Draft genome sequences of 9 LA-MRSA ST5 isolates obtained from humans after short term swine contact

USDA-ARS?s Scientific Manuscript database

Livestock associated methicillin resistant Staphylococcus aureus (LA-MRSA) sequence type 5 have raised concerns surrounding the potential for these isolates to colonize or cause disease in humans with swine contact. Here, we report draft genome sequences for 9 LA-MRSA ST5 isolates obtained from huma...

Complete Genome Sequence of Mycobacterium marinum ATCC 927T, Obtained Using Nanopore and Illumina Sequencing Technologies.

PubMed

Yoshida, Mitsunori; Fukano, Hanako; Miyamoto, Yuji; Shibayama, Keigo; Suzuki, Masato; Hoshino, Yoshihiko

2018-05-17

Mycobacterium marinum is a slowly growing, broad-host-range mycobacterial species. Here, we report the complete genome sequence of a Mycobacterium marinum type strain that was isolated from tubercles of diseased fish. This sequence will provide essential information for future taxonomic and comparative genome studies of its relatives. Copyright © 2018 Yoshida et al.

Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution

USDA-ARS?s Scientific Manuscript database

Genetic and genomic analyses of Upland cotton (Gossypium hirsutum) are difficult because it has a complex allotetraploid (AADD; 2n = 4x = 52) genome. Here we sequenced, assembled and analyzed the world's most important cultivated cotton genome with 246.2 gigabase (Gb) clean data obtained using whol...

Complete genome sequence of ‘Candidatus Liberibacter africanus’

USDA-ARS?s Scientific Manuscript database

The complete genome sequence of ‘Candidatus Liberibacter africanus’ (Laf), strain ptsapsy, was obtained by an Illumina HiSeq 2000. The Laf genome comprises 1,192,232 nucleotides, 34.5% GC content, 1,141 predicted coding sequences, 44 tRNAs, 3 complete copies of ribosomal RNA genes (16S, 23S and 5S) ...

Genome-Wide Search Identifies 1.9 Mb from the Polar Bear Y Chromosome for Evolutionary Analyses

PubMed Central

Bidon, Tobias; Schreck, Nancy; Hailer, Frank; Nilsson, Maria A.; Janke, Axel

2015-01-01

The male-inherited Y chromosome is the major haploid fraction of the mammalian genome, rendering Y-linked sequences an indispensable resource for evolutionary research. However, despite recent large-scale genome sequencing approaches, only a handful of Y chromosome sequences have been characterized to date, mainly in model organisms. Using polar bear (Ursus maritimus) genomes, we compare two different in silico approaches to identify Y-linked sequences: 1) Similarity to known Y-linked genes and 2) difference in the average read depth of autosomal versus sex chromosomal scaffolds. Specifically, we mapped available genomic sequencing short reads from a male and a female polar bear against the reference genome and identify 112 Y-chromosomal scaffolds with a combined length of 1.9 Mb. We verified the in silico findings for the longer polar bear scaffolds by male-specific in vitro amplification, demonstrating the reliability of the average read depth approach. The obtained Y chromosome sequences contain protein-coding sequences, single nucleotide polymorphisms, microsatellites, and transposable elements that are useful for evolutionary studies. A high-resolution phylogeny of the polar bear patriline shows two highly divergent Y chromosome lineages, obtained from analysis of the identified Y scaffolds in 12 previously published male polar bear genomes. Moreover, we find evidence of gene conversion among ZFX and ZFY sequences in the giant panda lineage and in the ancestor of ursine and tremarctine bears. Thus, the identification of Y-linked scaffold sequences from unordered genome sequences yields valuable data to infer phylogenomic and population-genomic patterns in bears. PMID:26019166

Draft genome sequence of Raoultella terrigena R1Gly, a diazotrophic endophyte

DOE PAGES

Schicklberger, M.; Shapiro, N.; Loqué, D.; ...

2015-06-11

Raoultella terrigena R1Gly is a diazotrophic endophyte isolated from surface-sterilized roots of Nicotiana tabacum. The whole-genome sequence was obtained to investigate the endophytic characteristics of this organism at the genetic level, as well as to compare this strain with its close relatives. To our knowledge, this is the first genome obtained from the Raoultella terrigena species and only the third genome from the Raoultella genus, after Raoultella ornitholytic and Raoultella planticola. This genome will provide a foundation for further comparative genomic, metagenomic, and functional studies of this genus.

Draft Genome Sequence of Ideonella sp. Strain A 288, Isolated from an Iron-Precipitating Biofilm

PubMed Central

Künzel, Sven; Szewzyk, Ulrich

2017-01-01

ABSTRACT Here, we report the draft genome sequence of the betaproteobacterium Ideonella sp. strain A_228. This isolate, obtained from a bog iron ore-containing floodplain area in Germany, provides valuable information about the genetic diversity of neutrophilic iron-depositing bacteria. The Illumina NextSeq technique was used to sequence the draft genome sequence of the strain. PMID:28818902

Whole Genome Complete Resequencing of Bacillus subtilis Natto by Combining Long Reads with High-Quality Short Reads

PubMed Central

Kamada, Mayumi; Hase, Sumitaka; Sato, Kengo; Toyoda, Atsushi; Fujiyama, Asao; Sakakibara, Yasubumi

2014-01-01

De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS) platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food “natto.” The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome. PMID:25329997

Draft genome sequences of two closely related aflatoxigenic Aspergillus species obtained from the Ivory Coast

USDA-ARS?s Scientific Manuscript database

The genomes of the A. ochraceoroseus and A. rambellii type strains were sequenced using a personal genome machine, followed by annotation of their genes. The genome size for A. ochraceoroseus was found to be approximately 23 Mb and contained 7,837 genes, while the A. rambellii genome was found to be...

First Complete Genome Sequence of Pepper vein yellows virus from Australia

PubMed Central

Maina, Solomon; Edwards, Owain R.

2016-01-01

We present here the first complete genomic RNA sequence of the polerovirus Pepper vein yellows virus (PeVYV) obtained from a pepper plant in Australia. We compare it with complete PeVYV genomes from Japan and China. The Australian genome was more closely related to the Japanese than the Chinese genome. PMID:27231375

Complete genome sequence and phylogenetic analyses of an aquabirnavirus isolated from a diseased marbled eel culture in Taiwan.

PubMed

Wen, Chiu-Ming

2017-08-01

An aquabirnavirus was isolated from diseased marbled eels (Anguilla marmorata; MEIPNV1310) with gill haemorrhages and associated mortality. Its genome segment sequences were obtained through next-generation sequencing and compared with published aquabirnavirus sequences. The results indicated that the genome sequence of MEIPNV1310 contains segment A (3099 nucleotides) and segment B (2789 nucleotides). Phylogenetic analysis showed that MEIPNV1310 is closely related to the infectious pancreatic necrosis Ab strain within genogroup II. This genome sequence is beneficial for studying the geographic distribution and evolution of aquabirnaviruses.

Next Generation Semiconductor Based Sequencing of the Donkey (Equus asinus) Genome Provided Comparative Sequence Data against the Horse Genome and a Few Millions of Single Nucleotide Polymorphisms

PubMed Central

Bertolini, Francesca; Scimone, Concetta; Geraci, Claudia; Schiavo, Giuseppina; Utzeri, Valerio Joe; Chiofalo, Vincenzo; Fontanesi, Luca

2015-01-01

Few studies investigated the donkey (Equus asinus) at the whole genome level so far. Here, we sequenced the genome of two male donkeys using a next generation semiconductor based sequencing platform (the Ion Proton sequencer) and compared obtained sequence information with the available donkey draft genome (and its Illumina reads from which it was originated) and with the EquCab2.0 assembly of the horse genome. Moreover, the Ion Torrent Personal Genome Analyzer was used to sequence reduced representation libraries (RRL) obtained from a DNA pool including donkeys of different breeds (Grigio Siciliano, Ragusano and Martina Franca). The number of next generation sequencing reads aligned with the EquCab2.0 horse genome was larger than those aligned with the draft donkey genome. This was due to the larger N50 for contigs and scaffolds of the horse genome. Nucleotide divergence between E. caballus and E. asinus was estimated to be ~ 0.52-0.57%. Regions with low nucleotide divergence were identified in several autosomal chromosomes and in the whole chromosome X. These regions might be evolutionally important in equids. Comparing Y-chromosome regions we identified variants that could be useful to track donkey paternal lineages. Moreover, about 4.8 million of single nucleotide polymorphisms (SNPs) in the donkey genome were identified and annotated combining sequencing data from Ion Proton (whole genome sequencing) and Ion Torrent (RRL) runs with Illumina reads. A higher density of SNPs was present in regions homologous to horse chromosome 12, in which several studies reported a high frequency of copy number variants. The SNPs we identified constitute a first resource useful to describe variability at the population genomic level in E. asinus and to establish monitoring systems for the conservation of donkey genetic resources. PMID:26151450

Next Generation Semiconductor Based Sequencing of the Donkey (Equus asinus) Genome Provided Comparative Sequence Data against the Horse Genome and a Few Millions of Single Nucleotide Polymorphisms.

PubMed

Bertolini, Francesca; Scimone, Concetta; Geraci, Claudia; Schiavo, Giuseppina; Utzeri, Valerio Joe; Chiofalo, Vincenzo; Fontanesi, Luca

2015-01-01

Few studies investigated the donkey (Equus asinus) at the whole genome level so far. Here, we sequenced the genome of two male donkeys using a next generation semiconductor based sequencing platform (the Ion Proton sequencer) and compared obtained sequence information with the available donkey draft genome (and its Illumina reads from which it was originated) and with the EquCab2.0 assembly of the horse genome. Moreover, the Ion Torrent Personal Genome Analyzer was used to sequence reduced representation libraries (RRL) obtained from a DNA pool including donkeys of different breeds (Grigio Siciliano, Ragusano and Martina Franca). The number of next generation sequencing reads aligned with the EquCab2.0 horse genome was larger than those aligned with the draft donkey genome. This was due to the larger N50 for contigs and scaffolds of the horse genome. Nucleotide divergence between E. caballus and E. asinus was estimated to be ~ 0.52-0.57%. Regions with low nucleotide divergence were identified in several autosomal chromosomes and in the whole chromosome X. These regions might be evolutionally important in equids. Comparing Y-chromosome regions we identified variants that could be useful to track donkey paternal lineages. Moreover, about 4.8 million of single nucleotide polymorphisms (SNPs) in the donkey genome were identified and annotated combining sequencing data from Ion Proton (whole genome sequencing) and Ion Torrent (RRL) runs with Illumina reads. A higher density of SNPs was present in regions homologous to horse chromosome 12, in which several studies reported a high frequency of copy number variants. The SNPs we identified constitute a first resource useful to describe variability at the population genomic level in E. asinus and to establish monitoring systems for the conservation of donkey genetic resources.

