoker [n = 8] or smoker [n = 11]). We identified 16 significantly altered regions from ArrayCGH data, three gain and four loss regions overlapped with the TCGA lung squamous cell carcinoma (LUSC) patients. Within these overlapped significant regions, we detected 15 genes that have been reported in the Cancer Gene census. We also found that the proto-oncogene GAB2 (11q14.1) was significantly amplified in non-smokers patients and vice versa in both ArrayCGH and TCGA data. Immunohistochemical analyses showed that GAB2 protein was relatively upregulated in non-smoker than smoker tissues (37.5% vs. 9.0%, P = 0.007). GAB2 amplification may have an important role in the development of lung SCC in non-smokers. GAB2 may represent a potential biomarker for lung SCC in non-smokers. PMID:28960030

GAB2 Amplification in Squamous Cell Lung Cancer of Non-Smokers.

PubMed

Park, Yu Rang; Bae, Soo Hyeon; Ji, Wonjun; Seo, Eul Ju; Lee, Jae Cheol; Kim, Hyeong Ryul; Jang, Se Jin; Choi, Chang Min

2017-11-01

Lung squamous cell cancer (SCC) is typically found in smokers and has a very low incidence in non-smokers, indicating differences in the tumor biology of lung SCC in smokers and non-smokers. However, the specific mutations that drive tumor growth in non-smokers have not been identified. To identify mutations in lung SCC of non-smokers, we performed a genetic analysis using arrays comparative genomic hybridization (ArrayCGH). We analyzed 19 patients with lung SCC who underwent surgical treatment between April 2005 and April 2015. Clinical characteristics were reviewed, and DNA was extracted from fresh frozen lung cancer specimens. All of copy number alterations from ArrayCGH were validated using The Cancer Genome Atlas (TCGA) copy number variation (CNV) data of lung SCC. We examined the frequency of copy number changes according to the smoking status (non-smoker [n = 8] or smoker [n = 11]). We identified 16 significantly altered regions from ArrayCGH data, three gain and four loss regions overlapped with the TCGA lung squamous cell carcinoma (LUSC) patients. Within these overlapped significant regions, we detected 15 genes that have been reported in the Cancer Gene census. We also found that the proto-oncogene GAB2 (11q14.1) was significantly amplified in non-smokers patients and vice versa in both ArrayCGH and TCGA data. Immunohistochemical analyses showed that GAB2 protein was relatively upregulated in non-smoker than smoker tissues (37.5% vs. 9.0%, P = 0.007). GAB2 amplification may have an important role in the development of lung SCC in non-smokers. GAB2 may represent a potential biomarker for lung SCC in non-smokers. © 2017 The Korean Academy of Medical Sciences.

MixHMM: Inferring Copy Number Variation and Allelic Imbalance Using SNP Arrays and Tumor Samples Mixed with Stromal Cells

PubMed Central

Schulz, Vincent; Chen, Min; Tuck, David

2010-01-01

Background Genotyping platforms such as single nucleotide polymorphism (SNP) arrays are powerful tools to study genomic aberrations in cancer samples. Allele specific information from SNP arrays provides valuable information for interpreting copy number variation (CNV) and allelic imbalance including loss-of-heterozygosity (LOH) beyond that obtained from the total DNA signal available from array comparative genomic hybridization (aCGH) platforms. Several algorithms based on hidden Markov models (HMMs) have been designed to detect copy number changes and copy-neutral LOH making use of the allele information on SNP arrays. However heterogeneity in clinical samples, due to stromal contamination and somatic alterations, complicates analysis and interpretation of these data. Methods We have developed MixHMM, a novel hidden Markov model using hidden states based on chromosomal structural aberrations. MixHMM allows CNV detection for copy numbers up to 7 and allows more complete and accurate description of other forms of allelic imbalance, such as increased copy number LOH or imbalanced amplifications. MixHMM also incorporates a novel sample mixing model that allows detection of tumor CNV events in heterogeneous tumor samples, where cancer cells are mixed with a proportion of stromal cells. Conclusions We validate MixHMM and demonstrate its advantages with simulated samples, clinical tumor samples and a dilution series of mixed samples. We have shown that the CNVs of cancer cells in a tumor sample contaminated with up to 80% of stromal cells can be detected accurately using Illumina BeadChip and MixHMM. Availability The MixHMM is available as a Python package provided with some other useful tools at http://genecube.med.yale.edu:8080/MixHMM. PMID:20532221

Application of array-comparative genomic hybridization in tetralogy of Fallot

PubMed Central

Liu, Lin; Wang, Hong-Dan; Cui, Cun-Ying; Wu, Dong; Li, Tao; Fan, Tai-Bing; Peng, Bang-Tian; Zhang, Lian-Zhong; Wang, Cheng-Zeng

2016-01-01

Abstract To explore the underlying pathogenesis and provide references for genetic counseling and prenatal gene diagnosis, we analyzed the chromosome karyotypes and genome-wide copy number variations (CNVs) in 86 patients with tetralogy of Fallot (TOF) by G-banding karyotype analysis and array-comparative genomic hybridization (aCGH), respectively. And then quantitative polymerase chain reaction was used to validate these candidate CNVs. Based on their different properties, CNVs were categorized into benign CNVs, suspiciously pathogenic CNVs, and indefinite CNVs. Data analysis was based on public databases such as UCSC, DECIPHER, DGV, ISCA, and OMIM. The karyotype was normal in all the 86 patients with TOF. CNVs were detected in 11 patients by aCGH and quantitative polymerase chain reaction. Patient no. 0001, 0010, and 0029 had 2.52-Mb deletion in the chromosome 22q11.21 region; patient no. 0008 had both 595- and 428-kb duplications, respectively, in 12p12.3p12.2 and 14q23.2q23.3 regions; patient no. 0009 had 1.46-Mb duplication in the 1q21.1q21.2 region; patient no. 0016 had 513-kb duplication in the 1q42.13 region; patient no. 0024 had 292-kb duplication in the 16q11.2 region; patient no. 0026 had 270-kb duplication in the 16q24.1 region; patient no. 0028 had 222-kb deletion in the 7q31.1 region; patient no. 0033 had 1.73-Mb duplication in the 17q12 region; and patient no. 0061 had 5.79-Mb deletion in the 1p36.33p36.31 region. aCGH can accurately detect CNVs in the patients with TOF. This is conducive to genetic counseling and prenatal diagnosis for TOF and provides a new clue and theoretical basis for exploring the pathogenesis of congenital heart disease. PMID:27930557

Application of array-comparative genomic hybridization in tetralogy of Fallot.

PubMed

Liu, Lin; Wang, Hong-Dan; Cui, Cun-Ying; Wu, Dong; Li, Tao; Fan, Tai-Bing; Peng, Bang-Tian; Zhang, Lian-Zhong; Wang, Cheng-Zeng

2016-12-01

To explore the underlying pathogenesis and provide references for genetic counseling and prenatal gene diagnosis, we analyzed the chromosome karyotypes and genome-wide copy number variations (CNVs) in 86 patients with tetralogy of Fallot (TOF) by G-banding karyotype analysis and array-comparative genomic hybridization (aCGH), respectively. And then quantitative polymerase chain reaction was used to validate these candidate CNVs. Based on their different properties, CNVs were categorized into benign CNVs, suspiciously pathogenic CNVs, and indefinite CNVs. Data analysis was based on public databases such as UCSC, DECIPHER, DGV, ISCA, and OMIM.The karyotype was normal in all the 86 patients with TOF. CNVs were detected in 11 patients by aCGH and quantitative polymerase chain reaction. Patient no. 0001, 0010, and 0029 had 2.52-Mb deletion in the chromosome 22q11.21 region; patient no. 0008 had both 595- and 428-kb duplications, respectively, in 12p12.3p12.2 and 14q23.2q23.3 regions; patient no. 0009 had 1.46-Mb duplication in the 1q21.1q21.2 region; patient no. 0016 had 513-kb duplication in the 1q42.13 region; patient no. 0024 had 292-kb duplication in the 16q11.2 region; patient no. 0026 had 270-kb duplication in the 16q24.1 region; patient no. 0028 had 222-kb deletion in the 7q31.1 region; patient no. 0033 had 1.73-Mb duplication in the 17q12 region; and patient no. 0061 had 5.79-Mb deletion in the 1p36.33p36.31 region.aCGH can accurately detect CNVs in the patients with TOF. This is conducive to genetic counseling and prenatal diagnosis for TOF and provides a new clue and theoretical basis for exploring the pathogenesis of congenital heart disease.

Assessment of data processing to improve reliability of microarray experiments using genomic DNA reference.

PubMed

Yang, Yunfeng; Zhu, Mengxia; Wu, Liyou; Zhou, Jizhong

2008-09-16

Using genomic DNA as common reference in microarray experiments has recently been tested by different laboratories. Conflicting results have been reported with regard to the reliability of microarray results using this method. To explain it, we hypothesize that data processing is a critical element that impacts the data quality. Microarray experiments were performed in a gamma-proteobacterium Shewanella oneidensis. Pair-wise comparison of three experimental conditions was obtained either with two labeled cDNA samples co-hybridized to the same array, or by employing Shewanella genomic DNA as a standard reference. Various data processing techniques were exploited to reduce the amount of inconsistency between both methods and the results were assessed. We discovered that data quality was significantly improved by imposing the constraint of minimal number of replicates, logarithmic transformation and random error analyses. These findings demonstrate that data processing significantly influences data quality, which provides an explanation for the conflicting evaluation in the literature. This work could serve as a guideline for microarray data analysis using genomic DNA as a standard reference.

Differential nuclear scaffold/matrix attachment marks expressed genes.

PubMed

Linnemann, Amelia K; Platts, Adrian E; Krawetz, Stephen A

2009-02-15

It is well established that nuclear architecture plays a key role in poising regions of the genome for transcription. This may be achieved using scaffold/matrix attachment regions (S/MARs) that establish loop domains. However, the relationship between changes in the physical structure of the genome as mediated by attachment to the nuclear scaffold/matrix and gene expression is not clearly understood. To define the role of S/MARs in organizing our genome and to resolve the often contradictory loci-specific studies, we have surveyed the S/MARs in HeLa S3 cells on human chromosomes 14-18 by array comparative genomic hybridization. Comparison of LIS (lithium 3,5-diiodosalicylate) extraction to identify SARs and 2 m NaCl extraction to identify MARs revealed that approximately one-half of the sites were in common. The results presented in this study suggest that SARs 5' of a gene are associated with transcript presence whereas MARs contained within a gene are associated with silenced genes. The varied functions of the S/MARs as revealed by the different extraction methods highlights their unique functional contribution.

Differential nuclear scaffold/matrix attachment marks expressed genes†

PubMed Central

Linnemann, Amelia K.; Platts, Adrian E.; Krawetz, Stephen A.

2009-01-01

It is well established that nuclear architecture plays a key role in poising regions of the genome for transcription. This may be achieved using scaffold/matrix attachment regions (S/MARs) that establish loop domains. However, the relationship between changes in the physical structure of the genome as mediated by attachment to the nuclear scaffold/matrix and gene expression is not clearly understood. To define the role of S/MARs in organizing our genome and to resolve the often contradictory loci-specific studies, we have surveyed the S/MARs in HeLa S3 cells on human chromosomes 14–18 by array comparative genomic hybridization. Comparison of LIS (lithium 3,5-diiodosalicylate) extraction to identify SARs and 2 m NaCl extraction to identify MARs revealed that approximately one-half of the sites were in common. The results presented in this study suggest that SARs 5′ of a gene are associated with transcript presence whereas MARs contained within a gene are associated with silenced genes. The varied functions of the S/MARs as revealed by the different extraction methods highlights their unique functional contribution. PMID:19017725

DNA Sequencing by Capillary Electrophoresis

PubMed Central

Karger, Barry L.; Guttman, Andras

2009-01-01

Sequencing of human and other genomes has been at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA sequencing methods have evolved from the labor intensive slab gel electrophoresis, through automated multicapillary electrophoresis systems using fluorophore labeling with multispectral imaging, to the “next generation” technologies of cyclic array, hybridization based, nanopore and single molecule sequencing. Deciphering the genetic blueprint and follow-up confirmatory sequencing of Homo sapiens and other genomes was only possible by the advent of modern sequencing technologies that was a result of step by step advances with a contribution of academics, medical personnel and instrument companies. While next generation sequencing is moving ahead at break-neck speed, the multicapillary electrophoretic systems played an essential role in the sequencing of the Human Genome, the foundation of the field of genomics. In this prospective, we wish to overview the role of capillary electrophoresis in DNA sequencing based in part of several of our articles in this journal. PMID:19517496

Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

PubMed

Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

2016-09-01

Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

Method for performing site-specific affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

1999-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

Miniaturized reaction vessel system, method for performing site-specific biochemical reactions and affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

2000-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

Method for performing site-specific affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, A.D.; Lysov, Y.P.; Dubley, S.A.

1999-05-18

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between the cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting the extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to the extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from the array. 14 figs.

Two circular chromosomes of unequal copy number make up the mitochondrial genome of the rotifer Brachionus plicatilis.

PubMed

Suga, Koushirou; Mark Welch, David B; Tanaka, Yukari; Sakakura, Yoshitaka; Hagiwara, Atsushi

2008-06-01

The monogonont rotifer Brachionus plicatilis is an emerging model system for a diverse array of questions in limnological ecosystem dynamics, the evolution of sexual recombination, cryptic speciation, and the phylogeny of basal metazoans. We sequenced the complete mitochondrial genome of B. plicatilis sensu strictu NH1L and found that it is composed of 2 circular chromosomes, designated mtDNA-I (11,153 bp) and mtDNA-II (12,672 bp). Hybridization to DNA isolated from mitochondria demonstrated that mtDNA-I is present at 4 times the copy number of mtDNA-II. The only nucleotide similarity between the 2 chromosomes is a 4.9-kbp region of 99.5% identity including a transfer RNA (tRNA) gene and an extensive noncoding region that contains putative D-loop and control sequence. The mtDNA-I chromosome encodes 4 proteins (ATP6, COB, NAD1, and NAD2), 13 tRNAs, and the large and small subunit ribosomal RNAs; mtDNA-II encodes 8 proteins (COX1-3, NAD3-6, and NAD4L) and 9 tRNAs. Gene order is not conserved between B. plicatilis and its closest relative with a sequenced mitochondrial genome, the acanthocephalan Leptorhynchoides thecatus, or other sequenced mitochondrial genomes. Polymerase chain reaction assays and Southern hybridization to DNA from 18 strains of Brachionus suggest that the 2-chromosome structure has been stable for millions of years. The novel organization of the B. plicatilis mitochondrial genome into 2 nearly equal chromosomes of 4-fold different copy number may provide insight into the evolution of metazoan mitochondria and the phylogenetics of rotifers and other basal animal phyla.

Molecular inversion probe assay for allelic quantitation

PubMed Central

Ji, Hanlee; Welch, Katrina

2010-01-01

Molecular inversion probe (MIP) technology has been demonstrated to be a robust platform for large-scale dual genotyping and copy number analysis. Applications in human genomic and genetic studies include the possibility of running dual germline genotyping and combined copy number variation ascertainment. MIPs analyze large numbers of specific genetic target sequences in parallel, relying on interrogation of a barcode tag, rather than direct hybridization of genomic DNA to an array. The MIP approach does not replace, but is complementary to many of the copy number technologies being performed today. Some specific advantages of MIP technology include: Less DNA required (37 ng vs. 250 ng), DNA quality less important, more dynamic range (amplifications detected up to copy number 60), allele specific information “cleaner” (less SNP crosstalk/contamination), and quality of markers better (fewer individual MIPs versus SNPs needed to identify copy number changes). MIPs can be considered a candidate gene (targeted whole genome) approach and can find specific areas of interest that otherwise may be missed with other methods. PMID:19488872

Species Origin of Genomic Factors in Nicotiana nudicaulis Watson Controlling Hybrid Lethality in Interspecific Hybrids between N. nudicaulis Watson and N. tabacum L

PubMed Central

Liu, Hongshuo; Marubashi, Wataru

2014-01-01

Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis×N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris×N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris×N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla×N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla×N. tabacum and N. sylvestris×N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis. PMID:24806486

Implementation of Quality Management in Core Service Laboratories

PubMed Central

Creavalle, T.; Haque, K.; Raley, C.; Subleski, M.; Smith, M.W.; Hicks, B.

2010-01-01

CF-28 The Genetics and Genomics group of the Advanced Technology Program of SAIC-Frederick exists to bring innovative genomic expertise, tools and analysis to NCI and the scientific community. The Sequencing Facility (SF) provides next generation short read (Illumina) sequencing capacity to investigators using a streamlined production approach. The Laboratory of Molecular Technology (LMT) offers a wide range of genomics core services including microarray expression analysis, miRNA analysis, array comparative genome hybridization, long read (Roche) next generation sequencing, quantitative real time PCR, transgenic genotyping, Sanger sequencing, and clinical mutation detection services to investigators from across the NIH. As the technology supporting this genomic research becomes more complex, the need for basic quality processes within all aspects of the core service groups becomes critical. The Quality Management group works alongside members of these labs to establish or improve processes supporting operations control (equipment, reagent and materials management), process improvement (reengineering/optimization, automation, acceptance criteria for new technologies and tech transfer), and quality assurance and customer support (controlled documentation/SOPs, training, service deficiencies and continual improvement efforts). Implementation and expansion of quality programs within unregulated environments demonstrates SAIC-Frederick's dedication to providing the highest quality products and services to the NIH community.

Genome-wide analyses of four major histone modifications in Arabidopsis hybrids at the germinating seed stage.

PubMed

Zhu, Anyu; Greaves, Ian K; Dennis, Elizabeth S; Peacock, W James

2017-02-07

Hybrid vigour (heterosis) has been used for decades in cropping agriculture, especially in the production of maize and rice, because hybrid varieties exceed their parents in plant biomass and seed yield. The molecular basis of hybrid vigour is not fully understood. Previous studies have suggested that epigenetic systems could play a role in heterosis. In this project, we investigated genome-wide patterns of four histone modifications in Arabidopsis hybrids in germinating seeds. We found that although hybrids have similar histone modification patterns to the parents in most regions of the genome, they have altered patterns at specific loci. A small subset of genes show changes in histone modifications in the hybrids that correlate with changes in gene expression. Our results also show that genome-wide patterns of histone modifications in geminating seeds parallel those at later developmental stages of seedlings. Ler/C24 hybrids showed similar genome-wide patterns of histone modifications as the parents at an early germination stage. However, a small subset of genes, such as FLC, showed correlated changes in histone modification and in gene expression in the hybrids. The altered patterns of histone modifications for those genes in hybrids could be related to some heterotic traits in Arabidopsis, such as flowering time, and could play a role in hybrid vigour establishment.

Chromosome 3p12.3-p14.2 and 3q26.2-q26.32 are genomic markers for prognosis of advanced nasopharyngeal carcinoma.

PubMed

Sheu, Jim Jinn-Chyuan; Lee, Chia-Huei; Ko, Jenq-Yuh; Tsao, George S W; Wu, Chung-Chun; Fang, Chih-Yeu; Tsai, Fuu-Jen; Hua, Chun-Hung; Chen, Chi-Long; Chen, Jen-Yang

2009-10-01

Nasopharyngeal carcinoma is an epithelial malignancy with a remarkable racial and geographic distribution. Previous cytogenetic studies have shown nasopharyngeal carcinoma to be characterized by gross genomic aberrations. However, identification of susceptible gene loci in advanced nasopharyngeal carcinoma has been poorly discussed. A genome-wide survey of gene copy number changes was initiated with two nasopharyngeal carcinoma cell lines by array-based comparative genomic hybridization analysis. These alterations were confirmed by a parallel analysis with the data from the gene expression microarray and were validated by quantitative PCR. Clinical association of the defined target genes was analyzed by fluorescence in situ hybridization on 48 metastatic tumors. A high percentage of genes were consistently altered in dosage and expression levels with gain on 3q26.2-q26.32 and losses on 3p12.3-p14.2 and 9p21.3-p23. Six candidate genes, GPR160 (3q26.2-q27), SKIL (3q26), ADAMTS9 (3p14.2-p14.3), LRIG1 (3p14), MPDZ (9p22-p24), and ADFP (9p22.1) were validated by quantitative PCR. Fluorescence in situ hybridization studies revealed amplification of GPR160 (in 25% of cases) and SKIL (33%); and deletion of ADAMTS9 (30%), LRIG1 (35%), MPDZ (15%), and ADFP (15%). Clinical association analyses indicated a poor survival rate with genetic alterations at the defined 3p deletion (P = 0.0012) and the 3q amplification regions (P = 0.0114). The combined microarray technologies suggested novel candidate oncogenes, amplification of GPR160 and SKIL at 3q26.2-q26.32, and deletion of tumor suppressor genes ADAMTS9 and LRIG1 at 3p12.3-p14.2. Altered expression of these genes may be responsible for malignant progression and could be used as potential markers for nasopharyngeal carcinoma.

Design of Hybrid Nanostructural Arrays to Manipulate SERS-Active Substrates by Nanosphere Lithography.

PubMed

Zhao, Xiaoyu; Wen, Jiahong; Zhang, Mengning; Wang, Dunhui; Wang, Yaxin; Chen, Lei; Zhang, Yongjun; Yang, Jinghai; Du, Youwei

2017-03-01

An easy-handling and low-cost method is utilized to controllably fabricate nanopattern arrays as the surface-enhanced Raman scattering (SERS) active substrates with high density of SERS-active areas (hot spots). A hybrid silver array of nanocaps and nanotriangles are prepared by combining magnetron sputtering and plasma etching. By adjusting the etching time of polystyrene (PS) colloid spheres array in silver nanobowls, the morphology of the arrays can be easily manipulated to control the formation and distribution of hot spots. The experimental results show that the hybrid nanostructural arrays have large enhancement factor, which is estimated to be seven times larger than that in the array of nanocaps and three times larger than that in the array of nanorings and nanoparticles. According to the results of finite-difference time-domain simulation, the excellent SERS performance of this array is ascribed to the high density of hot spots and enhanced electromagnetic field.

Copy number gain at 8q12.1-q22.1 is associated with a malignant tumor phenotype in salivary gland myoepitheliomas.

PubMed

Vékony, Hedy; Röser, Kerstin; Löning, Thomas; Ylstra, Bauke; Meijer, Gerrit A; van Wieringen, Wessel N; van de Wiel, Mark A; Carvalho, Beatriz; Kok, Klaas; Leemans, C René; van der Waal, Isaäc; Bloemena, Elisabeth

2009-02-01

Salivary gland myoepithelial tumors are relatively uncommon tumors with an unpredictable clinical course. More knowledge about their genetic profiles is necessary to identify novel predictors of disease. In this study, we subjected 27 primary tumors (15 myoepitheliomas and 12 myoepithelial carcinomas) to genome-wide microarray-based comparative genomic hybridization (array CGH). We set out to delineate known chromosomal aberrations in more detail and to unravel chromosomal differences between benign myoepitheliomas and myoepithelial carcinomas. Patterns of DNA copy number aberrations were analyzed by unsupervised hierarchical cluster analysis. Both benign and malignant tumors revealed a limited amount of chromosomal alterations (median of 5 and 7.5, respectively). In both tumor groups, high frequency gains (> or =20%) were found mainly at loci of growth factors and growth factor receptors (e.g., PDGF, FGF(R)s, and EGFR). In myoepitheliomas, high frequency losses (> or =20%) were detected at regions of proto-cadherins. Cluster analysis of the array CGH data identified three clusters. Differential copy numbers on chromosome arm 8q and chromosome 17 set the clusters apart. Cluster 1 contained a mixture of the two phenotypes (n = 10), cluster 2 included mostly benign tumors (n = 10), and cluster 3 only contained carcinomas (n = 7). Supervised analysis between malignant and benign tumors revealed a 36 Mbp-region at 8q being more frequently gained in malignant tumors (P = 0.007, FDR = 0.05). This is the first study investigating genomic differences between benign and malignant myoepithelial tumors of the salivary glands at a genomic level. Both unsupervised and supervised analysis of the genomic profiles revealed chromosome arm 8q to be involved in the malignant phenotype of salivary gland myoepitheliomas.

CLONAL INHERITANCE OF A DIPLOID NUCLEAR GENOME BY A HYBRID FRESHWATER MINNOW (PHOXINUS EOS-NEOGAEUS, PISCES: CYPRINIDAE).

PubMed

Goddard, Kathryn Ann; Dawley, Robert M

1990-07-01

Hybrids between the minnows Phoxinus eos and Phoxinus neogaeus coexist with a population of P. eos in East Inlet Pond, Coos Co., New Hampshire. Chromosome counts and flow cytometric analysis of erythrocyte DNA indicate that these hybrids include diploids, triploids, and diploid-triploid mosaics. The mosaics have both diploid and triploid cells in their bodies, even within the same tissues. All three hybrid types are heterozygous at seven putative loci for which P. eos and P. neogaeus are fixed for different allozymes, indicating that the hybrids carry one eos and one neogaeus haploid genome. The diploid hybrids are therefore P. eos-neogaeus, whereas the triploids and mosaics are derived from P. eos-neogaeus but have an extra eos or neogaeus genome in all or some of their cells. Diploid, triploid, and mosaic hybrids accept tissue grafts from diploid hybrids, indicating that all individuals carry the identical eos-neogaeus diploid genome. Thus, one P. eos-neogaeus clone exists at East Inlet Pond. Grafts among the triploids and mosaics or from these individuals to diploid hybrids are rejected, indicating that the third genome is different in each triploid and mosaic individual. In this study, diploid and mosaic hybrids, carrying the clonal eos-neogaeus genome, were bred in the laboratory with males of P. eos or P. neogaeus. Both diploid and mosaic hybrids produced diploid, triploid, and mosaic offspring, revealing the source of the three hybrid types present at East Inlet Pond. These offspring accepted grafts from P. eos-neogaeus individuals, indicating that they all had inherited the identical eos-neogaeus genome. Most grafts among triploid and mosaic progeny, or from these individuals to their diploid broodmates, were rejected, indicating that the third genome was different in each triploid and mosaic (as was observed in the wild hybrids) and was contributed by sperm from males of P. eos or P. neogaeus. Diploid progeny are produced if sperm serves only to stimulate embryogenesis; triploid or mosaic progeny are produced if the sperm genome is incorporated. Although based on a mode of reproduction that by definition results in a genetically identical community of individuals, i.e., gynogenesis, reproduction in hybrid Phoxinus results in a variety of genetically distinct individuals by the incorporation of sperm into approximately 50% of the diploid ova produced. © 1990 The Society for the Study of Evolution.

Primary adenocarcinoma of the thymus: an immunohistochemical and molecular study with review of the literature.

PubMed

Maghbool, Maryam; Ramzi, Mani; Nagel, Inga; Bejarano, Pablo; Siebert, Reiner; Saeedzadeh, Abolfazl; Daneshbod, Yahya

2013-05-31

Primary adenocarcinoma of thymus is extremely rare. This is a case of primary adenocarcinoma with intestinal differentiation and focal mucin production in the thymus. Thymic cyst was associated with this tumor. Intestinal differentiation was confirmed by immunohistochemical stain with positivity for CDX-2, CK20, villin, MOC31 and focal positivity of CK7. Array comperative genomic hybridization (CGH) analysis showed a complex pattern of chromosomal imbalances including homozygous deletion at the HLA locus in chromosomal region 6p21.32. This rare tumor shows a similar genetic aberration with other studied thymic epithelial tumors.

Prenatal diagnosis and molecular characterization of a novel locus for Dandy-Walker malformation on chromosome 7p21.3.

PubMed

Liao, Can; Fu, Fang; Li, Ru; Yang, Xin; Xu, Qing; Li, Dong-Zhi

2012-01-01

We present three foetuses with Dandy-Walker malformation, intra-uterine growth restriction and multiple congenital abnormalities, who were studied by array-based comparative genomic hybridization and revealed a novel locus on chromosome 7p21.3. The association of pure chromosome 7p aberrations with Dandy-Walker malformation has rarely been reported. The present study suggests that the critical region associated with Dandy-Walker malformation is restricted to 7p21.3, including the cerebellar disease associated genes NDUFA4 and PHF14. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The complex hybrid origins of the root knot nematodes revealed through comparative genomics

PubMed Central

Kumar, Sujai; Koutsovoulos, Georgios; Blaxter, Mark L.

2014-01-01

Root knot nematodes (RKN) can infect most of the world’s agricultural crop species and are among the most important of all plant pathogens. As yet however we have little understanding of their origins or the genomic basis of their extreme polyphagy. The most damaging pathogens reproduce by obligatory mitotic parthenogenesis and it has been suggested that these species originated from interspecific hybridizations between unknown parental taxa. We have sequenced the genome of the diploid meiotic parthenogen Meloidogyne floridensis, and use a comparative genomic approach to test the hypothesis that this species was involved in the hybrid origin of the tropical mitotic parthenogen Meloidogyne incognita. Phylogenomic analysis of gene families from M. floridensis, M. incognita and an outgroup species Meloidogyne hapla was carried out to trace the evolutionary history of these species’ genomes, and we demonstrate that M. floridensis was one of the parental species in the hybrid origins of M. incognita. Analysis of the M. floridensis genome itself revealed many gene loci present in divergent copies, as they are in M. incognita, indicating that it too had a hybrid origin. The triploid M. incognita is shown to be a complex double-hybrid between M. floridensis and a third, unidentified, parent. The agriculturally important RKN have very complex origins involving the mixing of several parental genomes by hybridization and their extreme polyphagy and success in agricultural environments may be related to this hybridization, producing transgressive variation on which natural selection can act. It is now clear that studying RKN variation via individual marker loci may fail due to the species’ convoluted origins, and multi-species population genomics is essential to understand the hybrid diversity and adaptive variation of this important species complex. This comparative genomic analysis provides a compelling example of the importance and complexity of hybridization in generating animal species diversity more generally. PMID:24860695

High-resolution mapping reveals hundreds of genetic incompatibilities in hybridizing fish species.

PubMed

Schumer, Molly; Cui, Rongfeng; Powell, Daniel L; Dresner, Rebecca; Rosenthal, Gil G; Andolfatto, Peter

2014-06-04

Hybridization is increasingly being recognized as a common process in both animal and plant species. Negative epistatic interactions between genes from different parental genomes decrease the fitness of hybrids and can limit gene flow between species. However, little is known about the number and genome-wide distribution of genetic incompatibilities separating species. To detect interacting genes, we perform a high-resolution genome scan for linkage disequilibrium between unlinked genomic regions in naturally occurring hybrid populations of swordtail fish. We estimate that hundreds of pairs of genomic regions contribute to reproductive isolation between these species, despite them being recently diverged. Many of these incompatibilities are likely the result of natural or sexual selection on hybrids, since intrinsic isolation is known to be weak. Patterns of genomic divergence at these regions imply that genetic incompatibilities play a significant role in limiting gene flow even in young species.

Copy number analysis of NIPBL in a cohort of 510 patients reveals rare copy number variants and a mosaic deletion.

PubMed

Cheng, Yu-Wei; Tan, Christopher A; Minor, Agata; Arndt, Kelly; Wysinger, Latrice; Grange, Dorothy K; Kozel, Beth A; Robin, Nathaniel H; Waggoner, Darrel; Fitzpatrick, Carrie; Das, Soma; Del Gaudio, Daniela

2014-03-01

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder characterized by growth retardation, intellectual disability, upper limb abnormalities, hirsutism, and characteristic facial features. In this study we explored the occurrence of intragenic NIPBL copy number variations (CNVs) in a cohort of 510 NIPBL sequence-negative patients with suspected CdLS. Copy number analysis was performed by custom exon-targeted oligonucleotide array-comparative genomic hybridization and/or MLPA. Whole-genome SNP array was used to further characterize rearrangements extending beyond the NIPBL gene. We identified NIPBL CNVs in 13 patients (2.5%) including one intragenic duplication and a deletion in mosaic state. Breakpoint sequences in two patients provided further evidence of a microhomology-mediated replicative mechanism as a potential predominant contributor to CNVs in NIPBL. Patients for whom clinical information was available share classical CdLS features including craniofacial and limb defects. Our experience in studying the frequency of NIBPL CNVs in the largest series of patients to date widens the mutational spectrum of NIPBL and emphasizes the clinical utility of performing NIPBL deletion/duplication analysis in patients with CdLS.

Synthesis of porous NiO/CeO2 hybrid nanoflake arrays as a platform for electrochemical biosensing

NASA Astrophysics Data System (ADS)

Cui, Jiewu; Luo, Jinbao; Peng, Bangguo; Zhang, Xinyi; Zhang, Yong; Wang, Yan; Qin, Yongqiang; Zheng, Hongmei; Shu, Xia; Wu, Yucheng

2015-12-01

Porous NiO/CeO2 hybrid nanoflake arrays fabricated by a facile hydrothermal method were employed as substrates for electrochemical biosensors. The resulting NiO/CeO2 hybrid nanoflake arrays with a large specific surface area and good biocompatibility presented an excellent platform for electrochemical biosensing.Porous NiO/CeO2 hybrid nanoflake arrays fabricated by a facile hydrothermal method were employed as substrates for electrochemical biosensors. The resulting NiO/CeO2 hybrid nanoflake arrays with a large specific surface area and good biocompatibility presented an excellent platform for electrochemical biosensing. Electronic supplementary information (ESI) available: Optical photographs of the as-prepared samples, SEM, TEM, EDS, XRD and BET data of the samples are presented, I-t curves of glucose biosensors based on NiO and NiO/CeO2 NFAs, EIS results of different electrodes. See DOI: 10.1039/c5nr05924k

Primary gamma ray selection in a hybrid timing/imaging Cherenkov array

NASA Astrophysics Data System (ADS)

Postnikov, E. B.; Grinyuk, A. A.; Kuzmichev, L. A.; Sveshnikova, L. G.

2017-06-01

This work is a methodical study on hybrid reconstruction techniques for hybrid imaging/timing Cherenkov observations. This type of hybrid array is to be realized at the gamma-observatory TAIGA intended for very high energy gamma-ray astronomy (> 30 TeV). It aims at combining the cost-effective timing-array technique with imaging telescopes. Hybrid operation of both of these techniques can lead to a relatively cheap way of development of a large area array. The joint approach of gamma event selection was investigated on both types of simulated data: the image parameters from the telescopes, and the shower parameters reconstructed from the timing array. The optimal set of imaging parameters and shower parameters to be combined is revealed. The cosmic ray background suppression factor depending on distance and energy is calculated. The optimal selection technique leads to cosmic ray background suppression of about 2 orders of magnitude on distances up to 450 m for energies greater than 50 TeV.

Graphene quantum dots-carbon nanotube hybrid arrays for supercapacitors

NASA Astrophysics Data System (ADS)

Hu, Yue; Zhao, Yang; Lu, Gewu; Chen, Nan; Zhang, Zhipan; Li, Hui; Shao, Huibo; Qu, Liangti

2013-05-01

Graphene quantum dots (GQDs) have been successfully deposited onto aligned carbon nanotubes (CNTs) by a benign electrochemical method and the capacitive properties of the as-formed GQD/CNT hybrid arrays were evaluated in symmetrical supercapacitors. It was found that supercapacitors fabricated from GQD/CNT hybrid arrays exhibited a high capacitance of 44 mF cm-2, representing a more than 200% improvement over that of bare CNT electrodes.

Graphene quantum dots-carbon nanotube hybrid arrays for supercapacitors.

PubMed

Hu, Yue; Zhao, Yang; Lu, Gewu; Chen, Nan; Zhang, Zhipan; Li, Hui; Shao, Huibo; Qu, Liangti

2013-05-17

Graphene quantum dots (GQDs) have been successfully deposited onto aligned carbon nanotubes (CNTs) by a benign electrochemical method and the capacitive properties of the as-formed GQD/CNT hybrid arrays were evaluated in symmetrical supercapacitors. It was found that supercapacitors fabricated from GQD/CNT hybrid arrays exhibited a high capacitance of 44 mF cm(-2), representing a more than 200% improvement over that of bare CNT electrodes.

Comparative genomics in chicken and Pekin duck using FISH mapping and microarray analysis

PubMed Central

2009-01-01

Background The availability of the complete chicken (Gallus gallus) genome sequence as well as a large number of chicken probes for fluorescent in-situ hybridization (FISH) and microarray resources facilitate comparative genomic studies between chicken and other bird species. In a previous study, we provided a comprehensive cytogenetic map for the turkey (Meleagris gallopavo) and the first analysis of copy number variants (CNVs) in birds. Here, we extend this approach to the Pekin duck (Anas platyrhynchos), an obvious target for comparative genomic studies due to its agricultural importance and resistance to avian flu. Results We provide a detailed molecular cytogenetic map of the duck genome through FISH assignment of 155 chicken clones. We identified one inter- and six intrachromosomal rearrangements between chicken and duck macrochromosomes and demonstrated conserved synteny among all microchromosomes analysed. Array comparative genomic hybridisation revealed 32 CNVs, of which 5 overlap previously designated "hotspot" regions between chicken and turkey. Conclusion Our results suggest extensive conservation of avian genomes across 90 million years of evolution in both macro- and microchromosomes. The data on CNVs between chicken and duck extends previous analyses in chicken and turkey and supports the hypotheses that avian genomes contain fewer CNVs than mammalian genomes and that genomes of evolutionarily distant species share regions of copy number variation ("CNV hotspots"). Our results will expedite duck genomics, assist marker development and highlight areas of interest for future evolutionary and functional studies. PMID:19656363

Accuracy of CNV Detection from GWAS Data.

PubMed

Zhang, Dandan; Qian, Yudong; Akula, Nirmala; Alliey-Rodriguez, Ney; Tang, Jinsong; Gershon, Elliot S; Liu, Chunyu

2011-01-13

Several computer programs are available for detecting copy number variants (CNVs) using genome-wide SNP arrays. We evaluated the performance of four CNV detection software suites--Birdsuite, Partek, HelixTree, and PennCNV-Affy--in the identification of both rare and common CNVs. Each program's performance was assessed in two ways. The first was its recovery rate, i.e., its ability to call 893 CNVs previously identified in eight HapMap samples by paired-end sequencing of whole-genome fosmid clones, and 51,440 CNVs identified by array Comparative Genome Hybridization (aCGH) followed by validation procedures, in 90 HapMap CEU samples. The second evaluation was program performance calling rare and common CNVs in the Bipolar Genome Study (BiGS) data set (1001 bipolar cases and 1033 controls, all of European ancestry) as measured by the Affymetrix SNP 6.0 array. Accuracy in calling rare CNVs was assessed by positive predictive value, based on the proportion of rare CNVs validated by quantitative real-time PCR (qPCR), while accuracy in calling common CNVs was assessed by false positive/false negative rates based on qPCR validation results from a subset of common CNVs. Birdsuite recovered the highest percentages of known HapMap CNVs containing >20 markers in two reference CNV datasets. The recovery rate increased with decreased CNV frequency. In the tested rare CNV data, Birdsuite and Partek had higher positive predictive values than the other software suites. In a test of three common CNVs in the BiGS dataset, Birdsuite's call was 98.8% consistent with qPCR quantification in one CNV region, but the other two regions showed an unacceptable degree of accuracy. We found relatively poor consistency between the two "gold standards," the sequence data of Kidd et al., and aCGH data of Conrad et al. Algorithms for calling CNVs especially common ones need substantial improvement, and a "gold standard" for detection of CNVs remains to be established.

Genomic profiling of plasma cell disorders in a clinical setting: integration of microarray and FISH, after CD138 selection of bone marrow

PubMed Central

Berry, Nadine Kaye; Bain, Nicole L; Enjeti, Anoop K; Rowlings, Philip

2014-01-01

Aim To evaluate the role of whole genome comparative genomic hybridisation microarray (array-CGH) in detecting genomic imbalances as compared to conventional karyotype (GTG-analysis) or myeloma specific fluorescence in situ hybridisation (FISH) panel in a diagnostic setting for plasma cell dyscrasia (PCD). Methods A myeloma-specific interphase FISH (i-FISH) panel was carried out on CD138 PC-enriched bone marrow (BM) from 20 patients having BM biopsies for evaluation of PCD. Whole genome array-CGH was performed on reference (control) and neoplastic (test patient) genomic DNA extracted from CD138 PC-enriched BM and analysed. Results Comparison of techniques demonstrated a much higher detection rate of genomic imbalances using array-CGH. Genomic imbalances were detected in 1, 19 and 20 patients using GTG-analysis, i-FISH and array-CGH, respectively. Genomic rearrangements were detected in one patient using GTG-analysis and seven patients using i-FISH, while none were detected using array-CGH. I-FISH was the most sensitive method for detecting gene rearrangements and GTG-analysis was the least sensitive method overall. All copy number aberrations observed in GTG-analysis were detected using array-CGH and i-FISH. Conclusions We show that array-CGH performed on CD138-enriched PCs significantly improves the detection of clinically relevant and possibly novel genomic abnormalities in PCD, and thus could be considered as a standard diagnostic technique in combination with IGH rearrangement i-FISH. PMID:23969274

Genomic profiling of plasma cell disorders in a clinical setting: integration of microarray and FISH, after CD138 selection of bone marrow.