Normalization of Complete Genome Characteristics: Application to Evolution from Primitive Organisms to Homo sapiens.

PubMed

Sorimachi, Kenji; Okayasu, Teiji; Ohhira, Shuji

2015-04-01

Normalized nucleotide and amino acid contents of complete genome sequences can be visualized as radar charts. The shapes of these charts depict the characteristics of an organism's genome. The normalized values calculated from the genome sequence theoretically exclude experimental errors. Further, because normalization is independent of both target size and kind, this procedure is applicable not only to single genes but also to whole genomes, which consist of a huge number of different genes. In this review, we discuss the applications of the normalization of the nucleotide and predicted amino acid contents of complete genomes to the investigation of genome structure and to evolutionary research from primitive organisms to Homo sapiens. Some of the results could never have been obtained from the analysis of individual nucleotide or amino acid sequences but were revealed only after the normalization of nucleotide and amino acid contents was applied to genome research. The discovery that genome structure was homogeneous was obtained only after normalization methods were applied to the nucleotide or predicted amino acid contents of genome sequences. Normalization procedures are also applicable to evolutionary research. Thus, normalization of the contents of whole genomes is a useful procedure that can help to characterize organisms.

Legume Genome Initiative at the University of Oklahoma

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

Bruce A. Roe

2004-02-27

Consolidated Appropriations Resolution, 2003 Conference Report for the Department of Energy's Biological and Environmental Research (BER) program provided $481,000 for the Legume Genome Initiative at the University of Oklahoma. These funds were used to support our research that is aimed at determining the entire sequence of the gene rich regions of the genome of the legume, Medicago truncatula, by allowing us to obtain a greater degree of finished BAC sequences from the draft sequences we have already obtained through research funded by the Noble Foundation. During the funding period we increased the number of Medicago truncatula BACs with finished (Bermudamore » standard) sequences from 109 to 359, and the total number of BACs for which we collected sequence data from 584 to 842, 359 of which reached phase 2 (ordered and oriented contigs). We also sequenced a series of pooled BAC clones that cover additional euchromatic (gene rich) genomic regions. This work resulted in 6 refereed publications, see below. Genes whose sequence was determined during this study included multiple members of the plant disease resistance (R-gene) family as well as several genes involved in flavinoid biosynthesis, nitrogen fixation and plant-microbial symbosis. This work also served as a prelude to obtaining NSF funding for the international collaborative effort to complete the entire sequence of the Medicago truncatula genomic euchromatic regions using a BAC based approach.« less

First full-length genome sequence of the polerovirus luffa aphid-borne yellows virus (LABYV) reveals the presence of at least two consensus sequences in an isolate from Thailand.

PubMed

Knierim, Dennis; Maiss, Edgar; Kenyon, Lawrence; Winter, Stephan; Menzel, Wulf

2015-10-01

Luffa aphid-borne yellows virus (LABYV) was proposed as the name for a previously undescribed polerovirus based on partial genome sequences obtained from samples of cucurbit plants collected in Thailand between 2008 and 2013. In this study, we determined the first full-length genome sequence of LABYV. Based on phylogenetic analysis and genome properties, it is clear that this virus represents a distinct species in the genus Polerovirus. Analysis of sequences from sample TH24, which was collected in 2010 from a luffa plant in Thailand, reveals the presence of two different full-length genome consensus sequences.

Complete genome sequence of chinese strain of ‘Candidatus Liberibacter asiaticus’

USDA-ARS?s Scientific Manuscript database

The complete genome sequence of ‘Candidatus Liberibacter asiaticus’ strain (Las) Guangxi-1(GX-1) was obtained by an Illumina HiSeq 2000. The GX-1 genome comprises 1,268,237 nucleotides, 36.5 % GC content, 1,141 predicted coding sequences, 44 tRNAs, 3 complete copies of ribosomal RNA genes (16S, 23S ...

Draft Genome Sequence of Thermotoga maritima A7A Reconstructed from Metagenomic Sequencing Analysis of a Hydrocarbon Reservoir in the Bass Strait, Australia

PubMed Central

Sutcliffe, Brodie; Rosewarne, Carly P.; Greenfield, Paul; Li, Dongmei

2013-01-01

The draft genome sequence of Thermotoga maritima A7A was obtained from a metagenomic assembly obtained from a high-temperature hydrocarbon reservoir in the Gippsland Basin, Australia. The organism is predicted to be a motile anaerobe with an array of catabolic enzymes for the degradation of numerous carbohydrates. PMID:24009120

Draft Genome Sequences of Endophytic Isolates of Klebsiella variicola and Klebsiella pneumoniae Obtained from the Same Sugarcane Plant

PubMed Central

2018-01-01

ABSTRACT Endophytic Klebsiella variicola KvMx2 and Klebsiella pneumoniae KpMx1 isolates obtained from the same sugarcane stem were used for whole-genome sequencing. The genomes revealed clear differences in essential genes for plant growth, development, and detoxification, as well as nitrogen fixation, catalases, cellulases, and shared virulence factors described in the K. pneumoniae pathogen. PMID:29567733

Genome-Wide Search Identifies 1.9 Mb from the Polar Bear Y Chromosome for Evolutionary Analyses.

PubMed

Bidon, Tobias; Schreck, Nancy; Hailer, Frank; Nilsson, Maria A; Janke, Axel

2015-05-27

The male-inherited Y chromosome is the major haploid fraction of the mammalian genome, rendering Y-linked sequences an indispensable resource for evolutionary research. However, despite recent large-scale genome sequencing approaches, only a handful of Y chromosome sequences have been characterized to date, mainly in model organisms. Using polar bear (Ursus maritimus) genomes, we compare two different in silico approaches to identify Y-linked sequences: 1) Similarity to known Y-linked genes and 2) difference in the average read depth of autosomal versus sex chromosomal scaffolds. Specifically, we mapped available genomic sequencing short reads from a male and a female polar bear against the reference genome and identify 112 Y-chromosomal scaffolds with a combined length of 1.9 Mb. We verified the in silico findings for the longer polar bear scaffolds by male-specific in vitro amplification, demonstrating the reliability of the average read depth approach. The obtained Y chromosome sequences contain protein-coding sequences, single nucleotide polymorphisms, microsatellites, and transposable elements that are useful for evolutionary studies. A high-resolution phylogeny of the polar bear patriline shows two highly divergent Y chromosome lineages, obtained from analysis of the identified Y scaffolds in 12 previously published male polar bear genomes. Moreover, we find evidence of gene conversion among ZFX and ZFY sequences in the giant panda lineage and in the ancestor of ursine and tremarctine bears. Thus, the identification of Y-linked scaffold sequences from unordered genome sequences yields valuable data to infer phylogenomic and population-genomic patterns in bears. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method.

PubMed

Gouveia, Juceli Gonzalez; Wolf, Ivan Rodrigo; de Moraes-Manécolo, Vivian Patrícia Oliveira; Bardella, Vanessa Belline; Ferracin, Lara Munique; Giuliano-Caetano, Lucia; da Rosa, Renata; Dias, Ana Lúcia

2016-12-01

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.

Whole Genome Sequencing of Greater Amberjack (Seriola dumerili) for SNP Identification on Aligned Scaffolds and Genome Structural Variation Analysis Using Parallel Resequencing

PubMed Central

Aokic, Jun-ya; Kawase, Junya; Hamada, Kazuhisa; Fujimoto, Hiroshi; Yamamoto, Ikki; Usuki, Hironori

2018-01-01

Greater amberjack (Seriola dumerili) is distributed in tropical and temperate waters worldwide and is an important aquaculture fish. We carried out de novo sequencing of the greater amberjack genome to construct a reference genome sequence to identify single nucleotide polymorphisms (SNPs) for breeding amberjack by marker-assisted or gene-assisted selection as well as to identify functional genes for biological traits. We obtained 200 times coverage and constructed a high-quality genome assembly using next generation sequencing technology. The assembled sequences were aligned onto a yellowtail (Seriola quinqueradiata) radiation hybrid (RH) physical map by sequence homology. A total of 215 of the longest amberjack sequences, with a total length of 622.8 Mbp (92% of the total length of the genome scaffolds), were lined up on the yellowtail RH map. We resequenced the whole genomes of 20 greater amberjacks and mapped the resulting sequences onto the reference genome sequence. About 186,000 nonredundant SNPs were successfully ordered on the reference genome. Further, we found differences in the genome structural variations between two greater amberjack populations using BreakDancer. We also analyzed the greater amberjack transcriptome and mapped the annotated sequences onto the reference genome sequence. PMID:29785397

Consequences of Normalizing Transcriptomic and Genomic Libraries of Plant Genomes Using a Duplex-Specific Nuclease and Tetramethylammonium Chloride

PubMed Central

Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce. PMID:23409088

Consequences of normalizing transcriptomic and genomic libraries of plant genomes using a duplex-specific nuclease and tetramethylammonium chloride.

PubMed

Matvienko, Marta; Kozik, Alexander; Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce.

Complete Genome Sequence of Bradyrhizobium sp. Strain CCGE-LA001, Isolated from Field Nodules of the Enigmatic Wild Bean Phaseolus microcarpus

PubMed Central

Servín-Garcidueñas, Luis E.; Rogel, Marco A.; Ormeño-Orrillo, Ernesto; Zayas-del Moral, Alejandra; Sánchez, Federico

2016-01-01

We present the complete genome sequence of Bradyrhizobium sp. strain CCGE-LA001, a nitrogen-fixing bacterium isolated from nodules of Phaseolus microcarpus. Strain CCGE-LA001 represents the first sequenced bradyrhizobial strain obtained from a wild Phaseolus sp. Its genome revealed a large and novel symbiotic island. PMID:26988045

Two Complete Genome Sequences of Phasey Bean Mild Yellows Virus, a Novel Member of the Luteoviridae from Australia

PubMed Central

Kehoe, Monica; Coutts, Brenda; van Leur, Joop; Filardo, Fiona; Thomas, John

2016-01-01

We present here the complete genome sequences of a novel polerovirus from Trifolium subterraneum (subterranean clover) and Cicer arietinum (chickpea) and compare these to a partial viral genome sequence obtained from Macroptilium lathyroides (phasey bean). We propose the name phasey bean mild yellows virus for this novel polerovirus. PMID:26847905

Complete genome sequence of Southern tomato virus naturally infecting tomatoes in Bangladesh using small RNA deep sequencing

USDA-ARS?s Scientific Manuscript database

The complete genome sequence of a Southern tomato virus (STV) isolate on tomato plants in a seed production field in Bangladesh was obtained for the first time using next generation sequencing. The identified isolate STV_BD-13 shares high degree of sequence identity (99%) with several known STV isol...

Draft Genome Sequences of Endophytic Isolates of Klebsiella variicola and Klebsiella pneumoniae Obtained from the Same Sugarcane Plant.

PubMed

Reyna-Flores, Fernando; Barrios-Camacho, Humberto; Dantán-González, Edgar; Ramírez-Trujillo, José Augusto; Lozano Aguirre Beltrán, Luis Fernando; Rodríguez-Medina, Nadia; Garza-Ramos, Ulises; Suárez-Rodríguez, Ramón

2018-03-22

Endophytic Klebsiella variicola KvMx2 and Klebsiella pneumoniae KpMx1 isolates obtained from the same sugarcane stem were used for whole-genome sequencing. The genomes revealed clear differences in essential genes for plant growth, development, and detoxification, as well as nitrogen fixation, catalases, cellulases, and shared virulence factors described in the K. pneumoniae pathogen. Copyright © 2018 Reyna-Flores et al.

Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method.

PubMed

Nakamura, Kosuke; Kondo, Kazunari; Akiyama, Hiroshi; Ishigaki, Takumi; Noguchi, Akio; Katsumata, Hiroshi; Takasaki, Kazuto; Futo, Satoshi; Sakata, Kozue; Fukuda, Nozomi; Mano, Junichi; Kitta, Kazumi; Tanaka, Hidenori; Akashi, Ryo; Nishimaki-Mogami, Tomoko

2016-08-15

Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities. Copyright © 2016 Elsevier Ltd. All rights reserved.

Strain-specific and pooled genome sequences for populations of Drosophila melanogaster from three continents.

PubMed Central

Bergman, Casey M.; Haddrill, Penelope R.

2015-01-01

To contribute to our general understanding of the evolutionary forces that shape variation in genome sequences in nature, we have sequenced genomes from 50 isofemale lines and six pooled samples from populations of Drosophila melanogaster on three continents. Analysis of raw and reference-mapped reads indicates the quality of these genomic sequence data is very high. Comparison of the predicted and experimentally-determined Wolbachia infection status of these samples suggests that strain or sample swaps are unlikely to have occurred in the generation of these data. Genome sequences are freely available in the European Nucleotide Archive under accession ERP009059. Isofemale lines can be obtained from the Drosophila Species Stock Center. PMID:25717372

Strain-specific and pooled genome sequences for populations of Drosophila melanogaster from three continents.

PubMed

Bergman, Casey M; Haddrill, Penelope R

2015-01-01

To contribute to our general understanding of the evolutionary forces that shape variation in genome sequences in nature, we have sequenced genomes from 50 isofemale lines and six pooled samples from populations of Drosophila melanogaster on three continents. Analysis of raw and reference-mapped reads indicates the quality of these genomic sequence data is very high. Comparison of the predicted and experimentally-determined Wolbachia infection status of these samples suggests that strain or sample swaps are unlikely to have occurred in the generation of these data. Genome sequences are freely available in the European Nucleotide Archive under accession ERP009059. Isofemale lines can be obtained from the Drosophila Species Stock Center.

A population of endogenous pararetrovirus genomes in carrizo citrange

USDA-ARS?s Scientific Manuscript database

The complete genomes of three related endogenous pararetroviruses (EPRVs) were obtained by 454 sequencing of nucleic acid extracts from ‘Carrizo’citrange, used as a citrus rootstock. Numerous homologous sequences have been found in the sweet orange genome. The new EPRVs are most closely related to...

Complete genome sequence of a tomato infecting tomato mottle mosaic virus in New York

USDA-ARS?s Scientific Manuscript database

Complete genome sequence of an emerging isolate of tomato mottle mosaic virus (ToMMV) infecting experimental nicotianan benthamiana plants in up-state New York was obtained using small RNA deep sequencing. ToMMV_NY-13 shared 99% sequence identity to ToMMV isolates from Mexico and Florida. Broader d...

Near complete genome sequence of Clostridium paradoxum strain JW-YL-7

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

Lancaster, Andrew; Utturkar, Sagar M.; Poole, Farris

2016-05-05

Clostridium paradoxum strain JW-YL-7 is a moderately thermophilic anaerobic alkaliphile isolated from the municipal sewage treatment plant in Athens, GA. We report the near-complete genome sequence of C. paradoxum strain JW-YL-7 obtained by using PacBio DNA sequencing and Pilon for sequence assembly refinement with Illumina data.

Two Complete Genome Sequences of Phasey Bean Mild Yellows Virus, a Novel Member of the Luteoviridae from Australia.

PubMed

Sharman, Murray; Kehoe, Monica; Coutts, Brenda; van Leur, Joop; Filardo, Fiona; Thomas, John

2016-02-04

We present here the complete genome sequences of a novel polerovirus from Trifolium subterraneum (subterranean clover) and Cicer arietinum (chickpea) and compare these to a partial viral genome sequence obtained from Macroptilium lathyroides (phasey bean). We propose the name phasey bean mild yellows virus for this novel polerovirus. Copyright © 2016 Sharman et al.

Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics.

PubMed

Timmermans, M J T N; Dodsworth, S; Culverwell, C L; Bocak, L; Ahrens, D; Littlewood, D T J; Pons, J; Vogler, A P

2010-11-01

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags ('barcodes'). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three 'bait' sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species 'barcodes' that currently use the cox1 gene only.

Tips and tricks for the assembly of a Corynebacterium pseudotuberculosis genome using a semiconductor sequencer.

PubMed

Ramos, Rommel Thiago Jucá; Carneiro, Adriana Ribeiro; Soares, Siomar de Castro; dos Santos, Anderson Rodrigues; Almeida, Sintia; Guimarães, Luis; Figueira, Flávia; Barbosa, Eudes; Tauch, Andreas; Azevedo, Vasco; Silva, Artur

2013-03-01

New sequencing platforms have enabled rapid decoding of complete prokaryotic genomes at relatively low cost. The Ion Torrent platform is an example of these technologies, characterized by lower coverage, generating challenges for the genome assembly. One particular problem is the lack of genomes that enable reference-based assembly, such as the one used in the present study, Corynebacterium pseudotuberculosis biovar equi, which causes high economic losses in the US equine industry. The quality treatment strategy incorporated into the assembly pipeline enabled a 16-fold greater use of the sequencing data obtained compared with traditional quality filter approaches. Data preprocessing prior to the de novo assembly enabled the use of known methodologies in the next-generation sequencing data assembly. Moreover, manual curation was proved to be essential for ensuring a quality assembly, which was validated by comparative genomics with other species of the genus Corynebacterium. The present study presents a modus operandi that enables a greater and better use of data obtained from semiconductor sequencing for obtaining the complete genome from a prokaryotic microorganism, C. pseudotuberculosis, which is not a traditional biological model such as Escherichia coli. © 2012 The Authors. Published by Society for Applied Microbiology and Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

High quality de novo sequencing and assembly of the Saccharomyces arboricolus genome

PubMed Central

2013-01-01

Background Comparative genomics is a formidable tool to identify functional elements throughout a genome. In the past ten years, studies in the budding yeast Saccharomyces cerevisiae and a set of closely related species have been instrumental in showing the benefit of analyzing patterns of sequence conservation. Increasing the number of closely related genome sequences makes the comparative genomics approach more powerful and accurate. Results Here, we report the genome sequence and analysis of Saccharomyces arboricolus, a yeast species recently isolated in China, that is closely related to S. cerevisiae. We obtained high quality de novo sequence and assemblies using a combination of next generation sequencing technologies, established the phylogenetic position of this species and considered its phenotypic profile under multiple environmental conditions in the light of its gene content and phylogeny. Conclusions We suggest that the genome of S. arboricolus will be useful in future comparative genomics analysis of the Saccharomyces sensu stricto yeasts. PMID:23368932

iMETHYL: an integrative database of human DNA methylation, gene expression, and genomic variation.

PubMed

Komaki, Shohei; Shiwa, Yuh; Furukawa, Ryohei; Hachiya, Tsuyoshi; Ohmomo, Hideki; Otomo, Ryo; Satoh, Mamoru; Hitomi, Jiro; Sobue, Kenji; Sasaki, Makoto; Shimizu, Atsushi

2018-01-01

We launched an integrative multi-omics database, iMETHYL (http://imethyl.iwate-megabank.org). iMETHYL provides whole-DNA methylation (~24 million autosomal CpG sites), whole-genome (~9 million single-nucleotide variants), and whole-transcriptome (>14 000 genes) data for CD4 + T-lymphocytes, monocytes, and neutrophils collected from approximately 100 subjects. These data were obtained from whole-genome bisulfite sequencing, whole-genome sequencing, and whole-transcriptome sequencing, making iMETHYL a comprehensive database.

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

PubMed

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

Preparing the next generation of genomicists: a laboratory-style course in medical genomics.