PubMed

Berry, Nadine Kaye; Bain, Nicole L; Enjeti, Anoop K; Rowlings, Philip

2014-01-01

To evaluate the role of whole genome comparative genomic hybridisation microarray (array-CGH) in detecting genomic imbalances as compared to conventional karyotype (GTG-analysis) or myeloma specific fluorescence in situ hybridisation (FISH) panel in a diagnostic setting for plasma cell dyscrasia (PCD). A myeloma-specific interphase FISH (i-FISH) panel was carried out on CD138 PC-enriched bone marrow (BM) from 20 patients having BM biopsies for evaluation of PCD. Whole genome array-CGH was performed on reference (control) and neoplastic (test patient) genomic DNA extracted from CD138 PC-enriched BM and analysed. Comparison of techniques demonstrated a much higher detection rate of genomic imbalances using array-CGH. Genomic imbalances were detected in 1, 19 and 20 patients using GTG-analysis, i-FISH and array-CGH, respectively. Genomic rearrangements were detected in one patient using GTG-analysis and seven patients using i-FISH, while none were detected using array-CGH. I-FISH was the most sensitive method for detecting gene rearrangements and GTG-analysis was the least sensitive method overall. All copy number aberrations observed in GTG-analysis were detected using array-CGH and i-FISH. We show that array-CGH performed on CD138-enriched PCs significantly improves the detection of clinically relevant and possibly novel genomic abnormalities in PCD, and thus could be considered as a standard diagnostic technique in combination with IGH rearrangement i-FISH.

Genomic heterozygosity and hybrid breakdown in cotton (Gossypium): different traits, different effects.

PubMed

Dai, Baosheng; Guo, Huanle; Huang, Cong; Zhang, Xianlong; Lin, Zhongxu

2016-04-12

Hybrid breakdown has been well documented in various species. Relationships between genomic heterozygosity and traits-fitness have been extensively explored especially in the natural populations. But correlations between genomic heterozygosity and vegetative and reproductive traits in cotton interspecific populations have not been studied. In the current study, two reciprocal F2 populations were developed using Gossypium hirsutum cv. Emian 22 and G. barbadense acc. 3-79 as parents to study hybrid breakdown in cotton. A total of 125 simple sequence repeat (SSR) markers were used to genotype the two F2 interspecific populations. To guarantee mutual independence among the genotyped markers, the 125 SSR markers were checked by the linkage disequilibrium analysis. To our knowledge, this is a novel approach to evaluate the individual genomic heterozygosity. After marker checking, 83 common loci were used to assess the extent of genomic heterozygosity. Hybrid breakdown was found extensively in the two interspecific F2 populations particularly on the reproductive traits because of the infertility and the bare seeds. And then, the relationships between the genomic heterozygosity and the vegetative reproductive traits were investigated. The only relationships between hybrid breakdown and heterozygosity were observed in the (Emian22 × 3-79) F2 population for seed index (SI) and boll number per plant (BN). The maternal cytoplasmic environment may have a significant effect on genomic heterozygosity and on correlations between heterozygosity and reproductive traits. A novel approach was used to evaluate genomic heterozygosity in cotton; and hybrid breakdown was observed in reproductive traits in cotton. These findings may offer new insight into hybrid breakdown in allotetraploid cotton interspecific hybrids, and may be useful for the development of interspecific hybrids for cotton genetic improvement.

Using Pool-seq to Search for Genomic Regions Affected by Hybrid Inviability in the copepod T. californicus.

PubMed

Lima, Thiago G; Willett, Christopher S

2018-05-11

The formation of reproductive barriers between allopatric populations involves the accumulation of incompatibilities that lead to intrinsic postzygotic isolation. The evolution of these incompatibilities is usually explained by the Dobzhansky-Muller model, where epistatic interactions that arise within the diverging populations, lead to deleterious interactions when they come together in a hybrid genome. These incompatibilities can lead to hybrid inviability, killing individuals with certain genotypic combinations, and causing the population's allele frequency to deviate from Mendelian expectations. Traditionally, hybrid inviability loci have been detected by genotyping individuals at different loci across the genome. However, this method becomes time consuming and expensive as the number of markers or individuals increases. Here, we test if a Pool-seq method can be used to scan the genome of F2 hybrids to detect genomic regions that are affected by hybrid inviability. We survey the genome of hybrids between 2 populations of the copepod Tigriopus californicus, and show that this method has enough power to detect even small changes in allele frequency caused by hybrid inviability. We show that allele frequency estimates in Pool-seq can be affected by the sampling of alleles from the pool of DNA during the library preparation and sequencing steps and that special considerations must be taken when aligning hybrid reads to a reference when the populations/species are divergent.

Genomic architecture of biomass heterosis in Arabidopsis.

PubMed

Yang, Mei; Wang, Xuncheng; Ren, Diqiu; Huang, Hao; Xu, Miqi; He, Guangming; Deng, Xing Wang

2017-07-25

Heterosis is most frequently manifested by the substantially increased vigorous growth of hybrids compared with their parents. Investigating genomic variations in natural populations is essential to understand the initial molecular mechanisms underlying heterosis in plants. Here, we characterized the genomic architecture associated with biomass heterosis in 200 Arabidopsis hybrids. The genome-wide heterozygosity of hybrids makes a limited contribution to biomass heterosis, and no locus shows an obvious overdominance effect in hybrids. However, the accumulation of significant genetic loci identified in genome-wide association studies (GWAS) in hybrids strongly correlates with better-parent heterosis (BPH). Candidate genes for biomass BPH fall into diverse biological functions, including cellular, metabolic, and developmental processes and stimulus-responsive pathways. Important heterosis candidates include WUSCHEL , ARGOS , and some genes that encode key factors involved in cell cycle regulation. Interestingly, transcriptomic analyses in representative Arabidopsis hybrid combinations reveal that heterosis candidate genes are functionally enriched in stimulus-responsive pathways, including responses to biotic and abiotic stimuli and immune responses. In addition, stimulus-responsive genes are repressed to low-parent levels in hybrids with high BPH, whereas middle-parent expression patterns are exhibited in hybrids with no BPH. Our study reveals a genomic architecture for understanding the molecular mechanisms of biomass heterosis in Arabidopsis , in which the accumulation of the superior alleles of genes involved in metabolic and cellular processes improve the development and growth of hybrids, whereas the overall repressed expression of stimulus-responsive genes prioritizes growth over responding to environmental stimuli in hybrids under normal conditions.

Identification of copy number variation in French dairy and beef breeds using next-generation sequencing.

PubMed

Letaief, Rabia; Rebours, Emmanuelle; Grohs, Cécile; Meersseman, Cédric; Fritz, Sébastien; Trouilh, Lidwine; Esquerré, Diane; Barbieri, Johanna; Klopp, Christophe; Philippe, Romain; Blanquet, Véronique; Boichard, Didier; Rocha, Dominique; Boussaha, Mekki

2017-10-24

Copy number variations (CNV) are known to play a major role in genetic variability and disease pathogenesis in several species including cattle. In this study, we report the identification and characterization of CNV in eight French beef and dairy breeds using whole-genome sequence data from 200 animals. Bioinformatics analyses to search for CNV were carried out using four different but complementary tools and we validated a subset of the CNV by both in silico and experimental approaches. We report the identification and localization of 4178 putative deletion-only, duplication-only and CNV regions, which cover 6% of the bovine autosomal genome; they were validated by two in silico approaches and/or experimentally validated using array-based comparative genomic hybridization and single nucleotide polymorphism genotyping arrays. The size of these variants ranged from 334 bp to 7.7 Mb, with an average size of ~ 54 kb. Of these 4178 variants, 3940 were deletions, 67 were duplications and 171 corresponded to both deletions and duplications, which were defined as potential CNV regions. Gene content analysis revealed that, among these variants, 1100 deletions and duplications encompassed 1803 known genes, which affect a wide spectrum of molecular functions, and 1095 overlapped with known QTL regions. Our study is a large-scale survey of CNV in eight French dairy and beef breeds. These CNV will be useful to study the link between genetic variability and economically important traits, and to improve our knowledge on the genomic architecture of cattle.

Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh

PubMed Central

Begum, Rabeya; Alam, Sheikh Shamimul; Menzel, Gerhard; Schmidt, Thomas

2009-01-01

Background and Aims Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera. Methods In order to isolate highly repetitive sequences, a c0t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH). Key Results Characterization of the c0t-1 library resulted in the detection of repetitive sequences including the (GA)n dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S–5·8S–25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species. Conclusions The repeated sequences isolated from D. moschatum c0t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species. PMID:19635741

Hybrid Arrays for Chemical Sensing

NASA Astrophysics Data System (ADS)

Kramer, Kirsten E.; Rose-Pehrsson, Susan L.; Johnson, Kevin J.; Minor, Christian P.

In recent years, multisensory approaches to environment monitoring for chemical detection as well as other forms of situational awareness have become increasingly popular. A hybrid sensor is a multimodal system that incorporates several sensing elements and thus produces data that are multivariate in nature and may be significantly increased in complexity compared to data provided by single-sensor systems. Though a hybrid sensor is itself an array, hybrid sensors are often organized into more complex sensing systems through an assortment of network topologies. Part of the reason for the shift to hybrid sensors is due to advancements in sensor technology and computational power available for processing larger amounts of data. There is also ample evidence to support the claim that a multivariate analytical approach is generally superior to univariate measurements because it provides additional redundant and complementary information (Hall, D. L.; Linas, J., Eds., Handbook of Multisensor Data Fusion, CRC, Boca Raton, FL, 2001). However, the benefits of a multisensory approach are not automatically achieved. Interpretation of data from hybrid arrays of sensors requires the analyst to develop an application-specific methodology to optimally fuse the disparate sources of data generated by the hybrid array into useful information characterizing the sample or environment being observed. Consequently, multivariate data analysis techniques such as those employed in the field of chemometrics have become more important in analyzing sensor array data. Depending on the nature of the acquired data, a number of chemometric algorithms may prove useful in the analysis and interpretation of data from hybrid sensor arrays. It is important to note, however, that the challenges posed by the analysis of hybrid sensor array data are not unique to the field of chemical sensing. Applications in electrical and process engineering, remote sensing, medicine, and of course, artificial intelligence and robotics, all share the same essential data fusion challenges. The design of a hybrid sensor array should draw on this extended body of knowledge. In this chapter, various techniques for data preprocessing, feature extraction, feature selection, and modeling of sensor data will be introduced and illustrated with data fusion approaches that have been implemented in applications involving data from hybrid arrays. The example systems discussed in this chapter involve the development of prototype sensor networks for damage control event detection aboard US Navy vessels and the development of analysis algorithms to combine multiple sensing techniques for enhanced remote detection of unexploded ordnance (UXO) in both ground surveys and wide area assessments.

High-throughput analysis of the satellitome illuminates satellite DNA evolution

NASA Astrophysics Data System (ADS)

Ruiz-Ruano, Francisco J.; López-León, María Dolores; Cabrero, Josefa; Camacho, Juan Pedro M.

2016-07-01

Satellite DNA (satDNA) is a major component yet the great unknown of eukaryote genomes and clearly underrepresented in genome sequencing projects. Here we show the high-throughput analysis of satellite DNA content in the migratory locust by means of the bioinformatic analysis of Illumina reads with the RepeatExplorer and RepeatMasker programs. This unveiled 62 satDNA families and we propose the term “satellitome” for the whole collection of different satDNA families in a genome. The finding that satDNAs were present in many contigs of the migratory locust draft genome indicates that they show many genomic locations invisible by fluorescent in situ hybridization (FISH). The cytological pattern of five satellites showing common descent (belonging to the SF3 superfamily) suggests that non-clustered satDNAs can become into clustered through local amplification at any of the many genomic loci resulting from previous dissemination of short satDNA arrays. The fact that all kinds of satDNA (micro- mini- and satellites) can show the non-clustered and clustered states suggests that all these elements are mostly similar, except for repeat length. Finally, the presence of VNTRs in bacteria, showing similar properties to non-clustered satDNAs in eukaryotes, suggests that this kind of tandem repeats show common properties in all living beings.

Genetic Dissection of Clonally Inherited Genomes of Poeciliopsis. I. Linkage Analysis and Preliminary Assessment of Deleterious Gene Loads

PubMed Central

Leslie, James F.; Vrijenhoek, Robert C.

1978-01-01

Theoretical considerations suggest that a high load of deleterious mutations should accumulate in asexual genomes. An ideal system for testing this hypothesis occurs in the hybrid all-female fish Poeciliopsis monacha-lucida. The hybrid genotype is retained between generations by an oogenetic process that transmits only a nonrecombinant haploid monacha genome to their ova. The hybrid genotype is re-established in nature by fertilization of these monacha eggs with sperm from a sexual species, P. lucida. The unique reproductive mechanism of these hybrids allows the genetic dissection of the clonal monacha genome by forced matings with males of P. monacha. The resultant F1 hybrids and their backcross progeny were examined to determine the amount and kinds of genetic changes that might have occurred in two clonal monacha genomes.—Using six allozyme markers, four similar linkage groups were identified in each clonal genome. Segregation and assortment at these loci revealed no apparent differences between monacha genomes from sexually and clonally reproducing species. Mortality of F1 and backcross progeny revealed differences between the two clonal genomes, suggesting that deleterious genes may accumulate in genomes sheltered from recombination. PMID:17248875

Genome Properties and Prospects of Genomic Prediction of Hybrid Performance in a Breeding Program of Maize

PubMed Central

Technow, Frank; Schrag, Tobias A.; Schipprack, Wolfgang; Bauer, Eva; Simianer, Henner; Melchinger, Albrecht E.

2014-01-01

Maize (Zea mays L.) serves as model plant for heterosis research and is the crop where hybrid breeding was pioneered. We analyzed genomic and phenotypic data of 1254 hybrids of a typical maize hybrid breeding program based on the important Dent × Flint heterotic pattern. Our main objectives were to investigate genome properties of the parental lines (e.g., allele frequencies, linkage disequilibrium, and phases) and examine the prospects of genomic prediction of hybrid performance. We found high consistency of linkage phases and large differences in allele frequencies between the Dent and Flint heterotic groups in pericentromeric regions. These results can be explained by the Hill–Robertson effect and support the hypothesis of differential fixation of alleles due to pseudo-overdominance in these regions. In pericentromeric regions we also found indications for consistent marker–QTL linkage between heterotic groups. With prediction methods GBLUP and BayesB, the cross-validation prediction accuracy ranged from 0.75 to 0.92 for grain yield and from 0.59 to 0.95 for grain moisture. The prediction accuracy of untested hybrids was highest, if both parents were parents of other hybrids in the training set, and lowest, if none of them were involved in any training set hybrid. Optimizing the composition of the training set in terms of number of lines and hybrids per line could further increase prediction accuracy. We conclude that genomic prediction facilitates a paradigm shift in hybrid breeding by focusing on the performance of experimental hybrids rather than the performance of parental lines in testcrosses. PMID:24850820

Simultaneous visualization of different genomes (J, JSt and St) in a Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid (Poaceae) and in its parental species by multicolour genomic in situ hybridization (mcGISH)

PubMed Central

Kruppa, Klaudia; Molnár-Láng, Márta

2016-01-01

Abstract Multicolour genomic in situ hybridization (mcGISH) using total genomic DNA probes from Thinopyrum bessarabicum (Săvulescu & Rayss, 1923) Á. Löve, 1984 (genome Jb or Eb, 2n = 14), and Pseudoroegneria spicata (Pursh, 1814) Á. Löve, 1980 (genome St, 2n = 14) was used to characterize the mitotic metaphase chromosomes of a synthetic hybrid of Thinopyrum intermedium (Host, 1805) Barkworth & D.R. Dewey, 1985 and Thinopyrum ponticum (Podpěra, 1902) Z.-W. Liu et R.-C.Wang, 1993 named „Agropyron glael” and produced by N.V. Tsitsin in the former Soviet Union. The mcGISH pattern of this synthetic hybrid was compared to its parental wheatgrass species. Hexaploid Thinopyrum intermedium contained 19 J, 9 JSt and 14 St chromosomes. The three analysed Thinopyrum ponticum accessions had different chromosome compositions: 43 J + 27 JSt (PI531737), 40 J + 30 JSt (VIR-44486) and 38 J + 32 JSt (D-3494). The synthetic hybrid carried 18 J, 28 JSt and 8 St chromosomes, including one pair of J-St translocation and/or decreased fluorescent intensity, resulting in unique hybridization patterns. Wheat line Mv9kr1 was crossed with the Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid in Hungary in order to transfer its advantageous agronomic traits (leaf rust and yellow rust resistance) into wheat. The chromosome composition of a wheat/A.glael F1 hybrid was 21 wheat + 28 wheatgrass (11 J + 14 JSt+ 3 S). In the present study, mcGISH involving the simultaneous use of St and J genomic DNA as probes provided information about the type of Thinopyrum chromosomes in a Thinopyrum intermedium/Thinopyrum ponticum synthetic hybrid called A. glael. PMID:27551349

Selection against recombinant hybrids maintains reproductive isolation in hybridizing Populus species despite F1 fertility and recurrent gene flow.

PubMed

Christe, Camille; Stölting, Kai N; Bresadola, Luisa; Fussi, Barbara; Heinze, Berthold; Wegmann, Daniel; Lexer, Christian

2016-06-01

Natural hybrid zones have proven to be precious tools for understanding the origin and maintenance of reproductive isolation (RI) and therefore species. Most available genomic studies of hybrid zones using whole- or partial-genome resequencing approaches have focused on comparisons of the parental source populations involved in genome admixture, rather than exploring fine-scale patterns of chromosomal ancestry across the full admixture gradient present between hybridizing species. We have studied three well-known European 'replicate' hybrid zones of Populus alba and P. tremula, two widespread, ecologically divergent forest trees, using up to 432 505 single-nucleotide polymorphisms (SNPs) from restriction site-associated DNA (RAD) sequencing. Estimates of fine-scale chromosomal ancestry, genomic divergence and differentiation across all 19 poplar chromosomes revealed strikingly contrasting results, including an unexpected preponderance of F1 hybrids in the centre of genomic clines on the one hand, and genomically localized, spatially variable shared variants consistent with ancient introgression between the parental species on the other. Genetic ancestry had a significant effect on survivorship of hybrid seedlings in a common garden trial, pointing to selection against early-generation recombinants. Our results indicate a role for selection against recombinant genotypes in maintaining RI in the face of apparent F1 fertility, consistent with the intragenomic 'coadaptation' model of barriers to introgression upon secondary contact. Whole-genome resequencing of hybridizing populations will clarify the roles of specific genetic pathways in RI between these model forest trees and may reveal which loci are affected most strongly by its cyclic breakdown. © 2016 John Wiley & Sons Ltd.

Genomic and transcriptomic alterations following intergeneric hybridization and polyploidization in the Chrysanthemum nankingense×Tanacetum vulgare hybrid and allopolyploid (Asteraceae).

PubMed

Qi, Xiangyu; Wang, Haibin; Song, Aiping; Jiang, Jiafu; Chen, Sumei; Chen, Fadi

2018-01-01

Allopolyploid formation involves two major events: interspecific hybridization and polyploidization. A number of species in the Asteraceae family are polyploids because of frequent hybridization. The effects of hybridization on genomics and transcriptomics in Chrysanthemum nankingense×Tanacetum vulgare hybrids have been reported. In this study, we obtained allopolyploids by applying a colchicine treatment to a synthesized C. nankingense × T. vulgare hybrid. Sequence-related amplified polymorphism (SRAP), methylation-sensitive amplification polymorphism (MSAP), and high-throughput RNA sequencing (RNA-Seq) technologies were used to investigate the genomic, epigenetic, and transcriptomic alterations in both the hybrid and allopolyploids. The genomic alterations in the hybrid and allopolyploids mainly involved the loss of parental fragments and the gain of novel fragments. The DNA methylation level of the hybrid was reduced by hybridization but was restored somewhat after polyploidization. There were more significant differences in gene expression between the hybrid/allopolyploid and the paternal parent than between the hybrid/allopolyploid and the maternal parent. Most differentially expressed genes (DEGs) showed down-regulation in the hybrid/allopolyploid relative to the parents. Among the non-additive genes, transgressive patterns appeared to be dominant, especially repression patterns. Maternal expression dominance was observed specifically for down-regulated genes. Many methylase and methyltransferase genes showed differential expression between the hybrid and parents and between the allopolyploid and parents. Our data indicate that hybridization may be a major factor affecting genomic and transcriptomic changes in newly formed allopolyploids. The formation of allopolyploids may not simply be the sum of hybridization and polyploidization changes but also may be influenced by the interaction between these processes.

High-resolution mapping and sequence analysis of 597 cDNA clones transcribed from the 1 Mb region in human chromosome 4q16.3 containing Huntington disease gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadano, S.; Ishida, Y.; Tomiyasu, H.

1994-09-01

To complete a transcription map of the 1 Mb region in human chromosome 4p16.3 containing the Huntington disease (HD) gene, the isolation of cDNA clones are being performed throughout. Our method relies on a direct screening of the cDNA libraries probed with single copy microclones from 3 YAC clones spanning 1 Mbp of the HD gene region. AC-DNAs were isolated by a preparative pulsed-field gel electrophoresis, amplified by both a single unique primer (SUP)-PCR and a linker ligation PCR, and 6 microclone-DNA libraries were generated. Then, 8,640 microclones from these libraries were independently amplified by PCR, and arrayed onto themore » membranes. 800-900 microclones that were not cross-hybridized with total human and yeast genomic DNA, TAC vector DNA, and ribosomal cDNA on a dot hybridization (putatively carrying single copy sequences) were pooled to make 9 probe pools. A total of {approximately}1.8x10{sup 7} plaques from the human brain cDNA libraries was screened with 9 pool-probes, and then 672 positive cDNA clones were obtained. So far, 597 cDNA clones were defined and arrayed onto a map of the 1 Mbp of the HD gene region by hybridization with HD region-specific cosmid contigs and YAC clones. Further characterization including a DNA sequencing and Northern blot analysis is currently underway.« less

Contribution of radiation hybrids to genome mapping in domestic animals.

PubMed

Faraut, T; de Givry, S; Hitte, C; Lahbib-Mansais, Y; Morisson, M; Milan, D; Schiex, T; Servin, B; Vignal, A; Galibert, F; Yerle, M

2009-01-01

Radiation hybrid mapping has emerged in the end of the 1990 s as a successful and complementary approach to map genomes, essentially because of its ability to bridge the gaps between genetic and clone-based physical maps, but also using comparative mapping approaches, between 'gene-rich' and 'gene-poor' maps. Since its early development in human, radiation hybrid mapping played a pivotal role in the process of mapping animal genomes, especially mammalian ones. We review here all the different steps involved in radiation hybrid mapping from the constitution of panels to the construction of maps. A description of its contribution to whole genome maps with a special emphasis on domestic animals will also be presented. Finally, current applications of radiation hybrid mapping in the context of whole genome assemblies will be described. (c) 2009 S. Karger AG, Basel.

Feasibility of microelectrode array (MEA) based on silicone-polyimide hybrid for retina prosthesis.

PubMed

Kim, Eui Tae; Kim, Cinoo; Lee, Seung Woo; Seo, Jong-Mo; Chung, Hum; Kim, Sung June

2009-09-01

To adopt micropatterning technology in manufacturing silicone elastomer-based microelectrode arrays for retinal stimulation, a silicone-polyimide hybrid microelectrode array was proposed and tested in vivo. Gold microelectrodes were created by semiconductor manufacturing technology based on polyimide and were hybridized with silicone elastomer by spin coating. The stability of the hybrid between the two materials was flex and blister tested. The feasibility of the hybrid electrode was evaluated in the rabbit eye by reviewing optical coherence tomography (OCT) findings after suprachoroidal implantation. The flex test showed no dehiscence between the two materials for 24 hours of alternative flexion and extension from -45.0 degrees to +45.0 degrees . During the blister test, delamination was observed at 8.33 +/- 1.36 psi of pressure stress; however, this property was improved to 11.50 +/- 1.04 psi by oxygen plasma treatment before hybridization. OCT examination revealed that the implanted electrodes were safely located in the suprachoroidal space during the 4-week follow-up period. The silicone-polyimide hybrid microelectrode array showed moderate physical properties, which are suitable for in vivo application. Appropriate pretreatment before hybridization improved electrode stability. In vivo testing indicated that this electrode is suitable as a stimulation electrode in artificial retina.

Use of fluorescence in situ hybridization as a tool for introgression analysis and chromosome identification in coffee (Coffea arabica L.).

PubMed

Herrera, Juan Carlos; D'Hont, Angelique; Lashermes, Philippe

2007-07-01

Fluorescence in situ hybridization (FISH) was used to study the presence of alien chromatin in interspecific hybrids and one introgressed line (S.288) derived from crosses between the cultivated species Coffea arabica and the diploid relatives C. canephora and C. liberica. In situ hybridization using genomic DNA from C. canephora and C. arabica as probes showed elevated cross hybridization along the hybrid genome, confirming the weak differentiation between parental genomes. According to our genomic in situ hybridization (GISH) data, the observed genomic resemblance between the modern C. canephora genome (C) and the C. canephora-derived subgenome of C. arabica (Ca) appears rather considerable. Poor discrimination between C and Ca chromosomes supports the idea of low structural modifications of both genomes since the C. arabica speciation, at least in the frequency and distribution of repetitive sequences. GISH was also used to identify alien chromatin segments on chromosome spreads of a C. liberica-introgressed line of C. arabica. Further, use of GISH together with BAC-FISH analysis gave us additional valuable information about the physical localization of the C. liberica fragments carrying the SH3 factor involved in resistance to the coffee leaf rust. Overall, our results illustrate that FISH analysis is a complementary tool for molecular cytogenetic studies in coffee, providing rapid localization of either specific chromosomes or alien chromatin in introgressed genotypes derived from diploid species displaying substantial genomic differentiation from C. arabica.

GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cronn, M.T.; Miyada, C.G.; Fucini, R.V.

1994-09-01

GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by amore » sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.« less

Evolutionary Conservation of a Coding Function for D4Z4, the Tandem DNA Repeat Mutated in Facioscapulohumeral Muscular Dystrophy

PubMed Central

Clapp, Jannine ; Mitchell, Laura M. ; Bolland, Daniel J. ; Fantes, Judy ; Corcoran, Anne E. ; Scotting, Paul J. ; Armour, John A. L. ; Hewitt, Jane E. 

2007-01-01

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within the polymorphic DNA tandem array D4Z4. Each D4Z4 repeat unit has an open reading frame (ORF), termed “DUX4,” containing two homeobox sequences. Because there has been no evidence of a transcript from the array, these deletions are thought to cause FSHD by a position effect on other genes. Here, we identify D4Z4 homologues in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other species and show that the DUX4 ORF is conserved. Phylogenetic analysis suggests that primate and Afrotherian D4Z4 arrays are orthologous and originated from a retrotransposed copy of an intron-containing DUX gene, DUXC. Reverse-transcriptase polymerase chain reaction and RNA fluorescence and tissue in situ hybridization data indicate transcription of the mouse array. Together with the conservation of the DUX4 ORF for >100 million years, this strongly supports a coding function for D4Z4 and necessitates re-examination of current models of the FSHD disease mechanism. PMID:17668377

Insertional translocation leading to a 4q13 duplication including the EPHA5 gene in two siblings with attention-deficit hyperactivity disorder.

PubMed

Matoso, Eunice; Melo, Joana B; Ferreira, Susana I; Jardim, Ana; Castelo, Teresa M; Weise, Anja; Carreira, Isabel M

2013-08-01

An insertional translocation (IT) can result in pure segmental aneusomy for the inserted genomic segment allowing to define a more accurate clinical phenotype. Here, we report on two siblings sharing an unbalanced IT inherited from the mother with a history of learning difficulty. An 8-year-old girl with developmental delay, speech disability, and attention-deficit hyperactivity disorder (ADHD), showed by GTG banding analysis a subtle interstitial alteration in 21q21. Oligonucleotide array comparative genomic hybridization (array-CGH) analysis showed a 4q13.1-q13.3 duplication spanning 8.6 Mb. Fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) clones confirmed the rearrangement, a der(21)ins(21;4)(q21;q13.1q13.3). The duplication described involves 50 RefSeq genes including the EPHA5 gene that encodes for the EphA5 receptor involved in embryonic development of the brain and also in synaptic remodeling and plasticity thought to underlie learning and memory. The same rearrangement was observed in a younger brother with behavioral problems and also exhibiting ADHD. ADHD is among the most heritable of neuropsychiatric disorders. There are few reports of patients with duplications involving the proximal region of 4q and a mild phenotype. To the best of our knowledge this is the first report of a duplication restricted to band 4q13. This abnormality could be easily missed in children who have nonspecific cognitive impairment. The presence of this behavioral disorder in the two siblings reinforces the hypothesis that the region involved could include genes involved in ADHD. Copyright © 2013 Wiley Periodicals, Inc.

Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media.

PubMed

Shamonki, Mousa I; Jin, Helen; Haimowitz, Zachary; Liu, Lian

2016-11-01

To assess whether preimplantation genetic screening (PGS) is possible by testing for free embryonic DNA in spent IVF media from embryos undergoing trophectoderm biopsy. Prospective cohort analysis. Academic fertility center. Seven patients undergoing IVF and 57 embryos undergoing trophectoderm biopsy for PGS. On day 3 of development, each embryo was placed in a separate media droplet. All biopsied embryos received a PGS result by array comparative genomic hybridization. Preimplantation genetic screening was performed on amplified DNA extracted from media and results were compared with PGS results for the corresponding biopsy. [1] Presence of DNA in spent IVF culture media. [2] Correlation between genetic screening result from spent media and corresponding biopsy. Fifty-five samples had detectable DNA ranging from 2-642 ng/μL after a 2-hour amplification. Six samples with the highest DNA levels underwent PGS, rendering one result with a derivative log ratio SD (DLRSD) of <0.85 (a quality control metric of oligonucleotide array comparative genomic hybridization). The fluid sample and trophectoderm results were identical demonstrating (45XY, -13). Three samples were reamplified 1 hour later and tested showing improving DLRSD. One of the three samples with a DLRSD of 0.85 demonstrated (46XY), consistent with the biopsy. Overnight DNA amplification showed DNA in all samples. We demonstrate two novel findings: the presence of free embryonic DNA in spent media and a result that is consistent with trophectoderm biopsy. Improvements in DNA collection, amplification, and testing may allow for PGS without biopsy in the future. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Six consecutive false positive cases from cell-free fetal DNA testing in a single referring centre

PubMed Central

Dugo, Nella; Padula, Francesco; Mobili, Luisa; Brizzi, Cristiana; D’Emidio, Laura; Cignini, Pietro; Mesoraca, Alvaro; Bizzoco, Domenico; Cima, Antonella; Giorlandino, Claudio

2014-01-01

Introduction recent studies have proposed the introduction of cell-free fetal DNA testing (NIPT-Non Invasive Prenatal Testing) in routine clinical practice emphasizing its high sensibility and specificity. In any case, false positive and false negative findings may result from placental mosaicism, because cell-free fetal DNA originates mainly from placenta. Case we report six cases of women who underwent chorionic villus sampling (CVS) or amniocentesis to confirm the results from NIPT: two Turner syndromes, two Triple X, one Patau syndrome, one Edward syndrome. Results using classic cytogenetic analysis and, also, Array - Comparative Genomic Hybridization (Array CGH) the karyotype of all 5 fetuses was found to be normal. Conclusion results from NIPT must always be confirmed by invasive prenatal diagnosis. It is mandatory to inform the patient that the CVS and amniocentesis still represent the only form of prenatal diagnostic test available. PMID:25332757

Genomic analysis using high density SNP based oligonucleotide arrays and MLPA provides a comprehensive analysis of INI1/SMARCB1 in malignant rhabdoid tumors

PubMed Central

Jackson, Eric M.; Sievert, Angela J.; Gai, Xiaowu; Hakonarson, Hakon; Judkins, Alexander R; Tooke, Laura; Perin, Juan Carlos; Xie, Hongbo; Shaikh, Tamim H.; Biegel, Jaclyn A.

2009-01-01

Translational Relevance Previous reports suggested that abnormalities of INI1 could be detected in 70–75% of malignant rhabdoid tumors. The mechanism of inactivation in the other 25% remained unclear. The goal of this study was to perform a high-resolution genomic analysis of a large series of rhabdoid tumors with the expectation of identifying additional loci related to the initiation or progression of these malignancies. We also developed a comprehensive set of assays, including a new MLPA assay, to interrogate the INI1 locus in 22q11.2. Intragenic deletions could be detected using the Illumina 550K Beadchip, whereas single exon deletions could be detected using MLPA. The current study demonstrates that with a multi-platform approach, alterations at the INI1 locus can be detected in almost all cases. Thus, appropriate molecular genetic testing can be used as an aid in the diagnosis and for treatment planning for most patients. Purpose A high-resolution genomic profiling and comprehensive targeted analysis of INI1/SMARCB1 of a large series of pediatric rhabdoid tumors was performed. The aim was to identify regions of copy number change and loss of heterozygosity that might pinpoint additional loci involved in the development or progression of rhabdoid tumors, and define the spectrum of genomic alterations of INI1 in this malignancy. Experimental Design A multi-platform approach, utilizing Illumina single nucleotide polymorphism (SNP) based oligonucleotide arrays, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), and coding sequence analysis was used to characterize genome wide copy number changes, loss of heterozygosity, and genomic alterations of INI1/SMARCB1 in a series of pediatric rhabdoid tumors. Results The bi-allelic alterations of INI1 that led to inactivation were elucidated in 50 of 51 tumors. INI1 inactivation was demonstrated by a variety of mechanisms, including deletions, mutations, and loss of heterozygosity. The results from the array studies highlighted the complexity of rearrangements of chromosome 22, compared to the low frequency of alterations involving the other chromosomes. Conclusions The results from the genome wide SNP-array analysis suggest that INI1 is the primary tumor suppressor gene involved in the development of rhabdoid tumors with no second locus identified. In addition, we did not identify hot spots for the breakpoints in sporadic tumors with deletions of chromosome 22q11.2. By employing a multimodality approach, the wide spectrum of alterations of INI1 can be identified in the majority of patients, which increases the clinical utility of molecular diagnostic testing. PMID:19276269

MicroArray Facility: a laboratory information management system with extended support for Nylon based technologies.

PubMed

Honoré, Paul; Granjeaud, Samuel; Tagett, Rebecca; Deraco, Stéphane; Beaudoing, Emmanuel; Rougemont, Jacques; Debono, Stéphane; Hingamp, Pascal

2006-09-20

High throughput gene expression profiling (GEP) is becoming a routine technique in life science laboratories. With experimental designs that repeatedly span thousands of genes and hundreds of samples, relying on a dedicated database infrastructure is no longer an option.GEP technology is a fast moving target, with new approaches constantly broadening the field diversity. This technology heterogeneity, compounded by the informatics complexity of GEP databases, means that software developments have so far focused on mainstream techniques, leaving less typical yet established techniques such as Nylon microarrays at best partially supported. MAF (MicroArray Facility) is the laboratory database system we have developed for managing the design, production and hybridization of spotted microarrays. Although it can support the widely used glass microarrays and oligo-chips, MAF was designed with the specific idiosyncrasies of Nylon based microarrays in mind. Notably single channel radioactive probes, microarray stripping and reuse, vector control hybridizations and spike-in controls are all natively supported by the software suite. MicroArray Facility is MIAME supportive and dynamically provides feedback on missing annotations to help users estimate effective MIAME compliance. Genomic data such as clone identifiers and gene symbols are also directly annotated by MAF software using standard public resources. The MAGE-ML data format is implemented for full data export. Journalized database operations (audit tracking), data anonymization, material traceability and user/project level confidentiality policies are also managed by MAF. MicroArray Facility is a complete data management system for microarray producers and end-users. Particular care has been devoted to adequately model Nylon based microarrays. The MAF system, developed and implemented in both private and academic environments, has proved a robust solution for shared facilities and industry service providers alike.

MicroArray Facility: a laboratory information management system with extended support for Nylon based technologies

PubMed Central

Honoré, Paul; Granjeaud, Samuel; Tagett, Rebecca; Deraco, Stéphane; Beaudoing, Emmanuel; Rougemont, Jacques; Debono, Stéphane; Hingamp, Pascal

2006-01-01

Background High throughput gene expression profiling (GEP) is becoming a routine technique in life science laboratories. With experimental designs that repeatedly span thousands of genes and hundreds of samples, relying on a dedicated database infrastructure is no longer an option. GEP technology is a fast moving target, with new approaches constantly broadening the field diversity. This technology heterogeneity, compounded by the informatics complexity of GEP databases, means that software developments have so far focused on mainstream techniques, leaving less typical yet established techniques such as Nylon microarrays at best partially supported. Results MAF (MicroArray Facility) is the laboratory database system we have developed for managing the design, production and hybridization of spotted microarrays. Although it can support the widely used glass microarrays and oligo-chips, MAF was designed with the specific idiosyncrasies of Nylon based microarrays in mind. Notably single channel radioactive probes, microarray stripping and reuse, vector control hybridizations and spike-in controls are all natively supported by the software suite. MicroArray Facility is MIAME supportive and dynamically provides feedback on missing annotations to help users estimate effective MIAME compliance. Genomic data such as clone identifiers and gene symbols are also directly annotated by MAF software using standard public resources. The MAGE-ML data format is implemented for full data export. Journalized database operations (audit tracking), data anonymization, material traceability and user/project level confidentiality policies are also managed by MAF. Conclusion MicroArray Facility is a complete data management system for microarray producers and end-users. Particular care has been devoted to adequately model Nylon based microarrays. The MAF system, developed and implemented in both private and academic environments, has proved a robust solution for shared facilities and industry service providers alike. PMID:16987406

GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes.

PubMed

van den Broek, Evert; van Lieshout, Stef; Rausch, Christian; Ylstra, Bauke; van de Wiel, Mark A; Meijer, Gerrit A; Fijneman, Remond J A; Abeln, Sanne

2016-01-01

Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs) of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large) series of tumor samples. 'GeneBreak' is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS). First, 'GeneBreak' collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, 'GeneBreak', is implemented in R ( www.cran.r-project.org ) and is available from Bioconductor ( www.bioconductor.org/packages/release/bioc/html/GeneBreak.html ).

Comparative genomics among Saccharomyces cerevisiae × Saccharomyces kudriavzevii natural hybrid strains isolated from wine and beer reveals different origins

PubMed Central

2012-01-01

Background Interspecific hybrids between S. cerevisiae × S. kudriavzevii have frequently been detected in wine and beer fermentations. Significant physiological differences among parental and hybrid strains under different stress conditions have been evidenced. In this study, we used comparative genome hybridization analysis to evaluate the genome composition of different S. cerevisiae × S. kudriavzevii natural hybrids isolated from wine and beer fermentations to infer their evolutionary origins and to figure out the potential role of common S. kudriavzevii gene fraction present in these hybrids. Results Comparative genomic hybridization (CGH) and ploidy analyses carried out in this study confirmed the presence of individual and differential chromosomal composition patterns for most S. cerevisiae × S. kudriavzevii hybrids from beer and wine. All hybrids share a common set of depleted S. cerevisiae genes, which also are depleted or absent in the wine strains studied so far, and the presence a common set of S. kudriavzevii genes, which may be associated with their capability to grow at low temperatures. Finally, a maximum parsimony analysis of chromosomal rearrangement events, occurred in the hybrid genomes, indicated the presence of two main groups of wine hybrids and different divergent lineages of brewing strains. Conclusion Our data suggest that wine and beer S. cerevisiae × S. kudriavzevii hybrids have been originated by different rare-mating events involving a diploid wine S. cerevisiae and a haploid or diploid European S. kudriavzevii strains. Hybrids maintain several S. kudriavzevii genes involved in cold adaptation as well as those related to S. kudriavzevii mitochondrial functions. PMID:22906207

Genome properties and prospects of genomic prediction of hybrid performance in a breeding program of maize.

PubMed

Technow, Frank; Schrag, Tobias A; Schipprack, Wolfgang; Bauer, Eva; Simianer, Henner; Melchinger, Albrecht E

2014-08-01

Maize (Zea mays L.) serves as model plant for heterosis research and is the crop where hybrid breeding was pioneered. We analyzed genomic and phenotypic data of 1254 hybrids of a typical maize hybrid breeding program based on the important Dent × Flint heterotic pattern. Our main objectives were to investigate genome properties of the parental lines (e.g., allele frequencies, linkage disequilibrium, and phases) and examine the prospects of genomic prediction of hybrid performance. We found high consistency of linkage phases and large differences in allele frequencies between the Dent and Flint heterotic groups in pericentromeric regions. These results can be explained by the Hill-Robertson effect and support the hypothesis of differential fixation of alleles due to pseudo-overdominance in these regions. In pericentromeric regions we also found indications for consistent marker-QTL linkage between heterotic groups. With prediction methods GBLUP and BayesB, the cross-validation prediction accuracy ranged from 0.75 to 0.92 for grain yield and from 0.59 to 0.95 for grain moisture. The prediction accuracy of untested hybrids was highest, if both parents were parents of other hybrids in the training set, and lowest, if none of them were involved in any training set hybrid. Optimizing the composition of the training set in terms of number of lines and hybrids per line could further increase prediction accuracy. We conclude that genomic prediction facilitates a paradigm shift in hybrid breeding by focusing on the performance of experimental hybrids rather than the performance of parental lines in test crosses. Copyright © 2014 by the Genetics Society of America.

Construction of two whole genome radiation hybrid panels for dromedary (Camelus dromedarius): 5000RAD and 15000RAD.

PubMed

Perelman, Polina L; Pichler, Rudolf; Gaggl, Anna; Larkin, Denis M; Raudsepp, Terje; Alshanbari, Fahad; Holl, Heather M; Brooks, Samantha A; Burger, Pamela A; Periasamy, Kathiravan

2018-01-31

The availability of genomic resources including linkage information for camelids has been very limited. Here, we describe the construction of a set of two radiation hybrid (RH) panels (5000 RAD and 15000 RAD ) for the dromedary (Camelus dromedarius) as a permanent genetic resource for camel genome researchers worldwide. For the 5000 RAD panel, a total of 245 female camel-hamster radiation hybrid clones were collected, of which 186 were screened with 44 custom designed marker loci distributed throughout camel genome. The overall mean retention frequency (RF) of the final set of 93 hybrids was 47.7%. For the 15000 RAD panel, 238 male dromedary-hamster radiation hybrid clones were collected, of which 93 were tested using 44 PCR markers. The final set of 90 clones had a mean RF of 39.9%. This 15000 RAD panel is an important high-resolution complement to the main 5000 RAD panel and an indispensable tool for resolving complex genomic regions. This valuable genetic resource of dromedary RH panels is expected to be instrumental for constructing a high resolution camel genome map. Construction of the set of RH panels is essential step toward chromosome level reference quality genome assembly that is critical for advancing camelid genomics and the development of custom genomic tools.

Relating hybrid advantage and genome replacement in unisexual salamanders.

PubMed

Charney, Noah D

2012-05-01

Unisexual vertebrates are model systems for understanding the evolution of sex. Many predominantly clonal lineages allow occasional genetic recombination, which may be sufficient to avoid the accumulation of deleterious mutations and parasites. Introgression of paternal DNA into an all-female lineage represents a one-way flow of genetic material. Over many generations, this could result in complete replacement of the unisexual genomes by those of the donor species. The process of genome replacement may be counteracted by contemporary dispersal or by positive selection on hybrid nuclear genomes in ecotones. I present a conceptual model that relates nuclear genome replacement, positive selection on hybrids and biogeography in unisexual systems. I execute an individual-based simulation of the fate of hybrid genotypes in contact with a single host species. I parameterize these models for unisexual salamanders in the Ambystoma genus, for which the frequency of genome replacement has been a source of ongoing debate. I find that, if genome replacement occurs at a rate greater than 1/10,000 in Ambystoma, then there must be compensating positive selection in order to maintain observed levels of hybrid nuclei. Future researchers studying unisexual systems may use this framework as a guide to evaluating the hybrid superiority hypothesis. © 2011 The Author. Evolution© 2011 The Society for the Study of Evolution.

Mechanisms Underlying Mammalian Hybrid Sterility in Two Feline Interspecies Models.

PubMed

Davis, Brian W; Seabury, Christopher M; Brashear, Wesley A; Li, Gang; Roelke-Parker, Melody; Murphy, William J

2015-10-01

The phenomenon of male sterility in interspecies hybrids has been observed for over a century, however, few genes influencing this recurrent phenotype have been identified. Genetic investigations have been primarily limited to a small number of model organisms, thus limiting our understanding of the underlying molecular basis of this well-documented "rule of speciation." We utilized two interspecies hybrid cat breeds in a genome-wide association study employing the Illumina 63 K single-nucleotide polymorphism array. Collectively, we identified eight autosomal genes/gene regions underlying associations with hybrid male sterility (HMS) involved in the function of the blood-testis barrier, gamete structural development, and transcriptional regulation. We also identified several candidate hybrid sterility regions on the X chromosome, with most residing in close proximity to complex duplicated regions. Differential gene expression analyses revealed significant chromosome-wide upregulation of X chromosome transcripts in testes of sterile hybrids, which were enriched for genes involved in chromatin regulation of gene expression. Our expression results parallel those reported in Mus hybrids, supporting the "Large X-Effect" in mammalian HMS and the potential epigenetic basis for this phenomenon. These results support the value of the interspecies feline model as a powerful tool for comparison to rodent models of HMS, demonstrating unique aspects and potential commonalities that underpin mammalian reproductive isolation. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Hybrid Vibrio cholerae El Tor Lacking SXT Identified as the Cause of a Cholera Outbreak in the Philippines

PubMed Central

Klinzing, David C.; Choi, Seon Young; Hasan, Nur A.; Matias, Ronald R.; Tayag, Enrique; Geronimo, Josefina; Skowronski, Evan; Rashed, Shah M.; Kawashima, Kent; Rosenzweig, C. Nicole; Gibbons, Henry S.; Torres, Brian C.; Liles, Veni; Alfon, Alicia C.; Juan, Maria Luisa; Natividad, Filipinas F.; Cebula, Thomas A.

2015-01-01

ABSTRACT Cholera continues to be a global threat, with high rates of morbidity and mortality. In 2011, a cholera outbreak occurred in Palawan, Philippines, affecting more than 500 people, and 20 individuals died. Vibrio cholerae O1 was confirmed as the etiological agent. Source attribution is critical in cholera outbreaks for proper management of the disease, as well as to control spread. In this study, three V. cholerae O1 isolates from a Philippines cholera outbreak were sequenced and their genomes analyzed to determine phylogenetic relatedness to V. cholerae O1 isolates from recent outbreaks of cholera elsewhere. The Philippines V. cholerae O1 isolates were determined to be V. cholerae O1 hybrid El Tor belonging to the seventh-pandemic clade. They clustered tightly, forming a monophyletic clade closely related to V. cholerae O1 hybrid El Tor from Asia and Africa. The isolates possess a unique multilocus variable-number tandem repeat analysis (MLVA) genotype (12-7-9-18-25 and 12-7-10-14-21) and lack SXT. In addition, they possess a novel 15-kb genomic island (GI-119) containing a predicted type I restriction-modification system. The CTXΦ-RS1 array of the Philippines isolates was similar to that of V. cholerae O1 MG116926, a hybrid El Tor strain isolated in Bangladesh in 1991. Overall, the data indicate that the Philippines V. cholerae O1 isolates are unique, differing from recent V. cholerae O1 isolates from Asia, Africa, and Haiti. Furthermore, the results of this study support the hypothesis that the Philippines isolates of V. cholerae O1 are indigenous and exist locally in the aquatic ecosystem of the Philippines. PMID:25900650

Hybrid incompatibilities are affected by dominance and dosage in the haplodiploid wasp Nasonia

PubMed Central

Beukeboom, Leo W.; Koevoets, Tosca; Morales, Hernán E.; Ferber, Steven; van de Zande, Louis

2015-01-01

Study of genome incompatibilities in species hybrids is important for understanding the genetic basis of reproductive isolation and speciation. According to Haldane's rule hybridization affects the heterogametic sex more than the homogametic sex. Several theories have been proposed that attribute asymmetry in hybridization effects to either phenotype (sex) or genotype (heterogamety). Here we investigate the genetic basis of hybrid genome incompatibility in the haplodiploid wasp Nasonia using the powerful features of haploid males and sex reversal. We separately investigate the effects of heterozygosity (ploidy level) and sex by generating sex reversed diploid hybrid males and comparing them to genotypically similar haploid hybrid males and diploid hybrid females. Hybrid effects of sterility were more pronounced than of inviability, and were particularly strong in haploid males, but weak to absent in diploid males and females, indicating a strong ploidy level but no sex specific effect. Molecular markers identified a number of genomic regions associated with hybrid inviability in haploid males that disappeared under diploidy in both hybrid males and females. Hybrid inviability was rescued by dominance effects at some genomic regions, but aggravated or alleviated by dosage effects at other regions, consistent with cytonuclear incompatibilities. Dosage effects underlying Bateson–Dobzhansky–Muller (BDM) incompatibilities need more consideration in explaining Haldane's rule in diploid systems. PMID:25926847

Did backcrossing contribute to the origin of hybrid edible bananas?

PubMed Central

De Langhe, Edmond; Hřibová, Eva; Carpentier, Sebastien; Doležel, Jaroslav; Swennen, Rony

2010-01-01

Background Bananas and plantains (Musa spp.) provide a staple food for many millions of people living in the humid tropics. The cultivated varieties (cultivars) are seedless parthenocarpic clones of which the origin remains unclear. Many are believed to be diploid and polyploid hybrids involving the A genome diploid M. acuminata and the B genome M. balbisiana, with the hybrid genomes consisting of a simple combination of the parental ones. Thus the genomic constitution of the diploids has been classified as AB, and that of the triploids as AAB or ABB. However, the morphology of many accessions is biased towards either the A or B phenotype and does not conform to predictions based on these genomic formulae. Scope On the basis of published cytotypes (mitochondrial and chloroplast genomes), we speculate here that the hybrid banana genomes are unbalanced with respect to the parental ones, and/or that inter-genome translocation chromosomes are relatively common. We hypothesize that the evolution under domestication of cultivated banana hybrids is more likely to have passed through an intermediate hybrid, which was then involved in a variety of backcrossing events. We present experimental data supporting our hypothesis and we propose a set of experimental approaches to test it, thereby indicating other possibilities for explaining some of the unbalanced genome expressions. Progress in this area would not only throw more light on the origin of one of the most important crops, but provide data of general relevance for the evolution under domestication of many other important clonal crops. At the same time, a complex origin of the cultivated banana hybrids would imply a reconsideration of current breeding strategies. PMID:20858591

Identification of a microdeletion at Xp22.13 in a Taiwanese family presenting with Nance-Horan syndrome.

PubMed

Liao, Hsiao-Mei; Niu, Dau-Ming; Chen, Yan-Jang; Fang, Jye-Siung; Chen, Shih-Jen; Chen, Chia-Hsiang

2011-01-01

Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital cataracts, dental anomalies and mental retardation. The disease has been linked to a novel gene termed NHS located at Xp22.13. The majority of pathogenic mutations of the disease include nonsense mutations and small deletions and insertions that lead to truncation of the NHS protein. In this study, we identified a microdeletion of ∼ 0.92 Mb at Xp22.13 detected by array-based comparative genomic hybridization in two brothers presenting congenital cataract, dental anomalies, facial dysmorphisms and mental retardation. The deleted region encompasses the REPS2, NHS, SCML1 and RAI2 genes, and was transmitted from their carrier mother who presented only mild cataract. Our findings are in line with several recent case reports to indicate that genomic rearrangement involving the NHS gene is an important genetic etiology underlying NHS.

Microdeletion of 19p13.3 in a girl with Peutz-Jeghers syndrome, intellectual disability, hypotonia, and distinctive features.

PubMed

Kuroda, Yukiko; Saito, Toshiyuki; Nagai, Jun-Ichi; Ida, Kazumi; Naruto, Takuya; Masuno, Mitsuo; Kurosawa, Kenji

2015-02-01

Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant disease characterized by gastrointestinal polyposis and mucocutaneous pigmentation. Germline point mutations in the serine/threonine kinase 11 (STK11) have been identified in about 70% of patients with PJS. Only a few large genomic deletions have been identified. We report on a girl with PJS and multiple congenital anomalies. She had intellectual disability, umbilical hernia, bilateral inguinal hernias, scoliosis, and distinct facial appearance including prominent mandible, smooth philtrum, and malformed ears. She developed lip pigmentation at the age of 12 years but had no gastrointestinal polyps. Array comparative genomic hybridization revealed an approximately 610 kb deletion at 19p13.3, encompassing STK11. Together with previous reports, the identification of common clinical features suggests that microdeletion at 19p13.3 encompassing STK11 constitutes a distinctive phenotype. © 2014 Wiley Periodicals, Inc.

The Nucleolus: In Genome Maintenance and Repair.

PubMed

Tsekrekou, Maria; Stratigi, Kalliopi; Chatzinikolaou, Georgia

2017-07-01

The nucleolus is the subnuclear membrane-less organelle where rRNA is transcribed and processed and ribosomal assembly occurs. During the last 20 years, however, the nucleolus has emerged as a multifunctional organelle, regulating processes that go well beyond its traditional role. Moreover, the unique organization of rDNA in tandem arrays and its unusually high transcription rates make it prone to unscheduled DNA recombination events and frequent RNA:DNA hybrids leading to DNA double strand breaks (DSBs). If not properly repaired, rDNA damage may contribute to premature disease onset and aging. Deregulation of ribosomal synthesis at any level from transcription and processing to ribosomal subunit assembly elicits a stress response and is also associated with disease onset. Here, we discuss how genome integrity is maintained within nucleoli and how such structures are functionally linked to nuclear DNA damage response and repair giving an emphasis on the newly emerging roles of the nucleolus in mammalian physiology and disease.

Causes of learning disability and epilepsy: a review.

PubMed

Prince, Elizabeth; Ring, Howard

2011-04-01

Although the association between learning disability and epilepsy is well known, until relatively recently specific processes underlying this association were relatively poorly understood. However, scientific advances in molecular biology are starting to guide researchers towards descriptions of genetic and pathophysiological processes that may explain why syndromes of epilepsy and learning disability often co-exist. This article will focus largely on three areas of advancing knowledge: insights gained from wider use of genome-wide array comparative genomic hybridization (aCGH), specific insights gained from detailed study of Rett syndrome and the role of abnormalities of astrocytic function in predisposing to both epilepsy and learning disability. The enormous complexity of the biological underpinnings of the co-occurrence of epilepsy and learning disability are becoming apparent. In the future it is likely that research into therapeutic approaches will include, amongst other approaches, investigations of gene structure and expression, the role of astrocytes and the stability of dendritic spines.

Analysis of cytoplasmic genomes in somatic hybrids between navel orange (Citrus sinensis Osb.) and 'Murcott' tangor.

PubMed

Kobayashi, S; Ohgawara, T; Fujiwara, K; Oiyama, I

1991-07-01

Somatic hybrid plants were produced by protoplast fusion of navel orange and 'Murcott' tangor. Hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA. All of the plants (16 clones) were normal, uniform, and had the amphidiploid chromosome number of 36 (2n=2x=18 for each parent). The cpDNA analysis showed that each of the 16 somatic hybrids contained either one parental chloroplast genome or the other. In all cases, the mitochondrial genomes of the regenerated somatic hybrids were of the navel orange type.

CNV-TV: a robust method to discover copy number variation from short sequencing reads.

PubMed

Duan, Junbo; Zhang, Ji-Gang; Deng, Hong-Wen; Wang, Yu-Ping

2013-05-02

Copy number variation (CNV) is an important structural variation (SV) in human genome. Various studies have shown that CNVs are associated with complex diseases. Traditional CNV detection methods such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) suffer from low resolution. The next generation sequencing (NGS) technique promises a higher resolution detection of CNVs and several methods were recently proposed for realizing such a promise. However, the performances of these methods are not robust under some conditions, e.g., some of them may fail to detect CNVs of short sizes. There has been a strong demand for reliable detection of CNVs from high resolution NGS data. A novel and robust method to detect CNV from short sequencing reads is proposed in this study. The detection of CNV is modeled as a change-point detection from the read depth (RD) signal derived from the NGS, which is fitted with a total variation (TV) penalized least squares model. The performance (e.g., sensitivity and specificity) of the proposed approach are evaluated by comparison with several recently published methods on both simulated and real data from the 1000 Genomes Project. The experimental results showed that both the true positive rate and false positive rate of the proposed detection method do not change significantly for CNVs with different copy numbers and lengthes, when compared with several existing methods. Therefore, our proposed approach results in a more reliable detection of CNVs than the existing methods.

Non-introgressive genome chimerisation by malsegregation in autodiploidised allotetraploids during meiosis of Saccharomyces kudriavzevii x Saccharomyces uvarum hybrids.

PubMed

Karanyicz, Edina; Antunovics, Zsuzsa; Kallai, Z; Sipiczki, M

2017-06-01

Saccharomyces strains with chimerical genomes consisting of mosaics of the genomes of different species ("natural hybrids") occur quite frequently among industrial and wine strains. The most widely endorsed hypothesis is that the mosaics are introgressions acquired via hybridisation and repeated backcrosses of the hybrids with one of the parental species. However, the interspecies hybrids are sterile, unable to mate with their parents. Here, we show by analysing synthetic Saccharomyces kudriavzevii x Saccharomyces uvarum hybrids that mosaic (chimeric) genomes can arise without introgressive backcrosses. These species are biologically separated by a double sterility barrier (sterility of allodiploids and F1 sterility of allotetraploids). F1 sterility is due to the diploidisation of the tetraploid meiosis resulting in MAT a /MAT α heterozygosity which suppresses mating in the spores. This barrier can occasionally be broken down by malsegregation of autosyndetically paired chromosomes carrying the MAT loci (loss of MAT heterozygosity). Subsequent malsegregation of additional autosyndetically paired chromosomes and occasional allosyndetic interactions chimerise the hybrid genome. Chromosomes are preferentially lost from the S. kudriavzevii subgenome. The uniparental transmission of the mitochondrial DNA to the hybrids indicates that nucleo-mitochondrial interactions might affect the direction of the genomic changes. We propose the name GARMe (Genome AutoReduction in Meiosis) for this process of genome reduction and chimerisation which involves no introgressive backcrossings. It opens a way to transfer genetic information between species and thus to get one step ahead after hybridisation in the production of yeast strains with beneficial combinations of properties of different species.

No evidence for mosaic pathogenic copy number variations in cardiac tissue from patients with congenital heart malformations.

PubMed

Winberg, Johanna; Berggren, Håkan; Malm, Torsten; Johansson, Sune; Johansson Ramgren, Jens; Nilsson, Boris; Liedén, Agne; Nordenskjöld, Agneta; Gustavsson, Peter; Nordgren, Ann

2015-03-01

The aim of this study was to investigate if pathogenic copy number variations (CNVs) are present in mosaic form in patients with congenital heart malformations. We have collected cardiac tissue and blood samples from 23 patients with congenital heart malformations that underwent cardiac surgery and screened for mosaic gene dose alterations restricted to cardiac tissue using array comparative genomic hybridization (array CGH). We did not find evidence of CNVs in mosaic form after array CGH analysis. Pathogenic CNVs that were present in both cardiac tissue and blood were detected in 2/23 patients (9%), and in addition we found several constitutional CNVs of unclear clinical significance. This is the first study investigating mosaicism for CNVs in heart tissue compared to peripheral blood and the results do not indicate that pathogenic mosaic copy number changes are common in patients with heart malformations. Importantly, in line with previous studies, our results show that constitutional pathogenic CNVs are important factors contributing to congenital heart malformations. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome

PubMed Central

Yu, Shoukai; Lemos, Bernardo

2016-01-01

Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. PMID:27797956

Reassessment of the Genome Size in Elaeis guineensis and Elaeis oleifera, and Its Interspecific Hybrid

PubMed Central

Camillo, Julceia; Leão, André P; Alves, Alexandre A; Formighieri, Eduardo F; Azevedo, Ana LS; Nunes, Juliana D; de Capdeville, Guy; de A Mattos, Jean K; Souza, Manoel T

2014-01-01

Aiming at generating a comprehensive genomic database on Elaeis spp., our group is leading several R&D initiatives with Elaeis guineensis (African oil palm) and Elaeis oleifera (American oil palm), including the whole-genome sequencing of the last. Genome size estimates currently available for this genus are controversial, as they indicate that American oil palm genome is about half the size of the African oil palm genome and that the genome of the interspecific hybrid is bigger than both the parental species genomes. We estimated the genome size of three E. guineensis genotypes, five E. oleifera genotypes, and two interspecific hybrids genotypes. On average, the genome size of E. guineensis is 4.32 ± 0.173 pg, while that of E. oleifera is 4.43 ± 0.018 pg. This indicates that both genomes are similar in size, even though E. oleifera is in fact bigger. As expected, the hybrid genome size is around the average of the two genomes, 4.40 ± 0.016 pg. Additionally, we demonstrate that both species present around 38% of GC content. As our results contradict the currently available data on Elaeis spp. genome sizes, we propose that the actual genome size of the Elaeis species is around 4 pg and that American oil palm possesses a larger genome than African oil palm. PMID:26203259

Comparative Genomic Analyses of the Human NPHP1 Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates

PubMed Central

Yuan, Bo; Liu, Pengfei; Gupta, Aditya; Beck, Christine R.; Tejomurtula, Anusha; Campbell, Ian M.; Gambin, Tomasz; Simmons, Alexandra D.; Withers, Marjorie A.; Harris, R. Alan; Rogers, Jeffrey; Schwartz, David C.; Lupski, James R.

2015-01-01

Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100) is an autosomal recessive disorder that can be caused by defects of NPHP1; the gene maps within the human 2q13 region where low copy repeats (LCRs) are abundant. Loss of function of NPHP1 is responsible for approximately 85% of the NPHP1 cases—about 80% of such individuals carry a large recurrent homozygous NPHP1 deletion that occurs via nonallelic homologous recombination (NAHR) between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the NPHP1 gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV) haplotypes at the NPHP1 locus that may protect a haploid genome from the NPHP1 deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the NPHP1 locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the NPHP1 deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual’s susceptibility to acquiring disease-associated alleles. PMID:26641089

[An updated review of 1p36 deletion (monosomy) syndrome].

PubMed

Bello, Sabina; Rodríguez-Moreno, Antonio

The Monosomy 1p36 deletion syndrome is part of the group of diseases known as Rare Diseases. The objective of the present work is to review the characteristics of Monosomy 1p36 deletion syndrome. The monosomy 1p36 deletion syndrome phenotype includes: dysmorphic craniofacial features; large anterior fontanelle, unibrow, deep-set eyes, epicanthus, wide nasal root/bridge, mandible hypoplasia, abnormal location of the pinna, philtrum and pointed chin; neurological alterations: seizures and hydrocephalus (in some cases). Cerebral malformations: ventricular hypertrophy, increased subarachnoid space, morphological alterations of corpus callosum, cortical atrophy, delays in myelinisation, periventricular leukomalacia and periventricular heterotopia. These alterations produce intellectual disability and delays in motor growth, communication skills, language, social and adaptive behaviour. It is Hearing and vision impairments are also observed in subjects with this syndrome, as well as alterations of cardiac, endocrine and urinary systems and alterations at skin and skeletal level. Approximately 100 cases have been documented since 1981. This rare disease is the most common subtelomeric-micro-deletion syndrome. In situ hybridization with fluorescence (FISH) and array-comparative genomic hybridization (CGH-array) are at present the two best diagnostic techniques. There is currently no effective medical treatment for this disease. Copyright © 2016 Sociedad Chilena de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.

The 'PUCE CAFE' Project: the First 15K Coffee Microarray, a New Tool for Discovering Candidate Genes correlated to Agronomic and Quality Traits

PubMed Central

2011-01-01

Background Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. Results The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. Conclusion We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research. PMID:21208403

The 'PUCE CAFE' Project: the first 15K coffee microarray, a new tool for discovering candidate genes correlated to agronomic and quality traits.

PubMed

Privat, Isabelle; Bardil, Amélie; Gomez, Aureliano Bombarely; Severac, Dany; Dantec, Christelle; Fuentes, Ivanna; Mueller, Lukas; Joët, Thierry; Pot, David; Foucrier, Séverine; Dussert, Stéphane; Leroy, Thierry; Journot, Laurent; de Kochko, Alexandre; Campa, Claudine; Combes, Marie-Christine; Lashermes, Philippe; Bertrand, Benoit

2011-01-05

Understanding the genetic elements that contribute to key aspects of coffee biology will have an impact on future agronomical improvements for this economically important tree. During the past years, EST collections were generated in Coffee, opening the possibility to create new tools for functional genomics. The "PUCE CAFE" Project, organized by the scientific consortium NESTLE/IRD/CIRAD, has developed an oligo-based microarray using 15,721 unigenes derived from published coffee EST sequences mostly obtained from different stages of fruit development and leaves in Coffea Canephora (Robusta). Hybridizations for two independent experiments served to compare global gene expression profiles in three types of tissue matter (mature beans, leaves and flowers) in C. canephora as well as in the leaves of three different coffee species (C. canephora, C. eugenoides and C. arabica). Microarray construction, statistical analyses and validation by Q-PCR analysis are presented in this study. We have generated the first 15 K coffee array during this PUCE CAFE project, granted by Génoplante (the French consortium for plant genomics). This new tool will help study functional genomics in a wide range of experiments on various plant tissues, such as analyzing bean maturation or resistance to pathogens or drought. Furthermore, the use of this array has proven to be valid in different coffee species (diploid or tetraploid), drastically enlarging its impact for high-throughput gene expression in the community of coffee research.

Genomic organization of the rat alpha 2u-globulin gene cluster.

PubMed

McFadyen, D A; Addison, W; Locke, J

1999-05-01

The alpha 2u-globulin are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many alpha 2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 alpha 2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the alpha 2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the alpha 2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing alpha 2u-globulin genes indicated that the alpha 2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the alpha 2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes.

Synthesis of NiMn-LDH Nanosheet@Ni3S2 Nanorod Hybrid Structures for Supercapacitor Electrode Materials with Ultrahigh Specific Capacitance.

PubMed

Yu, Shuai; Zhang, Yingxi; Lou, Gaobo; Wu, Yatao; Zhu, Xinqiang; Chen, Hao; Shen, Zhehong; Fu, Shenyuan; Bao, Binfu; Wu, Limin

2018-03-27

One of the key challenges for pseudocapacitive electrode materials with highly effective capacitance output and future practical applications is how to rationally construct hierarchical and ordered hybrid nanoarchitecture through the simple process. Herein, we design and synthesize a novel NiMn-layered double hydroxide nanosheet@Ni 3 S 2 nanorod hybrid array supported on porous nickel foam via a one-pot hydrothermal method. Benefited from the ultrathin and rough nature, the well-defined porous structure of the hybrid array, as well as the synergetic effect between NiMn-layered double hydroxide nanosheets and Ni 3 S 2 nanorods, the as-fabricated hybrid array-based electrode exhibits an ultrahigh specific capacitance of 2703 F g -1 at 3 A g -1 . Moreover, the asymmetric supercapacitor with this hybrid array as a positive electrode and wood-derived activated carbon as a negative electrode demonstrates high energy density (57 Wh Kg -1 at 738 W Kg -1 ) and very good electrochemical cycling stability.

Performance measurements of hybrid PIN diode arrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jernigan, J.G.; Arens, J.F.; Kramer, G.

We report on the successful effort to develop hybrid PIN diode arrays and to demonstrate their potential as components of vertex detectors. Hybrid pixel arrays have been fabricated by the Hughes Aircraft Co. by bump bonding readout chips developed by Hughes to an array of PIN diodes manufactured by Micron Semiconductor Inc. These hybrid pixel arrays were constructed in two configurations. One array format having 10 {times} 64 pixels, each 120 {mu}m square, and the other format having 256 {times} 256 pixels, each 30 {mu}m square. In both cases, the thickness of the PIN diode layer is 300 {mu}m. Measurementsmore » of detector performance show that excellent position resolution can be achieved by interpolation. By determining the centroid of the charge cloud which spreads charge into a number of neighboring pixels, a spatial resolution of a few microns has been attained. The noise has been measured to be about 300 electrons (rms) at room temperature, as expected from KTC and dark current considerations, yielding a signal-to-noise ratio of about 100 for minimum ionizing particles. 4 refs., 13 figs.« less

Characterization of the complete mitochondrial genome of the hybrid Epinephelus moara♀ × Epinephelus lanceolatus♂, and phylogenetic analysis in subfamily epinephelinae

NASA Astrophysics Data System (ADS)

Gao, Fengtao; Wei, Min; Zhu, Ying; Guo, Hua; Chen, Songlin; Yang, Guanpin

2017-06-01

This study presents the complete mitochondrial genome of the hybrid Epinephelus moara♀× Epinephelus lanceolatus♂. The genome is 16886 bp in length, and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, a light-strand replication origin and a control region. Additionally, phylogenetic analysis based on the nucleotide sequences of 13 conserved protein-coding genes using the maximum likelihood method indicated that the mitochondrial genome is maternally inherited. This study presents genomic data for studying phylogenetic relationships and breeding of hybrid Epinephelinae.

Molecular profiling reveals frequent gain of MYCN and anaplasia-specific loss of 4q and 14q in Wilms tumor.

PubMed

Williams, Richard D; Al-Saadi, Reem; Natrajan, Rachael; Mackay, Alan; Chagtai, Tasnim; Little, Suzanne; Hing, Sandra N; Fenwick, Kerry; Ashworth, Alan; Grundy, Paul; Anderson, James R; Dome, Jeffrey S; Perlman, Elizabeth J; Jones, Chris; Pritchard-Jones, Kathy

2011-12-01

Anaplasia in Wilms tumor, a distinctive histology characterized by abnormal mitoses, is associated with poor patient outcome. While anaplastic tumors frequently harbour TP53 mutations, little is otherwise known about their molecular biology. We have used array comparative genomic hybridization (aCGH) and cDNA microarray expression profiling to compare anaplastic and favorable histology Wilms tumors to determine their common and differentiating features. In addition to changes on 17p, consistent with TP53 deletion, recurrent anaplasia-specific genomic loss and under-expression were noted in several other regions, most strikingly 4q and 14q. Further aberrations, including gain of 1q and loss of 16q were common to both histologies. Focal gain of MYCN, initially detected by high resolution aCGH profiling in 6/61 anaplastic samples, was confirmed in a significant proportion of both tumor types by a genomic quantitative PCR survey of over 400 tumors. Overall, these results are consistent with a model where anaplasia, rather than forming an entirely distinct molecular entity, arises from the general continuum of Wilms tumor by the acquisition of additional genomic changes at multiple loci. Copyright © 2011 Wiley Periodicals, Inc.

Digestive tumor bank protocol: from surgical specimens to genomic studies of digestive cancers.

PubMed

Popescu, I; Stroescu, C; Dumitrascu, T; Herlea, V; Paslaru, Liliana; Lazar, V; Boissin, H; Taieb, J; Horeanga, Ionela

2006-01-01

Cancer is a complex polygenic and multifactorial disease, resulting from successive dynamic changes in the genome of somatic cells and from the accumulation of molecular alterations in both tumour cells and host cells. For the majority of cancers, including many malignancies of the gastrointestinal tract, our current means of diagnosis and treatment of the tumors are grossly insufficient. In recent years the development of several gene expression profiling methods such as comparative genomic hybridization (CGH), differential display, serial analysis of gene expression (SAGE) and DNA arrays, together with the sequencing of the human genome, has provided an opportunity to monitor and investigate the complete cascade of molecular events leading to tumor development and progression. Given the central role played by surgeons in the current management of patients with solid cancers, it is of paramount importance for them to know the principles characterizing this laboratory tools to critically assess the results originating from this biotechnology. We describe in this article the scientific partnership between Fundeni Clinical Institute Bucharest, Romania and RNtech Company, Paris, France for the development of a center of biological resources (Biobank) as well as the standardized protocol of working with the biological samples, the ongoing projects and the future perspectives.

Primary adenocarcinoma of the thymus: an immunohistochemical and molecular study with review of the literature

PubMed Central

2013-01-01

Background Primary adenocarcinoma of thymus is extremely rare. Case presentation This is a case of primary adenocarcinoma with intestinal differentiation and focal mucin production in the thymus. Thymic cyst was associated with this tumor. Intestinal differentiation was confirmed by immunohistochemical stain with positivity for CDX-2, CK20, villin, MOC31 and focal positivity of CK7. Array comperative genomic hybridization (CGH) analysis showed a complex pattern of chromosomal imbalances including homozygous deletion at the HLA locus in chromosomal region 6p21.32. Conclusion This rare tumor shows a similar genetic aberration with other studied thymic epithelial tumors. PMID:23725376

Case report: cytogenetic and molecular analysis of proximal interstitial deletion of 4p, review of the literature and comparison with wolf-hirschhorn syndrome.

PubMed

Bailey, Nathanael G; South, Sarah T; Hummel, Marybeth; Wenger, Sharon L

2010-01-01

We report on a two-year-old female with a de novo proximal interstitial deletion of the short arm of chromosome 4 and a tetralogy of Fallot malformation. The patient had a karyotype of 46,XX,del(4)(p14p15.33) that was further characterized by array comparative genomic hybridization (aCGH). Phenotypic abnormalities for our patient are compared with those of previously reported patients with similar proximal 4p deletions as well as more distal deletions. The functions of genes that are deleted within this segment are reviewed.

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

PubMed Central

Schena, M; Shalon, D; Heller, R; Chai, A; Brown, P O; Davis, R W

1996-01-01

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855227

Genomic models with genotype × environment interaction for predicting hybrid performance: an application in maize hybrids.

PubMed

Acosta-Pech, Rocío; Crossa, José; de Los Campos, Gustavo; Teyssèdre, Simon; Claustres, Bruno; Pérez-Elizalde, Sergio; Pérez-Rodríguez, Paulino

2017-07-01

A new genomic model that incorporates genotype × environment interaction gave increased prediction accuracy of untested hybrid response for traits such as percent starch content, percent dry matter content and silage yield of maize hybrids. The prediction of hybrid performance (HP) is very important in agricultural breeding programs. In plant breeding, multi-environment trials play an important role in the selection of important traits, such as stability across environments, grain yield and pest resistance. Environmental conditions modulate gene expression causing genotype × environment interaction (G × E), such that the estimated genetic correlations of the performance of individual lines across environments summarize the joint action of genes and environmental conditions. This article proposes a genomic statistical model that incorporates G × E for general and specific combining ability for predicting the performance of hybrids in environments. The proposed model can also be applied to any other hybrid species with distinct parental pools. In this study, we evaluated the predictive ability of two HP prediction models using a cross-validation approach applied in extensive maize hybrid data, comprising 2724 hybrids derived from 507 dent lines and 24 flint lines, which were evaluated for three traits in 58 environments over 12 years; analyses were performed for each year. On average, genomic models that include the interaction of general and specific combining ability with environments have greater predictive ability than genomic models without interaction with environments (ranging from 12 to 22%, depending on the trait). We concluded that including G × E in the prediction of untested maize hybrids increases the accuracy of genomic models.

Simultaneous visualization of different genomes (J, JSt and St) in a Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid (Poaceae) and in its parental species by multicolour genomic in situ hybridization (mcGISH).

PubMed

Kruppa, Klaudia; Molnár-Láng, Márta

2016-01-01

Multicolour genomic in situ hybridization (mcGISH) using total genomic DNA probes from Thinopyrum bessarabicum (Săvulescu & Rayss, 1923) Á. Löve, 1984 (genome J(b) or E(b), 2n = 14), and Pseudoroegneria spicata (Pursh, 1814) Á. Löve, 1980 (genome St, 2n = 14) was used to characterize the mitotic metaphase chromosomes of a synthetic hybrid of Thinopyrum intermedium (Host, 1805) Barkworth & D.R. Dewey, 1985 and Thinopyrum ponticum (Podpěra, 1902) Z.-W. Liu et R.-C.Wang, 1993 named "Agropyron glael" and produced by N.V. Tsitsin in the former Soviet Union. The mcGISH pattern of this synthetic hybrid was compared to its parental wheatgrass species. Hexaploid Thinopyrum intermedium contained 19 J, 9 J(St) and 14 St chromosomes. The three analysed Thinopyrum ponticum accessions had different chromosome compositions: 43 J + 27 J(St) (PI531737), 40 J + 30 J(St) (VIR-44486) and 38 J + 32 J(St) (D-3494). The synthetic hybrid carried 18 J, 28 J(St) and 8 St chromosomes, including one pair of J-St translocation and/or decreased fluorescent intensity, resulting in unique hybridization patterns. Wheat line Mv9kr1 was crossed with the Thinopyrum intermedium × Thinopyrum ponticum synthetic hybrid in Hungary in order to transfer its advantageous agronomic traits (leaf rust and yellow rust resistance) into wheat. The chromosome composition of a wheat/A.glael F1 hybrid was 21 wheat + 28 wheatgrass (11 J + 14 J(St)+ 3 S). In the present study, mcGISH involving the simultaneous use of St and J genomic DNA as probes provided information about the type of Thinopyrum chromosomes in a Thinopyrum intermedium/Thinopyrum ponticum synthetic hybrid called A. glael.

Identification of ecotype-specific marker genes for categorization of beer-spoiling Lactobacillus brevis.

PubMed

Behr, Jürgen; Geissler, Andreas J; Preissler, Patrick; Ehrenreich, Armin; Angelov, Angel; Vogel, Rudi F

2015-10-01

The tolerance to hop compounds, which is mainly associated with inhibition of bacterial growth in beer, is a multi-factorial trait. Any approaches to predict the physiological differences between beer-spoiling and non-spoiling strains on the basis of a single marker gene are limited. We identified ecotype-specific genes related to the ability to grow in Pilsner beer via comparative genome sequencing. The genome sequences of four different strains of Lactobacillus brevis were compared, including newly established genomes of two highly hop tolerant beer isolates, one strain isolated from faeces and one published genome of a silage isolate. Gene fragments exclusively occurring in beer-spoiling strains as well as sequences only occurring in non-spoiling strains were identified. Comparative genomic arrays were established and hybridized with a set of L. brevis strains, which are characterized by their ability to spoil beer. As result, a set of 33 and 4 oligonucleotide probes could be established specifically detecting beer-spoilers and non-spoilers, respectively. The detection of more than one of these marker sequences according to a genetic barcode enables scoring of L. brevis for their beer-spoiling potential and can thus assist in risk evaluation in brewing industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

P53 oncosuppressor influences selection of genomic imbalances in response to ionizing radiations in human osteosarcoma cell line SAOS-2.

PubMed

Zuffa, Elisa; Mancini, Manuela; Brusa, Gianluca; Pagnotta, Eleonora; Hattinger, Claudia Maria; Serra, Massimo; Remondini, Daniel; Castellani, Gastone; Corrado, Patrizia; Barbieri, Enza; Santucci, Maria Alessandra

2008-07-01

To investigate the impact of TP53 (tumor protein 53, p53) on genomic stability of osteosarcoma (OS). In first instance, we expressed in OS cell line SAOS-2 (lacking p53) a wild type (wt) p53 construct, whose protein undergoes nuclear import and activation in response to ionizing radiations (IR). Thereafter, we investigated genomic imbalances (amplifications and deletions at genes or DNA regions most frequently altered in human cancers) associated with radio-resistance relative to p53 expression by mean of an array-based comparative genomic hybridization (aCGH) strategy. Finally we investigated a putative marker of radio-induced oxidative stress, a 4,977 bp deletion at mitochondrial (mt) DNA usually referred to as 'common' deletion, by mean of a polimerase chain reaction (PCR) strategy. In radio-resistant subclones generated from wt p53-transfected SAOS-2 cells DNA deletions were remarkably reduced and the accumulation of 'common' deletion at mtDNA (that may let the persistence of oxidative damage by precluding detoxification from reactive oxygen species [ROS]) completely abrogated. The results of our study confirm that wt p53 has a role in protection of OS cell DNA integrity. Multiple mechanisms involved in p53 safeguard of genomic integrity and prevention of deletion outcome are discussed.

Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C.