PubMed

Linderman, Michael D; Bashir, Ali; Diaz, George A; Kasarskis, Andrew; Sanderson, Saskia C; Zinberg, Randi E; Mahajan, Milind; Shah, Hardik; Suckiel, Sabrina; Zweig, Micol; Schadt, Eric E

2015-08-12

The growing gap between the demand for genome sequencing and the supply of trained genomics professionals is creating an acute need to develop more effective genomics education. In response we developed "Practical Analysis of Your Personal Genome", a novel laboratory-style medical genomics course in which students have the opportunity to obtain and analyze their own whole genome. This report describes our motivations for and the content of a "practical" genomics course that incorporates personal genome sequencing and the lessons we learned during the first three iterations of this course.

Complete genome sequence of a novel genotype of squash mosaic virus

USDA-ARS?s Scientific Manuscript database

Complete genome sequence of a novel genotype of Squash mosaic virus (SqMV) infecting squash plants in Spain was obtained using deep sequencing of small ribonucleic acids and assembly. The low nucleotide sequence identities, with 87-88% on RNA1 and 84-86% on RNA2 to known SqMV isolates, suggest a new...

Genome Sequencing and Analysis of Yersina pestis KIM D27, an Avirulent Strain Exempt from Select Agent Regulation

PubMed Central

Losada, Liliana; Varga, John J.; Hostetler, Jessica; Radune, Diana; Kim, Maria; Durkin, Scott; Schneewind, Olaf; Nierman, William C.

2011-01-01

Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes. PMID:21559501

Genome sequencing and analysis of Yersina pestis KIM D27, an avirulent strain exempt from select agent regulation.

PubMed

Losada, Liliana; Varga, John J; Hostetler, Jessica; Radune, Diana; Kim, Maria; Durkin, Scott; Schneewind, Olaf; Nierman, William C

2011-04-29

Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes.

Complete genome sequence of Thioalkalivibrio sp. K90mix

PubMed Central

Muyzer, Gerard; Sorokin, Dimitry Y.; Mavromatis, Konstantinos; Lapidus, Alla; Foster, Brian; Sun, Hui; Ivanova, Natalia; Pati, Amrita; D'haeseleer, Patrik; Woyke, Tanja; Kyrpides, Nikos C.

2011-01-01

Thioalkalivibrio sp. K90mix is an obligately chemolithoautotrophic, natronophilic sulfur-oxidizing bacterium (SOxB) belonging to the family Ectothiorhodospiraceae within the Gammaproteobacteria. The strain was isolated from a mixture of sediment samples obtained from different soda lakes located in the Kulunda Steppe (Altai, Russia) based on its extreme potassium carbonate tolerance as an enrichment method. Here we report the complete genome sequence of strain K90mix and its annotation. The genome was sequenced within the Joint Genome Institute Community Sequencing Program, because of its relevance to the sustainable removal of sulfide from wastewater and gas streams. PMID:22675584

A first report and complete genome sequence of alfalfa enamovirus from Sudan

USDA-ARS?s Scientific Manuscript database

A full genome sequence of a viral pathogen, provisionally named alfalfa enamovirus 2 (AEV-2), was reconstructed from short reads obtained by Illumina RNA sequencing of alfalfa sample originating from Sudan. Ambiguous nucleotides in the resultant consensus assembly and identity of the predicted virus...

Complete genome sequence of Mycobacterium avium subsp. paratuberculosis, isolated from human breast milk

USDA-ARS?s Scientific Manuscript database

Mycobacterium avium subsp paratuberculosis is the etiologic agent of Johne’s disease. We report the draft genome sequences of six M. avium subsp paratuberculosis isolates obtained from diverse hosts including bison, cattle and sheep. These sequences will deepen our understanding of host association ...

Next Generation Sequencing of Actinobacteria for the Discovery of Novel Natural Products

PubMed Central

Gomez-Escribano, Juan Pablo; Alt, Silke; Bibb, Mervyn J.

2016-01-01

Like many fields of the biosciences, actinomycete natural products research has been revolutionised by next-generation DNA sequencing (NGS). Hundreds of new genome sequences from actinobacteria are made public every year, many of them as a result of projects aimed at identifying new natural products and their biosynthetic pathways through genome mining. Advances in these technologies in the last five years have meant not only a reduction in the cost of whole genome sequencing, but also a substantial increase in the quality of the data, having moved from obtaining a draft genome sequence comprised of several hundred short contigs, sometimes of doubtful reliability, to the possibility of obtaining an almost complete and accurate chromosome sequence in a single contig, allowing a detailed study of gene clusters and the design of strategies for refactoring and full gene cluster synthesis. The impact that these technologies are having in the discovery and study of natural products from actinobacteria, including those from the marine environment, is only starting to be realised. In this review we provide a historical perspective of the field, analyse the strengths and limitations of the most relevant technologies, and share the insights acquired during our genome mining projects. PMID:27089350

Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

PubMed Central

Rocher, Solen; Jean, Martine; Castonguay, Yves; Belzile, François

2015-01-01

Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids. PMID:26115486

Complete Sequence of the Intronless Mitochondrial Genome of the Saccharomyces cerevisiae Strain CW252

PubMed Central

2018-01-01

ABSTRACT The mitochondrial genomes of Saccharomyces cerevisiae strains contain up to 13 introns. An intronless recombinant genome introduced into the nuclear background of S. cerevisiae strain W303 gave the S. cerevisiae CW252 strain, which is used to model mitochondrial respiratory pathologies. The complete sequence of this mitochondrial genome was obtained using a hybrid assembling methodology. PMID:29700138

Draft genome sequence of Thauera sp. strain SWB20, isolated from a Singapore wastewater treatment facility using gel microdroplets

DOE PAGES

Dichosa, Armand E. K.; Davenport, Karen W.; Li, Po-E; ...

2015-03-19

In this study, we report here the genome sequence of Thauera sp. strain SWB20, isolated from a Singaporean wastewater treatment facility using gel microdroplets (GMDs) and single-cell genomics (SCG). This approach provided a single clonal microcolony that was sufficient to obtain a 4.9-Mbp genome assembly of an ecologically relevant Thauera species.

Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis.

PubMed

Sato, Kengo; Kuroki, Yoko; Kumita, Wakako; Fujiyama, Asao; Toyoda, Atsushi; Kawai, Jun; Iriki, Atsushi; Sasaki, Erika; Okano, Hideyuki; Sakakibara, Yasubumi

2015-11-20

The first draft of the common marmoset (Callithrix jacchus) genome was published by the Marmoset Genome Sequencing and Analysis Consortium. The draft was based on whole-genome shotgun sequencing, and the current assembly version is Callithrix_jacches-3.2.1, but there still exist 187,214 undetermined gap regions and supercontigs and relatively short contigs that are unmapped to chromosomes in the draft genome. We performed resequencing and assembly of the genome of common marmoset by deep sequencing with high-throughput sequencing technology. Several different sequence runs using Illumina sequencing platforms were executed, and 181 Gbp of high-quality bases including mate-pairs with long insert lengths of 3, 8, 20, and 40 Kbp were obtained, that is, approximately 60× coverage. The resequencing significantly improved the MGSAC draft genome sequence. The N50 of the contigs, which is a statistical measure used to evaluate assembly quality, doubled. As a result, 51% of the contigs (total length: 299 Mbp) that were unmapped to chromosomes in the MGSAC draft were merged with chromosomal contigs, and the improved genome sequence helped to detect 5,288 new genes that are homologous to human cDNAs and the gaps in 5,187 transcripts of the Ensembl gene annotations were completely filled.

Whole genome resequencing of a laboratory-adapted Drosophila melanogaster population sample

PubMed Central

Gilks, William P.; Pennell, Tanya M.; Flis, Ilona; Webster, Matthew T.; Morrow, Edward H.

2016-01-01

As part of a study into the molecular genetics of sexually dimorphic complex traits, we used high-throughput sequencing to obtain data on genomic variation in an outbred laboratory-adapted fruit fly ( Drosophila melanogaster) population. We successfully resequenced the whole genome of 220 hemiclonal females that were heterozygous for the same Berkeley reference line genome (BDGP6/dm6), and a unique haplotype from the outbred base population (LH M). The use of a static and known genetic background enabled us to obtain sequences from whole-genome phased haplotypes. We used a BWA-Picard-GATK pipeline for mapping sequence reads to the dm6 reference genome assembly, at a median depth-of coverage of 31X, and have made the resulting data publicly-available in the NCBI Short Read Archive (Accession number SRP058502). We used Haplotype Caller to discover and genotype 1,726,931 small genomic variants (SNPs and indels, <200bp). Additionally we detected and genotyped 167 large structural variants (1-100Kb in size) using GenomeStrip/2.0. Sequence and genotype data are publicly-available at the corresponding NCBI databases: Short Read Archive, dbSNP and dbVar (BioProject PRJNA282591). We have also released the unfiltered genotype data, and the code and logs for data processing and summary statistics ( https://zenodo.org/communities/sussex_drosophila_sequencing/). PMID:27928499

Microbial genomic taxonomy

PubMed Central

2013-01-01

A need for a genomic species definition is emerging from several independent studies worldwide. In this commentary paper, we discuss recent studies on the genomic taxonomy of diverse microbial groups and a unified species definition based on genomics. Accordingly, strains from the same microbial species share >95% Average Amino Acid Identity (AAI) and Average Nucleotide Identity (ANI), >95% identity based on multiple alignment genes, <10 in Karlin genomic signature, and > 70% in silico Genome-to-Genome Hybridization similarity (GGDH). Species of the same genus will form monophyletic groups on the basis of 16S rRNA gene sequences, Multilocus Sequence Analysis (MLSA) and supertree analysis. In addition to the established requirements for species descriptions, we propose that new taxa descriptions should also include at least a draft genome sequence of the type strain in order to obtain a clear outlook on the genomic landscape of the novel microbe. The application of the new genomic species definition put forward here will allow researchers to use genome sequences to define simultaneously coherent phenotypic and genomic groups. PMID:24365132

The Comprehensive Phytopathogen Genomics Resource: a web-based resource for data-mining plant pathogen genomes.