PubMed

Smukowski Heil, Caiti; Burton, Joshua N; Liachko, Ivan; Friedrich, Anne; Hanson, Noah A; Morris, Cody L; Schacherer, Joseph; Shendure, Jay; Thomas, James H; Dunham, Maitreya J

2018-01-01

Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi-C to the assembly of genomes from mixed populations. Here, we show the method's application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de-convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi-C-based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

The hybrid energy spectrum of Telescope Array's Middle Drum Detector and surface array

NASA Astrophysics Data System (ADS)

Abbasi, R. U.; Abe, M.; Abu-Zayyad, T.; Allen, M. G.; Anderson, R.; Azuma, R.; Barcikowski, E.; Belz, J. W.; Bergman, D. R.; Blake, S. A.; Cady, R.; Chae, M. J.; Cheon, B. G.; Chiba, J.; Chikawa, M.; Cho, W. R.; Fujii, T.; Fukushima, M.; Goto, T.; Hanlon, W.; Hayashi, Y.; Hayashida, N.; Hibino, K.; Honda, K.; Ikeda, D.; Inoue, N.; Ishii, T.; Ishimori, R.; Ito, H.; Ivanov, D.; Jui, C. C. H.; Kadota, K.; Kakimoto, F.; Kalashev, O.; Kasahara, K.; Kawai, H.; Kawakami, S.; Kawana, S.; Kawata, K.; Kido, E.; Kim, H. B.; Kim, J. H.; Kim, J. H.; Kitamura, S.; Kitamura, Y.; Kuzmin, V.; Kwon, Y. J.; Lan, J.; Lim, S. I.; Lundquist, J. P.; Machida, K.; Martens, K.; Matsuda, T.; Matsuyama, T.; Matthews, J. N.; Minamino, M.; Mukai, K.; Myers, I.; Nagasawa, K.; Nagataki, S.; Nakamura, T.; Nonaka, T.; Nozato, A.; Ogio, S.; Ogura, J.; Ohnishi, M.; Ohoka, H.; Oki, K.; Okuda, T.; Ono, M.; Oshima, A.; Ozawa, S.; Park, I. H.; Pshirkov, M. S.; Rodriguez, D. C.; Rubtsov, G.; Ryu, D.; Sagawa, H.; Sakurai, N.; Sampson, A. L.; Scott, L. M.; Shah, P. D.; Shibata, F.; Shibata, T.; Shimodaira, H.; Shin, B. K.; Shin, H. S.; Smith, J. D.; Sokolsky, P.; Springer, R. W.; Stokes, B. T.; Stratton, S. R.; Stroman, T. A.; Suzawa, T.; Takamura, M.; Takeda, M.; Takeishi, R.; Taketa, A.; Takita, M.; Tameda, Y.; Tanaka, H.; Tanaka, K.; Tanaka, M.; Thomas, S. B.; Thomson, G. B.; Tinyakov, P.; Tkachev, I.; Tokuno, H.; Tomida, T.; Troitsky, S.; Tsunesada, Y.; Tsutsumi, K.; Uchihori, Y.; Udo, S.; Urban, F.; Vasiloff, G.; Wong, T.; Yamane, R.; Yamaoka, H.; Yamazaki, K.; Yang, J.; Yashiro, K.; Yoneda, Y.; Yoshida, S.; Yoshii, H.; Zollinger, R.; Zundel, Z.

2015-08-01

The Telescope Array experiment studies ultra high energy cosmic rays using a hybrid detector. Fluorescence telescopes measure the longitudinal development of the extensive air shower generated when a primary cosmic ray particle interacts with the atmosphere. Meanwhile, scintillator detectors measure the lateral distribution of secondary shower particles that hit the ground. The Middle Drum (MD) fluorescence telescope station consists of 14 telescopes from the High Resolution Fly's Eye (HiRes) experiment, providing a direct link back to the HiRes measurements. Using the scintillator detector data in conjunction with the telescope data improves the geometrical reconstruction of the showers significantly, and hence, provides a more accurate reconstruction of the energy of the primary particle. The Middle Drum hybrid spectrum is presented and compared to that measured by the Middle Drum station in monocular mode. Further, the hybrid data establishes a link between the Middle Drum data and the surface array. A comparison between the Middle Drum hybrid energy spectrum and scintillator Surface Detector (SD) spectrum is also shown.

Radiation hybrid maps of D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes

USDA-ARS?s Scientific Manuscript database

The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high-resolution genome maps saturated with ordered markers to assist in anchoring and orienting BAC contigs/ sequence scaffolds for whole genome sequence assembly. Radiation hybrid (RH) mapping has proven to be an e...

Genomic patterns of introgression in rainbow and westslope cutthroat trout illuminated by overlapping paired-end RAD sequencing

USGS Publications Warehouse

Hohenlohe, Paul A.; Day, Mitch D.; Amish, Stephen J.; Miller, Michael R.; Kamps-Hughes, Nick; Boyer, Matthew C.; Muhlfeld, Clint C.; Allendorf, Fred W.; Johnson, Eric A.; Luikart, Gordon

2013-01-01

Rapid and inexpensive methods for genomewide single nucleotide polymorphism (SNP) discovery and genotyping are urgently needed for population management and conservation. In hybridized populations, genomic techniques that can identify and genotype thousands of species-diagnostic markers would allow precise estimates of population- and individual-level admixture as well as identification of 'super invasive' alleles, which show elevated rates of introgression above the genomewide background (likely due to natural selection). Techniques like restriction-site-associated DNA (RAD) sequencing can discover and genotype large numbers of SNPs, but they have been limited by the length of continuous sequence data they produce with Illumina short-read sequencing. We present a novel approach, overlapping paired-end RAD sequencing, to generate RAD contigs of >300–400 bp. These contigs provide sufficient flanking sequence for design of high-throughput SNP genotyping arrays and strict filtering to identify duplicate paralogous loci. We applied this approach in five populations of native westslope cutthroat trout that previously showed varying (low) levels of admixture from introduced rainbow trout (RBT). We produced 77 141 RAD contigs and used these data to filter and genotype 3180 previously identified species-diagnostic SNP loci. Our population-level and individual-level estimates of admixture were generally consistent with previous microsatellite-based estimates from the same individuals. However, we observed slightly lower admixture estimates from genomewide markers, which might result from natural selection against certain genome regions, different genomic locations for microsatellites vs. RAD-derived SNPs and/or sampling error from the small number of microsatellite loci (n = 7). We also identified candidate adaptive super invasive alleles from RBT that had excessively high admixture proportions in hybridized cutthroat trout populations.

Comparative structural analysis of Bru1 region homeologs in Saccharum spontaneum and S. officinarum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jisen; Sharma, Anupma; Yu, Qingyi

Here, sugarcane is a major sugar and biofuel crop, but genomic research and molecular breeding have lagged behind other major crops due to the complexity of auto-allopolyploid genomes. Sugarcane cultivars are frequently aneuploid with chromosome number ranging from 100 to 130, consisting of 70-80 % S. officinarum, 10-20 % S. spontaneum, and 10 % recombinants between these two species. Analysis of a genomic region in the progenitor autoploid genomes of sugarcane hybrid cultivars will reveal the nature and divergence of homologous chromosomes. As a result, to investigate the origin and evolution of haplotypes in the Bru1 genomic regions in sugarcanemore » cultivars, we identified two BAC clones from S. spontaneum and four from S. officinarum and compared to seven haplotype sequences from sugarcane hybrid R570. The results clarified the origin of seven homologous haplotypes in R570, four haplotypes originated from S. officinarum, two from S. spontaneum and one recombinant.. Retrotransposon insertions and sequences variations among the homologous haplotypes sequence divergence ranged from 18.2 % to 60.5 % with an average of 33. 7 %. Gene content and gene structure were relatively well conserved among the homologous haplotypes. Exon splitting occurred in haplotypes of the hybrid genome but not in its progenitor genomes. Tajima's D analysis revealed that S. spontaneum hapotypes in the Bru1 genomic regions were under strong directional selection. Numerous inversions, deletions, insertions and translocations were found between haplotypes within each genome. In conclusion, this is the first comparison among haplotypes of a modern sugarcane hybrid and its two progenitors. Tajima's D results emphasized the crucial role of this fungal disease resistance gene for enhancing the fitness of this species and indicating that the brown rust resistance gene in R570 is from S. spontaneum. Species-specific InDel, sequences similarity and phylogenetic analysis of homologous genes can be used for identifying the origin of S. spontaneum and S. officinarum haplotype in Saccharum hybrids. Comparison of exon splitting among the homologous haplotypes suggested that the genome rearrangements in Saccharum hybrids S. officinarum would be sufficient for proper genome assembly of this autopolyploid genome. Retrotransposon insertions and sequences variations among the homologous haplotypes sequence divergence may allow sequencing and assembling the autopolyploid Saccharum genomes and the auto-allopolyploid hybrid genomes using whole genome shotgun sequencing.« less

Comparative structural analysis of Bru1 region homeologs in Saccharum spontaneum and S. officinarum

DOE PAGES

Zhang, Jisen; Sharma, Anupma; Yu, Qingyi; ...

2016-06-10

Here, sugarcane is a major sugar and biofuel crop, but genomic research and molecular breeding have lagged behind other major crops due to the complexity of auto-allopolyploid genomes. Sugarcane cultivars are frequently aneuploid with chromosome number ranging from 100 to 130, consisting of 70-80 % S. officinarum, 10-20 % S. spontaneum, and 10 % recombinants between these two species. Analysis of a genomic region in the progenitor autoploid genomes of sugarcane hybrid cultivars will reveal the nature and divergence of homologous chromosomes. As a result, to investigate the origin and evolution of haplotypes in the Bru1 genomic regions in sugarcanemore » cultivars, we identified two BAC clones from S. spontaneum and four from S. officinarum and compared to seven haplotype sequences from sugarcane hybrid R570. The results clarified the origin of seven homologous haplotypes in R570, four haplotypes originated from S. officinarum, two from S. spontaneum and one recombinant.. Retrotransposon insertions and sequences variations among the homologous haplotypes sequence divergence ranged from 18.2 % to 60.5 % with an average of 33. 7 %. Gene content and gene structure were relatively well conserved among the homologous haplotypes. Exon splitting occurred in haplotypes of the hybrid genome but not in its progenitor genomes. Tajima's D analysis revealed that S. spontaneum hapotypes in the Bru1 genomic regions were under strong directional selection. Numerous inversions, deletions, insertions and translocations were found between haplotypes within each genome. In conclusion, this is the first comparison among haplotypes of a modern sugarcane hybrid and its two progenitors. Tajima's D results emphasized the crucial role of this fungal disease resistance gene for enhancing the fitness of this species and indicating that the brown rust resistance gene in R570 is from S. spontaneum. Species-specific InDel, sequences similarity and phylogenetic analysis of homologous genes can be used for identifying the origin of S. spontaneum and S. officinarum haplotype in Saccharum hybrids. Comparison of exon splitting among the homologous haplotypes suggested that the genome rearrangements in Saccharum hybrids S. officinarum would be sufficient for proper genome assembly of this autopolyploid genome. Retrotransposon insertions and sequences variations among the homologous haplotypes sequence divergence may allow sequencing and assembling the autopolyploid Saccharum genomes and the auto-allopolyploid hybrid genomes using whole genome shotgun sequencing.« less

EML4-ALK translocation in both metachronous second primary lung sarcomatoid carcinoma and lung adenocarcinoma: a case report.

PubMed

Alì, Greta; Proietti, Agnese; Niccoli, Cristina; Pelliccioni, Serena; Borrelli, Nicla; Giannini, Riccardo; Lupi, Cristiana; Valetto, Angelo; Bertini, Veronica; Lucchi, Marco; Mussi, Alfredo; Fontanini, Gabriella

2013-08-01

The EML4-ALK gene translocation was described in a non small cell lung cancer (NSCLC) subset, with a potent oncogenic activity. It represents one of the newest molecular targets in NSCLC. We report on the case of a metachronous second primary lung sarcomatoid carcinoma after resection of lung adenocarcinoma both with ALK translocation, in a non-smoking patient. EML4-ALK rearrangement was detected with immunohistochemistry and confirmed with fluorescent in situ hybridization (FISH). To assess the clonal relationship between the two tumors, both adenocarcinoma and sarcomatoid carcinoma were analyzed by array comparative genomic hybridization (aCGH). We observed different genomic profiles suggesting that the tumors arose independently and were thus multiple primaries. To the best of our knowledge, this is the first report concerning the presence of the EML4-ALK fusion gene in a sarcomatoid carcinoma of the lung. Crizotinib, the ALK tyrosine kinase inhibitor, is highly effective in ALK-rearranged NSCLC; therefore, it may be imperative to identify all NSCLC that harbor ALK translocations in the near future. Starting from our evidence, tumors with sarcomatoid histology may need to be screened for the presence of EML4-ALK rearrangement. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Partial Tetrasomy of Chromosome 22q11.1 Resulting from a Supernumerary Isodicentric Marker Chromosome in a Boy with Cat-eye Syndrome

PubMed Central

Kim, Jun Bum; Pai, Ki Soo; Yun, Jun-No; Park, Sang-Jin

2010-01-01

The 22q11 region has been implicated in chromosomal rearrangements that result in altered gene dosage, leading to three different congenital malformation syndromes: DiGeorge syndrome, cat-eye syndrome (CES), and der(22) syndrome. Although DiGeorge syndrome is a common genomic disorder on 22q11, CES is quite rare, and there has been no report of Korean CES cases with molecular cytogenetic confirmation. In this study, we present the phenotypic and genetic characteristics of a 3-month-old boy with CES. Clinical findings included micropthalmia, multiple colobomata, and renal and genital anomalies. Cytogenetic analyses showed the presence of a supernumerary marker chromosome, which was identified as a bisatellited and isodicentric chromosome derived from an acrocentric chromosome. The results of array comparative genomic hybridization and fluorescence in situ hybridization studies confirmed the karyotype as 47,XY,+mar.ish idic(22)(q11.1) (D22S43+).arr 22q11.1(15,500,000-15,900,000)x4, resulting in a partial tetrasomy of 22q11.1. To the best of our knowledge, this is the first report in Korea of CES confirmed by cytogenetic and molecular cytogenetic analyses. PMID:21165297

Partial tetrasomy of chromosome 22q11.1 resulting from a supernumerary isodicentric marker chromosome in a boy with cat-eye syndrome.

PubMed

Ko, Jung Min; Kim, Jun Bum; Pai, Ki Soo; Yun, Jun-No; Park, Sang-Jin

2010-12-01

The 22q11 region has been implicated in chromosomal rearrangements that result in altered gene dosage, leading to three different congenital malformation syndromes: DiGeorge syndrome, cat-eye syndrome (CES), and der(22) syndrome. Although DiGeorge syndrome is a common genomic disorder on 22q11, CES is quite rare, and there has been no report of Korean CES cases with molecular cytogenetic confirmation. In this study, we present the phenotypic and genetic characteristics of a 3-month-old boy with CES. Clinical findings included micropthalmia, multiple colobomata, and renal and genital anomalies. Cytogenetic analyses showed the presence of a supernumerary marker chromosome, which was identified as a bisatellited and isodicentric chromosome derived from an acrocentric chromosome. The results of array comparative genomic hybridization and fluorescence in situ hybridization studies confirmed the karyotype as 47,XY,+mar.ish idic(22)(q11.1) (D22S43+).arr 22q11.1(15,500,000-15,900,000)x4, resulting in a partial tetrasomy of 22q11.1. To the best of our knowledge, this is the first report in Korea of CES confirmed by cytogenetic and molecular cytogenetic analyses.

Development of Genetic Markers in Eucalyptus Species by Target Enrichment and Exome Sequencing

PubMed Central

Dasgupta, Modhumita Ghosh; Dharanishanthi, Veeramuthu; Agarwal, Ishangi; Krutovsky, Konstantin V.

2015-01-01

The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs) and insertions/ deletions (InDels) were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family– based QTL and association analysis in Eucalyptus. PMID:25602379

Identification of novel deletions of 15q11q13 in Angelman syndrome by array-CGH: molecular characterization and genotype-phenotype correlations.

PubMed

Sahoo, Trilochan; Bacino, Carlos A; German, Jennifer R; Shaw, Chad A; Bird, Lynne M; Kimonis, Virginia; Anselm, Irinia; Waisbren, Susan; Beaudet, Arthur L; Peters, Sarika U

2007-09-01

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by mental retardation, absent speech, ataxia, and a happy disposition. Deletions of the 15q11q13 region are found in approximately 70% of AS patients. The deletions are sub-classified into class I and class II based on their sizes of approximately 6.8 and approximately 6.0, respectively, with two different proximal breakpoints and a common distal breakpoint. Utilizing a chromosome 15-specific comparative genomic hybridization genomic microarray (array-CGH), we have identified, determined the deletion sizes, and mapped the breakpoints in a cohort of 44 cases, to relate those breakpoints to the genomic architecture and derive more precise genotype-phenotype correlations. Interestingly four patients of the 44 studied (9.1%) had novel and unusually large deletions, and are reported here. This is the first report of very large deletions of 15q11q13 resulting in AS; the largest deletion being >10.6 Mb. These novel deletions involve three different distal breakpoints, two of which have been earlier shown to be involved in the generation of isodicentric 15q chromosomes (idic15). Additionally, precise determination of the deletion breakpoints reveals the presence of directly oriented low-copy repeats (LCRs) flanking the recurrent and novel breakpoints. The LCRs are adequate in size, orientation, and homology to enable abnormal recombination events leading to deletions and duplications. This genomic organization provides evidence for a common mechanism for the generation of both common and rare deletion types. Larger deletions result in a loss of several genes outside the common Angelman syndrome-Prader-Willi syndrome (AS-PWS) critical interval, and a more severe phenotype.

Arrays of probes for positional sequencing by hybridization

DOEpatents

Cantor, Charles R [Boston, MA; Prezetakiewiczr, Marek [East Boston, MA; Smith, Cassandra L [Boston, MA; Sano, Takeshi [Waltham, MA

2008-01-15

This invention is directed to methods and reagents useful for sequencing nucleic acid targets utilizing sequencing by hybridization technology comprising probes, arrays of probes and methods whereby sequence information is obtained rapidly and efficiently in discrete packages. That information can be used for the detection, identification, purification and complete or partial sequencing of a particular target nucleic acid. When coupled with a ligation step, these methods can be performed under a single set of hybridization conditions. The invention also relates to the replication of probe arrays and methods for making and replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.

Genome size and invasiveness traits in the hybrid meadow knapweed complex (Centaurea x moncktonii) in eastern North America

USDA-ARS?s Scientific Manuscript database

Hybridization and genomic admixture between divergent populations or species may be an important driver of plant invasiveness. Recent studies have emphasized the critical role that reductions in genome size may play in facilitating the rapid evolution of invasiveness, and small genome size has been ...

Segregation distortion causes large-scale differences between male and female genomes in hybrid ants.

PubMed

Kulmuni, Jonna; Seifert, Bernhard; Pamilo, Pekka

2010-04-20

Hybridization in isolated populations can lead either to hybrid breakdown and extinction or in some cases to speciation. The basis of hybrid breakdown lies in genetic incompatibilities between diverged genomes. In social Hymenoptera, the consequences of hybridization can differ from those in other animals because of haplodiploidy and sociality. Selection pressures differ between sexes because males are haploid and females are diploid. Furthermore, sociality and group living may allow survival of hybrid genotypes. We show that hybridization in Formica ants has resulted in a stable situation in which the males form two highly divergent gene pools whereas all the females are hybrids. This causes an exceptional situation with large-scale differences between male and female genomes. The genotype differences indicate strong transmission ratio distortion depending on offspring sex, whereby the mother transmits some alleles exclusively to her daughters and other alleles exclusively to her sons. The genetic differences between the sexes and the apparent lack of multilocus hybrid genotypes in males can be explained by recessive incompatibilities which cause the elimination of hybrid males because of their haploid genome. Alternatively, differentiation between sexes could be created by prezygotic segregation into male-forming and female-forming gametes in diploid females. Differentiation between sexes is stable and maintained throughout generations. The present study shows a unique outcome of hybridization and demonstrates that hybridization has the potential of generating evolutionary novelties in animals.

Distinguishing between incomplete lineage sorting and genomic introgressions: complete fixation of allospecific mitochondrial DNA in a sexually reproducing fish (Cobitis; Teleostei), despite clonal reproduction of hybrids.

PubMed

Choleva, Lukas; Musilova, Zuzana; Kohoutova-Sediva, Alena; Paces, Jan; Rab, Petr; Janko, Karel

2014-01-01

Distinguishing between hybrid introgression and incomplete lineage sorting causing incongruence among gene trees in that they exhibit topological differences requires application of statistical approaches that are based on biologically relevant models. Such study is especially challenging in hybrid systems, where usual vectors mediating interspecific gene transfers--hybrids with Mendelian heredity--are absent or unknown. Here we study a complex of hybridizing species, which are known to produce clonal hybrids, to discover how one of the species, Cobitis tanaitica, has achieved a pattern of mito-nuclear mosaic genome over the whole geographic range. We appplied three distinct methods, including the method using solely the information on gene tree topologies, and found that the contrasting mito-nuclear signal might not have resulted from the retention of ancestral polymorphism. Instead, we found two signs of hybridization events related to C. tanaitica; one concerning nuclear gene flow and the other suggested mitochondrial capture. Interestingly, clonal inheritance (gynogenesis) of contemporary hybrids prevents genomic introgressions and non-clonal hybrids are either absent or too rare to be detected among European Cobitis. Our analyses therefore suggest that introgressive hybridizations are rather old episodes, mediated by previously existing hybrids whose inheritance was not entirely clonal. Cobitis complex thus supports the view that the type of resulting hybrids depends on a level of genomic divergence between sexual species.

Autism-specific copy number variants further implicate the phosphatidylinositol signaling pathway and the glutamatergic synapse in the etiology of the disorder.

PubMed

Cuscó, Ivon; Medrano, Andrés; Gener, Blanca; Vilardell, Mireia; Gallastegui, Fátima; Villa, Olaya; González, Eva; Rodríguez-Santiago, Benjamín; Vilella, Elisabet; Del Campo, Miguel; Pérez-Jurado, Luis A

2009-05-15

Autism spectrum disorders (ASDs) constitute a group of severe neurodevelopmental conditions with complex multifactorial etiology. In order to explore the hypothesis that submicroscopic genomic rearrangements underlie some ASD cases, we have analyzed 96 Spanish patients with idiopathic ASD after extensive clinical and laboratory screening, by array comparative genomic hybridization (aCGH) using a homemade bacterial artificial chromosome (BAC) array. Only 13 of the 238 detected copy number alterations, ranging in size from 89 kb to 2.4 Mb, were present specifically in the autistic population (12 out of 96 individuals, 12.5%). Following validation by additional molecular techniques, we have characterized these novel candidate regions containing 24 different genes including alterations in two previously reported regions of chromosome 7 associated with the ASD phenotype. Some of the genes located in ASD-specific copy number variants act in common pathways, most notably the phosphatidylinositol signaling and the glutamatergic synapse, both known to be affected in several genetic syndromes related with autism and previously associated with ASD. Our work supports the idea that the functional alteration of genes in related neuronal networks is involved in the etiology of the ASD phenotype and confirms a significant diagnostic yield for aCGH, which should probably be included in the diagnostic workup of idiopathic ASD.

A challenge to the striking genotypic heterogeneity of retinitis pigmentosa: a better understanding of the pathophysiology using the newest genetic strategies

PubMed Central

Sorrentino, F S; Gallenga, C E; Bonifazzi, C; Perri, P

2016-01-01

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by a complex association between tremendous genotypic multiplicity and great phenotypic heterogeneity. The severity of the clinical manifestation depends on penetrance and expressivity of the disease-gene. Also, various interactions between gene expression and environmental factors have been hypothesized. More than 250 genes with ~4500 causative mutations have been reported to be involved in different RP-related mechanisms. Nowadays, not more than the 50% of RPs are attributable to identified genes, whereas the rest of molecular defects are still undetectable, especially in populations where few genetic screenings have been performed. Therefore, new genetic strategies can be a remarkably useful tool to aid clinical diagnosis, potentially modifying treatment options, and family counseling. Genome-wide analytical techniques (array comparative genomic hybridization and single-nucleotide polymorphism genotyping) and DNA sequencing strategies (arrayed primer extension, Sanger sequencing, and ultra high-throughput sequencing) are successfully used to early make molecular diagnosis detecting single or multiple mutations in the huge heterogeneity of RPs. To date, further research needs to be carried out to better investigate the genotype/phenotype correlation, putting together genetic and clinical findings to provide detailed information concerning the risk of RP development and novel effective treatments. PMID:27564722

Application of Whole Genome Expression Analysis to Assess Bacterial Responses to Environmental Conditions

NASA Astrophysics Data System (ADS)

Vukanti, R. V.; Mintz, E. M.; Leff, L. G.

2005-05-01

Bacterial responses to environmental signals are multifactorial and are coupled to changes in gene expression. An understanding of bacterial responses to environmental conditions is possible using microarray expression analysis. In this study, the utility of microarrays for examining changes in gene expression in Escherichia coli under different environmental conditions was assessed. RNA was isolated, hybridized to Affymetrix E. coli Genome 2.0 chips and analyzed using Affymetrix GCOS and Genespring software. Major limiting factors were obtaining enough quality RNA (107-108 cells to get 10μg RNA)and accounting for differences in growth rates under different conditions. Stabilization of RNA prior to isolation and taking extreme precautions while handling RNA were crucial. In addition, use of this method in ecological studies is limited by availability and cost of commercial arrays; choice of primers for cDNA synthesis, reproducibility, complexity of results generated and need to validate findings. This method may be more widely applicable with the development of better approaches for RNA recovery from environmental samples and increased number of available strain-specific arrays. Diligent experimental design and verification of results with real-time PCR or northern blots is needed. Overall, there is a great potential for use of this technology to discover mechanisms underlying organisms' responses to environmental conditions.

Identification of copy number variants in horses.

PubMed

Doan, Ryan; Cohen, Noah; Harrington, Jessica; Veazey, Kylee; Veazy, Kylee; Juras, Rytis; Cothran, Gus; McCue, Molly E; Skow, Loren; Dindot, Scott V

2012-05-01

Copy number variants (CNVs) represent a substantial source of genetic variation in mammals. However, the occurrence of CNVs in horses and their subsequent impact on phenotypic variation is unknown. We performed a study to identify CNVs in 16 horses representing 15 distinct breeds (Equus caballus) and an individual gray donkey (Equus asinus) using a whole-exome tiling array and the array comparative genomic hybridization methodology. We identified 2368 CNVs ranging in size from 197 bp to 3.5 Mb. Merging identical CNVs from each animal yielded 775 CNV regions (CNVRs), involving 1707 protein- and RNA-coding genes. The number of CNVs per animal ranged from 55 to 347, with median and mean sizes of CNVs of 5.3 kb and 99.4 kb, respectively. Approximately 6% of the genes investigated were affected by a CNV. Biological process enrichment analysis indicated CNVs primarily affected genes involved in sensory perception, signal transduction, and metabolism. CNVs also were identified in genes regulating blood group antigens, coat color, fecundity, lactation, keratin formation, neuronal homeostasis, and height in other species. Collectively, these data are the first report of copy number variation in horses and suggest that CNVs are common in the horse genome and may modulate biological processes underlying different traits observed among horses and horse breeds.

High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination

PubMed Central

2013-01-01

Background The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies. Results In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable. Conclusion This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome. PMID:23597128

A functional hybrid memristor crossbar-array/CMOS system for data storage and neuromorphic applications.

PubMed

Kim, Kuk-Hwan; Gaba, Siddharth; Wheeler, Dana; Cruz-Albrecht, Jose M; Hussain, Tahir; Srinivasa, Narayan; Lu, Wei

2012-01-11

Crossbar arrays based on two-terminal resistive switches have been proposed as a leading candidate for future memory and logic applications. Here we demonstrate a high-density, fully operational hybrid crossbar/CMOS system composed of a transistor- and diode-less memristor crossbar array vertically integrated on top of a CMOS chip by taking advantage of the intrinsic nonlinear characteristics of the memristor element. The hybrid crossbar/CMOS system can reliably store complex binary and multilevel 1600 pixel bitmap images using a new programming scheme. © 2011 American Chemical Society

Loss of Heterozygosity Drives Adaptation in Hybrid Yeast.

PubMed

Smukowski Heil, Caiti S; DeSevo, Christopher G; Pai, Dave A; Tucker, Cheryl M; Hoang, Margaret L; Dunham, Maitreya J

2017-07-01

Hybridization is often considered maladaptive, but sometimes hybrids can invade new ecological niches and adapt to novel or stressful environments better than their parents. The genomic changes that occur following hybridization that facilitate genome resolution and/or adaptation are not well understood. Here, we examine hybrid genome evolution using experimental evolution of de novo interspecific hybrid yeast Saccharomyces cerevisiae × Saccharomyces uvarum and their parentals. We evolved these strains in nutrient-limited conditions for hundreds of generations and sequenced the resulting cultures identifying numerous point mutations, copy number changes, and loss of heterozygosity (LOH) events, including species-biased amplification of nutrient transporters. We focused on a particularly interesting example, in which we saw repeated LOH at the high-affinity phosphate transporter gene PHO84 in both intra- and interspecific hybrids. Using allele replacement methods, we tested the fitness of different alleles in hybrid and S. cerevisiae strain backgrounds and found that the LOH is indeed the result of selection on one allele over the other in both S. cerevisiae and the hybrids. This is an example where hybrid genome resolution is driven by positive selection on existing heterozygosity and demonstrates that even infrequent outcrossing may have lasting impacts on adaptation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The More the Merrier: Recent Hybridization and Polyploidy in Cardamine[W

PubMed Central

Mandáková, Terezie; Kovařík, Aleš; Zozomová-Lihová, Judita; Shimizu-Inatsugi, Rie; Shimizu, Kentaro K.; Mummenhoff, Klaus; Marhold, Karol; Lysak, Martin A.

2013-01-01

This article describes the use of cytogenomic and molecular approaches to explore the origin and evolution of Cardamine schulzii, a textbook example of a recent allopolyploid, in its ∼110-year history of human-induced hybridization and allopolyploidy in the Swiss Alps. Triploids are typically viewed as bridges between diploids and tetraploids but rarely as parental genomes of high-level hybrids and polyploids. The genome of the triploid semifertile hybrid Cardamine × insueta (2n = 24, RRA) was shown to combine the parental genomes of two diploid (2n = 2x = 16) species, Cardamine amara (AA) and Cardamine rivularis (RR). These parental genomes have remained structurally stable within the triploid genome over the >100 years since its origin. Furthermore, we provide compelling evidence that the alleged recent polyploid C. schulzii is not an autohexaploid derivative of C. × insueta. Instead, at least two hybridization events involving C. × insueta and the hypotetraploid Cardamine pratensis (PPPP, 2n = 4x−2 = 30) have resulted in the origin of the trigenomic hypopentaploid (2n = 5x−2 = 38, PPRRA) and hypohexaploid (2n = 6x−2 = 46, PPPPRA). These data show that the semifertile triploid hybrid can promote a merger of three different genomes and demonstrate how important it is to reexamine the routinely repeated textbook examples using modern techniques. PMID:24082009

Development and Use of Integrated Microarray-Based Genomic Technologies for Assessing Microbial Community Composition and Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, J.; Wu, L.; Gentry, T.

2006-04-05

To effectively monitor microbial populations involved in various important processes, a 50-mer-based oligonucleotide microarray was developed based on known genes and pathways involved in: biodegradation, metal resistance and reduction, denitrification, nitrification, nitrogen fixation, methane oxidation, methanogenesis, carbon polymer decomposition, and sulfate reduction. This array contains approximately 2000 unique and group-specific probes with <85% similarity to their non-target sequences. Based on artificial probes, our results showed that at hybridization conditions of 50 C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appearedmore » to be specific to their corresponding target genes. Detection limits were about 5-10ng genomic DNA in the absence of background DNA, and 50-100ng ({approx}1.3{sup o} 10{sup 7} cells) in the presence background DNA. Strong linear relationships between signal intensity and target DNA and RNA concentration were observed (r{sup 2} = 0.95-0.99). Application of this microarray to naphthalene-amended enrichments and soil microcosms demonstrated that composition of the microflora varied depending on incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in enrichments, the genes involved in naphthalene degradation from Gram-negative microorganisms such as Ralstonia, Comamonas, and Burkholderia were most abundant in the soil microcosms (as well as those for polyaromatic hydrocarbon and nitrotoluene degradation). Although naphthalene degradation is widely known and studied in Pseudomonas, Pseudomonas genes were not detected in either system. Real-time PCR analysis of 4 representative genes was consistent with microarray-based quantification (r{sup 2} = 0.95). Currently, we are also applying this microarray to the study of several different microbial communities and processes at the NABIR-FRC in Oak Ridge, TN. One project involves the monitoring of the development and dynamics of the microbial community of a fluidized bed reactor (FBR) used for reducing nitrate and the other project monitors microbial community responses to stimulation of uranium reducing populations via ethanol donor additions in situ and in a model system. Additionally, we are developing novel strategies for increasing microarray hybridization sensitivity. Finally, great improvements to our methods of probe design were made by the development of a new computer program, CommOligo. CommOligo designs unique and group-specific oligo probes for whole-genomes, metagenomes, and groups of environmental sequences and uses a new global alignment algorithm to design single or multiple probes for each gene or group. We are now using this program to design a more comprehensive functional gene array for environmental studies. Overall, our results indicate that the 50mer-based microarray technology has potential as a specific and quantitative tool to reveal the composition of microbial communities and their dynamics important to processes within contaminated environments.« less

A comparative analysis of chromosome pairing at metaphase I in interspecific hybrids between durum wheat (Triticum turgidum L.) and the most widespread Aegilops species.

PubMed

Cifuentes, M; Garcia-Agüero, V; Benavente, E

2010-07-01

Homoeologous metaphase I (MI) associations in hybrids between durum wheat and its wild allotetraploid relatives Aegilops neglecta, Ae. triuncialis and Ae. ventricosa have been characterized by a genomic in situ hybridization procedure that allows simultaneous discrimination of A, B and wild species genomes. Earlier results in equivalent hybrids with the wild species Ae. cylindrica and Ae. geniculata have also been considered to comparatively assay the MI pairing pattern of the durum wheat x Aegilops interspecific combinations more likely to occur in nature. The general picture can be drawn as follows. A and B wheat genomes pair with each other less than the 2 wild constituent genomes do in any of the hybrid combinations examined. Interspecific wheat-wild associations account for 60-70% of total MI pairing in all hybrids, except in that derived from Ae. triuncialis, but the A genome is always the wheat partner most frequently involved in MI pairing with the wild homoeologues. Hybrids with Ae. cylindrica, Ae. geniculata and Ae. ventricosa showed similar reduced levels of MI association and virtually identical MI pairing patterns. However, certain recurrent differences were found when the pattern of homoeologous pairing of hybrids from either Ae. triuncialis or Ae. neglecta was contrasted to that observed in the other durum wheat hybrid combinations. In the former case, a remarkable preferential pairing between the wild species constituent genomes U(t) and C(t) seems to be the reason, whereas a general promotion of homoeologous pairing, qualitatively similar to that observed under the effect of the ph1c mutation, appears to occur in the hybrid with Ae. neglecta. It is further discussed whether the results reported here can be extrapolated to the corresponding bread wheat hybrid combinations. Copyright 2010 S. Karger AG, Basel.

Different selective pressures lead to different genomic outcomes as newly-formed hybrid yeasts evolve.

PubMed

Piotrowski, Jeff S; Nagarajan, Saisubramanian; Kroll, Evgueny; Stanbery, Alison; Chiotti, Kami E; Kruckeberg, Arthur L; Dunn, Barbara; Sherlock, Gavin; Rosenzweig, Frank

2012-04-02

Interspecific hybridization occurs in every eukaryotic kingdom. While hybrid progeny are frequently at a selective disadvantage, in some instances their increased genome size and complexity may result in greater stress resistance than their ancestors, which can be adaptively advantageous at the edges of their ancestors' ranges. While this phenomenon has been repeatedly documented in the field, the response of hybrid populations to long-term selection has not often been explored in the lab. To fill this knowledge gap we crossed the two most distantly related members of the Saccharomyces sensu stricto group, S. cerevisiae and S. uvarum, and established a mixed population of homoploid and aneuploid hybrids to study how different types of selection impact hybrid genome structure. As temperature was raised incrementally from 31°C to 46.5°C over 500 generations of continuous culture, selection favored loss of the S. uvarum genome, although the kinetics of genome loss differed among independent replicates. Temperature-selected isolates exhibited greater inherent and induced thermal tolerance than parental species and founding hybrids, and also exhibited ethanol resistance. In contrast, as exogenous ethanol was increased from 0% to 14% over 500 generations of continuous culture, selection favored euploid S. cerevisiae x S. uvarum hybrids. Ethanol-selected isolates were more ethanol tolerant than S. uvarum and one of the founding hybrids, but did not exhibit resistance to temperature stress. Relative to parental and founding hybrids, temperature-selected strains showed heritable differences in cell wall structure in the forms of increased resistance to zymolyase digestion and Micafungin, which targets cell wall biosynthesis. This is the first study to show experimentally that the genomic fate of newly-formed interspecific hybrids depends on the type of selection they encounter during the course of evolution, underscoring the importance of the ecological theatre in determining the outcome of the evolutionary play.

High-MTF hybrid ferroelectric IRFPA

NASA Astrophysics Data System (ADS)

Evans, Scott B.; Hayden, Terrence

1998-07-01

Low cost, uncooled hybrid infrared focal plane arrays (IRFPA's) are in full-scale production at Raytheon Systems Company (RSC), formerly Texas Instruments Defense Systems and Electronics Group. Detectors consist of reticulated ceramic barium strontium titanate (BST) arrays of 320 X 240 pixels on 48.5 micrometer pitch. The principal performance shortcoming of the hybrid arrays has been low MTF due to thermal crosstalk between pixels. In the past two years, significant improvements have been made to increase MTF making hybrids more competitive in performance with monolithic arrays. The improvements are (1) the reduction of the thickness of the IR absorbing layer electrode that maintains electrical continuity and increases thermal isolation between pixels, (2) reduction of the electrical crosstalk from the ROIC, and (3) development of a process to increase the thermal path-length between pixels called 'elevated optical coat.' This paper describes all three activities and their efficacy. Also discussed is the uncooled IRFPA production capability at RSC.

Multiple Origins of the Pathogenic Yeast Candida orthopsilosis by Separate Hybridizations between Two Parental Species.

PubMed

Schröder, Markus S; Martinez de San Vicente, Kontxi; Prandini, Tâmara H R; Hammel, Stephen; Higgins, Desmond G; Bagagli, Eduardo; Wolfe, Kenneth H; Butler, Geraldine

2016-11-01

Mating between different species produces hybrids that are usually asexual and stuck as diploids, but can also lead to the formation of new species. Here, we report the genome sequences of 27 isolates of the pathogenic yeast Candida orthopsilosis. We find that most isolates are diploid hybrids, products of mating between two unknown parental species (A and B) that are 5% divergent in sequence. Isolates vary greatly in the extent of homogenization between A and B, making their genomes a mosaic of highly heterozygous regions interspersed with homozygous regions. Separate phylogenetic analyses of SNPs in the A- and B-derived portions of the genome produces almost identical trees of the isolates with four major clades. However, the presence of two mutually exclusive genotype combinations at the mating type locus, and recombinant mitochondrial genomes diagnostic of inter-clade mating, shows that the species C. orthopsilosis does not have a single evolutionary origin but was created at least four times by separate interspecies hybridizations between parents A and B. Older hybrids have lost more heterozygosity. We also identify two isolates with homozygous genomes derived exclusively from parent A, which are pure non-hybrid strains. The parallel emergence of the same hybrid species from multiple independent hybridization events is common in plant evolution, but is much less documented in pathogenic fungi.

An initial comparative map of copy number variations in the goat (Capra hircus) genome

PubMed Central

2010-01-01

Background The goat (Capra hircus) represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. Results We identified a total of 161 CNVs (an average of 17.9 CNVs per goat), with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs): on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome). These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P < 0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Genes with environmental functions were over-represented in goat CNVRs as reported in other mammals. Conclusions We describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative recurrent interspecies CNVs between these two ruminant species. Several goat CNVs affect genes with important biological functions. Further studies are needed to evaluate the functional relevance of these CNVs and their effects on behavior, production, and disease resistance traits in goats. PMID:21083884

A 1.37-Mb 12p11.22-p11.21 deletion coincident with a 367-kb 22q11.2 duplication detected by array comparative genomic hybridization in an adolescent girl with autism and difficulty in self-care of menstruation.

PubMed

Chen, Chih-Ping; Lin, Shuan-Pei; Chern, Schu-Rern; Wu, Peih-Shan; Su, Jun-Wei; Lee, Chen-Chi; Wang, Wayseen

2014-03-01

To present an array comparative genomic hybridization (aCGH) characterization of a 12p11.22-p11.21 microdeletion and 22q11.2 microduplication in an adolescent girl with autism, mental retardation, facial dysmorphism, microcephaly, behavior problems, and an apparently balanced reciprocal translocation of t(8;12)(q24.3;p11.2). A 13-year-old girl was referred to the hospital because of autism, mental retardation, and difficulty in the self-care of her menstruation. Cytogenetic analysis revealed an apparently balanced reciprocal translocation and a karyotype of 46,XX,t(8;12) (q24.3;p11.2)dn. The girl manifested microcephaly, hypertelorism, flat facial profile, prominent forehead, thick scalp hair, upslanting palpebral fissures, broad nasal bridge, bulbous nose, right simian crease, bilateral clinodactyly of the fifth fingers, bilateral pes cavus, learning difficulties, mental retardation, emotional instability, cognitive impairment, behavior problems, jumping-like gaits, and autistic spectrum disorder. aCGH was performed to evaluate genomic imbalance in this patient. aCGH analysis revealed a 1.37-Mb 12p11.22-p11.21 microdeletion or arr [hg 19] 12p11.22-p11.21 (30,645,008-32,014,774)×1 and a 367-kb 22q11.21 microduplication or arr [hg 19] 22q11.21 (18,657,470-19,024,306)×3. The 1.37-Mb 12p11.22-p11.21 microdeletion encompassed 26 genes including IPO8, CAPRIN2, and DDX11, and the 367-kb 22q11.21 microduplication encompassed 20 genes including USP18, DGCR6, PRODH, and DGCR2. An apparently balanced translocation may be in fact affected by concurrent deletion and duplication in two different chromosomal regions. Our presentation provides information on diagnostic phenotype of 12p11.22-p11.21 microdeletion and 22q11.2 microduplication. Copyright © 2014. Published by Elsevier B.V.

Characterization of genome in tetraploid StY species of Elymus (Triticeae: Poaceae) using sequential FISH and GISH.

PubMed

Liu, Ruijuan; Wang, Richard R-C; Yu, Feng; Lu, Xingwang; Dou, Quanwen

2017-08-01

Genomes of ten species of Elymus, either presumed or known as tetraploid StY, were characterized using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH). These tetraploid species could be grouped into three categories. Type I included StY genome reported species-Roegneria pendulina, R. nutans, R. glaberrima, R. ciliaris, and Elymus nevskii, and StY genome presumed species-R. sinica, R. breviglumis, and R. dura, whose genome could be separated into two sets based on different GISH intensities. Type I genome constitution was deemed as putative StY. The St genome were mainly characterized with intense hybridization with pAs1, fewer AAG sites, and linked distribution of 5S rDNA and 18S-26S rDNA, while the Y genome with less intense hybridization with pAs1, more varied AAG sites, and isolated distribution of 5S rDNA and 18S-26S rDNA. Nevertheless, further genomic variations were detected among the different StY species. Type II included E. alashanicus, whose genome could be easily separated based on GISH pattern. FISH and GISH patterns suggested that E. alashanicus comprised a modified St genome and an unknown genome. Type III included E. longearistatus, whose genome could not be separated by GISH and was designated as St l Y l . Notably, a close relationship between S l and Y l genomes was observed.

MECP2 duplications in six patients with complex sex chromosome rearrangements

PubMed Central

Breman, Amy M; Ramocki, Melissa B; Kang, Sung-Hae L; Williams, Misti; Freedenberg, Debra; Patel, Ankita; Bader, Patricia I; Cheung, Sau Wai

2011-01-01

Duplications of the Xq28 chromosome region resulting in functional disomy are associated with a distinct clinical phenotype characterized by infantile hypotonia, severe developmental delay, progressive neurological impairment, absent speech, and proneness to infections. Increased expression of the dosage-sensitive MECP2 gene is considered responsible for the severe neurological impairments observed in affected individuals. Although cytogenetically visible duplications of Xq28 are well documented in the published literature, recent advances using array comparative genomic hybridization (CGH) led to the detection of an increasing number of microduplications spanning MECP2. In rare cases, duplication results from intrachromosomal rearrangement between the X and Y chromosomes. We report six cases with sex chromosome rearrangements involving duplication of MECP2. Cases 1–4 are unbalanced rearrangements between X and Y, resulting in MECP2 duplication. The additional Xq material was translocated to Yp in three cases (cases 1–3), and to the heterochromatic region of Yq12 in one case (case 4). Cases 5 and 6 were identified by array CGH to have a loss in copy number at Xp and a gain in copy number at Xq28 involving the MECP2 gene. In both cases, fluorescent in situ hybridization (FISH) analysis revealed a recombinant X chromosome containing the duplicated material from Xq28 on Xp, resulting from a maternal pericentric inversion. These cases add to a growing number of MECP2 duplications that have been detected by array CGH, while demonstrating the value of confirmatory chromosome and FISH studies for the localization of the duplicated material and the identification of complex rearrangements. PMID:21119712

A handy motion driven hybrid energy harvester: dual Halbach array based electromagnetic and triboelectric generators

NASA Astrophysics Data System (ADS)

Salauddin, M.; Park, J. Y.

2016-11-01

In this work, we have proposed and experimentally validated of hybrid electromagnetic and triboelectric energy harvester using dual Halbach magnets array excited by human handy motion. Hybrid electromagnetic (EM) and triboelectric (TE) generator that can deliver an output performance much higher than that of the individual energy-harvesting unit due to the combination operation of EM and TE mechanisms under the same mechanical movements. A Halbach array concentrates the magnetic flux lines on one side of the array while suppressing the flux lines on the other side. Dual Halbach array allows the concentrated magnetic flux lines to interact with the same coil in a way where maximum flux linkage occurs. When an external mechanical vibration is applied to the hybrid structure in the axial direction of the harvester, the suspended mass (two sided dual-Halbach-array frame) starts to oscillate within the magnetic springs and TEG part. Therefore, the TEG part, the Al film and microstructure PDMS film are collected into full contact with each other, generating triboelectric charges due to the various triboelectricities between them. A prototype of the hybrid harvester has been fabricated and tested. The EMG is capable of delivering maximum 11.5mW peak power at 32.5Ω matching load resistance and the TEG delivering 88μW peak power at 10MΩ load resistance.

Genome-Wide Prediction of the Performance of Three-Way Hybrids in Barley.

PubMed

Li, Zuo; Philipp, Norman; Spiller, Monika; Stiewe, Gunther; Reif, Jochen C; Zhao, Yusheng

2017-03-01

Predicting the grain yield performance of three-way hybrids is challenging. Three-way crosses are relevant for hybrid breeding in barley ( L.) and maize ( L.) adapted to East Africa. The main goal of our study was to implement and evaluate genome-wide prediction approaches of the performance of three-way hybrids using data of single-cross hybrids for a scenario in which parental lines of the three-way hybrids originate from three genetically distinct subpopulations. We extended the ridge regression best linear unbiased prediction (RRBLUP) and devised a genomic selection model allowing for subpopulation-specific marker effects (GSA-RRBLUP: general and subpopulation-specific additive RRBLUP). Using an empirical barley data set, we showed that applying GSA-RRBLUP tripled the prediction ability of three-way hybrids from 0.095 to 0.308 compared with RRBLUP, modeling one additive effect for all three subpopulations. The experimental findings were further substantiated with computer simulations. Our results emphasize the potential of GSA-RRBLUP to improve genome-wide hybrid prediction of three-way hybrids for scenarios of genetically diverse parental populations. Because of the advantages of the GSA-RRBLUP model in dealing with hybrids from different parental populations, it may also be a promising approach to boost the prediction ability for hybrid breeding programs based on genetically diverse heterotic groups. Copyright © 2017 Crop Science Society of America.

Next generation haplotyping to decipher nuclear genomic interspecific admixture in Citrus species: analysis of chromosome 2.

PubMed

Curk, Franck; Ancillo, Gema; Garcia-Lor, Andres; Luro, François; Perrier, Xavier; Jacquemoud-Collet, Jean-Pierre; Navarro, Luis; Ollitrault, Patrick

2014-12-29

The most economically important Citrus species originated by natural interspecific hybridization between four ancestral taxa (Citrus reticulata, Citrus maxima, Citrus medica, and Citrus micrantha) and from limited subsequent interspecific recombination as a result of apomixis and vegetative propagation. Such reticulate evolution coupled with vegetative propagation results in mosaic genomes with large chromosome fragments from the basic taxa in frequent interspecific heterozygosity. Modern breeding of these species is hampered by their complex heterozygous genomic structures that determine species phenotype and are broken by sexual hybridisation. Nevertheless, a large amount of diversity is present in the citrus gene pool, and breeding to allow inclusion of desirable traits is of paramount importance. However, the efficient mobilization of citrus biodiversity in innovative breeding schemes requires previous understanding of Citrus origins and genomic structures. Haplotyping of multiple gene fragments along the whole genome is a powerful approach to reveal the admixture genomic structure of current species and to resolve the evolutionary history of the gene pools. In this study, the efficiency of parallel sequencing with 454 methodology to decipher the hybrid structure of modern citrus species was assessed by analysis of 16 gene fragments on chromosome 2. 454 amplicon libraries were established using the Fluidigm array system for 48 genotypes and 16 gene fragments from chromosome 2. Haplotypes were established from the reads of each accession and phylogenetic analyses were performed using the haplotypic data for each gene fragment. The length of 454 reads and the level of differentiation between the ancestral taxa of modern citrus allowed efficient haplotype phylogenetic assignations for 12 of the 16 gene fragments. The analysis of the mixed genomic structure of modern species and cultivars (i) revealed C. maxima introgressions in modern mandarins, (ii) was consistent with previous hypotheses regarding the origin of secondary species, and (iii) provided a new picture of the evolution of chromosome 2. 454 sequencing was an efficient strategy to establish haplotypes with significant phylogenetic assignations in Citrus, providing a new picture of the mixed structure on chromosome 2 in 48 citrus genotypes.

Comparative genomics of wild type yeast strains unveils important genome diversity

PubMed Central

Carreto, Laura; Eiriz, Maria F; Gomes, Ana C; Pereira, Patrícia M; Schuller, Dorit; Santos, Manuel AS

2008-01-01

Background Genome variability generates phenotypic heterogeneity and is of relevance for adaptation to environmental change, but the extent of such variability in natural populations is still poorly understood. For example, selected Saccharomyces cerevisiae strains are variable at the ploidy level, have gene amplifications, changes in chromosome copy number, and gross chromosomal rearrangements. This suggests that genome plasticity provides important genetic diversity upon which natural selection mechanisms can operate. Results In this study, we have used wild-type S. cerevisiae (yeast) strains to investigate genome variation in natural and artificial environments. We have used comparative genome hybridization on array (aCGH) to characterize the genome variability of 16 yeast strains, of laboratory and commercial origin, isolated from vineyards and wine cellars, and from opportunistic human infections. Interestingly, sub-telomeric instability was associated with the clinical phenotype, while Ty element insertion regions determined genomic differences of natural wine fermentation strains. Copy number depletion of ASP3 and YRF1 genes was found in all wild-type strains. Other gene families involved in transmembrane transport, sugar and alcohol metabolism or drug resistance had copy number changes, which also distinguished wine from clinical isolates. Conclusion We have isolated and genotyped more than 1000 yeast strains from natural environments and carried out an aCGH analysis of 16 strains representative of distinct genotype clusters. Important genomic variability was identified between these strains, in particular in sub-telomeric regions and in Ty-element insertion sites, suggesting that this type of genome variability is the main source of genetic diversity in natural populations of yeast. The data highlights the usefulness of yeast as a model system to unravel intraspecific natural genome diversity and to elucidate how natural selection shapes the yeast genome. PMID:18983662

A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome.

PubMed

Yu, Shoukai; Lemos, Bernardo

2016-12-31

Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Arabidopsis gene expression patterns during spaceflight

NASA Astrophysics Data System (ADS)

Paul, A.-L.; Ferl, R. J.

The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

Gene-enriched draft genome of the cattle tick Rhipicephalus microplus: Assembly by the hybrid Pacific Biosciences/Illumina approach enabled analysis of the highly repetitive genome

USDA-ARS?s Scientific Manuscript database

The genome of the cattle tick R. microplus, an ectoparasite with global distribution, is estimated to be 7.1 Gbp and consists of ~70% repetitive DNA. We report the first assembly of a tick genome that utilized a hybrid sequencing and assembly approach to capture the repetitive fractions of the genom...

aCGH Local Copy Number Aberrations Associated with Overall Copy Number Genomic Instability in Colorectal Cancer: Coordinate Involvement of the Regions Including BCR and ABL

PubMed Central

Bartos, Jeremy D.; Gaile, Daniel P.; McQuaid, Devin E.; Conroy, Jeffrey M.; Darbary, Huferesh; Nowak, Norma J.; Block, Annemarie; Petrelli, Nicholas J.; Mittelman, Arnold; Stoler, Daniel L.; Anderson, Garth R.

2007-01-01

In order to identify small regions of the genome whose specific copy number alteration is associated with high genomic instability in the form of overall genome-wide copy number aberrations, we have analyzed array-based comparative genomic hybridization (aCGH) data from 33 sporadic colorectal carcinomas. Copy number changes of a small number of specific regions were significantly correlated with elevated overall amplifications and deletions scattered throughout the entire genome. One significant region at 9q34 includes the c-ABL gene Another region spanning 22q11–13 includes the breakpoint cluster region (BCR) of the Philadelphia chromosome Coordinate 22q11–13 alterations were observed in nine of eleven tumors with the 9q34 alteration Additional regions on 1q and 14q were associated with overall genome-wide copy number changes, while copy number aberrations on chromosome 7p, 7q, and 13q21.1–31.3 were found associated with this instability only in tumors from patients with a smoking history Our analysis demonstrates there are a small number of regions of the genome where gain or loss is commonly associated with a tumor’s overall level of copy number aberrations Our finding BCR and ABL located within two of the instability-associated regions, and the involvement of these two regions occurring coordinately, suggests a system akin to the BCR-ABL translocation of CML may be involved in genomic instability in about one-third of human colorectal carcinomas. PMID:17196995

Genome wide profiling in oral squamous cell carcinoma identifies a four genetic marker signature of prognostic significance

PubMed Central

Vincent-Chong, Vui King; Salahshourifar, Iman; Woo, Kar Mun; Anwar, Arif; Razali, Rozaimi; Gudimella, Ranganath; Rahman, Zainal Ariff Abdul; Ismail, Siti Mazlipah; Kallarakkal, Thomas George; Ramanathan, Anand; Wan Mustafa, Wan Mahadzir; Abraham, Mannil Thomas; Tay, Keng Kiong; Zain, Rosnah Binti

2017-01-01

Background Cancers of the oral cavity are primarily oral squamous cell carcinomas (OSCCs). Many of the OSCCs present at late stages with an exceptionally poor prognosis. A probable limitation in management of patients with OSCC lies in the insufficient knowledge pertaining to the linkage between copy number alterations in OSCC and oral tumourigenesis thereby resulting in an inability to deliver targeted therapy. Objectives The current study aimed to identify copy number alterations (CNAs) in OSCC using array comparative genomic hybridization (array CGH) and to correlate the CNAs with clinico-pathologic parameters and clinical outcomes. Materials and methods Using array CGH, genome-wide profiling was performed on 75 OSCCs. Selected genes that were harboured in the frequently amplified and deleted regions were validated using quantitative polymerase chain reaction (qPCR). Thereafter, pathway and network functional analysis were carried out using Ingenuity Pathway Analysis (IPA) software. Results Multiple chromosomal regions including 3q, 5p, 7p, 8q, 9p, 10p, 11q were frequently amplified, while 3p and 8p chromosomal regions were frequently deleted. These findings were in confirmation with our previous study using ultra-dense array CGH. In addition, amplification of 8q, 11q, 7p and 9p and deletion of 8p chromosomal regions showed a significant correlation with clinico-pathologic parameters such as the size of the tumour, metastatic lymph nodes and pathological staging. Co-amplification of 7p, 8q, 9p and 11q regions that harbored amplified genes namely CCND1, EGFR, TPM2 and LRP12 respectively, when combined, continues to be an independent prognostic factor in OSCC. Conclusion Amplification of 3q, 5p, 7p, 8q, 9p, 10p, 11q and deletion of 3p and 8p chromosomal regions were recurrent among OSCC patients. Co-alteration of 7p, 8q, 9p and 11q was found to be associated with clinico-pathologic parameters and poor survival. These regions contain genes that play critical roles in tumourigenesis pathways. PMID:28384287

Genome-wide polymorphisms and development of a microarray platform to detect genetic variations in Plasmodium yoelii.

PubMed

Nair, Sethu C; Pattaradilokrat, Sittiporn; Zilversmit, Martine M; Dommer, Jennifer; Nagarajan, Vijayaraj; Stephens, Melissa T; Xiao, Wenming; Tan, John C; Su, Xin-Zhuan

2014-01-01

The rodent malaria parasite Plasmodium yoelii is an important model for studying malaria immunity and pathogenesis. One approach for studying malaria disease phenotypes is genetic mapping, which requires typing a large number of genetic markers from multiple parasite strains and/or progeny from genetic crosses. Hundreds of microsatellite (MS) markers have been developed to genotype the P. yoelii genome; however, typing a large number of MS markers can be labor intensive, time consuming, and expensive. Thus, development of high-throughput genotyping tools such as DNA microarrays that enable rapid and accurate large-scale genotyping of the malaria parasite will be highly desirable. In this study, we sequenced the genomes of two P. yoelii strains (33X and N67) and obtained a large number of single nucleotide polymorphisms (SNPs). Based on the SNPs obtained, we designed sets of oligonucleotide probes to develop a microarray that could interrogate ∼11,000 SNPs across the 14 chromosomes of the parasite in a single hybridization. Results from hybridizations of DNA samples of five P. yoelii strains or cloned lines (17XNL, YM, 33X, N67 and N67C) and two progeny from a genetic cross (N67×17XNL) to the microarray showed that the array had a high call rate (∼97%) and accuracy (99.9%) in calling SNPs, providing a simple and reliable tool for typing the P. yoelii genome. Our data show that the P. yoelii genome is highly polymorphic, although isogenic pairs of parasites were also detected. Additionally, our results indicate that the 33X parasite is a progeny of 17XNL (or YM) and an unknown parasite. The highly accurate and reliable microarray developed in this study will greatly facilitate our ability to study the genetic basis of important traits and the disease it causes. Published by Elsevier B.V.

Genome‐scale diversity and niche adaptation analysis of Lactococcus lactis by comparative genome hybridization using multi‐strain arrays

PubMed Central

Siezen, Roland J.; Bayjanov, Jumamurat R.; Felis, Giovanna E.; van der Sijde, Marijke R.; Starrenburg, Marjo; Molenaar, Douwe; Wels, Michiel; van Hijum, Sacha A. F. T.; van Hylckama Vlieg, Johan E. T.

2011-01-01

Summary Lactococcus lactis produces lactic acid and is widely used in the manufacturing of various fermented dairy products. However, the species is also frequently isolated from non‐dairy niches, such as fermented plant material. Recently, these non‐dairy strains have gained increasing interest, as they have been described to possess flavour‐forming activities that are rarely found in dairy isolates and have diverse metabolic properties. We performed an extensive whole‐genome diversity analysis on 39 L. lactis strains, isolated from dairy and plant sources. Comparative genome hybridization analysis with multi‐strain microarrays was used to assess presence or absence of genes and gene clusters in these strains, relative to all L. lactis sequences in public databases, whereby chromosomal and plasmid‐encoded genes were computationally analysed separately. Nearly 3900 chromosomal orthologous groups (chrOGs) were defined on basis of four sequenced chromosomes of L. lactis strains (IL1403, KF147, SK11, MG1363). Of these, 1268 chrOGs are present in at least 35 strains and represent the presently known core genome of L. lactis, and 72 chrOGs appear to be unique for L. lactis. Nearly 600 and 400 chrOGs were found to be specific for either the subspecies lactis or subspecies cremoris respectively. Strain variability was found in presence or absence of gene clusters related to growth on plant substrates, such as genes involved in the consumption of arabinose, xylan, α‐galactosides and galacturonate. Further niche‐specific differences were found in gene clusters for exopolysaccharides biosynthesis, stress response (iron transport, osmotolerance) and bacterial defence mechanisms (nisin biosynthesis). Strain variability of functions encoded on known plasmids included proteolysis, lactose fermentation, citrate uptake, metal ion resistance and exopolysaccharides biosynthesis. The present study supports the view of L. lactis as a species with a very flexible genome. PMID:21338475

Comparison between fluorescent in-situ hybridisation and array comparative genomic hybridisation in preimplantation genetic diagnosis in translocation carriers.

PubMed

Lee, Vivian C Y; Chow, Judy F C; Lau, Estella Y L; Yeung, William S B; Ho, P C; Ng, Ernest H Y

2015-02-01

To compare the pregnancy outcome of the fluorescent in-situ hybridisation and array comparative genomic hybridisation in preimplantation genetic diagnosis of translocation carriers. Historical cohort. A teaching hospital in Hong Kong. All preimplantation genetic diagnosis treatment cycles performed for translocation carriers from 2001 to 2013. Overall, 101 treatment cycles for preimplantation genetic diagnosis in translocation were included: 77 cycles for reciprocal translocation and 24 cycles for Robertsonian translocation. Fluorescent in-situ hybridisation and array comparative genomic hybridisation were used in 78 and 11 cycles, respectively. The ongoing pregnancy rate per initiated cycle after array comparative genomic hybridisation was significantly higher than that after fluorescent in-situ hybridisation in all translocation carriers (36.4% vs 9.0%; P=0.010). The miscarriage rate was comparable with both techniques. The testing method (array comparative genomic hybridisation or fluorescent in-situ hybridisation) was the only significant factor affecting the ongoing pregnancy rate after controlling for the women's age, type of translocation, and clinical information of the preimplantation genetic diagnosis cycles by logistic regression (odds ratio=1.875; P=0.023; 95% confidence interval, 1.090-3.226). This local retrospective study confirmed that comparative genomic hybridisation is associated with significantly higher pregnancy rates versus fluorescent in-situ hybridisation in translocation carriers. Array comparative genomic hybridisation should be the technique of choice in preimplantation genetic diagnosis cycles in translocation carriers.

Complete characterization of wheat-alien metaphase I pairing in interspecific hybrids between durum wheat (Triticum turgidum L.) and jointed goatgrass (Aegilops cylindrica Host).

PubMed

Cifuentes, Marta; Benavente, Elena

2009-05-01

The pattern of homoeologous metaphase I (MI) pairing has been fully characterized in durum wheat x Aegilops cylindrica hybrids (2n = 4x = 28, ABC(c)D(c)) by an in situ hybridization procedure that has permitted individual discrimination of every wheat and wild constituent genome. One of the three hybrid genotypes examined carried the ph1c mutation. In all cases, MI associations between chromosomes of both species represented around two-third of total. Main results from the analysis are as follows (a) the A genome chromosomes are involved in wheat-wild MI pairing more frequently than the B genome partners, irrespective of the alien genome considered; (b) both durum wheat genomes pair preferentially with the D(c) genome of jointed goatgrass. These findings are discussed in relation to the potential of genetic transference between wheat crops and this weedy relative. It can also be highlighted that inactivation of Ph1 provoked a relatively higher promotion of MI associations involving B genome.

Multi-segment detector array for hybrid reflection-mode ultrasound and optoacoustic tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Merčep, Elena; Burton, Neal C.; Deán-Ben, Xosé Luís.; Razansky, Daniel

2017-02-01

The complementary contrast of the optoacoustic (OA) and pulse-echo ultrasound (US) modalities makes the combined usage of these imaging technologies highly advantageous. Due to the different physical contrast mechanisms development of a detector array optimally suited for both modalities is one of the challenges to efficient implementation of a single OA-US imaging device. We demonstrate imaging performance of the first hybrid detector array whose novel design, incorporating array segments of linear and concave geometry, optimally supports image acquisition in both reflection-mode ultrasonography and optoacoustic tomography modes. Hybrid detector array has a total number of 256 elements and three segments of different geometry and variable pitch size: a central 128-element linear segment with pitch of 0.25mm, ideally suited for pulse-echo US imaging, and two external 64-elements segments with concave geometry and 0.6mm pitch optimized for OA image acquisition. Interleaved OA and US image acquisition with up to 25 fps is facilitated through a custom-made multiplexer unit. Spatial resolution of the transducer was characterized in numerical simulations and validated in phantom experiments and comprises 230 and 300 μm in the respective OA and US imaging modes. Imaging performance of the multi-segment detector array was experimentally shown in a series of imaging sessions with healthy volunteers. Employing mixed array geometries allows at the same time achieving excellent OA contrast with a large field of view, and US contrast for complementary structural features with reduced side-lobes and improved resolution. The newly designed hybrid detector array that comprises segments of linear and concave geometries optimally fulfills requirements for efficient US and OA imaging and may expand the applicability of the developed hybrid OPUS imaging technology and accelerate its clinical translation.

Hybridization and genome size evolution: timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species

PubMed Central

Baack, Eric J.; Whitney, Kenneth D.; Rieseberg, Loren H.

2008-01-01

Summary Hybridization and polyploidy can induce rapid genomic changes, including the gain or loss of DNA, but the magnitude and timing of such changes are not well understood. The homoploid hybrid system in Helianthus (three hybrid-derived species and their two parents) provides an opportunity to examine the link between hybridization and genome size changes in a replicated fashion. Flow cytometry was used to estimate the nuclear DNA content in multiple populations of three homoploid hybrid Helianthus species (Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus), the parental species (Helianthus annuus and Helianthus petiolaris), synthetic hybrids, and natural hybrid-zone populations. Results confirm that hybrid-derived species have 50% more nuclear DNA than the parental species. Despite multiple origins, hybrid species were largely consistent in their DNA content across populations, although H. deserticola showed significant interpopulation differences. First- and sixth-generation synthetic hybrids and hybrid-zone plants did not show an increase from parental DNA content. First-generation hybrids differed in DNA content according to the maternal parent. In summary, hybridization by itself does not lead to increased nuclear DNA content in Helianthus, and the evolutionary forces responsible for the repeated increases in DNA content seen in the hybrid-derived species remain mysterious. PMID:15998412

Vive la résistance: genome-wide selection against introduced alleles in invasive hybrid zones

USGS Publications Warehouse

Kovach, Ryan P.; Hand, Brian K.; Hohenlohe, Paul A.; Cosart, Ted F.; Boyer, Matthew C.; Neville, Helen H.; Muhlfeld, Clint C.; Amish, Stephen J.; Carim, Kellie; Narum, Shawn R.; Lowe, Winsor H.; Allendorf, Fred W.; Luikart, Gordon

2016-01-01

Evolutionary and ecological consequences of hybridization between native and invasive species are notoriously complicated because patterns of selection acting on non-native alleles can vary throughout the genome and across environments. Rapid advances in genomics now make it feasible to assess locus-specific and genome-wide patterns of natural selection acting on invasive introgression within and among natural populations occupying diverse environments. We quantified genome-wide patterns of admixture across multiple independent hybrid zones of native westslope cutthroat trout and invasive rainbow trout, the world's most widely introduced fish, by genotyping 339 individuals from 21 populations using 9380 species-diagnostic loci. A significantly greater proportion of the genome appeared to be under selection favouring native cutthroat trout (rather than rainbow trout), and this pattern was pervasive across the genome (detected on most chromosomes). Furthermore, selection against invasive alleles was consistent across populations and environments, even in those where rainbow trout were predicted to have a selective advantage (warm environments). These data corroborate field studies showing that hybrids between these species have lower fitness than the native taxa, and show that these fitness differences are due to selection favouring many native genes distributed widely throughout the genome.

Beyond Genomic Prediction: Combining Different Types of omics Data Can Improve Prediction of Hybrid Performance in Maize.

PubMed

Schrag, Tobias A; Westhues, Matthias; Schipprack, Wolfgang; Seifert, Felix; Thiemann, Alexander; Scholten, Stefan; Melchinger, Albrecht E

2018-04-01

The ability to predict the agronomic performance of single-crosses with high precision is essential for selecting superior candidates for hybrid breeding. With recent technological advances, thousands of new parent lines, and, consequently, millions of new hybrid combinations are possible in each breeding cycle, yet only a few hundred can be produced and phenotyped in multi-environment yield trials. Well established prediction approaches such as best linear unbiased prediction (BLUP) using pedigree data and whole-genome prediction using genomic data are limited in capturing epistasis and interactions occurring within and among downstream biological strata such as transcriptome and metabolome. Because mRNA and small RNA (sRNA) sequences are involved in transcriptional, translational and post-translational processes, we expect them to provide information influencing several biological strata. However, using sRNA data of parent lines to predict hybrid performance has not yet been addressed. Here, we gathered genomic, transcriptomic (mRNA and sRNA) and metabolomic data of parent lines to evaluate the ability of the data to predict the performance of untested hybrids for important agronomic traits in grain maize. We found a considerable interaction for predictive ability between predictor and trait, with mRNA data being a superior predictor for grain yield and genomic data for grain dry matter content, while sRNA performed relatively poorly for both traits. Combining mRNA and genomic data as predictors resulted in high predictive abilities across both traits and combining other predictors improved prediction over that of the individual predictors alone. We conclude that downstream "omics" can complement genomics for hybrid prediction, and, thereby, contribute to more efficient selection of hybrid candidates. Copyright © 2018 by the Genetics Society of America.

Genomic Prediction of Single Crosses in the Early Stages of a Maize Hybrid Breeding Pipeline.

PubMed

Kadam, Dnyaneshwar C; Potts, Sarah M; Bohn, Martin O; Lipka, Alexander E; Lorenz, Aaron J

2016-09-19

Prediction of single-cross performance has been a major goal of plant breeders since the beginning of hybrid breeding. Recently, genomic prediction has shown to be a promising approach, but only limited studies have examined the accuracy of predicting single-cross performance. Moreover, no studies have examined the potential of predicting single crosses among random inbreds derived from a series of biparental families, which resembles the structure of germplasm comprising the initial stages of a hybrid maize breeding pipeline. The main objectives of this study were to evaluate the potential of genomic prediction for identifying superior single crosses early in the hybrid breeding pipeline and optimize its application. To accomplish these objectives, we designed and analyzed a novel population of single crosses representing the Iowa Stiff Stalk Synthetic/Non-Stiff Stalk heterotic pattern commonly used in the development of North American commercial maize hybrids. The performance of single crosses was predicted using parental combining ability and covariance among single crosses. Prediction accuracies were estimated using cross-validation and ranged from 0.28 to 0.77 for grain yield, 0.53 to 0.91 for plant height, and 0.49 to 0.94 for staygreen, depending on the number of tested parents of the single cross and genomic prediction method used. The genomic estimated general and specific combining abilities showed an advantage over genomic covariances among single crosses when one or both parents of the single cross were untested. Overall, our results suggest that genomic prediction of single crosses in the early stages of a hybrid breeding pipeline holds great potential to re-design hybrid breeding and increase its efficiency. Copyright © 2016 Author et al.

Analysis of X chromosome genomic DNA sequence copy number variation associated with premature ovarian failure (POF)

PubMed Central

Quilter, C.R.; Karcanias, A.C.; Bagga, M.R.; Duncan, S.; Murray, A.; Conway, G.S.; Sargent, C.A.; Affara, N.A.

2013-01-01

BACKGROUND Premature ovarian failure (POF) is a heterogeneous disease defined as amenorrhoea for >6 months before age 40, with an FSH serum level >40 mIU/ml (menopausal levels). While there is a strong genetic association with POF, familial studies have also indicated that idiopathic POF may also be genetically linked. Conventional cytogenetic analyses have identified regions of the X chromosome that are strongly associated with ovarian function, as well as several POF candidate genes. Cryptic chromosome abnormalities that have been missed might be detected by array comparative genomic hybridization. METHODS In this study, samples from 42 idiopathic POF patients were subjected to a complete end-to-end X/Y chromosome tiling path array to achieve a detailed copy number variation (CNV) analysis of X chromosome involvement in POF. The arrays also contained a 1 Mb autosomal tiling path as a reference control. Quantitative PCR for selected genes contained within the CNVs was used to confirm the majority of the changes detected. The expression pattern of some of these genes in human tissue RNA was examined by reverse transcription (RT)–PCR. RESULTS A number of CNVs were identified on both Xp and Xq, with several being shared among the POF cases. Some CNVs fall within known polymorphic CNV regions, and others span previously identified POF candidate regions and genes. CONCLUSIONS The new data reported in this study reveal further discrete X chromosome intervals not previously associated with the disease and therefore implicate new clusters of candidate genes. Further studies will be required to elucidate their involvement in POF. PMID:20570974

Use of the Genomic Subtractive Hybridization Technique To Develop a Real-Time PCR Assay for Quantitative Detection of Prevotella spp. in Oral Biofilm Samples

PubMed Central

Nagashima, Shiori; Yoshida, Akihiro; Suzuki, Nao; Ansai, Toshihiro; Takehara, Tadamichi

2005-01-01

Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species. PMID:15956428

Efficient Graph Based Assembly of Short-Read Sequences on Hybrid Core Architecture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sczyrba, Alex; Pratap, Abhishek; Canon, Shane

2011-03-22

Advanced architectures can deliver dramatically increased throughput for genomics and proteomics applications, reducing time-to-completion in some cases from days to minutes. One such architecture, hybrid-core computing, marries a traditional x86 environment with a reconfigurable coprocessor, based on field programmable gate array (FPGA) technology. In addition to higher throughput, increased performance can fundamentally improve research quality by allowing more accurate, previously impractical approaches. We will discuss the approach used by Convey?s de Bruijn graph constructor for short-read, de-novo assembly. Bioinformatics applications that have random access patterns to large memory spaces, such as graph-based algorithms, experience memory performance limitations on cache-based x86more » servers. Convey?s highly parallel memory subsystem allows application-specific logic to simultaneously access 8192 individual words in memory, significantly increasing effective memory bandwidth over cache-based memory systems. Many algorithms, such as Velvet and other de Bruijn graph based, short-read, de-novo assemblers, can greatly benefit from this type of memory architecture. Furthermore, small data type operations (four nucleotides can be represented in two bits) make more efficient use of logic gates than the data types dictated by conventional programming models.JGI is comparing the performance of Convey?s graph constructor and Velvet on both synthetic and real data. We will present preliminary results on memory usage and run time metrics for various data sets with different sizes, from small microbial and fungal genomes to very large cow rumen metagenome. For genomes with references we will also present assembly quality comparisons between the two assemblers.« less

Analysis of plastome and chondriome genome types in potato somatic hybrids from Solanum tuberosum × Solanum etuberosum.

PubMed

Tiwari, Jagesh K; Chandel, Poonam; Singh, Bir Pal; Bhardwaj, Vinay

2014-01-01

Cytoplasm types of the potato somatic hybrids from Solanum tuberosum × Solanum etuberosum were analysed using chloroplast (cp) and mitochondrial (mt) organelle genomes-specific markers. Of the 29 markers (15 cpDNA and 14 mtDNA) amplified in the 26 genotypes, 5 cpDNA (H3, NTCP4, NTCP8, NTCP9, and ALC1/ALC3) and 13 mtDNA markers showed polymorphism. The cluster analysis based on the mtDNA markers detected higher diversity compared with the cpDNA markers. Presence of new mtDNA fragments of the markers, namely, T11-2, Nsm1, pumD, Nsm3, and Nsm4, were observed, while monomorphic loci revealed highly conserved genomic regions in the somatic hybrids. The study revealed that the somatic hybrids had diverse cytoplasm types consisting predominantly of T-, W-, and C-, with a few A- and S-type cp genomes; and α-, β-, and γ-type mt genomes. Somatic hybridization has unique potential to widen the cytoplasm types of the cultivated gene pools from wild species through introgression by breeding methods.

CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli.

PubMed Central

Kipling, D; Mitchell, A R; Masumoto, H; Wilson, H E; Nicol, L; Cooke, H J

1995-01-01

Minor satellite DNA, found at Mus musculus centromeres, is not present in the genome of the Asian mouse Mus caroli. This repetitive sequence family is speculated to have a role in centromere function by providing an array of binding sites for the centromere-associated protein CENP-B. The apparent absence of CENP-B binding sites in the M. caroli genome poses a major challenge to this hypothesis. Here we describe two abundant satellite DNA sequences present at M. caroli centromeres. These satellites are organized as tandem repeat arrays, over 1 Mb in size, of either 60- or 79-bp monomers. All autosomes carry both satellites and small amounts of a sequence related to the M. musculus major satellite. The Y chromosome contains small amounts of both major satellite and the 60-bp satellite, whereas the X chromosome carries only major satellite sequences. M. caroli chromosomes segregate in M. caroli x M. musculus interspecific hybrid cell lines, indicating that the two sets of chromosomes can interact with the same mitotic spindle. Using a polyclonal CENP-B antiserum, we demonstrate that M. caroli centromeres can bind murine CENP-B in such an interspecific cell line, despite the absence of canonical 17-bp CENP-B binding sites in the M. caroli genome. Sequence analysis of the 79-bp M. caroli satellite reveals a 17-bp motif that contains all nine bases previously shown to be necessary for in vitro binding of CENP-B. This M. caroli motif binds CENP-B from HeLa cell nuclear extract in vitro, as indicated by gel mobility shift analysis. We therefore suggest that this motif also causes CENP-B to associate with M. caroli centromeres in vivo. Despite the sequence differences, M. caroli presents a third, novel mammalian centromeric sequence producing an array of binding sites for CENP-B. PMID:7623797

Genome instability in Novel Lolium multiflorum x L. arundinaceum hybrids

USDA-ARS?s Scientific Manuscript database

We have identified a method whereby Lolium multiflorum (Lm) or L. arundinaceum (Fa) genomes are preferentially eliminated through a mitotic loss behavior in interspecific Lm x Fa F1 hybrids,generating either dihaploid Lm lines or Fa lines. The genome instability has been visualized phenotypically an...

Genome analysis of 7 Kengyilia (Triticeae Poaceae) species with FISH and GISH

USDA-ARS?s Scientific Manuscript database

Genome composition of and genetic relationships among seven Kengyilia species were assessed using a technique of sequential FISH (fluorescence in situ hybridization) and GISH (genomic in situ hybridization). Five of these 7 species, K. kokonorica, K. rigidula, K. hirsula, K. grandiglumis, and K. th...

Energy and charge transfer effects in two-dimensional van der Waals hybrid nanostructures on periodic gold nanopost array

NASA Astrophysics Data System (ADS)

Kim, Jun Young; Kim, Sun Gyu; Youn, Jong Won; Lee, Yongjun; Kim, Jeongyong; Joo, Jinsoo

2018-05-01

Two-dimensional (2D) semiconducting MoS2 and WSe2 flakes grown by chemical vapor deposition were mechanically hybridized. A hexagonal boron nitride (h-BN) dielectric flake was inserted between MoS2 and WSe2 flakes to investigate the nanoscale optical properties of 2D van der Waals hybrid nanostructures. The fabricated MoS2/WSe2 and MoS2/h-BN/WSe2 van der Waals hybrid nanostructures were loaded on a periodic gold nanopost (Au-NPo) array to study energy and charge transfer effects at the surface plasmon resonance (SPR) condition. Nanoscale photoluminescence (PL) spectra of the 2D hybrid nanostructures were measured using a high-resolution laser confocal microscope (LCM). A shift of the LCM PL peak of the MoS2/WSe2 n-p hybrid nanostructures was observed owing to the charge transfer. In contrast, the shift of the LCM PL peak of the MoS2/h-BN/WSe2 n-insulator-p hybrid nanostructure was not considerable, as the inserted h-BN dielectric layer prevented the charge transfer. The intensity of the LCM PL peak of the MoS2/h-BN/WSe2 hybrid nanostructure considerably increased once the nanostructure was loaded on the Au-NPo array, owing to the energy transfer between the 2D materials and the Au-NPo array at the SPR condition, which was confirmed by the increase in the LCM Raman intensity.

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

PubMed

Blattmann, Claudia; Thiemann, Markus; Stenzinger, Albrecht; Roth, Eva K; Dittmar, Anne; Witt, Hendrik; Lehner, Burkhard; Renker, Eva; Jugold, Manfred; Eichwald, Viktoria; Weichert, Wilko; Huber, Peter E; Kulozik, Andreas E

2015-04-30

Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

Patterns of Reproductive Isolation in Eucalyptus-A Phylogenetic Perspective.