PubMed

Hamilton, John P; Neeno-Eckwall, Eric C; Adhikari, Bishwo N; Perna, Nicole T; Tisserat, Ned; Leach, Jan E; Lévesque, C André; Buell, C Robin

2011-01-01

The Comprehensive Phytopathogen Genomics Resource (CPGR) provides a web-based portal for plant pathologists and diagnosticians to view the genome and trancriptome sequence status of 806 bacterial, fungal, oomycete, nematode, viral and viroid plant pathogens. Tools are available to search and analyze annotated genome sequences of 74 bacterial, fungal and oomycete pathogens. Oomycete and fungal genomes are obtained directly from GenBank, whereas bacterial genome sequences are downloaded from the A Systematic Annotation Package (ASAP) database that provides curation of genomes using comparative approaches. Curated lists of bacterial genes relevant to pathogenicity and avirulence are also provided. The Plant Pathogen Transcript Assemblies Database provides annotated assemblies of the transcribed regions of 82 eukaryotic genomes from publicly available single pass Expressed Sequence Tags. Data-mining tools are provided along with tools to create candidate diagnostic markers, an emerging use for genomic sequence data in plant pathology. The Plant Pathogen Ribosomal DNA (rDNA) database is a resource for pathogens that lack genome or transcriptome data sets and contains 131 755 rDNA sequences from GenBank for 17 613 species identified as plant pathogens and related genera. Database URL: http://cpgr.plantbiology.msu.edu.

The solution space of sorting by DCJ.

PubMed

Braga, Marília D V; Stoye, Jens

2010-09-01

In genome rearrangements, the double cut and join (DCJ) operation, introduced by Yancopoulos et al. in 2005, allows one to represent most rearrangement events that could happen in multichromosomal genomes, such as inversions, translocations, fusions, and fissions. No restriction on the genome structure considering linear and circular chromosomes is imposed. An advantage of this general model is that it leads to considerable algorithmic simplifications compared to other genome rearrangement models. Recently, several works concerning the DCJ operation have been published, and in particular, an algorithm was proposed to find an optimal DCJ sequence for sorting one genome into another one. Here we study the solution space of this problem and give an easy-to-compute formula that corresponds to the exact number of optimal DCJ sorting sequences for a particular subset of instances of the problem. We also give an algorithm to count the number of optimal sorting sequences for any instance of the problem. Another interesting result is the demonstration of the possibility of obtaining one optimal sorting sequence by properly replacing any pair of consecutive operations in another optimal sequence. As a consequence, any optimal sorting sequence can be obtained from one other by applying such replacements successively, but the problem of finding the shortest number of replacements between two sorting sequences is still open.

Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform.

PubMed

Rutvisuttinunt, Wiriya; Chinnawirotpisan, Piyawan; Simasathien, Sriluck; Shrestha, Sanjaya K; Yoon, In-Kyu; Klungthong, Chonticha; Fernandez, Stefan

2013-11-01

Active global surveillance and characterization of influenza viruses are essential for better preparation against possible pandemic events. Obtaining comprehensive information about the influenza genome can improve our understanding of the evolution of influenza viruses and emergence of new strains, and improve the accuracy when designing preventive vaccines. This study investigated the use of deep sequencing by the next-generation sequencing (NGS) Illumina MiSeq Platform to obtain complete genome sequence information from influenza virus isolates. The influenza virus isolates were cultured from 6 respiratory acute clinical specimens collected in Thailand and Nepal. DNA libraries obtained from each viral isolate were mixed and all were sequenced simultaneously. Total information of 2.6 Gbases was obtained from a 455±14 K/mm2 density with 95.76% (8,571,655/8,950,724 clusters) of the clusters passing quality control (QC) filters. Approximately 93.7% of all sequences from Read1 and 83.5% from Read2 contained high quality sequences that were ≥Q30, a base calling QC score standard. Alignments analysis identified three seasonal influenza A H3N2 strains, one 2009 pandemic influenza A H1N1 strain and two influenza B strains. The nearly entire genomes of all six virus isolates yielded equal or greater than 600-fold sequence coverage depth. MiSeq Platform identified seasonal influenza A H3N2, 2009 pandemic influenza A H1N1and influenza B in the DNA library mixtures efficiently. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Bioinformatics prediction of siRNAs as potential antiviral agents against dengue viruses

PubMed Central

Villegas-Rosales, Paula M; Méndez-Tenorio, Alfonso; Ortega-Soto, Elizabeth; Barrón, Blanca L

2012-01-01

Dengue virus (DENV 1-4) represents the major emerging arthropod-borne viral infection in the world. Currently, there is neither an available vaccine nor a specific treatment. Hence, there is a need of antiviral drugs for these viral infections; we describe the prediction of short interfering RNA (siRNA) as potential therapeutic agents against the four DENV serotypes. Our strategy was to carry out a series of multiple alignments using ClustalX program to find conserved sequences among the four DENV serotype genomes to obtain a consensus sequence for siRNAs design. A highly conserved sequence among the four DENV serotypes, located in the encoding sequence for NS4B and NS5 proteins was found. A total of 2,893 complete DENV genomes were downloaded from the NCBI, and after a depuration procedure to identify identical sequences, 220 complete DENV genomes were left. They were edited to select the NS4B and NS5 sequences, which were aligned to obtain a consensus sequence. Three different servers were used for siRNA design, and the resulting siRNAs were aligned to identify the most prevalent sequences. Three siRNAs were chosen, one targeted the genome region that codifies for NS4B protein and the other two; the region for NS5 protein. Predicted secondary structure for DENV genomes was used to demonstrate that the siRNAs were able to target the viral genome forming double stranded structures, necessary to activate the RNA silencing machinery. PMID:22829722

Microsatellite analysis in the genome of Acanthaceae: An in silico approach.

PubMed

Kaliswamy, Priyadharsini; Vellingiri, Srividhya; Nathan, Bharathi; Selvaraj, Saravanakumar

2015-01-01

Acanthaceae is one of the advanced and specialized families with conventionally used medicinal plants. Simple sequence repeats (SSRs) play a major role as molecular markers for genome analysis and plant breeding. The microsatellites existing in the complete genome sequences would help to attain a direct role in the genome organization, recombination, gene regulation, quantitative genetic variation, and evolution of genes. The current study reports the frequency of microsatellites and appropriate markers for the Acanthaceae family genome sequences. The whole nucleotide sequences of Acanthaceae species were obtained from National Center for Biotechnology Information database and screened for the presence of SSRs. SSR Locator tool was used to predict the microsatellites and inbuilt Primer3 module was used for primer designing. Totally 110 repeats from 108 sequences of Acanthaceae family plant genomes were identified, and the occurrence of dinucleotide repeats was found to be abundant in the genome sequences. The essential amino acid isoleucine was found rich in all the sequences. We also designed the SSR-based primers/markers for 59 sequences of this family that contains microsatellite repeats in their genome. The identified microsatellites and primers might be useful for breeding and genetic studies of plants that belong to Acanthaceae family in the future.

Complete Genome Sequence of a Naturally Occurring Simian Foamy Virus Isolate from Rhesus Macaque (SFVmmu_K3T).

PubMed

Nandakumar, Subhiksha; Bae, Eunhae H; Khan, Arifa S

2017-08-17

The full-length genome sequence of a simian foamy virus (SFVmmu_K3T), isolated from a rhesus macaque ( Macaca mulatta ), was obtained using high-throughput sequencing. SFVmmu_K3T consisted of 12,983 bp and had a genomic organization similar to that of other SFVs, with long terminal repeats (LTRs) and open reading frames for Gag, Pol, Env, Tas, and Bet.

Draft Genome Sequences of Gammaproteobacterial Methanotrophs Isolated from Marine Ecosystems: TABLE 1 

DOE PAGES

Flynn, James D.; Hirayama, Hisako; Sakai, Yasuyoshi; ...

2016-01-21

The genome sequences ofMethylobacter marinusA45,Methylobactersp. strain BBA5.1, andMethylomarinum vadiIT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems.

Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species

PubMed Central

Hirakawa, Hideki; Shirasawa, Kenta; Kosugi, Shunichi; Tashiro, Kosuke; Nakayama, Shinobu; Yamada, Manabu; Kohara, Mistuyo; Watanabe, Akiko; Kishida, Yoshie; Fujishiro, Tsunakazu; Tsuruoka, Hisano; Minami, Chiharu; Sasamoto, Shigemi; Kato, Midori; Nanri, Keiko; Komaki, Akiko; Yanagi, Tomohiro; Guoxin, Qin; Maeda, Fumi; Ishikawa, Masami; Kuhara, Satoru; Sato, Shusei; Tabata, Satoshi; Isobe, Sachiko N.

2014-01-01

Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species. PMID:24282021

GI-POP: a combinational annotation and genomic island prediction pipeline for ongoing microbial genome projects.

PubMed

Lee, Chi-Ching; Chen, Yi-Ping Phoebe; Yao, Tzu-Jung; Ma, Cheng-Yu; Lo, Wei-Cheng; Lyu, Ping-Chiang; Tang, Chuan Yi

2013-04-10

Sequencing of microbial genomes is important because of microbial-carrying antibiotic and pathogenetic activities. However, even with the help of new assembling software, finishing a whole genome is a time-consuming task. In most bacteria, pathogenetic or antibiotic genes are carried in genomic islands. Therefore, a quick genomic island (GI) prediction method is useful for ongoing sequencing genomes. In this work, we built a Web server called GI-POP (http://gipop.life.nthu.edu.tw) which integrates a sequence assembling tool, a functional annotation pipeline, and a high-performance GI predicting module, in a support vector machine (SVM)-based method called genomic island genomic profile scanning (GI-GPS). The draft genomes of the ongoing genome projects in contigs or scaffolds can be submitted to our Web server, and it provides the functional annotation and highly probable GI-predicting results. GI-POP is a comprehensive annotation Web server designed for ongoing genome project analysis. Researchers can perform annotation and obtain pre-analytic information include possible GIs, coding/non-coding sequences and functional analysis from their draft genomes. This pre-analytic system can provide useful information for finishing a genome sequencing project. Copyright © 2012 Elsevier B.V. All rights reserved.

Phased genotyping-by-sequencing enhances analysis of genetic diversity and reveals divergent copy number variants in maize

USDA-ARS?s Scientific Manuscript database

High-throughput sequencing of reduced representation genomic libraries has ushered in an era of genotyping-by-sequencing (GBS), where genome-wide genotype data can be obtained for nearly any species. However, there remains a need for imputation-free GBS methods for genotyping large samples taken fr...