PubMed

Larcombe, Matthew J; Holland, Barbara; Steane, Dorothy A; Jones, Rebecca C; Nicolle, Dean; Vaillancourt, René E; Potts, Brad M

2015-07-01

We assess phylogenetic patterns of hybridization in the speciose, ecologically and economically important genus Eucalyptus, in order to better understand the evolution of reproductive isolation. Eucalyptus globulus pollen was applied to 99 eucalypt species, mainly from the large commercially important subgenus, Symphyomyrtus. In the 64 species that produce seeds, hybrid compatibility was assessed at two stages, hybrid-production (at approximately 1 month) and hybrid-survival (at 9 months), and compared with phylogenies based on 8,350 genome-wide DArT (diversity arrays technology) markers. Model fitting was used to assess the relationship between compatibility and genetic distance, and whether or not the strength of incompatibility "snowballs" with divergence. There was a decline in compatibility with increasing genetic distance between species. Hybridization was common within two closely related clades (one including E. globulus), but rare between E. globulus and species in two phylogenetically distant clades. Of three alternative models tested (linear, slowdown, and snowball), we found consistent support for a snowball model, indicating that the strength of incompatibility accelerates relative to genetic distance. Although we can only speculate about the genetic basis of this pattern, it is consistent with a Dobzhansky-Muller-model prediction that incompatibilities should snowball with divergence due to negative epistasis. Different rates of compatibility decline in the hybrid-production and hybrid-survival measures suggest that early-acting postmating barriers developed first and are stronger than later-acting barriers. We estimated that complete reproductive isolation can take up to 21-31 My in Eucalyptus. Practical implications for hybrid eucalypt breeding and genetic risk assessment in Australia are discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Stabilization process in Saccharomyces intra and interspecific hybrids in fermentative conditions.

PubMed

Pérez-Través, Laura; Lopes, Christian A; Barrio, Eladio; Querol, Amparo

2014-12-01

We evaluated the genetic stabilization of artificial intra- (Saccharomyces cerevisiae) and interspecific (S. cerevisiae × S. kudriavzevii) hybrids under wine fermentative conditions. Large-scale transitions in genome size and genome reorganizations were observed during this process. Interspecific hybrids seem to need fewer generations to reach genetic stability than intraspecific hybrids. The largest number of molecular patterns recovered among the derived clones was observed for intraspecific hybrids, particularly for those obtained by rare-mating. Molecular marker analyses revealed that unstable clones could change during the industrial process to obtain active dry yeast. When no changes in molecular markers and ploidy were observed after this process, no changes in genetic composition were confirmed by comparative genome hybridization, considering the clone as a stable hybrid. According to our results, under these conditions, fermentation steps 3 and 5 (30-50 generations) would suffice to obtain genetically stable interspecific and intraspecific hybrids, respectively. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

The genomic mosaicism of hybrid speciation

PubMed Central

Elgvin, Tore O.; Trier, Cassandra N.; Tørresen, Ole K.; Hagen, Ingerid J.; Lien, Sigbjørn; Nederbragt, Alexander J.; Ravinet, Mark; Jensen, Henrik; Sætre, Glenn-Peter

2017-01-01

Hybridization is widespread in nature and, in some instances, can result in the formation of a new hybrid species. We investigate the genetic foundation of this poorly understood process through whole-genome analysis of the hybrid Italian sparrow and its progenitors. We find overall balanced yet heterogeneous levels of contribution from each parent species throughout the hybrid genome and identify areas of novel divergence in the hybrid species exhibiting signals consistent with balancing selection. High-divergence areas are disproportionately located on the Z chromosome and overrepresented in gene networks relating to key traits separating the focal species, which are likely involved in reproductive barriers and/or species-specific adaptations. Of special interest are genes and functional groups known to affect body patterning, beak morphology, and the immune system, which are important features of diversification and fitness. We show that a combination of mosaic parental inheritance and novel divergence within the hybrid lineage has facilitated the origin and maintenance of an avian hybrid species. PMID:28630911

Prenatal diagnosis and molecular cytogenetic characterization of de novo pure partial trisomy 6p associated with microcephaly, craniosynostosis and abnormal maternal serum biochemistry.

PubMed

Chen, Chih-Ping; Chen, Ming; Chen, Chen-Yu; Chern, Schu-Rern; Wu, Peih-Shan; Chang, Shun-Ping; Kuo, Yu-Ling; Chen, Wen-Lin; Pan, Chen-Wen; Wang, Wayseen

2014-02-25

We present prenatal diagnosis and molecular cytogenetic characterization of de novo pure trisomy 6p22.3 → p25.3 encompassing BMP6 in a fetus associated with microcephaly and craniosynostosis on prenatal ultrasound, abnormal maternal serum biochemistry of a low PAPP-A level in the first-trimester combined test, and a karyotype of 46,XX,der(22)t(6;22)(p22.3;p13)dn. The present case demonstrates the usefulness of rapid prenatal identification of the origin of the extra chromosome material on the short arm of an acrocentric chromosome by spectral karyotyping, fluorescence in situ hybridization and array comparative genomic hybridization. We review the phenotypic abnormality of craniosynostosis in previously reported patients with partial trisomy 6p. We discuss the genotype-phenotype correlation of the involved gene of BMP6 in this case. Copyright © 2013 Elsevier B.V. All rights reserved.

Alagille syndrome with deletion 20p12.2-p12.3 and hypoplastic left heart.

PubMed

Robert, Marie Leema P; Lopez, Tony; Crolla, John; Huang, Shuwen; Owen, Carol; Burvill-Holmes, Lisa; Stumper, Oliver; Turnpenny, Peter D

2007-10-01

We report a male patient born at 37-weeks gestation, weighing 1.885 kg (<0.4th centile). Shortly after birth, he was diagnosed with hypoplastic left heart syndrome for which he underwent a Norwood procedure. Subsequently, he developed problems with failure to thrive and developmental delay. At the age of 4 years his delay in growth and development led to further investigations, which revealed a small de-novo interstitial deletion of chromosome 20p12.2. JAGGED1 haploinsufficiency was confirmed by fluorescence in situ hybridization. Array-comparative genomic hybridization analysis confirmed and quantified an approximate 5.4 Mb interstitial deletion involving the chromosomal region 20p12.2-p12.3. This precise interstitial deletion has not been previously reported. Further clinical evaluation revealed posterior embryotoxon and butterfly vertebrae. He has normal liver function tests, facial features consistent with Alagille syndrome, and mild learning difficulties. To our knowledge this is the first report of Alagille syndrome associated with hypoplastic left heart syndrome.

Periventricular heterotopia in a boy with interstitial deletion of chromosome 4p.

PubMed

Gawlik-Kuklinska, Katarzyna; Wierzba, Jolanta; Wozniak, Agnieszka; Iliszko, Mariola; Debiec-Rychter, Maria; Dubaniewicz-Wybieralska, Miroslawa; Limon, Janusz

2008-01-01

We report on a 4-year-old boy with a proximal interstitial deletion in the short arm of chromosome 4p with the karyotype 46,XY,del(4)(p14p15.32),inv(9)(p13q13). For a precise delineation of the deleted region, an array-based comparative genomic hybridization (a-CGH) analysis was performed. The proband's phenotype and cytogenetic findings are compared with previously reported cases with proximal 4p deletion syndrome. The syndrome is associated with normal growth, varying degrees of mental retardation, characteristic facial appearance and minor dysmorphic features. Additionally, our patient developed a seizure disorder due to abnormal neuronal migration, i.e., periventricular heterotopia.

Rapid quantification and taxonomic classification of environmentalDNA from both prokaryotic and eukaryotic origins using a microarray

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeSantis, Todd Z.; Stone, Carol E.; Murray, Sonya R.

2005-02-22

A microarray has been designed using 62,358 probes matched to both prokaryotic and eukaryotic small-subunit ribosomal RNA genes. The array categorized environmental DNA to specific phylogenetic clusters in under 9 h. To a background of DNA generated from natural outdoor aerosols, known quantities of rRNA gene copies from distinct organisms were added producing corresponding hybridization intensity scores that correlated well with their concentrations (r=0.917). Reproducible differences in microbial community composition were observed by altering the genomic DNA extraction method. Notably, gentle extractions produced peak intensities for Mycoplasmatales and Burkholderiales, whereas a vigorous disruption produced peak intensities for Vibrionales,Clostridiales, and Bacillales.

Parallel gene analysis with allele-specific padlock probes and tag microarrays

PubMed Central

Banér, Johan; Isaksson, Anders; Waldenström, Erik; Jarvius, Jonas; Landegren, Ulf; Nilsson, Mats

2003-01-01

Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes. PMID:12930977

Centromere reference models for human chromosomes X and Y satellite arrays

PubMed Central

Miga, Karen H.; Newton, Yulia; Jain, Miten; Altemose, Nicolas; Willard, Huntington F.; Kent, W. James

2014-01-01

The human genome sequence remains incomplete, with multimegabase-sized gaps representing the endogenous centromeres and other heterochromatic regions. Available sequence-based studies within these sites in the genome have demonstrated a role in centromere function and chromosome pairing, necessary to ensure proper chromosome segregation during cell division. A common genomic feature of these regions is the enrichment of long arrays of near-identical tandem repeats, known as satellite DNAs, which offer a limited number of variant sites to differentiate individual repeat copies across millions of bases. This substantial sequence homogeneity challenges available assembly strategies and, as a result, centromeric regions are omitted from ongoing genomic studies. To address this problem, we utilize monomer sequence and ordering information obtained from whole-genome shotgun reads to model two haploid human satellite arrays on chromosomes X and Y, resulting in an initial characterization of 3.83 Mb of centromeric DNA within an individual genome. To further expand the utility of each centromeric reference sequence model, we evaluate sites within the arrays for short-read mappability and chromosome specificity. Because satellite DNAs evolve in a concerted manner, we use these centromeric assemblies to assess the extent of sequence variation among 366 individuals from distinct human populations. We thus identify two satellite array variants in both X and Y centromeres, as determined by array length and sequence composition. This study provides an initial sequence characterization of a regional centromere and establishes a foundation to extend genomic characterization to these sites as well as to other repeat-rich regions within complex genomes. PMID:24501022

Identification of a duplication within the GDF9 gene and novel candidate genes for primary ovarian insufficiency (POI) by a customized high-resolution array comparative genomic hybridization platform.

PubMed

Norling, A; Hirschberg, A L; Rodriguez-Wallberg, K A; Iwarsson, E; Wedell, A; Barbaro, M

2014-08-01

Can high-resolution array comparative genomic hybridization (CGH) analysis of DNA samples from women with primary ovarian insufficiency (POI) improve the diagnosis of the condition and identify novel candidate genes for POI? A mutation affecting the regulatory region of growth differentiation factor 9 (GDF9) was identified for the first time together with several novel candidate genes for POI. Most patients with POI do not receive a molecular diagnosis despite a significant genetic component in the pathogenesis. We performed a case-control study. Twenty-six patients were analyzed by array CGH for identification of copy number variants. Novel changes were investigated in 95 controls and in a separate population of 28 additional patients with POI. The experimental procedures were performed during a 1-year period. DNA samples from 26 patients with POI were analyzed by a customized 1M array-CGH platform with whole genome coverage and probe enrichment targeting 78 genes in sex development. By PCR amplification and sequencing, the breakpoint of an identified partial GDF9 gene duplication was characterized. A multiplex ligation-dependent probe amplification (MLPA) probe set for specific identification of deletions/duplications affecting GDF9 was developed. An MLPA probe set for the identification of additional cases or controls carrying novel candidate regions identified by array-CGH was developed. Sequencing of three candidate genes was performed. Eleven unique copy number changes were identified in a total of 11 patients, including a tandem duplication of 475 bp, containing part of the GDF9 gene promoter region. The duplicated region contains three NOBOX-binding elements and an E-box, important for GDF9 gene regulation. This aberration is likely causative of POI. Fifty-four patients were investigated for copy number changes within GDF9, but no additional cases were found. Ten aberrations constituting novel candidate regions were detected, including a second DNAH6 deletion in a patient with POI. Other identified candidate genes were TSPYL6, SMARCC1, CSPG5 and ZFR2. This is a descriptive study and no functional experiments were performed. The study illustrates the importance of analyzing small copy number changes in addition to sequence alterations in the genetic investigation of patients with POI. Also, promoter regions should be included in the investigation. The study was supported by grants from the Swedish Research council (project no 12198 to A.W. and project no 20324 to A.L.H.), Stockholm County Council (E.I., A.W. and K.R.W.), Foundation Frimurare Barnhuset (A.N., A.W. and M.B.), Karolinska Institutet (A.N., A.L.H., E.I., A.W. and M.B.), Novo Nordic Foundation (A.W.) and Svenska Läkaresällskapet (M.B.). The funding sources had no involvement in the design or analysis of the study. The authors have no competing interests to declare. Not applicable. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Evaluation and Validation of Assembling Corrected PacBio Long Reads for Microbial Genome Completion via Hybrid Approaches.

PubMed

Lin, Hsin-Hung; Liao, Yu-Chieh

2015-01-01

Despite the ever-increasing output of next-generation sequencing data along with developing assemblers, dozens to hundreds of gaps still exist in de novo microbial assemblies due to uneven coverage and large genomic repeats. Third-generation single-molecule, real-time (SMRT) sequencing technology avoids amplification artifacts and generates kilobase-long reads with the potential to complete microbial genome assembly. However, due to the low accuracy (~85%) of third-generation sequences, a considerable amount of long reads (>50X) are required for self-correction and for subsequent de novo assembly. Recently-developed hybrid approaches, using next-generation sequencing data and as few as 5X long reads, have been proposed to improve the completeness of microbial assembly. In this study we have evaluated the contemporary hybrid approaches and demonstrated that assembling corrected long reads (by runCA) produced the best assembly compared to long-read scaffolding (e.g., AHA, Cerulean and SSPACE-LongRead) and gap-filling (SPAdes). For generating corrected long reads, we further examined long-read correction tools, such as ECTools, LSC, LoRDEC, PBcR pipeline and proovread. We have demonstrated that three microbial genomes including Escherichia coli K12 MG1655, Meiothermus ruber DSM1279 and Pdeobacter heparinus DSM2366 were successfully hybrid assembled by runCA into near-perfect assemblies using ECTools-corrected long reads. In addition, we developed a tool, Patch, which implements corrected long reads and pre-assembled contigs as inputs, to enhance microbial genome assemblies. With the additional 20X long reads, short reads of S. cerevisiae W303 were hybrid assembled into 115 contigs using the verified strategy, ECTools + runCA. Patch was subsequently applied to upgrade the assembly to a 35-contig draft genome. Our evaluation of the hybrid approaches shows that assembling the ECTools-corrected long reads via runCA generates near complete microbial genomes, suggesting that genome assembly could benefit from re-analyzing the available hybrid datasets that were not assembled in an optimal fashion.

Analysis of genome-wide copy number variations in Chinese indigenous and western pig breeds by 60 K SNP genotyping arrays.

PubMed

Wang, Yanan; Tang, Zhonglin; Sun, Yaqi; Wang, Hongyang; Wang, Chao; Yu, Shaobo; Liu, Jing; Zhang, Yu; Fan, Bin; Li, Kui; Liu, Bang

2014-01-01

Copy number variations (CNVs) represent a substantial source of structural variants in mammals and contribute to both normal phenotypic variability and disease susceptibility. Although low-resolution CNV maps are produced in many domestic animals, and several reports have been published about the CNVs of porcine genome, the differences between Chinese and western pigs still remain to be elucidated. In this study, we used Porcine SNP60 BeadChip and PennCNV algorithm to perform a genome-wide CNV detection in 302 individuals from six Chinese indigenous breeds (Tongcheng, Laiwu, Luchuan, Bama, Wuzhishan and Ningxiang pigs), three western breeds (Yorkshire, Landrace and Duroc) and one hybrid (Tongcheng×Duroc). A total of 348 CNV Regions (CNVRs) across genome were identified, covering 150.49 Mb of the pig genome or 6.14% of the autosomal genome sequence. In these CNVRs, 213 CNVRs were found to exist only in the six Chinese indigenous breeds, and 60 CNVRs only in the three western breeds. The characters of CNVs in four Chinese normal size breeds (Luchuan, Tongcheng and Laiwu pigs) and two minipig breeds (Bama and Wuzhishan pigs) were also analyzed in this study. Functional annotation suggested that these CNVRs possess a great variety of molecular function and may play important roles in phenotypic and production traits between Chinese and western breeds. Our results are important complementary to the CNV map in pig genome, which provide new information about the diversity of Chinese and western pig breeds, and facilitate further research on porcine genome CNVs.

Analysis of Genome-Wide Copy Number Variations in Chinese Indigenous and Western Pig Breeds by 60 K SNP Genotyping Arrays

PubMed Central

Sun, Yaqi; Wang, Hongyang; Wang, Chao; Yu, Shaobo; Liu, Jing; Zhang, Yu; Fan, Bin; Li, Kui; Liu, Bang

2014-01-01

Copy number variations (CNVs) represent a substantial source of structural variants in mammals and contribute to both normal phenotypic variability and disease susceptibility. Although low-resolution CNV maps are produced in many domestic animals, and several reports have been published about the CNVs of porcine genome, the differences between Chinese and western pigs still remain to be elucidated. In this study, we used Porcine SNP60 BeadChip and PennCNV algorithm to perform a genome-wide CNV detection in 302 individuals from six Chinese indigenous breeds (Tongcheng, Laiwu, Luchuan, Bama, Wuzhishan and Ningxiang pigs), three western breeds (Yorkshire, Landrace and Duroc) and one hybrid (Tongcheng×Duroc). A total of 348 CNV Regions (CNVRs) across genome were identified, covering 150.49 Mb of the pig genome or 6.14% of the autosomal genome sequence. In these CNVRs, 213 CNVRs were found to exist only in the six Chinese indigenous breeds, and 60 CNVRs only in the three western breeds. The characters of CNVs in four Chinese normal size breeds (Luchuan, Tongcheng and Laiwu pigs) and two minipig breeds (Bama and Wuzhishan pigs) were also analyzed in this study. Functional annotation suggested that these CNVRs possess a great variety of molecular function and may play important roles in phenotypic and production traits between Chinese and western breeds. Our results are important complementary to the CNV map in pig genome, which provide new information about the diversity of Chinese and western pig breeds, and facilitate further research on porcine genome CNVs. PMID:25198154

Novel origins of copy number variation in the dog genome

PubMed Central

2012-01-01

Background Copy number variants (CNVs) account for substantial variation between genomes and are a major source of normal and pathogenic phenotypic differences. The dog is an ideal model to investigate mutational mechanisms that generate CNVs as its genome lacks a functional ortholog of the PRDM9 gene implicated in recombination and CNV formation in humans. Here we comprehensively assay CNVs using high-density array comparative genomic hybridization in 50 dogs from 17 dog breeds and 3 gray wolves. Results We use a stringent new method to identify a total of 430 high-confidence CNV loci, which range in size from 9 kb to 1.6 Mb and span 26.4 Mb, or 1.08%, of the assayed dog genome, overlapping 413 annotated genes. Of CNVs observed in each breed, 98% are also observed in multiple breeds. CNVs predicted to disrupt gene function are significantly less common than expected by chance. We identify a significant overrepresentation of peaks of GC content, previously shown to be enriched in dog recombination hotspots, in the vicinity of CNV breakpoints. Conclusions A number of the CNVs identified by this study are candidates for generating breed-specific phenotypes. Purifying selection seems to be a major factor shaping structural variation in the dog genome, suggesting that many CNVs are deleterious. Localized peaks of GC content appear to be novel sites of CNV formation in the dog genome by non-allelic homologous recombination, potentially activated by the loss of PRDM9. These sequence features may have driven genome instability and chromosomal rearrangements throughout canid evolution. PMID:22916802

Exploring the origin of the D genome of oat by fluorescence in situ hybridization.

PubMed

Luo, Xiaomei; Zhang, Haiqin; Kang, Houyang; Fan, Xing; Wang, Yi; Sha, Lina; Zhou, Yonghong

2014-09-01

Further understanding of the origin of cultivated oat would accelerate its genetic improvement. In particular, it would be useful to clarify which diploid progenitor contributed the D genome of this allohexaploid species. In this study, we demonstrate that the landmarks produced by fluorescence in situ hybridization (FISH) of species of Avena using probes derived from Avena sativa can be used to explore the origin of the D genome. Selected sets of probes were hybridized in several sequential experiments performed on exactly the same chromosome spreads, with multiple probes of cytological preparations. Probes pITS and A3-19 showed there might be a similar distribution of pITS between the Ac and D genomes. These results indicated that the Ac genome is closely related to the D genome, and that Avena canariensis (AcAc) could be the D-genome donor of cultivated oat.

Identification and characterization of karyotype in Passiflora hybrids using FISH and GISH.

PubMed

Silva, Gonçalo Santos; Souza, Margarete Magalhães; de Melo, Cláusio Antônio Ferreira; Urdampilleta, Juan Domingo; Forni-Martins, Eliana Regina

2018-04-27

A great interest exists in the production of hybrid plants of the genus Passiflora given the beauty and exotic features of its flowers which have ornamental value. Hybrid paternity confirmation is therefore important for assuring germplasm origin, and is typically carried out by molecular marker segregation. The aim of this study was to karyotypically characterize the chromosome heritance patterns of the progeny resultant from a cross of P. gardneri and P. gibertii using classical cytogenetics, chromosome banding, and molecular cytogenetics. All analyzed genotypes showed the same diploid chromosome number as the genitor species: 2n = 18. Classical and CMA 3 and DAPI staining allowed for chromosome counting and satellite identification (secondary constrictions). Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were used to characterize subgenomes by either identifying rDNA-specific genome patterns or parental genomes, respectively. The heritance of chromosomal markers presenting rDNA sites from each parent for genome identification confirmed that all obtained plants were hybrids. These results will improve breeding programs involving the species of this genus. Apart from confirming hybridization, GISH allowed the visualization of recombination between the homeologous chromosome and the introgression of sequences of interest.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform.

PubMed

Salehi, Reza; Tsoi, Stephen C M; Colazo, Marcos G; Ambrose, Divakar J; Robert, Claude; Dyck, Michael K

2017-01-30

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Novel SNP array analysis and exome sequencing detect a homozygous exon 7 deletion of MEGF10 causing early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD)

PubMed Central

Pierson, Tyler Mark; Markello, Thomas; Accardi, John; Wolfe, Lynne; Adams, David; Sincan, Murat; Tarazi, Noor M.; Fajardo, Karin Fuentes; Cherukuri, Praveen F.; Bajraktari, Ilda; Meilleur, Katy G.; Donkervoort, Sandra; Jain, Mina; Hu, Ying; Lehky, Tanya J.; Cruz, Pedro; Mullikin, James C.; Bonnemann, Carsten; Gahl, William A.; Boerkoel, Cornelius F.; Tifft, Cynthia J.

2013-01-01

Early-onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD) is a myopathic disorder associated with mutations in MEGF10. By novel analysis of SNP array hybridization and exome sequence coverage, we diagnosed a 10-year old girl with EMARDD following identification of a novel homozygous deletion of exon 7 in MEGF10. In contrast to previously reported EMARDD patients, her weakness was more prominent proximally than distally, and involved her legs more than her arms. MRI of her pelvis and thighs showed muscle atrophy and fatty replacement. Ultrasound of several muscle groups revealed dense homogenous increases in echogenicity. Cloning and sequencing of the deletion breakpoint identified features suggesting the mutation arose by fork stalling and template switching. These findings constitute the first genomic deletion causing EMARDD, expand the clinical phenotype, and provide new insight into the pattern and histology of its muscular pathology. PMID:23453856

Homoeolog-specific transcriptional bias in allopolyploid wheat

PubMed Central

2010-01-01

Background Interaction between parental genomes is accompanied by global changes in gene expression which, eventually, contributes to growth vigor and the broader phenotypic diversity of allopolyploid species. In order to gain a better understanding of the effects of allopolyploidization on the regulation of diverged gene networks, we performed a genome-wide analysis of homoeolog-specific gene expression in re-synthesized allohexaploid wheat created by the hybridization of a tetraploid derivative of hexaploid wheat with the diploid ancestor of the wheat D genome Ae. tauschii. Results Affymetrix wheat genome arrays were used for both the discovery of divergent homoeolog-specific mutations and analysis of homoeolog-specific gene expression in re-synthesized allohexaploid wheat. More than 34,000 detectable parent-specific features (PSF) distributed across the wheat genome were used to assess AB genome (could not differentiate A and B genome contributions) and D genome parental expression in the allopolyploid transcriptome. In re-synthesized polyploid 81% of PSFs detected mid-parent levels of gene expression, and only 19% of PSFs showed the evidence of non-additive expression. Non-additive expression in both AB and D genomes was strongly biased toward up-regulation of parental type of gene expression with only 6% and 11% of genes, respectively, being down-regulated. Of all the non-additive gene expression, 84% can be explained by differences in the parental genotypes used to make the allopolyploid. Homoeolog-specific co-regulation of several functional gene categories was found, particularly genes involved in photosynthesis and protein biosynthesis in wheat. Conclusions Here, we have demonstrated that the establishment of interactions between the diverged regulatory networks in allopolyploids is accompanied by massive homoeolog-specific up- and down-regulation of gene expression. This study provides insights into interactions between homoeologous genomes and their role in growth vigor, development, and fertility of allopolyploid species. PMID:20849627

A high-resolution radiation hybrid map of the bovine genome

USDA-ARS?s Scientific Manuscript database

We are building high-resolution radiation hybrid maps of all 29 bovine autosomes and chromosome X, using a 58,000-marker genotyping assay, and a 12,000-rad whole-genome radiation hybrid (RH) panel. To accommodate the large number of markers, and to automate the map building procedure, a software pip...

Soft Lithography for Oligonucleotide Arrays Fabrication

DTIC Science & Technology

2001-10-25

adenosine; Abbreviated T, C, G, A respectively), the other synthesis reagents and solvents except oxidation agent (seen in Table 1) were purchased...dried by cold blowing before hybridization. Oligonucleotide arrays were hybridized in 200 nM 3’-TCC TCC GAT TCA GAG AGT CC- HEX (PE Biosystems... citrate buffer), 0.1% SDS in 0.1xSSC respectively. The probe array was scanned on the Scanarray Microarray Systems (Packard Biochip Technologies, USA

The Landscape of Somatic Chromosomal Copy Number Aberrations in GEM Models of Prostate Carcinoma

PubMed Central

Bianchi-Frias, Daniella; Hernandez, Susana A.; Coleman, Roger; Wu, Hong; Nelson, Peter S.

2015-01-01

Human prostate cancer (PCa) is known to harbor recurrent genomic aberrations consisting of chromosomal losses, gains, rearrangements and mutations that involve oncogenes and tumor suppressors. Genetically engineered mouse (GEM) models have been constructed to assess the causal role of these putative oncogenic events and provide molecular insight into disease pathogenesis. While GEM models generally initiate neoplasia by manipulating a single gene, expression profiles of GEM tumors typically comprise hundreds of transcript alterations. It is unclear whether these transcriptional changes represent the pleiotropic effects of single oncogenes, and/or cooperating genomic or epigenomic events. Therefore, it was determined if structural chromosomal alterations occur in GEM models of PCa and whether the changes are concordant with human carcinomas. Whole genome array-based comparative genomic hybridization (CGH) was used to identify somatic chromosomal copy number aberrations (SCNAs) in the widely used TRAMP, Hi-Myc, Pten-null and LADY GEM models. Interestingly, very few SCNAs were identified and the genomic architecture of Hi-Myc, Pten-null and LADY tumors were essentially identical to the germline. TRAMP neuroendocrine carcinomas contained SCNAs, which comprised three recurrent aberrations including a single copy loss of chromosome 19 (encoding Pten). In contrast, cell lines derived from the TRAMP, Hi-Myc, and Pten-null tumors were notable for numerous SCNAs that included copy gains of chromosome 15 (encoding Myc) and losses of chromosome 11 (encoding p53). PMID:25298407

Parents' Experience with Pediatric Microarray: Transferrable Lessons in the Era of Genomic Counseling.

PubMed

Hayeems, R Z; Babul-Hirji, R; Hoang, N; Weksberg, R; Shuman, C

2016-04-01

Advances in genome-based microarray and sequencing technologies hold tremendous promise for understanding, better-managing and/or preventing disease and disease-related risk. Chromosome microarray technology (array based comparative genomic hybridization [aCGH]) is widely utilized in pediatric care to inform diagnostic etiology and medical management. Less clear is how parents experience and perceive the value of this technology. This study explored parents' experiences with aCGH in the pediatric setting, focusing on how they make meaning of various types of test results. We conducted in-person or telephone-based semi-structured interviews with parents of 21 children who underwent aCGH testing in 2010. Transcripts were coded and analyzed thematically according to the principles of interpretive description. We learned that parents expect genomic tests to be of personal use; their experiences with aCGH results characterize this use as intrinsic in the test's ability to provide a much sought-after answer for their child's condition, and instrumental in its ability to guide care, access to services, and family planning. In addition, parents experience uncertainty regardless of whether aCGH results are of pathogenic, uncertain, or benign significance; this triggers frustration, fear, and hope. Findings reported herein better characterize the notion of personal utility and highlight the pervasive nature of uncertainty in the context of genomic testing. Empiric research that links pre-test counseling content and psychosocial outcomes is warranted to optimize patient care.

Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Proudnikov, D.; Kirillov, E.; Chumakov, K.

2000-01-01

This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements.more » Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.« less

Experimental study of surface insulated-standard hybrid tungsten planar wire array Z-pinches at "QiangGuang-I" facility

NASA Astrophysics Data System (ADS)

Sheng, Liang; Peng, Bodong; Li, Yang; Yuan, Yuan; Li, Mo; Zhang, Mei; Zhao, Chen; Zhao, Jizhen; Wang, Liangping

2016-01-01

The experimental results of the insulated-standard hybrid wire array Z pinches carried out on "QiangGuang-I" facility at Northwest Institute of Nuclear Technology were presented and discussed. The surface insulating can impose a significant influence on the dynamics and radiation characteristics of the hybrid wire array Z pinches, especially on the early stage (t/timp < 0.6). The expansion of insulated wires at the ablation stage is suppressed, while the streams stripped from the insulated wires move faster than that from the standard wires. The foot radiation of X-ray is enhanced by increment of the number of insulated wires, 19.6 GW, 33.6 GW, and 68.6 GW for shots 14037S, 14028H, and 14039I, respectively. The surface insulation also introduces nonhomogeneity along the single wire—the streams move much faster near the electrodes. The colliding boundary of the hybrid wire array Z pinches is bias to the insulated side approximately 0.6 mm.

The Genome Sequence of Saccharomyces eubayanus and the Domestication of Lager-Brewing Yeasts

PubMed Central

Baker, EmilyClare; Wang, Bing; Bellora, Nicolas; Peris, David; Hulfachor, Amanda Beth; Koshalek, Justin A.; Adams, Marie; Libkind, Diego; Hittinger, Chris Todd

2015-01-01

The dramatic phenotypic changes that occur in organisms during domestication leave indelible imprints on their genomes. Although many domesticated plants and animals have been systematically compared with their wild genetic stocks, the molecular and genomic processes underlying fungal domestication have received less attention. Here, we present a nearly complete genome assembly for the recently described yeast species Saccharomyces eubayanus and compare it to the genomes of multiple domesticated alloploid hybrids of S. eubayanus × S. cerevisiae (S. pastorianus syn. S. carlsbergensis), which are used to brew lager-style beers. We find that the S. eubayanus subgenomes of lager-brewing yeasts have experienced increased rates of evolution since hybridization, and that certain genes involved in metabolism may have been particularly affected. Interestingly, the S. eubayanus subgenome underwent an especially strong shift in selection regimes, consistent with more extensive domestication of the S. cerevisiae parent prior to hybridization. In contrast to recent proposals that lager-brewing yeasts were domesticated following a single hybridization event, the radically different neutral site divergences between the subgenomes of the two major lager yeast lineages strongly favor at least two independent origins for the S. cerevisiae × S. eubayanus hybrids that brew lager beers. Our findings demonstrate how this industrially important hybrid has been domesticated along similar evolutionary trajectories on multiple occasions. PMID:26269586

Entamoeba histolytica: construction and applications of subgenomic databases.

PubMed

Hofer, Margit; Duchêne, Michael

2005-07-01

Knowledge about the influence of environmental stress such as the action of chemotherapeutic agents on gene expression in Entamoeba histolytica is limited. We plan to use oligonucleotide microarray hybridization to approach these questions. As the basis for our array, sequence data from the genome project carried out by the Institute for Genomic Research (TIGR) and the Sanger Institute were used to annotate parts of the parasite genome. Three subgenomic databases containing enzymes, cytoskeleton genes, and stress genes were compiled with the help of the ExPASy proteomics website and the BLAST servers at the two genome project sites. The known sequences from reference species, mostly human and Escherichia coli, were searched against TIGR and Sanger E. histolytica sequence contigs and the homologs were copied into a Microsoft Access database. In a similar way, two additional databases of cytoskeletal genes and stress genes were generated. Metabolic pathways could be assembled from our enzyme database, but sometimes they were incomplete as is the case for the sterol biosynthesis pathway. The raw databases contained a significant number of duplicate entries which were merged to obtain curated non-redundant databases. This procedure revealed that some E. histolytica genes may have several putative functions. Representative examples such as the case of the delta-aminolevulinate synthase/serine palmitoyltransferase are discussed.

Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR.

PubMed

Hughesman, Curtis B; Lu, X J David; Liu, Kelly Y P; Zhu, Yuqi; Towle, Rebecca M; Haynes, Charles; Poh, Catherine F

2017-09-19

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

PubMed

Sun, Huihui; Li, Fanfan; Liu, Jie; Yang, Fayu; Zeng, Zhenhai; Lv, Xiujuan; Tu, Mengjun; Liu, Yeqing; Ge, Xianglian; Liu, Changbao; Zhao, Junzhao; Zhang, Zongduan; Qu, Jia; Song, Zongming; Gu, Feng

2018-06-15

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H-Related 3 Gene in Atypical Hemolytic Uremic Syndrome.

PubMed

Challis, Rachel C; Araujo, Geisilaine S R; Wong, Edwin K S; Anderson, Holly E; Awan, Atif; Dorman, Anthony M; Waldron, Mary; Wilson, Valerie; Brocklebank, Vicky; Strain, Lisa; Morgan, B Paul; Harris, Claire L; Marchbank, Kevin J; Goodship, Timothy H J; Kavanagh, David

2016-06-01

The regulators of complement activation cluster at chromosome 1q32 contains the complement factor H (CFH) and five complement factor H-related (CFHR) genes. This area of the genome arose from several large genomic duplications, and these low-copy repeats can cause genome instability in this region. Genomic disorders affecting these genes have been described in atypical hemolytic uremic syndrome, arising commonly through nonallelic homologous recombination. We describe a novel CFH/CFHR3 hybrid gene secondary to a de novo 6.3-kb deletion that arose through microhomology-mediated end joining rather than nonallelic homologous recombination. We confirmed a transcript from this hybrid gene and showed a secreted protein product that lacks the recognition domain of factor H and exhibits impaired cell surface complement regulation. The fact that the formation of this hybrid gene arose as a de novo event suggests that this cluster is a dynamic area of the genome in which additional genomic disorders may arise. Copyright © 2016 by the American Society of Nephrology.

Characterization of a Genomic Signature of Pregnancy in the Breast

PubMed Central

Belitskaya-Lévy, Ilana; Zeleniuch-Jacquotte, Anne; Russo, Jose; Russo, Irma H.; Bordás, Pal; Åhman, Janet; Afanasyeva, Yelena; Johansson, Robert; Lenner, Per; Li, Xiaochun; de Cicco, Ricardo López; Peri, Suraj; Ross, Eric; Russo, Patricia A.; Santucci-Pereira, Julia; Sheriff, Fathima S.; Slifker, Michael; Hallmans, Göran; Toniolo, Paolo; Arslan, Alan A.

2012-01-01

The objective of the current study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts; scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression and significance analysis for microarrays were used to identify statistically significant differences in expression of genes. The False Discovery Rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at a FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell cycle control, adhesion and differentiation. The results provide persuasive evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer. PMID:21622728

The Nucleolus: In Genome Maintenance and Repair

PubMed Central

Tsekrekou, Maria; Stratigi, Kalliopi; Chatzinikolaou, Georgia

2017-01-01

The nucleolus is the subnuclear membrane-less organelle where rRNA is transcribed and processed and ribosomal assembly occurs. During the last 20 years, however, the nucleolus has emerged as a multifunctional organelle, regulating processes that go well beyond its traditional role. Moreover, the unique organization of rDNA in tandem arrays and its unusually high transcription rates make it prone to unscheduled DNA recombination events and frequent RNA:DNA hybrids leading to DNA double strand breaks (DSBs). If not properly repaired, rDNA damage may contribute to premature disease onset and aging. Deregulation of ribosomal synthesis at any level from transcription and processing to ribosomal subunit assembly elicits a stress response and is also associated with disease onset. Here, we discuss how genome integrity is maintained within nucleoli and how such structures are functionally linked to nuclear DNA damage response and repair giving an emphasis on the newly emerging roles of the nucleolus in mammalian physiology and disease. PMID:28671574

CNV-seq, a new method to detect copy number variation using high-throughput sequencing.

PubMed

Xie, Chao; Tammi, Martti T

2009-03-06

DNA copy number variation (CNV) has been recognized as an important source of genetic variation. Array comparative genomic hybridization (aCGH) is commonly used for CNV detection, but the microarray platform has a number of inherent limitations. Here, we describe a method to detect copy number variation using shotgun sequencing, CNV-seq. The method is based on a robust statistical model that describes the complete analysis procedure and allows the computation of essential confidence values for detection of CNV. Our results show that the number of reads, not the length of the reads is the key factor determining the resolution of detection. This favors the next-generation sequencing methods that rapidly produce large amount of short reads. Simulation of various sequencing methods with coverage between 0.1x to 8x show overall specificity between 91.7 - 99.9%, and sensitivity between 72.2 - 96.5%. We also show the results for assessment of CNV between two individual human genomes.

Genome-scale transcriptional analyses of first-generation interspecific sunflower hybrids reveals broad regulatory compatibility

PubMed Central

2013-01-01

Background Interspecific hybridization creates individuals harboring diverged genomes. The interaction of these genomes can generate successful evolutionary novelty or disadvantageous genomic conflict. Annual sunflowers Helianthus annuus and H. petiolaris have a rich history of hybridization in natural populations. Although first-generation hybrids generally have low fertility, hybrid swarms that include later generation and fully fertile backcross plants have been identified, as well as at least three independently-originated stable hybrid taxa. We examine patterns of transcript accumulation in the earliest stages of hybridization of these species via analyses of transcriptome sequences from laboratory-derived F1 offspring of an inbred H. annuus cultivar and a wild H. petiolaris accession. Results While nearly 14% of the reference transcriptome showed significant accumulation differences between parental accessions, total F1 transcript levels showed little evidence of dominance, as midparent transcript levels were highly predictive of transcript accumulation in F1 plants. Allelic bias in F1 transcript accumulation was detected in 20% of transcripts containing sufficient polymorphism to distinguish parental alleles; however the magnitude of these biases were generally smaller than differences among parental accessions. Conclusions While analyses of allelic bias suggest that cis regulatory differences between H. annuus and H. petiolaris are common, their effect on transcript levels may be more subtle than trans-acting regulatory differences. Overall, these analyses found little evidence of regulatory incompatibility or dominance interactions between parental genomes within F1 hybrid individuals, although it is unclear whether this is a legacy or an enabler of introgression between species. PMID:23701699

A stable hybrid containing haploid genomes of two obligate diploid Candida species.

PubMed

Chakraborty, Uttara; Mohamed, Aiyaz; Kakade, Pallavi; Mugasimangalam, Raja C; Sadhale, Parag P; Sanyal, Kaustuv

2013-08-01

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.

Comparative studies of copy number variation detection methods for next-generation sequencing technologies.

PubMed

Duan, Junbo; Zhang, Ji-Gang; Deng, Hong-Wen; Wang, Yu-Ping

2013-01-01

Copy number variation (CNV) has played an important role in studies of susceptibility or resistance to complex diseases. Traditional methods such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) suffer from low resolution of genomic regions. Following the emergence of next generation sequencing (NGS) technologies, CNV detection methods based on the short read data have recently been developed. However, due to the relatively young age of the procedures, their performance is not fully understood. To help investigators choose suitable methods to detect CNVs, comparative studies are needed. We compared six publicly available CNV detection methods: CNV-seq, FREEC, readDepth, CNVnator, SegSeq and event-wise testing (EWT). They are evaluated both on simulated and real data with different experiment settings. The receiver operating characteristic (ROC) curve is employed to demonstrate the detection performance in terms of sensitivity and specificity, box plot is employed to compare their performances in terms of breakpoint and copy number estimation, Venn diagram is employed to show the consistency among these methods, and F-score is employed to show the overlapping quality of detected CNVs. The computational demands are also studied. The results of our work provide a comprehensive evaluation on the performances of the selected CNV detection methods, which will help biological investigators choose the best possible method.

Micro-dressing of a carbon nanotube array with MoS2 gauze

NASA Astrophysics Data System (ADS)

Lim, Sharon Xiaodai; Woo, Kah Whye; Ng, Junju; Lu, Junpeng; Kwang, Siu Yi; Zhang, Zheng; Tok, Eng Soon; Sow, Chorng-Haur

2015-10-01

Few-layer MoS2 film has been successfully assembled over an array of CNTs. Using different focused laser beams with different wavelengths, site selective patterning of either the MoS2 film or the supporting CNT array is achieved. This paves the way for applications and investigations into the fundamental properties of the hybrid MoS2/CNT material with a controlled architecture. Through Raman mapping, straining and electron doping of the MoS2 film as a result of interaction with the supporting CNT array are detected. The role of the MoS2 film was further emphasized with a lower work function being detected from Ultra-violet Photoelectron Spectrsocopy (UPS) measurements of the hybrid material, compared to the CNT array. The effect of the changes in the work function was illustrated through the optoelectronic behavior of the hybrid material. At 0 V, 3.49 nA of current is measured upon illuminating the sample with a broad laser beam emitting laser light with a wavelength of 532 nm. With a strong response to external irradiation of different wavelengths, and changes to the power of the excitation source, the hybrid material has shown potential for applications in optoelectronic devices.

Molecular cytogenetic analysis of Xq critical regions in premature ovarian failure

PubMed Central

2013-01-01

Background One of the frequent reasons for unsuccessful conception is premature ovarian failure/primary ovarian insufficiency (POF/POI) that is defined as the loss of functional follicles below the age of 40 years. Among the genetic causes the most common one involves the X chromosome, as in Turner syndrome, partial X deletion and X-autosome translocations. Here we report a case of a 27-year-old female patient referred to genetic counselling because of premature ovarian failure. The aim of this case study to perform molecular genetic and cytogenetic analyses in order to identify the exact genetic background of the pathogenic phenotype. Results For premature ovarian failure disease diagnostics we performed the Fragile mental retardation 1 gene analysis using Southern blot technique and Repeat Primed PCR in order to identify the relationship between the Fragile mental retardation 1 gene premutation status and the premature ovarion failure disease. At this early onset, the premature ovarian failure affected patient we detected one normal allele of Fragile mental retardation 1 gene and we couldn’t verify the methylated allele, therefore we performed the cytogenetic analyses using G-banding and fluorescent in situ hybridization methods and a high resolution molecular cytogenetic method, the array comparative genomic hybridization technique. For this patient applying the G-banding, we identified a large deletion on the X chromosome at the critical region (ChrX q21.31-q28) which is associated with the premature ovarian failure phenotype. In order to detect the exact breakpoints, we used a special cytogenetic array ISCA plus CGH array and we verified a 67.355 Mb size loss at the critical region which include total 795 genes. Conclusions We conclude for this case study that the karyotyping is definitely helpful in the evaluation of premature ovarian failure patients, to identify the non submicroscopic chromosomal rearrangement, and using the array CGH technique we can contribute to the most efficient detection and mapping of exact deletion breakpoints of the deleted Xq region. PMID:24359613

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels.

PubMed

Zhang, He; Liu, Lian; Li, Cheuk-Wing; Fu, Huayang; Chen, Yao; Yang, Mengsu

2011-11-15

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/μL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence. Copyright © 2011 Elsevier B.V. All rights reserved.

Genome-wide patterns of copy number variation in the diversified chicken genomes using next-generation sequencing.

PubMed

Yi, Guoqiang; Qu, Lujiang; Liu, Jianfeng; Yan, Yiyuan; Xu, Guiyun; Yang, Ning

2014-11-07

Copy number variation (CNV) is important and widespread in the genome, and is a major cause of disease and phenotypic diversity. Herein, we performed a genome-wide CNV analysis in 12 diversified chicken genomes based on whole genome sequencing. A total of 8,840 CNV regions (CNVRs) covering 98.2 Mb and representing 9.4% of the chicken genome were identified, ranging in size from 1.1 to 268.8 kb with an average of 11.1 kb. Sequencing-based predictions were confirmed at a high validation rate by two independent approaches, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR). The Pearson's correlation coefficients between sequencing and aCGH results ranged from 0.435 to 0.755, and qPCR experiments revealed a positive validation rate of 91.71% and a false negative rate of 22.43%. In total, 2,214 (25.0%) predicted CNVRs span 2,216 (36.4%) RefSeq genes associated with specific biological functions. Besides two previously reported copy number variable genes EDN3 and PRLR, we also found some promising genes with potential in phenotypic variation. Two genes, FZD6 and LIMS1, related to disease susceptibility/resistance are covered by CNVRs. The highly duplicated SOCS2 may lead to higher bone mineral density. Entire or partial duplication of some genes like POPDC3 may have great economic importance in poultry breeding. Our results based on extensive genetic diversity provide a more refined chicken CNV map and genome-wide gene copy number estimates, and warrant future CNV association studies for important traits in chickens.

Segmental Duplications and Copy-Number Variation in the Human Genome

PubMed Central

Sharp, Andrew J. ; Locke, Devin P. ; McGrath, Sean D. ; Cheng, Ze ; Bailey, Jeffrey A. ; Vallente, Rhea U. ; Pertz, Lisa M. ; Clark, Royden A. ; Schwartz, Stuart ; Segraves, Rick ; Oseroff, Vanessa V. ; Albertson, Donna G. ; Pinkel, Daniel ; Eichler, Evan E. 

2005-01-01

The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P < .000001), which suggests that segmental duplications are a major catalyst of large-scale variation in the human genome. Importantly, segmental duplications themselves were also significantly enriched >4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders. PMID:15918152

Genetic Progression of High Grade Prostatic Intraepithelial Neoplasia to Prostate Cancer.

PubMed

Jung, Seung-Hyun; Shin, Sun; Kim, Min Sung; Baek, In-Pyo; Lee, Ji Youl; Lee, Sung Hak; Kim, Tae-Min; Lee, Sug Hyung; Chung, Yeun-Jun

2016-05-01

Although high grade prostatic intraepithelial neoplasia (HGPIN) is considered a neoplastic lesion that precedes prostate cancer (PCA), the genomic structures of HGPIN remain unknown. Identification of the genomic landscape of HGPIN and the genomic differences between HGPIN and PCA that may drive the progression to PCA. We analyzed 20 regions of paired HGPIN and PCA from six patients using whole-exome sequencing and array-comparative genomic hybridization. Somatic mutation and copy number alteration (CNA) profiles of paired HGPIN and PCA were measured and compared. The number of total mutations and CNAs of HGPINs were significantly fewer than those of PCAs. Mutations in FOXA1 and CNAs (1q and 8q gains) were detected in both HGPIN and PCA ('common'), suggesting their roles in early PCA development. Mutations in SPOP, KDM6A, and KMT2D were 'PCA-specific', suggesting their roles in HGPIN progression to PCA. The 8p loss was either 'common' or 'PCA-specific'. In-silico estimation of evolutionary ages predicted that HGPIN genomes were much younger than PCA genomes. Our data show that PCAs are direct descendants of HGPINs in most cases that require more genomic alterations to progress to PCA. The nature of heterogeneous HGPIN population that might attenuate genomic signals should further be studied. HGPIN genomes harbor relatively fewer mutations and CNAs than PCA but require additional hits for the progression. In this study, we suggest a systemic diagram from high grade prostatic intraepithelial neoplasia (HGPIN) to prostate cancer (PCA). Our results provide a clue to explain the long latency from HGPIN to PCA and provide useful information for the genetic diagnosis of HGPIN and PCA. Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Comparison of GWAS models to identify non-additive genetic control of flowering time in sunflower hybrids.

PubMed

Bonnafous, Fanny; Fievet, Ghislain; Blanchet, Nicolas; Boniface, Marie-Claude; Carrère, Sébastien; Gouzy, Jérôme; Legrand, Ludovic; Marage, Gwenola; Bret-Mestries, Emmanuelle; Munos, Stéphane; Pouilly, Nicolas; Vincourt, Patrick; Langlade, Nicolas; Mangin, Brigitte

2018-02-01

This study compares five models of GWAS, to show the added value of non-additive modeling of allelic effects to identify genomic regions controlling flowering time of sunflower hybrids. Genome-wide association studies are a powerful and widely used tool to decipher the genetic control of complex traits. One of the main challenges for hybrid crops, such as maize or sunflower, is to model the hybrid vigor in the linear mixed models, considering the relatedness between individuals. Here, we compared two additive and three non-additive association models for their ability to identify genomic regions associated with flowering time in sunflower hybrids. A panel of 452 sunflower hybrids, corresponding to incomplete crossing between 36 male lines and 36 female lines, was phenotyped in five environments and genotyped for 2,204,423 SNPs. Intra-locus effects were estimated in multi-locus models to detect genomic regions associated with flowering time using the different models. Thirteen quantitative trait loci were identified in total, two with both model categories and one with only non-additive models. A quantitative trait loci on LG09, detected by both the additive and non-additive models, is located near a GAI homolog and is presented in detail. Overall, this study shows the added value of non-additive modeling of allelic effects for identifying genomic regions that control traits of interest and that could participate in the heterosis observed in hybrids.

Array comparative genomic hybridization screening in IVF significantly reduces number of embryos available for cryopreservation

PubMed Central

Liu, Jiaen; Yang, Zhihong; Salem, Shala A; Rahil, Tayyab; Collins, Gary S; Liu, Xiaohong; Salem, Rifaat D

2012-01-01

Objective During IVF, non-transferred embryos are usually selected for cryopreservation on the basis of morphological criteria. This investigation evaluated an application for array comparative genomic hybridization (aCGH) in assessment of surplus embryos prior to cryopreservation. Methods First-time IVF patients undergoing elective single embryo transfer and having at least one extra non-transferred embryo suitable for cryopreservation were offered enrollment in the study. Patients were randomized into two groups: Patients in group A (n=55) had embryos assessed first by morphology and then by aCGH, performed on cells obtained from trophectoderm biopsy on post-fertilization day 5. Only euploid embryos were designated for cryopreservation. Patients in group B (n=48) had embryos assessed by morphology alone, with only good morphology embryos considered suitable for cryopreservation. Results Among biopsied embryos in group A (n=425), euploidy was confirmed in 226 (53.1%). After fresh single embryo transfer, 64 (28.3%) surplus euploid embryos were cryopreserved for 51 patients (92.7%). In group B, 389 good morphology blastocysts were identified and a single top quality blastocyst was selected for fresh transfer. All group B patients (48/48) had at least one blastocyst remaining for cryopreservation. A total of 157 (40.4%) blastocysts were frozen in this group, a significantly larger proportion than was cryopreserved in group A (p=0.017, by chi-squared analysis). Conclusion While aCGH and subsequent frozen embryo transfer are currently used to screen embryos, this is the first investigation to quantify the impact of aCGH specifically on embryo cryopreservation. Incorporation of aCGH screening significantly reduced the total number of cryopreserved blastocysts compared to when suitability for freezing was determined by morphology only. IVF patients should be counseled that the benefits of aCGH screening will likely come at the cost of sharply limiting the number of surplus embryos available for cryopreservation. PMID:22816070

Rescue karyotyping: a case series of array-based comparative genomic hybridization evaluation of archival conceptual tissue

PubMed Central

2014-01-01

Background Determination of fetal aneuploidy is central to evaluation of recurrent pregnancy loss (RPL). However, obtaining this information at the time of a miscarriage is not always possible or may not have been ordered. Here we report on “rescue karyotyping”, wherein DNA extracted from archived paraffin-embedded pregnancy loss tissue from a prior dilation and curettage (D&C) is evaluated by array-based comparative genomic hybridization (aCGH). Methods A retrospective case series was conducted at an academic medical center. Patients included had unexplained RPL and a prior pregnancy loss for which karyotype information would be clinically informative but was unavailable. After extracting DNA from slides of archived tissue, aCGH with a reduced stringency approach was performed, allowing for analysis of partially degraded DNA. Statistics were computed using STATA v12.1 (College Station, TX). Results Rescue karyotyping was attempted on 20 specimens from 17 women. DNA was successfully extracted in 16 samples (80.0%), enabling analysis at either high or low resolution. The longest interval from tissue collection to DNA extraction was 4.2 years. There was no significant difference in specimen sufficiency for analysis in the collection-to-extraction interval (p = 0.14) or gestational age at pregnancy loss (p = 0.32). Eight specimens showed copy number variants: 3 trisomies, 2 partial chromosomal deletions, 1 mosaic abnormality and 2 unclassified variants. Conclusions Rescue karyotyping using aCGH on DNA extracted from paraffin-embedded tissue provides the opportunity to obtain critical fetal cytogenetic information from a prior loss, even if it occurred years earlier. Given the ubiquitous archiving of paraffin embedded tissue obtained during a D&C and the ease of obtaining results despite long loss-to-testing intervals or early gestational age at time of fetal demise, this may provide a useful technique in the evaluation of couples with recurrent pregnancy loss. PMID:24589081

Partial trisomy 16p (16p12.2→pter) and partial monosomy 22q (22q13.31 →qter) presenting with fetal ascites and ventriculomegaly: prenatal diagnosis and array comparative genomic hybridization characterization.

PubMed

Chen, Chih-Ping; Su, Yi-Ning; Young, Richard Shih-Hung; Tsai, Fuu-Jen; Wu, Pei-Chen; Chern, Schu-Rern; Town, Dai-Dyi; Pan, Chen-Wen; Wang, Wayseen

2010-12-01

To present prenatal diagnosis and array comparative genomic hybridization (aCGH) characterization of partial trisomy 16p (16p12.2→pter) and partial monosomy 22q (22q13.31→qter) presenting with fetal ascites and ventriculomegaly in the second trimester. A 31-year-old woman, gravida 2, para 1, was referred to the hospital at 20 weeks of gestation because of fetal ascites. Amniocentesis revealed a derivative chromosome 22. Subsequent parental karyotyping revealed that the father carried a balanced reciprocal translocation between 16p12 and 22q13. Bacterial artificial chromosome-based aCGH using amniocyte DNA demonstrated partial trisomy 16p and partial monosomy 22q [arr cgh 16p13.3p12.2 (CTD-3077J14→RP11-650D5)x3, 22q13.31q13.33 (RP1-111J24→CTD-3035C16)x1]. Oligonucleotide-based aCGH showed a 20.9-Mb duplication of distal 16p and an approximate 3.7-Mb deletion of distal 22q. Level II ultrasound revealed fetal ascites and ventriculomegaly. The pregnancy was terminated and a malformed male fetus was delivered with craniofacial dysmorphism and abnormalities of the digits. The fetal karyotype was 46,XY,der(22)t(16;22)(p12.2;q13.31)pat. The paternal karyotype was 46,XY,t(16;22)(p12.2;q13.31). Partial trisomy 16p can be associated with fetal ascites and ventriculomegaly in the second trimester. Prenatal sonographic detection of fetal ascites in association with ventriculomegaly should alert chromosomal abnormalities and prompt cytogenetic investigation, which may lead to the identification of an unexpected parental translocation involving chromosomal segments associated with cerebral and vascular abnormalities. Copyright © 2010 Taiwan Association of Obstetric & Gynecology. Published by Elsevier B.V. All rights reserved.

Partial monosomy 13q (13q21.32--->qter) and partial trisomy 8p (8p1--->pter) presenting with anencephaly and increased nuchal translucency: array comparative genomic hybridization characterization.

PubMed

Chen, Chih-Ping; Su, Yi-Ning; Tsai, Fuu-Jen; Lin, Ming-Huei; Wu, Pei-Chen; Chern, Schu-Rern; Lee, Chen-Chi; Pan, Chen-Wen; Wang, Wayseen

2011-06-01

To present array comparative genomic hybridization (aCGH) characterization of partial monosomy 13q (13q21.32→qter) and partial trisomy 8p (8p12→pter) presenting with anencephaly and increased nuchal translucency (NT). A 34-year-old primigravid woman was referred to the hospital at 12 weeks of gestation for termination of the pregnancy because of major structural abnormalities of the fetus. Prenatal ultrasound revealed a malformed fetus with anencephaly and an increased NT thickness of 5mm at 12 weeks of gestation. Cytogenetic analysis of the fetus revealed a derivative chromosome 13. The mother was subsequently found to carry a balanced reciprocal translocation between 8p12 and 13q21. Bacterial artificial chromosome-based aCGH using fetal DNA demonstrated partial trisomy 8p and partial monosomy 13q [arr cgh 8p23.3p12 (RP11-1150M5→RP11-1145H12)×3, 13q21.32q34 (RP11-326B4→RP11-450H16)×1]. Oligonucleotide-based aCGH showed a 36.7-Mb duplication of distal 8p and a 48.4-Mb deletion of distal 13q. The fetal karyotype was 46,XY,der(13) t(8;13)(p12;q21.32)mat. The maternal karyotype was 46,XX,t(8;13)(p12;q21.32). The 13q deletion syndrome can be associated with neural tube defects and increased NT in the first trimester. Prenatal sonographic detection of neural tube defects should alert chromosomal abnormalities and prompt cytogenetic investigation, which may lead to the identification of an unexpected parental translocation involving chromosomal segments associated with neural tube development. Copyright © 2011. Published by Elsevier B.V.

Search for Rare Copy-Number Variants in Congenital Heart Defects Identifies Novel Candidate Genes and a Potential Role for FOXC1 in Patients With Coarctation of the Aorta.

PubMed

Sanchez-Castro, Marta; Eldjouzi, Hadja; Charpentier, Eric; Busson, Pierre-François; Hauet, Quentin; Lindenbaum, Pierre; Delasalle-Guyomarch, Béatrice; Baudry, Adrien; Pichon, Olivier; Pascal, Cécile; Lefort, Bruno; Bajolle, Fanny; Pezard, Philippe; Schott, Jean-Jacques; Dina, Christian; Redon, Richard; Gournay, Véronique; Bonnet, Damien; Le Caignec, Cédric

2016-02-01

Congenital heart defects are the most frequent malformations among newborns and a frequent cause of morbidity and mortality. Although genetic variation contributes to congenital heart defects, their precise molecular bases remain unknown in the majority of patients. We analyzed, by high-resolution array comparative genomic hybridization, 316 children with sporadic, nonsyndromic congenital heart defects, including 76 coarctation of the aorta, 159 transposition of the great arteries, and 81 tetralogy of Fallot, as well as their unaffected parents. We identified by array comparative genomic hybridization, and validated by quantitative real-time polymerase chain reaction, 71 rare de novo (n=8) or inherited (n=63) copy-number variants (CNVs; 50 duplications and 21 deletions) in patients. We identified 113 candidate genes for congenital heart defects within these CNVs, including BTRC, CHRNB3, CSRP2BP, ERBB2, ERMARD, GLIS3, PLN, PTPRJ, RLN3, and TCTE3. No de novo CNVs were identified in patients with transposition of the great arteries in contrast to coarctation of the aorta and tetralogy of Fallot (P=0.002; Fisher exact test). A search for transcription factor binding sites showed that 93% of the rare CNVs identified in patients with coarctation of the aorta contained at least 1 gene with FOXC1-binding sites. This significant enrichment (P<0.0001; permutation test) was not observed for the CNVs identified in patients with transposition of the great arteries and tetralogy of Fallot. We hypothesize that these CNVs may alter the expression of genes regulated by FOXC1. Foxc1 belongs to the forkhead transcription factors family, which plays a critical role in cardiovascular development in mice. These data suggest that deregulation of FOXC1 or its downstream genes play a major role in the pathogenesis of coarctation of the aorta in humans. © 2015 American Heart Association, Inc.

Autism-specific copy number variants further implicate the phosphatidylinositol signaling pathway and the glutamatergic synapse in the etiology of the disorder

PubMed Central

Cuscó, Ivon; Medrano, Andrés; Gener, Blanca; Vilardell, Mireia; Gallastegui, Fátima; Villa, Olaya; González, Eva; Rodríguez-Santiago, Benjamín; Vilella, Elisabet; Del Campo, Miguel; Pérez-Jurado, Luis A.

2009-01-01

Autism spectrum disorders (ASDs) constitute a group of severe neurodevelopmental conditions with complex multifactorial etiology. In order to explore the hypothesis that submicroscopic genomic rearrangements underlie some ASD cases, we have analyzed 96 Spanish patients with idiopathic ASD after extensive clinical and laboratory screening, by array comparative genomic hybridization (aCGH) using a homemade bacterial artificial chromosome (BAC) array. Only 13 of the 238 detected copy number alterations, ranging in size from 89 kb to 2.4 Mb, were present specifically in the autistic population (12 out of 96 individuals, 12.5%). Following validation by additional molecular techniques, we have characterized these novel candidate regions containing 24 different genes including alterations in two previously reported regions of chromosome 7 associated with the ASD phenotype. Some of the genes located in ASD-specific copy number variants act in common pathways, most notably the phosphatidylinositol signaling and the glutamatergic synapse, both known to be affected in several genetic syndromes related with autism and previously associated with ASD. Our work supports the idea that the functional alteration of genes in related neuronal networks is involved in the etiology of the ASD phenotype and confirms a significant diagnostic yield for aCGH, which should probably be included in the diagnostic workup of idiopathic ASD. PMID:19246517

A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

PubMed

Möhlendick, Birte; Bartenhagen, Christoph; Behrens, Bianca; Honisch, Ellen; Raba, Katharina; Knoefel, Wolfram T; Stoecklein, Nikolas H

2013-01-01

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

Metabolic Capacity of Sinorhizobium (Ensifer) meliloti Strains as Determined by Phenotype MicroArray Analysis▿ †

PubMed Central

Biondi, Emanuele G.; Tatti, Enrico; Comparini, Diego; Giuntini, Elisa; Mocali, Stefano; Giovannetti, Luciana; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo

2009-01-01

Sinorhizobium meliloti is a soil bacterium that fixes atmospheric nitrogen in plant roots. The high genetic diversity of its natural populations has been the subject of extensive analysis. Recent genomic studies of several isolates revealed a high content of variable genes, suggesting a correspondingly large phenotypic differentiation among strains of S. meliloti. Here, using the Phenotype MicroArray (PM) system, hundreds of different growth conditions were tested in order to compare the metabolic capabilities of the laboratory reference strain Rm1021 with those of four natural S. meliloti isolates previously analyzed by comparative genomic hybridization (CGH). The results of PM analysis showed that most phenotypic differences involved carbon source utilization and tolerance to osmolytes and pH, while fewer differences were scored for nitrogen, phosphorus, and sulfur source utilization. Only the variability of the tested strain in tolerance to sodium nitrite and ammonium sulfate of pH 8 was hypothesized to be associated with the genetic polymorphisms detected by CGH analysis. Colony and cell morphologies and the ability to nodulate Medicago truncatula plants were also compared, revealing further phenotypic diversity. Overall, our results suggest that the study of functional (phenotypic) variability of S. meliloti populations is an important and complementary step in the investigation of genetic polymorphism of rhizobia and may help to elucidate rhizobial evolutionary dynamics, including adaptation to diverse environments. PMID:19561177

Rapid construction of genome map for large yellow croaker (Larimichthys crocea) by the whole-genome mapping in BioNano Genomics Irys system.

PubMed

Xiao, Shijun; Li, Jiongtang; Ma, Fengshou; Fang, Lujing; Xu, Shuangbin; Chen, Wei; Wang, Zhi Yong

2015-09-03

Large yellow croaker (Larimichthys crocea) is an important commercial fish in China and East-Asia. The annual product of the species from the aqua-farming industry is about 90 thousand tons. In spite of its economic importance, genetic studies of economic traits and genomic selections of the species are hindered by the lack of genomic resources. Specifically, a whole-genome physical map of large yellow croaker is still missing. The traditional BAC-based fingerprint method is extremely time- and labour-consuming. Here we report the first genome map construction using the high-throughput whole-genome mapping technique by nanochannel arrays in BioNano Genomics Irys system. For an optimal marker density of ~10 per 100 kb, the nicking endonuclease Nt.BspQ1 was chosen for the genome map generation. 645,305 DNA molecules with a total length of ~112 Gb were labelled and detected, covering more than 160X of the large yellow croaker genome. Employing IrysView package and signature patterns in raw DNA molecules, a whole-genome map of large yellow croaker was assembled into 686 maps with a total length of 727 Mb, which was consistent with the estimated genome size. The N50 length of the whole-genome map, including 126 maps, was up to 1.7 Mb. The excellent hybrid alignment with large yellow croaker draft genome validated the consensus genome map assembly and highlighted a promising application of whole-genome mapping on draft genome sequence super-scaffolding. The genome map data of large yellow croaker are accessible on lycgenomics.jmu.edu.cn/pm. Using the state-of-the-art whole-genome mapping technique in Irys system, the first whole-genome map for large yellow croaker has been constructed and thus highly facilitates the ongoing genomic and evolutionary studies for the species. To our knowledge, this is the first public report on genome map construction by the whole-genome mapping for aquatic-organisms. Our study demonstrates a promising application of the whole-genome mapping on genome maps construction for other non-model organisms in a fast and reliable manner.

Centromere separation and association in the nuclei of an interspecific hybrid between Torenia fournieri and T. baillonii (Scrophulariaceae) during mitosis and meiosis.

PubMed

Kikuchi, Shinji; Tanaka, Hiroyuki; Wako, Toshiyuki; Tsujimoto, Hisashi

2007-10-01

In the nuclei of some interspecific hybrid and allopolyploid plant species, each genome occupies a separate spatial domain. To analyze this phenomenon, we studied localization of the centromeres in the nuclei of a hybrid between Torenia fournieri and T. baillonii during mitosis and meiosis using three-dimensional fluorescence in situ hybridization (3D-FISH) probed with species-specific centromere repeats. Centromeres of each genome were located separately in undifferentiated cells but not differentiated cells, suggesting that cell division might be the possible force causing centromere separation. However, no remarkable difference of dividing distance was detected between chromatids with different centromeres in anaphase and telophase, indicating that tension of the spindle fiber attached to each chromatid is not the cause of centromere separation in Torenia. In differentiated cells, centromeres in both genomes were not often observed for the expected chromosome number, indicating centromere association. In addition, association of centromeres from the same genome was observed at a higher frequency than between different genomes. This finding suggests that centromeres within one genome are spatially separated from those within the other. This close position may increase possibility of association between centromeres of the same genome. In meiotic prophase, all centromeres irrespective of the genome were associated in a certain portion of the nucleus. Since centromere association in the interspecific hybrid and amphiploid was tighter than that in the diploid parents, it is possible that this phenomenon may be involved in sorting and pairing of homologous chromosomes.

Jumping genes: Genomic ballast or powerhouse of biological diversification.

PubMed

Choudhury, Rimjhim Roy; Parisod, Christian

2017-09-01

Studying hybridization has the potential to elucidate challenging questions in evolutionary biology such as the nature of adaptive genetic variation and reproductive isolation. A growing body of work highlights that the merging of divergent genomes goes beyond the reshuffling of standing variation from related species and promotes mutations (Abbott et al., ). However, to what extent such genome instability generates evolutionary significant variation remains largely elusive. In this issue of Molecular Ecology, Dennenmoser et al. () report considerable dynamics of transposable elements (TEs) in a recent invasive fish species of hybrid origin (Cottus; Figure ). It adds to the recent examples from plants to support TE-specific genome variation following hybridization. Insights from early, as well as established, hybrids are largely coherent with increased TE activity, and this fish system thus represents an inspiring opportunity to further address the possible association between genome dynamics and "rapid evolution of hybrid species." This work based on genome (re)sequencing contrasts with prior transcriptomics or PCR-based studies of TEs and illustrates how unprecedented amount of information promises a better understanding of the multiple patterns of variation across eukaryotic genomes; provided that we get the better of methodological advances. As discussed here, unbiased assessment of TE variation from genome surveys indeed remains a challenge precluding firm conclusions to be reached about the evolutionary significance of TEs. Despite methodological and conceptual developments that appear necessary to unambiguously uncover the unexplored iceberg below the known tip, the role of coding genes vs. TEs in promoting adaptation and speciation might be clarified in a not so remote future. © 2017 John Wiley & Sons Ltd.

Blocked impurity band hybrid infrared focal plane arrays for astronomy

NASA Technical Reports Server (NTRS)

Reynolds, D. B.; Seib, D. H.; Stetson, S. B.; Herter, T.; Rowlands, N.

1989-01-01

High-performance infrared hybrid focal plane arrays using 10- x 50-element Si:As blocked-impurity-band (BIB) detectors (cutoff wavelength = 28 microns) and matching switched MOSFET multiplexers have been developed and characterized for space astronomy. Use of impurity-band-conduction technology provides detectors which are nuclear-radiation-hard and free of the many anomalies associated with conventional silicon photoconductive detectors. Emphasis in the present work is on recent advances in detector material quality which have led to significantly improved detector and hybrid characteristics. Results demonstrating increased quantum efficiency (particularly at short-wavelength infrared), obtained by varying the BIB detector properties (infrared active layer thickness and arsenic doping profile), are summarized. Measured read noise and dark current for different temperatures are reported. The hybrid array performance achieved demonstrates that BIB detectors are well suited for use in astronomical instrumentation.

Hybridization Reveals the Evolving Genomic Architecture of Speciation

PubMed Central

Kronforst, Marcus R.; Hansen, Matthew E.B.; Crawford, Nicholas G.; Gallant, Jason R.; Zhang, Wei; Kulathinal, Rob J.; Kapan, Durrell D.; Mullen, Sean P.

2014-01-01

SUMMARY The rate at which genomes diverge during speciation is unknown, as are the physical dynamics of the process. Here, we compare full genome sequences of 32 butterflies, representing five species from a hybridizing Heliconius butterfly community, to examine genome-wide patterns of introgression and infer how divergence evolves during the speciation process. Our analyses reveal that initial divergence is restricted to a small fraction of the genome, largely clustered around known wing-patterning genes. Over time, divergence evolves rapidly, due primarily to the origin of new divergent regions. Furthermore, divergent genomic regions display signatures of both selection and adaptive introgression, demonstrating the link between microevolutionary processes acting within species and the origin of species across macroevolutionary timescales. Our results provide a uniquely comprehensive portrait of the evolving species boundary due to the role that hybridization plays in reducing the background accumulation of divergence at neutral sites. PMID:24183670

aCGH Analysis to Estimate Genetic Variations among Domesticated Chickens

PubMed Central

Lin, Mengjie

2016-01-01

Chickens have been familiar to humans since ancient times and have been used not only for culinary purposes but also for cultural purposes including ritual ceremonies and traditional entertainment. The various chicken breeds developed for these purposes often display distinct morphological and/or behavioural traits. For example, the Japanese Shamo is larger and more aggressive than other domesticated chickens, reflecting its role as a fighting cock breed, whereas Japanese Naganakidori breeds, which have long-crowing behaviour, were bred instead for their entertaining and aesthetic qualities. However, the genetic backgrounds of these distinct morphological and behavioural traits remain unclear. Therefore, the question arises as to which genomic regions in these chickens were acted upon by selective pressures through breeding. We compared the entire genomes of six chicken breeds domesticated for various cultural purposes by utilizing array comparative genomic hybridization. From these analyses, we identified 782 regions that underwent insertions, deletions, or mutations, representing man-made selection pressure in these chickens. Furthermore, we found that a number of genes diversified in domesticated chickens bred for cultural or entertainment purposes were different from those diversified in chickens bred for food, such as broilers and layers. PMID:27525263

Integrated analysis of epigenomic and genomic changes by DNA methylation dependent mechanisms provides potential novel biomarkers for prostate cancer.

PubMed

White-Al Habeeb, Nicole M A; Ho, Linh T; Olkhov-Mitsel, Ekaterina; Kron, Ken; Pethe, Vaijayanti; Lehman, Melanie; Jovanovic, Lidija; Fleshner, Neil; van der Kwast, Theodorus; Nelson, Colleen C; Bapat, Bharati

2014-09-15

Epigenetic silencing mediated by CpG methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with tumor progression may identify potential prognostic markers for prostate cancer (PCa). We treated two PCa cell lines, 22Rv1 and DU-145 with the demethylating agent 5-Aza 2'-deoxycitidine (DAC) and global methylation status was analyzed by performing methylation-sensitive restriction enzyme based differential methylation hybridization strategy followed by genome-wide CpG methylation array profiling. In addition, we examined gene expression changes using a custom microarray. Gene Set Enrichment Analysis (GSEA) identified the most significantly dysregulated pathways. In addition, we assessed methylation status of candidate genes that showed reduced CpG methylation and increased gene expression after DAC treatment, in Gleason score (GS) 8 vs. GS6 patients using three independent cohorts of patients; the publically available The Cancer Genome Atlas (TCGA) dataset, and two separate patient cohorts. Our analysis, by integrating methylation and gene expression in PCa cell lines, combined with patient tumor data, identified novel potential biomarkers for PCa patients. These markers may help elucidate the pathogenesis of PCa and represent potential prognostic markers for PCa patients.

Competitive hybridization models

NASA Astrophysics Data System (ADS)

Cherepinsky, Vera; Hashmi, Ghazala; Mishra, Bud

2010-11-01

Microarray technology, in its simplest form, allows one to gather abundance data for target DNA molecules, associated with genomes or gene-expressions, and relies on hybridizing the target to many short probe oligonucleotides arrayed on a surface. While for such multiplexed reactions conditions are optimized to make the most of each individual probe-target interaction, subsequent analysis of these experiments is based on the implicit assumption that a given experiment yields the same result regardless of whether it was conducted in isolation or in parallel with many others. It has been discussed in the literature that this assumption is frequently false, and its validity depends on the types of probes and their interactions with each other. We present a detailed physical model of hybridization as a means of understanding probe interactions in a multiplexed reaction. Ultimately, the model can be derived from a system of ordinary differential equations (ODE’s) describing kinetic mass action with conservation-of-mass equations completing the system. We examine pairwise probe interactions in detail and present a model of “competition” between the probes for the target—especially, when the target is effectively in short supply. These effects are shown to be predictable from the affinity constants for each of the four probe sequences involved, namely, the match and mismatch sequences for both probes. These affinity constants are calculated from the thermodynamic parameters such as the free energy of hybridization, which are in turn computed according to the nearest neighbor (NN) model for each probe and target sequence. Simulations based on the competitive hybridization model explain the observed variability in the signal of a given probe when measured in parallel with different groupings of other probes or individually. The results of the simulations can be used for experiment design and pooling strategies, based on which probes have been shown to have a strong effect on each other’s signal in the in silico experiment. These results are aimed at better design of multiplexed reactions on arrays used in genotyping (e.g., HLA typing, SNP, or CNV detection, etc.) and mutation analysis (e.g., cystic fibrosis, cancer, autism, etc.).

Interspecific hybridization as a genomic stressor inducing mobilization of transposable elements in Drosophila

PubMed Central

Guerreiro, Maria Pilar García

2014-01-01

Transposable elements (TEs) are DNA sequences able to be mobilized in host genomes. They are currently recognized as the major mutation inducers because of their insertion in the target, their effect on neighboring regions, or their ectopic recombination. A large number of factors including chemical and physical factors as well as intraspecific crosses have traditionally been identified as inducers of transposition. Besides environmental factors, interspecific crosses have also been proposed as promoters of transposition of particular TEs in plants and different animals. Our previous published work includes a genome-wide survey with the set of genomic TEs and shows that interspecific hybridization between the species Drosophila buzzatii and Drosophila koepferae induces genomic instability by transposition bursts. A high percentage of this instability corresponds to TEs belonging to classes I and II. The detailed study of three TEs (Osvaldo, Helena, and Galileo), representative of the different TE families, shows an increase of transposition in hybrids compared with parental species, that varies depending on the element. This study suggests ample variation in TE regulation mechanisms and the question is why this variation occurs. Interspecific hybridization is a genomic stressor that disrupts the stability of TEs probably contributing to a relaxation of the mechanisms controlling TEs in the Drosophila genome. In this commentary paper we will discuss these results and the molecular mechanisms that could explain these increases of transposition rates observed in interspecific Drosophila hybrids. PMID:25136509

Whole Genome Analyses of a Well-Differentiated Liposarcoma Reveals Novel SYT1 and DDR2 Rearrangements

PubMed Central

Egan, Jan B.; Barrett, Michael T.; Champion, Mia D.; Middha, Sumit; Lenkiewicz, Elizabeth; Evers, Lisa; Francis, Princy; Schmidt, Jessica; Shi, Chang-Xin; Van Wier, Scott; Badar, Sandra; Ahmann, Gregory; Kortuem, K. Martin; Boczek, Nicole J.; Fonseca, Rafael; Craig, David W.; Carpten, John D.; Borad, Mitesh J.; Stewart, A. Keith

2014-01-01

Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2. PMID:24505276

Oligonucleotide Array for Identification and Detection of Pythium Species†

PubMed Central

Tambong, J. T.; de Cock, A. W. A. M.; Tinker, N. A.; Lévesque, C. A.

2006-01-01

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies. PMID:16597974

Development of a gallium-doped germanium far-infrared photoconductor direct hybrid two-dimensional array.

PubMed

Fujiwara, Mikio; Hirao, Takanori; Kawada, Mitsunobu; Shibai, Hiroshi; Matsuura, Shuji; Kaneda, Hidehiro; Patrashin, Mikhail; Nakagawa, Takao

2003-04-20

To our knowledge, we are the first to successfully report a direct hybrid two-dimensional (2D) detector array in the far-infrared region. Gallium-doped germanium (Ge:Ga) has been used extensively to produce sensitive far-infrared detectors with a cutoff wavelength of approximately equal to 110 microm (2.7 THz). It is widely used in the fields of astronomy and molecular and solid spectroscopy. However, Ge:Ga photoconductors must be cooled below 4.2 K to reduce thermal noise, and this operating condition makes it difficult to develop a large format array because of the need for a warm amplifier. Development of Ge:Ga photoconductor arrays to take 2D terahertz images is now an important target in such research fields as space astronomy. We present the design of a 20 x 3 Ge:Ga far-infrared photoconductor array directly hybridized to a Si p-type metal-oxide-semiconductor readout integrated circuit using indium-bump technology. The main obstacles in creating this 2D array were (1) fabricating a monolithic Ge:Ga 2D array with a longitudinal configuration, (2) developing a cryogenic capacitive transimpedance amplifer, and (3) developing a technology for connecting the detector to the electronics. With this technology, a prototype Ge:Ga photoconductor with a direct hybrid structure has shown a responsivity as high as 14.6 A/W and a minimum detectable power of 5.6 x 10(-17) W for an integration time of 0.14 s when it was cooled to 2.1 K. Its noise is limited by the readout circuit with 20 microV/Hz(1/2) at 1 Hz. Vibration and cooling tests demonstrated that this direct hybrid structure is strong enough for spaceborne instruments. This detector array will be installed on the Japanese infrared satellite ASTRO-F.

Genomic and environmental selection patterns in two distinct lettuce crop–wild hybrid crosses

PubMed Central

Hartman, Yorike; Uwimana, Brigitte; Hooftman, Danny A P; Schranz, Michael E; van de Wiel, Clemens C M; Smulders, Marinus J M; Visser, Richard G F; van Tienderen, Peter H

2013-01-01

Genomic selection patterns and hybrid performance influence the chance that crop (trans)genes can spread to wild relatives. We measured fitness(-related) traits in two different field environments employing two different crop–wild crosses of lettuce. We performed quantitative trait loci (QTL) analyses and estimated the fitness distribution of early- and late-generation hybrids. We detected consistent results across field sites and crosses for a fitness QTL at linkage group 7, where a selective advantage was conferred by the wild allele. Two fitness QTL were detected on linkage group 5 and 6, which were unique to one of the crop–wild crosses. Average hybrid fitness was lower than the fitness of the wild parent, but several hybrid lineages outperformed the wild parent, especially in a novel habitat for the wild type. In early-generation hybrids, this may partly be due to heterosis effects, whereas in late-generation hybrids transgressive segregation played a major role. The study of genomic selection patterns can identify crop genomic regions under negative selection across multiple environments and cultivar–wild crosses that might be applicable in transgene mitigation strategies. At the same time, results were cultivar-specific, so that a case-by-case environmental risk assessment is still necessary, decreasing its general applicability. PMID:23789025

Genomic and environmental selection patterns in two distinct lettuce crop-wild hybrid crosses.

PubMed

Hartman, Yorike; Uwimana, Brigitte; Hooftman, Danny A P; Schranz, Michael E; van de Wiel, Clemens C M; Smulders, Marinus J M; Visser, Richard G F; van Tienderen, Peter H

2013-06-01

Genomic selection patterns and hybrid performance influence the chance that crop (trans)genes can spread to wild relatives. We measured fitness(-related) traits in two different field environments employing two different crop-wild crosses of lettuce. We performed quantitative trait loci (QTL) analyses and estimated the fitness distribution of early- and late-generation hybrids. We detected consistent results across field sites and crosses for a fitness QTL at linkage group 7, where a selective advantage was conferred by the wild allele. Two fitness QTL were detected on linkage group 5 and 6, which were unique to one of the crop-wild crosses. Average hybrid fitness was lower than the fitness of the wild parent, but several hybrid lineages outperformed the wild parent, especially in a novel habitat for the wild type. In early-generation hybrids, this may partly be due to heterosis effects, whereas in late-generation hybrids transgressive segregation played a major role. The study of genomic selection patterns can identify crop genomic regions under negative selection across multiple environments and cultivar-wild crosses that might be applicable in transgene mitigation strategies. At the same time, results were cultivar-specific, so that a case-by-case environmental risk assessment is still necessary, decreasing its general applicability.