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

PubMed

Goya, Stephanie; Valinotto, Laura E; Tittarelli, Estefania; Rojo, Gabriel L; Nabaes Jodar, Mercedes S; Greninger, Alexander L; Zaiat, Jonathan J; Marti, Marcelo A; Mistchenko, Alicia S; Viegas, Mariana

2018-01-01

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ≥ E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1-3 (HPIV1-3) were also obtained with the selected optimal methodology.

Organizational heterogeneity of vertebrate genomes.

PubMed

Frenkel, Svetlana; Kirzhner, Valery; Korol, Abraham

2012-01-01

Genomes of higher eukaryotes are mosaics of segments with various structural, functional, and evolutionary properties. The availability of whole-genome sequences allows the investigation of their structure as "texts" using different statistical and computational methods. One such method, referred to as Compositional Spectra (CS) analysis, is based on scoring the occurrences of fixed-length oligonucleotides (k-mers) in the target DNA sequence. CS analysis allows generating species- or region-specific characteristics of the genome, regardless of their length and the presence of coding DNA. In this study, we consider the heterogeneity of vertebrate genomes as a joint effect of regional variation in sequence organization superimposed on the differences in nucleotide composition. We estimated compositional and organizational heterogeneity of genome and chromosome sequences separately and found that both heterogeneity types vary widely among genomes as well as among chromosomes in all investigated taxonomic groups. The high correspondence of heterogeneity scores obtained on three genome fractions, coding, repetitive, and the remaining part of the noncoding DNA (the genome dark matter--GDM) allows the assumption that CS-heterogeneity may have functional relevance to genome regulation. Of special interest for such interpretation is the fact that natural GDM sequences display the highest deviation from the corresponding reshuffled sequences.

Comparison of the genomic sequence of the microminipig, a novel breed of swine, with the genomic database for conventional pig.

PubMed

Miura, Naoki; Kucho, Ken-Ichi; Noguchi, Michiko; Miyoshi, Noriaki; Uchiumi, Toshiki; Kawaguchi, Hiroaki; Tanimoto, Akihide

2014-01-01

The microminipig, which weighs less than 10 kg at an early stage of maturity, has been reported as a potential experimental model animal. Its extremely small size and other distinct characteristics suggest the possibility of a number of differences between the genome of the microminipig and that of conventional pigs. In this study, we analyzed the genomes of two healthy microminipigs using a next-generation sequencer SOLiD™ system. We then compared the obtained genomic sequences with a genomic database for the domestic pig (Sus scrofa). The mapping coverage of sequenced tag from the microminipig to conventional pig genomic sequences was greater than 96% and we detected no clear, substantial genomic variance from these data. The results may indicate that the distinct characteristics of the microminipig derive from small-scale alterations in the genome, such as Single Nucleotide Polymorphisms or translational modifications, rather than large-scale deletion or insertion polymorphisms. Further investigation of the entire genomic sequence of the microminipig with methods enabling deeper coverage is required to elucidate the genetic basis of its distinct phenotypic traits. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum.

PubMed

Guo, Yaqiong; Tang, Kevin; Rowe, Lori A; Li, Na; Roellig, Dawn M; Knipe, Kristine; Frace, Michael; Yang, Chunfu; Feng, Yaoyu; Xiao, Lihua

2015-04-18

Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis-associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome. Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45-767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5' and 3' ends of chromosome 6 and the gp60 region, largely the result of genetic recombination. The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.

The Complete Mitochondrial Genome of Gossypium hirsutum and Evolutionary Analysis of Higher Plant Mitochondrial Genomes

PubMed Central

Su, Aiguo; Geng, Jianing; Grover, Corrinne E.; Hu, Songnian; Hua, Jinping

2013-01-01

Background Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes. Methodology/Principal Findings We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes. Conclusion The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species. PMID:23940520

The complete mitochondrial genome of Gossypium hirsutum and evolutionary analysis of higher plant mitochondrial genomes.

PubMed

Liu, Guozheng; Cao, Dandan; Li, Shuangshuang; Su, Aiguo; Geng, Jianing; Grover, Corrinne E; Hu, Songnian; Hua, Jinping

2013-01-01

Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes. We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes. The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.

Metagenome-Assembled Genome Sequences of Acetobacterium sp. Strain MES1 and Desulfovibrio sp. Strain MES5 from a Cathode-Associated Acetogenic Microbial Community.

PubMed

Ross, Daniel E; Marshall, Christopher W; May, Harold D; Norman, R Sean

2017-09-07

Draft genome sequences of Acetobacterium sp. strain MES1 and Desulfovibrio sp. strain MES5 were obtained from the metagenome of a cathode-associated community enriched within a microbial electrosynthesis system (MES). The draft genome sequences provide insight into the functional potential of these microorganisms within an MES and a foundation for future comparative analyses. Copyright © 2017 Ross et al.

A Workshop Report on Wheat Genome Sequencing

PubMed Central

Gill, Bikram S.; Appels, Rudi; Botha-Oberholster, Anna-Maria; Buell, C. Robin; Bennetzen, Jeffrey L.; Chalhoub, Boulos; Chumley, Forrest; Dvořák, Jan; Iwanaga, Masaru; Keller, Beat; Li, Wanlong; McCombie, W. Richard; Ogihara, Yasunari; Quetier, Francis; Sasaki, Takuji

2004-01-01

Sponsored by the National Science Foundation and the U.S. Department of Agriculture, a wheat genome sequencing workshop was held November 10–11, 2003, in Washington, DC. It brought together 63 scientists of diverse research interests and institutions, including 45 from the United States and 18 from a dozen foreign countries (see list of participants at http://www.ksu.edu/igrow). The objectives of the workshop were to discuss the status of wheat genomics, obtain feedback from ongoing genome sequencing projects, and develop strategies for sequencing the wheat genome. The purpose of this report is to convey the information discussed at the workshop and provide the basis for an ongoing dialogue, bringing forth comments and suggestions from the genetics community. PMID:15514080

Exome-wide DNA capture and next generation sequencing in domestic and wild species.

PubMed

Cosart, Ted; Beja-Pereira, Albano; Chen, Shanyuan; Ng, Sarah B; Shendure, Jay; Luikart, Gordon

2011-07-05

Gene-targeted and genome-wide markers are crucial to advance evolutionary biology, agriculture, and biodiversity conservation by improving our understanding of genetic processes underlying adaptation and speciation. Unfortunately, for eukaryotic species with large genomes it remains costly to obtain genome sequences and to develop genome resources such as genome-wide SNPs. A method is needed to allow gene-targeted, next-generation sequencing that is flexible enough to include any gene or number of genes, unlike transcriptome sequencing. Such a method would allow sequencing of many individuals, avoiding ascertainment bias in subsequent population genetic analyses.We demonstrate the usefulness of a recent technology, exon capture, for genome-wide, gene-targeted marker discovery in species with no genome resources. We use coding gene sequences from the domestic cow genome sequence (Bos taurus) to capture (enrich for), and subsequently sequence, thousands of exons of B. taurus, B. indicus, and Bison bison (wild bison). Our capture array has probes for 16,131 exons in 2,570 genes, including 203 candidate genes with known function and of interest for their association with disease and other fitness traits. We successfully sequenced and mapped exon sequences from across the 29 autosomes and X chromosome in the B. taurus genome sequence. Exon capture and high-throughput sequencing identified thousands of putative SNPs spread evenly across all reference chromosomes, in all three individuals, including hundreds of SNPs in our targeted candidate genes. This study shows exon capture can be customized for SNP discovery in many individuals and for non-model species without genomic resources. Our captured exome subset was small enough for affordable next-generation sequencing, and successfully captured exons from a divergent wild species using the domestic cow genome as reference.

Measuring Sister Chromatid Cohesion Protein Genome Occupancy in Drosophila melanogaster by ChIP-seq.

PubMed

Dorsett, Dale; Misulovin, Ziva

2017-01-01

This chapter presents methods to conduct and analyze genome-wide chromatin immunoprecipitation of the cohesin complex and the Nipped-B cohesin loading factor in Drosophila cells using high-throughput DNA sequencing (ChIP-seq). Procedures for isolation of chromatin, immunoprecipitation, and construction of sequencing libraries for the Ion Torrent Proton high throughput sequencer are detailed, and computational methods to calculate occupancy as input-normalized fold-enrichment are described. The results obtained by ChIP-seq are compared to those obtained by ChIP-chip (genomic ChIP using tiling microarrays), and the effects of sequencing depth on the accuracy are analyzed. ChIP-seq provides similar sensitivity and reproducibility as ChIP-chip, and identifies the same broad regions of occupancy. The locations of enrichment peaks, however, can differ between ChIP-chip and ChIP-seq, and low sequencing depth can splinter broad regions of occupancy into distinct peaks.

Complete mitochondrial genome of the fennec fox (Vulpes zerda).

PubMed

Yang, Xiufeng; Zhao, Chao; Zhang, Honghai; Zhang, Jin; Chen, Lei; Sha, Weilai; Liu, Guangshuai

2016-01-01

In this study, the complete mitochondrial genome of the fennec fox (Vulpes zerda) was sequenced using blood samples obtained from a female individual in Shanghai wildlife Park. Sequence analysis showed that the content of T (26.7%) in total composition was no more than C (27.2%), which is different from most of Canide individuals sequenced previously.

Complete Mitochondrial Genome Sequence of Aethina tumida (Coleoptera: Nitidulidae), a Beekeeping Pest.

PubMed

Duquesne, Véronique; Delcont, Aurélie; Huleux, Anthéa; Beven, Véronique; Touzain, Fabrice; Ribière-Chabert, Magali

2017-11-02

We report here the full mitochondrial genome sequence of Aethina tumida , a Nitidulidae species beetle, that is a pest of bee hives. The obtained sequence is 16,576 bp in length and contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNAs. Copyright © 2017 Duquesne et al.

Advanced Applications of Next-Generation Sequencing Technologies to Orchid Biology.

PubMed

Yeh, Chuan-Ming; Liu, Zhong-Jian; Tsai, Wen-Chieh

2018-01-01

Next-generation sequencing technologies are revolutionizing biology by permitting, transcriptome sequencing, whole-genome sequencing and resequencing, and genome-wide single nucleotide polymorphism profiling. Orchid research has benefited from this breakthrough, and a few orchid genomes are now available; new biological questions can be approached and new breeding strategies can be designed. The first part of this review describes the unique features of orchid biology. The second part provides an overview of the current next-generation sequencing platforms, many of which are already used in plant laboratories. The third part summarizes the state of orchid transcriptome and genome sequencing and illustrates current achievements. The genetic sequences currently obtained will not only provide a broad scope for the study of orchid biology, but also serves as a starting point for uncovering the mystery of orchid evolution.

High-Throughput Next-Generation Sequencing of Polioviruses

PubMed Central

Montmayeur, Anna M.; Schmidt, Alexander; Zhao, Kun; Magaña, Laura; Iber, Jane; Castro, Christina J.; Chen, Qi; Henderson, Elizabeth; Ramos, Edward; Shaw, Jing; Tatusov, Roman L.; Dybdahl-Sissoko, Naomi; Endegue-Zanga, Marie Claire; Adeniji, Johnson A.; Oberste, M. Steven; Burns, Cara C.

2016-01-01

ABSTRACT The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance. PMID:27927929

Estimation of the genome sizes of the chigger mites Leptotrombidium pallidum and Leptotrombidium scutellare based on quantitative PCR and k-mer analysis

PubMed Central

2014-01-01

Background Leptotrombidium pallidum and Leptotrombidium scutellare are the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. Before these organisms can be subjected to whole-genome sequencing, it is necessary to estimate their genome sizes to obtain basic information for establishing the strategies that should be used for genome sequencing and assembly. Method The genome sizes of L. pallidum and L. scutellare were estimated by a method based on quantitative real-time PCR. In addition, a k-mer analysis of the whole-genome sequences obtained through Illumina sequencing was conducted to verify the mutual compatibility and reliability of the results. Results The genome sizes estimated using qPCR were 191 ± 7 Mb for L. pallidum and 262 ± 13 Mb for L. scutellare. The k-mer analysis-based genome lengths were estimated to be 175 Mb for L. pallidum and 286 Mb for L. scutellare. The estimates from these two independent methods were mutually complementary and within a similar range to those of other Acariform mites. Conclusions The estimation method based on qPCR appears to be a useful alternative when the standard methods, such as flow cytometry, are impractical. The relatively small estimated genome sizes should facilitate whole-genome analysis, which could contribute to our understanding of Arachnida genome evolution and provide key information for scrub typhus prevention and mite vector competence. PMID:24947244

High-throughput physical mapping of chromosomes using automated in situ hybridization.

PubMed

George, Phillip; Sharakhova, Maria V; Sharakhov, Igor V

2012-06-28

Projects to obtain whole-genome sequences for 10,000 vertebrate species and for 5,000 insect and related arthropod species are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented. Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations. Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila, allows the user to visualize more details on chromosomes than the regular squashing technique. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

When Genomics Is Not Enough: Experimental Evidence for a Decrease in LINE-1 Activity During the Evolution of Australian Marsupials

PubMed Central

Gallus, Susanne; Lammers, Fritjof

2016-01-01

The autonomous transposable element LINE-1 is a highly abundant element that makes up between 15% and 20% of therian mammal genomes. Since their origin before the divergence of marsupials and placental mammals, LINE-1 elements have contributed actively to the genome landscape. A previous in silico screen of the Tasmanian devil genome revealed a lack of functional coding LINE-1 sequences. In this study we present the results of an in vitro analysis from a partial LINE-1 reverse transcriptase coding sequence in five marsupial species. Our experimental screen supports the in silico findings of the genome-wide degradation of LINE-1 sequences in the Tasmanian devil, and identifies a high frequency of degraded LINE-1 sequences in other Australian marsupials. The comparison between the experimentally obtained LINE-1 sequences and reference genome assemblies suggests that conclusions from in silico analyses of retrotransposition activity can be influenced by incomplete genome assemblies from short reads. PMID:27389686

Analysis of the Genome and Chromium Metabolism-Related Genes of Serratia sp. S2.

PubMed

Dong, Lanlan; Zhou, Simin; He, Yuan; Jia, Yan; Bai, Qunhua; Deng, Peng; Gao, Jieying; Li, Yingli; Xiao, Hong

2018-05-01

This study is to investigate the genome sequence of Serratia sp. S2. The genomic DNA of Serratia sp. S2 was extracted and the sequencing library was constructed. The sequencing was carried out by Illumina 2000 and complete genomic sequences were obtained. Gene function annotation and bioinformatics analysis were performed by comparing with the known databases. The genome size of Serratia sp. S2 was 5,604,115 bp and the G+C content was 57.61%. There were 5373 protein coding genes, and 3732, 3614, and 3942 genes were respectively annotated into the GO, KEGG, and COG databases. There were 12 genes related to chromium metabolism in the Serratia sp. S2 genome. The whole genome sequence of Serratia sp. S2 is submitted to the GenBank database with gene accession number of LNRP00000000. Our findings may provide theoretical basis for the subsequent development of new biotechnology to repair environmental chromium pollution.

Draft Genome Sequence of Brevibacterium linens AE038-8, an Extremely Arsenic-Resistant Bacterium

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

Maizel, Daniela; Utturkar, Sagar M.; Brown, Steven D.

To understand the arsenic biogeocycles in the groundwaters at Tucumán, Argentina, we isolated Brevibacterium linens sp. strain AE38-8, obtained from arsenic-contaminated well water. This strain is extremely resistant to arsenicals and has arsenic resistance (ars) genes in its genome. Here, we report the draft genome sequence of B. linens AE38-8.

Draft Genome Sequence of Brevibacterium linens AE038-8, an Extremely Arsenic-Resistant Bacterium

DOE PAGES

Maizel, Daniela; Utturkar, Sagar M.; Brown, Steven D.; ...

2015-04-16

To understand the arsenic biogeocycles in the groundwaters at Tucumán, Argentina, we isolated Brevibacterium linens sp. strain AE38-8, obtained from arsenic-contaminated well water. This strain is extremely resistant to arsenicals and has arsenic resistance (ars) genes in its genome. Here, we report the draft genome sequence of B. linens AE38-8.

Construction of a large collection of small genome variations in French dairy and beef breeds using whole-genome sequences.

PubMed

Boussaha, Mekki; Michot, Pauline; Letaief, Rabia; Hozé, Chris; Fritz, Sébastien; Grohs, Cécile; Esquerré, Diane; Duchesne, Amandine; Philippe, Romain; Blanquet, Véronique; Phocas, Florence; Floriot, Sandrine; Rocha, Dominique; Klopp, Christophe; Capitan, Aurélien; Boichard, Didier

2016-11-15

In recent years, several bovine genome sequencing projects were carried out with the aim of developing genomic tools to improve dairy and beef production efficiency and sustainability. In this study, we describe the first French cattle genome variation dataset obtained by sequencing 274 whole genomes representing several major dairy and beef breeds. This dataset contains over 28 million single nucleotide polymorphisms (SNPs) and small insertions and deletions. Comparisons between sequencing results and SNP array genotypes revealed a very high genotype concordance rate, which indicates the good quality of our data. To our knowledge, this is the first large-scale catalog of small genomic variations in French dairy and beef cattle. This resource will contribute to the study of gene functions and population structure and also help to improve traits through genotype-guided selection.

Microsatellite analysis in the genome of Acanthaceae: An in silico approach

PubMed Central

Kaliswamy, Priyadharsini; Vellingiri, Srividhya; Nathan, Bharathi; Selvaraj, Saravanakumar

2015-01-01

Background: Acanthaceae is one of the advanced and specialized families with conventionally used medicinal plants. Simple sequence repeats (SSRs) play a major role as molecular markers for genome analysis and plant breeding. The microsatellites existing in the complete genome sequences would help to attain a direct role in the genome organization, recombination, gene regulation, quantitative genetic variation, and evolution of genes. Objective: The current study reports the frequency of microsatellites and appropriate markers for the Acanthaceae family genome sequences. Materials and Methods: The whole nucleotide sequences of Acanthaceae species were obtained from National Center for Biotechnology Information database and screened for the presence of SSRs. SSR Locator tool was used to predict the microsatellites and inbuilt Primer3 module was used for primer designing. Results: Totally 110 repeats from 108 sequences of Acanthaceae family plant genomes were identified, and the occurrence of dinucleotide repeats was found to be abundant in the genome sequences. The essential amino acid isoleucine was found rich in all the sequences. We also designed the SSR-based primers/markers for 59 sequences of this family that contains microsatellite repeats in their genome. Conclusion: The identified microsatellites and primers might be useful for breeding and genetic studies of plants that belong to Acanthaceae family in the future. PMID:25709226

Draft Genome Sequence of Thermoanaerobacter sp. Strain A7A, Reconstructed from a Metagenome Obtained from a High-Temperature Hydrocarbon Reservoir in the Bass Strait, Australia

PubMed Central

Li, Dongmei; Greenfield, Paul; Rosewarne, Carly P.

2013-01-01

The draft genome sequence of Thermoanaerobacter sp. strain A7A was reconstructed from a metagenome of a microbial consortium obtained from the Tuna oil field in the Gippsland Basin, Australia. The organism is a strict anaerobe that is predicted to ferment a range of simple sugars and undertake sulfur reduction. PMID:24029756

First Complete Genomic Sequence of a Rabies Virus from the Republic of Tajikistan Obtained Directly from a Flinders Technology Associates Card

PubMed Central

Goharriz, H.; Marston, D. A.; Sharifzoda, F.; Ellis, R. J.; Horton, D. L.; Khakimov, T.; Whatmore, A.; Khamroev, K.; Makhmadshoev, A. N.; Bazarov, M.; Fooks, A. R.

2017-01-01

ABSTRACT A brain homogenate derived from a rabid dog in the district of Tojikobod, Republic of Tajikistan, was applied to a Flinders Technology Associates (FTA) card. A full-genome sequence of rabies virus (RABV) was generated from the FTA card directly without extraction, demonstrating the utility of these cards for readily obtaining genetic data. PMID:28684566

First Complete Genomic Sequence of a Rabies Virus from the Republic of Tajikistan Obtained Directly from a Flinders Technology Associates Card.