Genomic islands of differentiation in two songbird species reveal candidate genes for hybrid female sterility.

PubMed

Mořkovský, Libor; Janoušek, Václav; Reif, Jiří; Rídl, Jakub; Pačes, Jan; Choleva, Lukáš; Janko, Karel; Nachman, Michael W; Reifová, Radka

2018-02-01

Hybrid sterility is a common first step in the evolution of postzygotic reproductive isolation. According to Haldane's Rule, it affects predominantly the heterogametic sex. While the genetic basis of hybrid male sterility in organisms with heterogametic males has been studied for decades, the genetic basis of hybrid female sterility in organisms with heterogametic females has received much less attention. We investigated the genetic basis of reproductive isolation in two closely related avian species, the common nightingale (Luscinia megarhynchos) and the thrush nightingale (L. luscinia), that hybridize in a secondary contact zone and produce viable hybrid progeny. In accordance with Haldane's Rule, hybrid females are sterile, while hybrid males are fertile, allowing gene flow to occur between the species. Using transcriptomic data from multiple individuals of both nightingale species, we identified genomic islands of high differentiation (F ST ) and of high divergence (D xy ), and we analysed gene content and patterns of molecular evolution within these islands. Interestingly, we found that these islands were enriched for genes related to female meiosis and metabolism. The islands of high differentiation and divergence were also characterized by higher levels of linkage disequilibrium than the rest of the genome in both species indicating that they might be situated in genomic regions of low recombination. This study provides one of the first insights into genetic basis of hybrid female sterility in organisms with heterogametic females. © 2018 John Wiley & Sons Ltd.

Formation of diploid and triploid hybrid groupers (hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂) and their 5S gene analysis.

PubMed

Huang, Wen; Qin, Qinbo; Yang, Huirong; Li, Shuisheng; Hu, Chaoqun; Wang, Yude; Zhang, Yong; Liu, Shaojun; Lin, Haoran

2016-10-07

Interspecies hybridization is widely used to achieve heterosis or hybrid vigor, which has been observed and harnessed by breeders for centuries. Natural allopolyploid hybrids generally exhibit more superior heterosis than both the diploid progenies and their parental species. However, polyploid formation processes have been long ignored, the genetic basis of heterosis in polyploids remains elusive. In the present study, triploid hybrids had been demonstrated to contain two sets of chromosomes from mother species and one set from father species. Cellular polyploidization process in the embryos had been traced. The triploid hybrids might be formed by failure formation of the second polarized genome during the second meiosis stage. Four spindle centers were observed in anaphase stage of the first cell division. Three spindle centers were observed in side of cell plate after the first cell division. The 5S rDNA genes of four types of groupers were cloned and analyzed. The diploid and triploid hybrids had been proved to contain the tandem chimera structures which were recombined by maternal and paternal monomer units. The results indicated that genome re-fusion had occurred in the hybrid progenies. To further elucidate the genetic patterns of diploid and triploid hybrids, fluorescence chromosome location had been carried out, maternal 5S gene (M-386) were used as the probe. The triploid hybrids contained fewer fluorescence loci numbers than the maternal species. The results indicated that participation of paternal 5S gene in the triploid hybrid genome had degraded the match rates of M-386 probe. Our study is the first to investigate the cellular formation processes of natural allopolyploids in hybrid fish, the cellular polyploidization process may be caused by failure formation of the second polarized genome during the meiosis, and our results will provide the molecular basis of hybrid vigor in interspecies hybridization.

RUSSIAS HYBRID APPROACH: WHAT CAN NATO DO TO SHARPEN ITS SWORD AGAINST IT

DTIC Science & Technology

2016-02-16

to maintain its survival and territorial integrity. Russian President Vladimir Putin prefers to cloak his sword under an array of hybrid tools such...prefers to cloak his “sharp sword” under an array of hybrid tools such as misinformation, cyber attacks and special purpose forces intended to create...Ukraine, there are diplomatic and informational actions that NATO can employ to blunt Putin’s cloaked sword. Russia’s Struggle for Survival and

The genome sequence of Saccharomyces eubayanus and the domestication of lager-brewing yeasts

DOE PAGES

Baker, Emily Clare; Wang, Bing; Bellora, Nicolas; ...

2015-08-11

The dramatic phenotypic changes that occur in organisms during domestication leave indelible imprints on their genomes. Although many domesticated plants and animals have been systematically compared with their wild genetic stocks, the molecular and genomic processes underlying fungal domestication have received less attention. Here, we present a nearly complete genome assembly for the recently described yeast species Saccharomyces eubayanus and compare it to the genomes of multiple domesticated alloploid hybrids of S. eubayanus × S. cerevisiae ( S. pastorianus syn. S. carlsbergensis), which are used to brew lager-style beers. We find that the S. eubayanus subgenomes of lager-brewing yeasts havemore » experienced increased rates of evolution since hybridization, and that certain genes involved in metabolism may have been particularly affected. Interestingly, the S. eubayanus subgenome underwent an especially strong shift in selection regimes, consistent with more extensive domestication of the S. cerevisiae parent prior to hybridization. In contrast to recent proposals that lager-brewing yeasts were domesticated following a single hybridization event, the radically different neutral site divergences between the subgenomes of the two major lager yeast lineages strongly favor at least two independent origins for the S. cerevisiae × S. eubayanus hybrids that brew lager beers. In conclusion, our findings demonstrate how this industrially important hybrid has been domesticated along similar evolutionary trajectories on multiple occasions.« less

The Genome Sequence of Saccharomyces eubayanus and the Domestication of Lager-Brewing Yeasts.

PubMed

Baker, EmilyClare; Wang, Bing; Bellora, Nicolas; Peris, David; Hulfachor, Amanda Beth; Koshalek, Justin A; Adams, Marie; Libkind, Diego; Hittinger, Chris Todd

2015-11-01

The dramatic phenotypic changes that occur in organisms during domestication leave indelible imprints on their genomes. Although many domesticated plants and animals have been systematically compared with their wild genetic stocks, the molecular and genomic processes underlying fungal domestication have received less attention. Here, we present a nearly complete genome assembly for the recently described yeast species Saccharomyces eubayanus and compare it to the genomes of multiple domesticated alloploid hybrids of S. eubayanus × S. cerevisiae (S. pastorianus syn. S. carlsbergensis), which are used to brew lager-style beers. We find that the S. eubayanus subgenomes of lager-brewing yeasts have experienced increased rates of evolution since hybridization, and that certain genes involved in metabolism may have been particularly affected. Interestingly, the S. eubayanus subgenome underwent an especially strong shift in selection regimes, consistent with more extensive domestication of the S. cerevisiae parent prior to hybridization. In contrast to recent proposals that lager-brewing yeasts were domesticated following a single hybridization event, the radically different neutral site divergences between the subgenomes of the two major lager yeast lineages strongly favor at least two independent origins for the S. cerevisiae × S. eubayanus hybrids that brew lager beers. Our findings demonstrate how this industrially important hybrid has been domesticated along similar evolutionary trajectories on multiple occasions. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Development and validation of a 20K single nucleotide polymorphism (SNP) whole genome genotyping array for apple (Malus × domestica Borkh).

PubMed

Bianco, Luca; Cestaro, Alessandro; Sargent, Daniel James; Banchi, Elisa; Derdak, Sophia; Di Guardo, Mario; Salvi, Silvio; Jansen, Johannes; Viola, Roberto; Gut, Ivo; Laurens, Francois; Chagné, David; Velasco, Riccardo; van de Weg, Eric; Troggio, Michela

2014-01-01

High-density SNP arrays for genome-wide assessment of allelic variation have made high resolution genetic characterization of crop germplasm feasible. A medium density array for apple, the IRSC 8K SNP array, has been successfully developed and used for screens of bi-parental populations. However, the number of robust and well-distributed markers contained on this array was not sufficient to perform genome-wide association analyses in wider germplasm sets, or Pedigree-Based Analysis at high precision, because of rapid decay of linkage disequilibrium. We describe the development of an Illumina Infinium array targeting 20K SNPs. The SNPs were predicted from re-sequencing data derived from the genomes of 13 Malus × domestica apple cultivars and one accession belonging to a crab apple species (M. micromalus). A pipeline for SNP selection was devised that avoided the pitfalls associated with the inclusion of paralogous sequence variants, supported the construction of robust multi-allelic SNP haploblocks and selected up to 11 entries within narrow genomic regions of ±5 kb, termed focal points (FPs). Broad genome coverage was attained by placing FPs at 1 cM intervals on a consensus genetic map, complementing them with FPs to enrich the ends of each of the chromosomes, and by bridging physical intervals greater than 400 Kbps. The selection also included ∼3.7K validated SNPs from the IRSC 8K array. The array has already been used in other studies where ∼15.8K SNP markers were mapped with an average of ∼6.8K SNPs per full-sib family. The newly developed array with its high density of polymorphic validated SNPs is expected to be of great utility for Pedigree-Based Analysis and Genomic Selection. It will also be a valuable tool to help dissect the genetic mechanisms controlling important fruit quality traits, and to aid the identification of marker-trait associations suitable for the application of Marker Assisted Selection in apple breeding programs.

Development and Validation of a 20K Single Nucleotide Polymorphism (SNP) Whole Genome Genotyping Array for Apple (Malus × domestica Borkh)

PubMed Central

Bianco, Luca; Cestaro, Alessandro; Sargent, Daniel James; Banchi, Elisa; Derdak, Sophia; Di Guardo, Mario; Salvi, Silvio; Jansen, Johannes; Viola, Roberto; Gut, Ivo; Laurens, Francois; Chagné, David; Velasco, Riccardo; van de Weg, Eric; Troggio, Michela

2014-01-01

High-density SNP arrays for genome-wide assessment of allelic variation have made high resolution genetic characterization of crop germplasm feasible. A medium density array for apple, the IRSC 8K SNP array, has been successfully developed and used for screens of bi-parental populations. However, the number of robust and well-distributed markers contained on this array was not sufficient to perform genome-wide association analyses in wider germplasm sets, or Pedigree-Based Analysis at high precision, because of rapid decay of linkage disequilibrium. We describe the development of an Illumina Infinium array targeting 20K SNPs. The SNPs were predicted from re-sequencing data derived from the genomes of 13 Malus × domestica apple cultivars and one accession belonging to a crab apple species (M. micromalus). A pipeline for SNP selection was devised that avoided the pitfalls associated with the inclusion of paralogous sequence variants, supported the construction of robust multi-allelic SNP haploblocks and selected up to 11 entries within narrow genomic regions of ±5 kb, termed focal points (FPs). Broad genome coverage was attained by placing FPs at 1 cM intervals on a consensus genetic map, complementing them with FPs to enrich the ends of each of the chromosomes, and by bridging physical intervals greater than 400 Kbps. The selection also included ∼3.7K validated SNPs from the IRSC 8K array. The array has already been used in other studies where ∼15.8K SNP markers were mapped with an average of ∼6.8K SNPs per full-sib family. The newly developed array with its high density of polymorphic validated SNPs is expected to be of great utility for Pedigree-Based Analysis and Genomic Selection. It will also be a valuable tool to help dissect the genetic mechanisms controlling important fruit quality traits, and to aid the identification of marker-trait associations suitable for the application of Marker Assisted Selection in apple breeding programs. PMID:25303088

Identification of misexpressed genetic elements in hybrids between Drosophila-related species.

PubMed

Lopez-Maestre, Hélène; Carnelossi, Elias A G; Lacroix, Vincent; Burlet, Nelly; Mugat, Bruno; Chambeyron, Séverine; Carareto, Claudia M A; Vieira, Cristina

2017-01-16

Crosses between close species can lead to genomic disorders, often considered to be the cause of hybrid incompatibility, one of the initial steps in the speciation process. How these incompatibilities are established and what are their causes remain unclear. To understand the initiation of hybrid incompatibility, we performed reciprocal crosses between two species of Drosophila (D. mojavensis and D. arizonae) that diverged less than 1 Mya. We performed a genome-wide transcriptomic analysis on ovaries from parental lines and on hybrids from reciprocal crosses. Using an innovative procedure of co-assembling transcriptomes, we show that parental lines differ in the expression of their genes and transposable elements. Reciprocal hybrids presented specific gene categories and few transposable element families misexpressed relative to the parental lines. Because TEs are mainly silenced by piwi-interacting RNAs (piRNAs), we hypothesize that in hybrids the deregulation of specific TE families is due to the absence of such small RNAs. Small RNA sequencing confirmed our hypothesis and we therefore propose that TEs can indeed be major players of genome differentiation and be implicated in the first steps of genomic incompatibilities through small RNA regulation.

Identification of misexpressed genetic elements in hybrids between Drosophila-related species

PubMed Central

Lopez-Maestre, Hélène; Carnelossi, Elias A. G.; Lacroix, Vincent; Burlet, Nelly; Mugat, Bruno; Chambeyron, Séverine; Carareto, Claudia M. A.; Vieira, Cristina

2017-01-01

Crosses between close species can lead to genomic disorders, often considered to be the cause of hybrid incompatibility, one of the initial steps in the speciation process. How these incompatibilities are established and what are their causes remain unclear. To understand the initiation of hybrid incompatibility, we performed reciprocal crosses between two species of Drosophila (D. mojavensis and D. arizonae) that diverged less than 1 Mya. We performed a genome-wide transcriptomic analysis on ovaries from parental lines and on hybrids from reciprocal crosses. Using an innovative procedure of co-assembling transcriptomes, we show that parental lines differ in the expression of their genes and transposable elements. Reciprocal hybrids presented specific gene categories and few transposable element families misexpressed relative to the parental lines. Because TEs are mainly silenced by piwi-interacting RNAs (piRNAs), we hypothesize that in hybrids the deregulation of specific TE families is due to the absence of such small RNAs. Small RNA sequencing confirmed our hypothesis and we therefore propose that TEs can indeed be major players of genome differentiation and be implicated in the first steps of genomic incompatibilities through small RNA regulation. PMID:28091568

The variable genomic architecture of isolation between hybridizing species of house mice.

PubMed

Teeter, Katherine C; Thibodeau, Lisa M; Gompert, Zachariah; Buerkle, C Alex; Nachman, Michael W; Tucker, Priscilla K

2010-02-01

Studies of the genetics of hybrid zones can provide insight into the genomic architecture of species boundaries. By examining patterns of introgression of multiple loci across a hybrid zone, it may be possible to identify regions of the genome that have experienced selection. Here, we present a comparison of introgression in two replicate transects through the house mouse hybrid zone through central Europe, using data from 41 single nucleotide markers. Using both genomic and geographic clines, we found many differences in patterns of introgression between the two transects, as well as some similarities. We found that many loci may have experienced the effects of selection at linked sites, including selection against hybrid genotypes, as well as positive selection in the form of genotypes introgressed into a foreign genetic background. We also found many positive associations of conspecific alleles among unlinked markers, which could be caused by epistatic interactions. Different patterns of introgression in the two transects highlight the challenge of using hybrid zones to identify genes underlying isolation and raise the possibility that the genetic basis of isolation between these species may be dependent on the local population genetic make-up or the local ecological setting.

The chromosomal constitution of fish hybrid lineage revealed by 5S rDNA FISH.

PubMed

Zhang, Chun; Ye, Lihai; Chen, Yiyi; Xiao, Jun; Wu, Yanhong; Tao, Min; Xiao, Yamei; Liu, Shaojun

2015-12-03

The establishment of the bisexual fertile fish hybrid lineage including the allodiploid and allotetraploid hybrids, from interspecific hybridization of red crucian carp (Carassius auratus red var. 2n = 100, 2n = AA) (♀) × common carp (Cyprinus carpio L. 2n = 100, 2n = BB) (♂), provided a good platform to investigate genetic relationship between the parents and their hybrid progenies. The chromosomal inheritance of diploid and allotetraploid hybrid progenies in successive generations, was studied by applying 5S rDNA fluorescence in situ hybridization. Signals of 5S rDNA distinguished the chromosomal constitution of common carp (B-genome) from red crucian carp (A-genome), in which two strong signals were observed on the first submetacentric chromosome, while no major signal was found in common carp. After fish hybridization, one strong signal of 5S rDNA was detected in the same locus on the chromosome of diploid hybrids. As expected, two strong signals were observed in 4nF3 tetraploid hybrids offspring and it is worth mentioning that two strong signals were detected in a separating bivalent of a primary spermatocyte in 4nF3. Furthermore, the mitosis of heterozygous chromosomes was shown normal and stable with blastular tissue histological studies. We revealed that 5S rDNA signal can be applied to discern A-genome from B-genome, and that 5S rDNA bearing chromosomes can be stably passed down in successive generations. Our work provided a significant method in fish breeding and this is important for studies in fish evolutionary biology.

Tips on hybridizing, washing, and scanning affymetrix microarrays.

PubMed

Ares, Manuel

2014-02-01

Starting in the late 1990s, Affymetrix, Inc. produced a commercial system for hybridizing, washing, and scanning microarrays that was designed to be easy to operate and reproducible. The system used arrays packaged in a plastic cassette or chamber in which the prefabricated array was mounted and could be filled with fluid through resealable membrane ports either by hand or by an automated "fluidics station" specially designed to handle the arrays. A special rotating hybridization oven and a specially designed scanner were also required. Primarily because of automation and standardization the Affymetrix system was and still remains popular. Here, we provide a skeleton protocol with the potential pitfalls identified. It is designed to augment the protocols provided by Affymetrix.

Inter-sectional hybrids obtained from reciprocal crosses between Begonia semperflorens (section Begonia) and B. ‘Orange Rubra’ (section Gaerdita × section Pritzelia)

PubMed Central

Chen, Yen-Ming; Mii, Masahiro

2012-01-01

Inter-sectional hybrids were successfully obtained by the reciprocal crosses between 11 cultivars (including 6 diploids and 5 tetraploids) of Begonia semperflorens (SS & SSSS genomes) and B. ‘Orange Rubra’ (RR genome) with the aid of in vitro culture of mature or immature seeds on MS medium containing 0.1 mg l−1 α-naphthylacetic acid, 0.1 mg l−1 6-benzyladenine, 10 mg l−1 gibberellic acid, 30 g l−1 sucrose and 2.5 g l−1 gellan gum. Embryo rescue as ovary culture with immature seeds 12th–16th day after pollination (DAP) generally gave higher efficiency of plantlet formation, but in some cross combinations, culture of mature seeds (30 DAP) resulted in higher yield of plantlets. Flow cytometric analysis revealed that they were consisted of the plants with various genomic combinations (RS, RR, RSS, RRS, RRSS and RRRRSS) as estimated by the DNA contents of both parents. Hybridity of these plants with various genomic combinations including RR was confirmed by random amplified polymorphic DNA analysis. These results suggested that unreduced gamete formation and spontaneous chromosome doubling were involved in the hybrid formation of various ploidy levels and genomic combinations. These hybrids showed various levels of intermediate traits between both parents according to the genomic compositions, and some of them had desirable characters of both parents. PMID:23136522

Inter-sectional hybrids obtained from reciprocal crosses between Begonia semperflorens (section Begonia) and B. 'Orange Rubra' (section Gaerdita × section Pritzelia).

PubMed

Chen, Yen-Ming; Mii, Masahiro

2012-06-01

Inter-sectional hybrids were successfully obtained by the reciprocal crosses between 11 cultivars (including 6 diploids and 5 tetraploids) of Begonia semperflorens (SS & SSSS genomes) and B. 'Orange Rubra' (RR genome) with the aid of in vitro culture of mature or immature seeds on MS medium containing 0.1 mg l(-1) α-naphthylacetic acid, 0.1 mg l(-1) 6-benzyladenine, 10 mg l(-1) gibberellic acid, 30 g l(-1) sucrose and 2.5 g l(-1) gellan gum. Embryo rescue as ovary culture with immature seeds 12(th)-16(th) day after pollination (DAP) generally gave higher efficiency of plantlet formation, but in some cross combinations, culture of mature seeds (30 DAP) resulted in higher yield of plantlets. Flow cytometric analysis revealed that they were consisted of the plants with various genomic combinations (RS, RR, RSS, RRS, RRSS and RRRRSS) as estimated by the DNA contents of both parents. Hybridity of these plants with various genomic combinations including RR was confirmed by random amplified polymorphic DNA analysis. These results suggested that unreduced gamete formation and spontaneous chromosome doubling were involved in the hybrid formation of various ploidy levels and genomic combinations. These hybrids showed various levels of intermediate traits between both parents according to the genomic compositions, and some of them had desirable characters of both parents.

A bicontinental origin of polyploid Australian/New Zealand Lepidium species (Brassicaceae)? Evidence from genomic in situ hybridization.

PubMed

Dierschke, Tom; Mandáková, Terezie; Lysak, Martin A; Mummenhoff, Klaus

2009-09-01

Incongruence between chloroplast and nuclear DNA phylogenies, and single additive nucleotide positions in internal transcribed spacer (ITS) sequences of polyploid Australian/New Zealand (NZ) Lepidium species have been used to suggest a bicontinental hybrid origin. This pattern was explained by two trans-oceanic dispersals of Lepidium species from California and Africa and subsequent hybridization followed by homogenization of the ribosomal DNA sequence either to the Californian (C-clade) or to the African ITS-type (A-clade) in two different ITS-lineages of Australian/NZ Lepidium polyploids. Genomic in situ hybridization (GISH) was used to unravel the genomic origin of polyploid Australian/NZ Lepidium species. Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) probes was applied to test the purported ITS evolution, and to facilitate chromosome counting in high-numbered polyploids. In Australian/NZ A-clade Lepidium polyploids, GISH identified African and Australian/NZ C-clade species as putative ancestral genomes. Neither the African nor the Californian genome were detected in Australian/NZ C-clade species and the Californian genome was not detected in Australian/NZ A-clade species. Five of the eight polyploid species (from 7x to 11x) displayed a diploid-like set of rDNA loci. Even the undecaploid species Lepidium muelleriferdinandi (2n = 11x = 88) showed only one pair of each rDNA repeat. In A-clade allopolyploids, in situ rDNA localization combined with GISH corroborated the presence of the African ITS-type. The nuclear genomes of African and Australian/NZ C-clade species were detected by GISH in allopolyploid Australian/NZ Lepidium species of the A-clade, supporting their hybrid origin. The presumed hybrid origin of Australian/NZ C-clade taxa could not be confirmed. Hence, it is assumed that Californian ancestral taxa experienced rapid radiation in Australia/NZ into extant C-clade polyploid taxa followed by hybridization with African species. As a result, A-clade allopolyploid Lepidium species share the Californian chloroplast type and the African ITS-type with the C-clade Australian/NZ polyploid and African diploid species, respectively.

Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2000-12-01

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

2001-07-05

Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less

Chromosome elimination, addition and introgression in intertribal partial hybrids between Brassica rapa and Isatis indigotica

PubMed Central

Tu, Yuqin; Sun, Jian; Ge, Xianhong; Li, Zaiyun

2009-01-01

Background and Aims Partial hybrids with female-parent-type phenotypes and chromosome numbers but altered genomic compositions have been reported in wide crosses of several plants. In order to introgress desirable genes from a wild relative, Isatis indigotica (a dye and medicinal plant; 2n = 14), into Brassica crops, intertribal sexual hybridizations were carried out with B. rapa (2n = 20), and the resulting hybrids and their progenies were characterized. Methods Using genomic in situ hybridization (GISH) and amplified fragment length polymorphism (AFLP), chromosomal/genomic components of the hybrids and their progenies were analysed. Key Results Many hybrid plants were obtained from the mature seeds harvested from the B. rapa × I. indigotica cross, and these exhibited different morphological traits. However, the majority of them did not survive and only three plants grew to maturity. These three hybrids showed poor growth and much smaller stature than the two parents, but had some morphological traits and chemical composition of I. indigotica. One plant had 2n = 10, the haploid chromosome number of B. rapa, and was absolutely sterile. The other two plants had 20 and 22 somatic chromosomes and were male sterile but produced seeds following pollinations with B. rapa. All back-cross progenies over several generations maintained a B. rapa-type phenotype and also displayed some variations in morphological characters and fatty acid compositions. They were all 2n = 20 and showed good seed-set. The hybrid with 2n = 22 produced some progeny plants with 2n = 21 and 2n = 22. GISH detected two chromosomes of I. indigotica in the hybrid with 2n = 22 but none in the one with 2n = 20. AFLP bands specific for I. indigotica, novel for two parents or absent in B. rapa, were detected in the two hybrids and their progenies. These progeny plants were novel B. rapa types with an altered genomic constitution or alien additions. Conclusions Complete or partial chromosome elimination and diploidization with genomic rearrangements were considered to lead to the formation of partial hybrids in this cross. PMID:19258339

Genome-scale transcriptional study of hybrid effects and regulatory divergence in an F1 hybrid Ruellia (Wild Petunias: Acanthaceae) and its parents.

PubMed

Zhuang, Yongbin; Tripp, Erin A

2017-01-17

New combinations of divergent genomes can give rise to novel genetic functions in resulting hybrid progeny. Such functions may yield opportunities for ecological divergence, contributing ultimately to reproductive isolation and evolutionary longevity of nascent hybrid lineages. In plants, the degree to which transgressive genotypes contribute to floral novelty remains a question of key interest. Here, we generated an F 1 hybrid plant between the red-flowered Ruellia elegans and yellow flowered R. speciosa. RNA-seq technology was used to explore differential gene expression between the hybrid and its two parents, with emphasis on genetic elements involved in the production of floral anthocyanin pigments. The hybrid was purple flowered and produced novel floral delphinidin pigments not manufactured by either parent. We found that nearly a fifth of all 86,475 unigenes expressed were unique to the hybrid. The majority of hybrid unigenes (80.97%) showed a pattern of complete dominance to one parent or the other although this ratio was uneven, suggesting asymmetrical influence of parental genomes on the progeny transcriptome. However, 8.87% of all transcripts within the hybrid were expressed at significantly higher or lower mean levels than observed for either parent. A total of 28 unigenes coding putatively for eight core enzymes in the anthocyanin pathway were recovered, along with three candidate MYBs involved in anthocyanin regulation. Our results suggest that models of gene evolution that explain phenotypic novelty and hybrid establishment in plants may need to include transgressive effects. Additionally, our results lend insight into the potential for floral novelty that derives from unions of divergent genomes. These findings serve as a starting point to further investigate molecular mechanisms involved in flower color transitions in Ruellia.

Anchored enrichment dataset for true flies (order Diptera) reveals insights into the phylogeny of flower flies (family Syrphidae).

PubMed

Young, Andrew Donovan; Lemmon, Alan R; Skevington, Jeffrey H; Mengual, Ximo; Ståhls, Gunilla; Reemer, Menno; Jordaens, Kurt; Kelso, Scott; Lemmon, Emily Moriarty; Hauser, Martin; De Meyer, Marc; Misof, Bernhard; Wiegmann, Brian M

2016-06-29

Anchored hybrid enrichment is a form of next-generation sequencing that uses oligonucleotide probes to target conserved regions of the genome flanked by less conserved regions in order to acquire data useful for phylogenetic inference from a broad range of taxa. Once a probe kit is developed, anchored hybrid enrichment is superior to traditional PCR-based Sanger sequencing in terms of both the amount of genomic data that can be recovered and effective cost. Due to their incredibly diverse nature, importance as pollinators, and historical instability with regard to subfamilial and tribal classification, Syrphidae (flower flies or hoverflies) are an ideal candidate for anchored hybrid enrichment-based phylogenetics, especially since recent molecular phylogenies of the syrphids using only a few markers have resulted in highly unresolved topologies. Over 6200 syrphids are currently known and uncovering their phylogeny will help us to understand how these species have diversified, providing insight into an array of ecological processes, from the development of adult mimicry, the origin of adult migration, to pollination patterns and the evolution of larval resource utilization. We present the first use of anchored hybrid enrichment in insect phylogenetics on a dataset containing 30 flower fly species from across all four subfamilies and 11 tribes out of 15. To produce a phylogenetic hypothesis, 559 loci were sampled to produce a final dataset containing 217,702 sites. We recovered a well resolved topology with bootstrap support values that were almost universally >95 %. The subfamily Eristalinae is recovered as paraphyletic, with the strongest support for this hypothesis to date. The ant predators in the Microdontinae are sister to all other syrphids. Syrphinae and Pipizinae are monophyletic and sister to each other. Larval predation on soft-bodied hemipterans evolved only once in this family. Anchored hybrid enrichment was successful in producing a robustly supported phylogenetic hypothesis for the syrphids. Subfamilial reconstruction is concordant with recent phylogenetic hypotheses, but with much higher support values. With the newly designed probe kit this analysis could be rapidly expanded with further sampling, opening the door to more comprehensive analyses targeting problem areas in syrphid phylogenetics and ecology.

Experimental study of surface insulated-standard hybrid tungsten planar wire array Z-pinches at “QiangGuang-I” facility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheng, Liang; Peng, Bodong; Yuan, Yuan

The experimental results of the insulated-standard hybrid wire array Z pinches carried out on “QiangGuang-I” facility at Northwest Institute of Nuclear Technology were presented and discussed. The surface insulating can impose a significant influence on the dynamics and radiation characteristics of the hybrid wire array Z pinches, especially on the early stage (t/t{sub imp} < 0.6). The expansion of insulated wires at the ablation stage is suppressed, while the streams stripped from the insulated wires move faster than that from the standard wires. The foot radiation of X-ray is enhanced by increment of the number of insulated wires, 19.6 GW, 33.6 GW, and 68.6 GWmore » for shots 14037S, 14028H, and 14039I, respectively. The surface insulation also introduces nonhomogeneity along the single wire—the streams move much faster near the electrodes. The colliding boundary of the hybrid wire array Z pinches is bias to the insulated side approximately 0.6 mm.« less

Comparison of the Roche Cobas(®) 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia.

PubMed

Phillips, Samuel; Garland, Suzanne M; Tan, Jeffery H; Quinn, Michael A; Tabrizi, Sepehr N

2015-01-01

The recently FDA (U.S. food and drug administration) approved Roche Cobas(®) 4800 (Cobas) human papillomavirus (HPV) has limited performance data compared to current HPV detection methods for test of cure in women undergoing treatment for high grade lesions. Evaluation of Cobas HPV assay using historical samples from women undergoing treatment for cervical dysplasia. A selection of 407 samples was tested on the Cobas assay and compared to previous results from Hybrid Capture 2, HPV Amplicor and Roche Linear Array. Overall, a correlation between high-risk HPV positivity and high grade histological diagnosis was 90.6% by the Cobas, 86.1% by Hybrid Capture 2, 92.9% by HPV Amplicor and 91.8% by Roche Linear Array. The Cobas HPV assay is comparative to both the HPV Amplicor and Roche Linear Array assays and better than Hybrid capture 2 assay in the detection of High-Risk HPV in women undergoing treatment for cervical dysplasia. Copyright © 2014 Elsevier B.V. All rights reserved.

Canine urothelial carcinoma: genomically aberrant and comparatively relevant

PubMed Central

Shapiro, S. G.; Raghunath, S.; Williams, C.; Motsinger-Reif, A. A.; Cullen, J. M.; Liu, T.; Albertson, D.; Ruvolo, M.; Lucas, A. Bergstrom; Jin, J.; Knapp, D. W.; Schiffman, J. D.

2015-01-01

Urothelial carcinoma (UC), also referred to as transitional cell carcinoma (TCC), is the most common bladder malignancy in both human and canine populations. In human UC, numerous studies have demonstrated the prevalence of chromosomal imbalances. Although the histopathology of the disease is similar in both species, studies evaluating the genomic profile of canine UC are lacking, limiting the discovery of key comparative molecular markers associated with driving UC pathogenesis. In the present study, we evaluated 31 primary canine UC biopsies by oligonucleotide array comparative genomic hybridization (oaCGH). Results highlighted the presence of three highly recurrent numerical aberrations: gain of dog chromosome (CFA) 13 and 36 and loss of CFA 19. Regional gains of CFA 13 and 36 were present in 97% and 84% of cases, respectively, and losses on CFA 19 were present in 77% of cases. Fluorescence in situ hybridization (FISH), using targeted bacterial artificial chromosome (BAC) clones and custom Agilent SureFISH probes, was performed to detect and quantify these regions in paraffin-embedded biopsy sections and urine-derived urothelial cells. The data indicate that these three aberrations are potentially diagnostic of UC. Comparison of our canine oaCGH data with that of 285 human cases identified a series of shared copy number aberrations. Using an informatics approach to interrogate the frequency of copy number aberrations across both species, we identified those that had the highest joint probability of association with UC. The most significant joint region contained the gene PABPC1, which should be considered further for its role in UC progression. In addition, cross-species filtering of genome-wide copy number data highlighted several genes as high-profile candidates for further analysis, including CDKN2A, S100A8/9, and LRP1B. We propose that these common aberrations are indicative of an evolutionarily conserved mechanism of pathogenesis and harbor genes key to urothelial neoplasia, warranting investigation for diagnostic, prognostic, and therapeutic applications. PMID:25783786

Canine urothelial carcinoma: genomically aberrant and comparatively relevant.

PubMed

Shapiro, S G; Raghunath, S; Williams, C; Motsinger-Reif, A A; Cullen, J M; Liu, T; Albertson, D; Ruvolo, M; Bergstrom Lucas, A; Jin, J; Knapp, D W; Schiffman, J D; Breen, M

2015-06-01

Urothelial carcinoma (UC), also referred to as transitional cell carcinoma (TCC), is the most common bladder malignancy in both human and canine populations. In human UC, numerous studies have demonstrated the prevalence of chromosomal imbalances. Although the histopathology of the disease is similar in both species, studies evaluating the genomic profile of canine UC are lacking, limiting the discovery of key comparative molecular markers associated with driving UC pathogenesis. In the present study, we evaluated 31 primary canine UC biopsies by oligonucleotide array comparative genomic hybridization (oaCGH). Results highlighted the presence of three highly recurrent numerical aberrations: gain of dog chromosome (CFA) 13 and 36 and loss of CFA 19. Regional gains of CFA 13 and 36 were present in 97 % and 84 % of cases, respectively, and losses on CFA 19 were present in 77 % of cases. Fluorescence in situ hybridization (FISH), using targeted bacterial artificial chromosome (BAC) clones and custom Agilent SureFISH probes, was performed to detect and quantify these regions in paraffin-embedded biopsy sections and urine-derived urothelial cells. The data indicate that these three aberrations are potentially diagnostic of UC. Comparison of our canine oaCGH data with that of 285 human cases identified a series of shared copy number aberrations. Using an informatics approach to interrogate the frequency of copy number aberrations across both species, we identified those that had the highest joint probability of association with UC. The most significant joint region contained the gene PABPC1, which should be considered further for its role in UC progression. In addition, cross-species filtering of genome-wide copy number data highlighted several genes as high-profile candidates for further analysis, including CDKN2A, S100A8/9, and LRP1B. We propose that these common aberrations are indicative of an evolutionarily conserved mechanism of pathogenesis and harbor genes key to urothelial neoplasia, warranting investigation for diagnostic, prognostic, and therapeutic applications.

Hybrid structures based on gold nanoparticles and semiconductor quantum dots for biosensor applications.

PubMed

Kurochkina, Margarita; Konshina, Elena; Oseev, Aleksandr; Hirsch, Soeren

2018-01-01

The luminescence amplification of semiconductor quantum dots (QD) in the presence of self-assembled gold nanoparticles (Au NPs) is one of way for creating biosensors with highly efficient transduction. The objective of this study was to fabricate the hybrid structures based on semiconductor CdSe/ZnS QDs and Au NP arrays and to use them as biosensors of protein. In this paper, the hybrid structures based on CdSe/ZnS QDs and Au NP arrays were fabricated using spin coating processes. Au NP arrays deposited on a glass wafer were investigated by optical microscopy and absorption spectroscopy depending on numbers of spin coating layers and their baking temperature. Bovine serum albumin (BSA) was used as the target protein analyte in a phosphate buffer. A confocal laser scanning microscope was used to study the luminescent properties of Au NP/QD hybrid structures and to test BSA. The dimensions of Au NP aggregates increased and the space between them decreased with increasing processing temperature. At the same time, a blue shift of the plasmon resonance peak in the absorption spectra of Au NP arrays was observed. The deposition of CdSe/ZnS QDs with a core diameter of 5 nm on the surface of the Au NP arrays caused an increase in absorption and a red shift of the plasmon peak in the spectra. The exciton-plasmon enhancement of the QDs' photoluminescence intensity has been obtained at room temperature for hybrid structures with Au NPs array pretreated at temperatures of 100°C and 150°C. It has been found that an increase in the weight content of BSA increases the photoluminescence intensity of such hybrid structures. The ability of the qualitative and quantitative determination of protein content in solution using the Au NP/QD structures as an optical biosensor has been shown experimentally.

Hybrid structures based on gold nanoparticles and semiconductor quantum dots for biosensor applications

PubMed Central

Kurochkina, Margarita; Konshina, Elena; Oseev, Aleksandr; Hirsch, Soeren

2018-01-01

Background The luminescence amplification of semiconductor quantum dots (QD) in the presence of self-assembled gold nanoparticles (Au NPs) is one of way for creating biosensors with highly efficient transduction. Aims The objective of this study was to fabricate the hybrid structures based on semiconductor CdSe/ZnS QDs and Au NP arrays and to use them as biosensors of protein. Methods In this paper, the hybrid structures based on CdSe/ZnS QDs and Au NP arrays were fabricated using spin coating processes. Au NP arrays deposited on a glass wafer were investigated by optical microscopy and absorption spectroscopy depending on numbers of spin coating layers and their baking temperature. Bovine serum albumin (BSA) was used as the target protein analyte in a phosphate buffer. A confocal laser scanning microscope was used to study the luminescent properties of Au NP/QD hybrid structures and to test BSA. Results The dimensions of Au NP aggregates increased and the space between them decreased with increasing processing temperature. At the same time, a blue shift of the plasmon resonance peak in the absorption spectra of Au NP arrays was observed. The deposition of CdSe/ZnS QDs with a core diameter of 5 nm on the surface of the Au NP arrays caused an increase in absorption and a red shift of the plasmon peak in the spectra. The exciton–plasmon enhancement of the QDs’ photoluminescence intensity has been obtained at room temperature for hybrid structures with Au NPs array pretreated at temperatures of 100°C and 150°C. It has been found that an increase in the weight content of BSA increases the photoluminescence intensity of such hybrid structures. Conclusion The ability of the qualitative and quantitative determination of protein content in solution using the Au NP/QD structures as an optical biosensor has been shown experimentally. PMID:29731613

Evaluation of SNP Data from the Malus Infinium Array Identifies Challenges for Genetic Analysis of Complex Genomes of Polyploid Origin.

PubMed

Troggio, Michela; Surbanovski, Nada; Bianco, Luca; Moretto, Marco; Giongo, Lara; Banchi, Elisa; Viola, Roberto; Fernández, Felicdad Fernández; Costa, Fabrizio; Velasco, Riccardo; Cestaro, Alessandro; Sargent, Daniel James

2013-01-01

High throughput arrays for the simultaneous genotyping of thousands of single-nucleotide polymorphisms (SNPs) have made the rapid genetic characterisation of plant genomes and the development of saturated linkage maps a realistic prospect for many plant species of agronomic importance. However, the correct calling of SNP genotypes in divergent polyploid genomes using array technology can be problematic due to paralogy, and to divergence in probe sequences causing changes in probe binding efficiencies. An Illumina Infinium II whole-genome genotyping array was recently developed for the cultivated apple and used to develop a molecular linkage map for an apple rootstock progeny (M432), but a large proportion of segregating SNPs were not mapped in the progeny, due to unexpected genotype clustering patterns. To investigate the causes of this unexpected clustering we performed BLAST analysis of all probe sequences against the 'Golden Delicious' genome sequence and discovered evidence for paralogous annealing sites and probe sequence divergence for a high proportion of probes contained on the array. Following visual re-evaluation of the genotyping data generated for 8,788 SNPs for the M432 progeny using the array, we manually re-scored genotypes at 818 loci and mapped a further 797 markers to the M432 linkage map. The newly mapped markers included the majority of those that could not be mapped previously, as well as loci that were previously scored as monomorphic, but which segregated due to divergence leading to heterozygosity in probe annealing sites. An evaluation of the 8,788 probes in a diverse collection of Malus germplasm showed that more than half the probes returned genotype clustering patterns that were difficult or impossible to interpret reliably, highlighting implications for the use of the array in genome-wide association studies.