PubMed

Goharriz, H; Marston, D A; Sharifzoda, F; Ellis, R J; Horton, D L; Khakimov, T; Whatmore, A; Khamroev, K; Makhmadshoev, A N; Bazarov, M; Fooks, A R; Banyard, A C

2017-07-06

A brain homogenate derived from a rabid dog in the district of Tojikobod, Republic of Tajikistan, was applied to a Flinders Technology Associates (FTA) card. A full-genome sequence of rabies virus (RABV) was generated from the FTA card directly without extraction, demonstrating the utility of these cards for readily obtaining genetic data. © Crown copyright 2017.

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data.

PubMed

de Andrade, Roberto R S; Vaslin, Maite F S

2014-03-07

Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/.

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data

PubMed Central

2014-01-01

Background Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. Methods In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. Results The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. Conclusions SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. Availability and implementation SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/. PMID:24607237

An Exploration into Fern Genome Space.

PubMed

Wolf, Paul G; Sessa, Emily B; Marchant, Daniel Blaine; Li, Fay-Wei; Rothfels, Carl J; Sigel, Erin M; Gitzendanner, Matthew A; Visger, Clayton J; Banks, Jo Ann; Soltis, Douglas E; Soltis, Pamela S; Pryer, Kathleen M; Der, Joshua P

2015-08-26

Ferns are one of the few remaining major clades of land plants for which a complete genome sequence is lacking. Knowledge of genome space in ferns will enable broad-scale comparative analyses of land plant genes and genomes, provide insights into genome evolution across green plants, and shed light on genetic and genomic features that characterize ferns, such as their high chromosome numbers and large genome sizes. As part of an initial exploration into fern genome space, we used a whole genome shotgun sequencing approach to obtain low-density coverage (∼0.4X to 2X) for six fern species from the Polypodiales (Ceratopteris, Pteridium, Polypodium, Cystopteris), Cyatheales (Plagiogyria), and Gleicheniales (Dipteris). We explore these data to characterize the proportion of the nuclear genome represented by repetitive sequences (including DNA transposons, retrotransposons, ribosomal DNA, and simple repeats) and protein-coding genes, and to extract chloroplast and mitochondrial genome sequences. Such initial sweeps of fern genomes can provide information useful for selecting a promising candidate fern species for whole genome sequencing. We also describe variation of genomic traits across our sample and highlight some differences and similarities in repeat structure between ferns and seed plants. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The complete mitochondrial genome sequence of the maned wolf (Chrysocyon brachyurus).

PubMed

Zhao, Chao; Yang, Xiufeng; Zhang, Honghai; Zhang, Jin; Chen, Lei; Sha, Weilai; Liu, Guangshuai

2016-01-01

In this study, the complete mitochondrial genome of the maned wolf (Chrysocyon brachyurus), the unique species in Chrysocyon, was sequenced and reported for the first time using blood samples obtained from a female individual in Shanghai Zoo, China. Sequence analysis showed that the genome structure was in accordance with other Canidae species and it contained 12 S rRNA gene, 16 S rRNA gene, 22 tRNA genes, 13 protein-coding genes and 1 control region.

The draft genome of a diploid cotton Gossypium raimondii

USDA-ARS?s Scientific Manuscript database

We have sequenced and assembled the draft genome of Gossypium raimondii, whose progenitor is considered the contributor of the D-subgenome to the economically important natural textile fiber producer, G. hirsutum. Next-generation Illumina pair-end (PE) sequencing strategies were employed to obtain ...

A high resolution radiation hybrid map of wheat chromosome 4A

USDA-ARS?s Scientific Manuscript database

Bread wheat has a large and complex allohexaploid genome with low recombination level at chromosome centromeric and peri-centromeric regions. This significantly hampers ordering of markers, contigs of physical maps and sequence scaffolds and impedes obtaining of high-quality reference genome sequenc...

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

PubMed Central

Nowrousian, Minou; Stajich, Jason E.; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D.; Pöggeler, Stefanie; Read, Nick D.; Seiler, Stephan; Smith, Kristina M.; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-01-01

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology. PMID:20386741

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

PubMed

Nowrousian, Minou; Stajich, Jason E; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D; Pöggeler, Stefanie; Read, Nick D; Seiler, Stephan; Smith, Kristina M; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-04-08

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

The Mitochondrial Genome Sequence and Molecular Phylogeny of the Turkey, Meleagris gallopavo

PubMed Central

Guan, Xiaojing; Silva, Pradeepa; Gyenai, Kwaku B.; Xu, Jun; Geng, Tuoyu; Tu, Zhijian; Samuels, David C.; Smith, Edward J.

2009-01-01

Summary The mitochondrial genome (mtGenome) has been very little studied in the turkey (Meleagris gallopavo), for which there is no publicly available whole genome mitochondrial sequence. Here, we used PCR-based methods with 19 pairs of primers designed from the chicken and other species to develop a complete turkey mtGenome sequence. A total length of 16, 717 bp of the whole turkey mtGenome was obtained, with 85% similarity to chicken mtGenome. There were 13 genes and 24 RNA (22 tRNA and 2 rRNA) annotated. The mtGenome-based phylogenetic analysis suggests that the turkey is most closely related to the chicken, Gallus gallus, and quail, Corturnix japonica. Given the importance of the mitochondria genome, the present work adds to the growing genomic resources needed to define the genetic mechanisms that underlie some economic traits in the turkey. PMID:19067672

The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

PubMed

Pfeiffer, Friedhelm; Zamora-Lagos, Maria-Antonia; Blettinger, Martin; Yeroslaviz, Assa; Dahl, Andreas; Gruber, Stephan; Habermann, Bianca H

2018-01-05

Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity. Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel. Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain's genetic profile from pathogenic to environmental.

The contribution of the DNA microarray technology to gene expression profiling in Leishmania spp.: a retrospective.

PubMed

Alonso, Ana; Larraga, Vicente; Alcolea, Pedro J

2018-05-07

The first genome project of any living organism excluding viruses, the gammaproteobacteria Haemophilus influenzae, was completed in 1995. Until the last decade, genome sequencing was very tedious because genome survey sequences (GSS) and/or expressed sequence tags (ESTs) belonging to plasmid, cosmid and artificial chromosome genome libraries had to be sequenced and assembled in silico. Nowadays, no genome is completely assembled actually, because gaps and unassembled contigs are always remaining. However, most represent the whole genome of the organism of origin from a practical point of view. The first genome sequencing projects of trypanosomatid parasites were completed in 2005 following those strategies, and belong to Leishmania major, Trypanosoma cruzi and T. brucei. The functional genomics era rapidly developed on the basis of the microarray technology and has been evolving. In the case of the genus Leishmania, substantial biological information about differentiation in the digenetic life cycle of the parasite has been obtained. Later on, next generation sequencing has revolutionized genome sequencing and functional genomics, leading to more sensitive, accurate results by using much less resources. This new technology is more advantageous, but does not invalidate microarray results. In fact, promising vaccine candidates and drug targets have been found on the basis of microarray-based screening and preliminary proof-of-concept tests. Copyright © 2018. Published by Elsevier B.V.

Draft Genome Sequences of Six Mycobacterium immunogenum, Strains Obtained from a Chloraminated Drinking Water Distribution System Simulator

EPA Science Inventory

We report the draft genome sequences of six Mycobacterium immunogenum isolated from a chloraminated drinking water distribution system simulator subjected to changes in operational parameters. M. immunogenum, a rapidly growing mycobacteria previously reported as the cause of hyp...

Genome Survey Sequencing for the Characterization of the Genetic Background of Rosa roxburghii Tratt and Leaf Ascorbate Metabolism Genes.

PubMed

Lu, Min; An, Huaming; Li, Liangliang

2016-01-01

Rosa roxburghii Tratt is an important commercial horticultural crop in China that is recognized for its nutritional and medicinal values. In spite of the economic significance, genomic information on this rose species is currently unavailable. In the present research, a genome survey of R. roxburghii was carried out using next-generation sequencing (NGS) technologies. Total 30.29 Gb sequence data was obtained by HiSeq 2500 sequencing and an estimated genome size of R. roxburghii was 480.97 Mb, in which the guanine plus cytosine (GC) content was calculated to be 38.63%. All of these reads were technically assembled and a total of 627,554 contigs with a N50 length of 1.484 kb and furthermore 335,902 scaffolds with a total length of 409.36 Mb were obtained. Transposable elements (TE) sequence of 90.84 Mb which comprised 29.20% of the genome, and 167,859 simple sequence repeats (SSRs) were identified from the scaffolds. Among these, the mono-(66.30%), di-(25.67%), and tri-(6.64%) nucleotide repeats contributed to nearly 99% of the SSRs, and sequence motifs AG/CT (28.81%) and GAA/TTC (14.76%) were the most abundant among the dinucleotide and trinucleotide repeat motifs, respectively. Genome analysis predicted a total of 22,721 genes which have an average length of 2311.52 bp, an average exon length of 228.15 bp, and average intron length of 401.18 bp. Eleven genes putatively involved in ascorbate metabolism were identified and its expression in R. roxburghii leaves was validated by quantitative real-time PCR (qRT-PCR). This is the first report of genome-wide characterization of this rose species.

Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

PubMed Central

Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

2015-01-01

Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

Genome Survey Sequencing of Luffa Cylindrica L. and Microsatellite High Resolution Melting (SSR-HRM) Analysis for Genetic Relationship of Luffa Genotypes.

PubMed

An, Jianyu; Yin, Mengqi; Zhang, Qin; Gong, Dongting; Jia, Xiaowen; Guan, Yajing; Hu, Jin

2017-09-11

Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica , about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N 50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (≥5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both "Zheda 23" and "Zheda 83". Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species.

Gekko japonicus genome reveals evolution of adhesive toe pads and tail regeneration

PubMed Central

Liu, Yan; Zhou, Qian; Wang, Yongjun; Luo, Longhai; Yang, Jian; Yang, Linfeng; Liu, Mei; Li, Yingrui; Qian, Tianmei; Zheng, Yuan; Li, Meiyuan; Li, Jiang; Gu, Yun; Han, Zujing; Xu, Man; Wang, Yingjie; Zhu, Changlai; Yu, Bin; Yang, Yumin; Ding, Fei; Jiang, Jianping; Yang, Huanming; Gu, Xiaosong

2015-01-01

Reptiles are the most morphologically and physiologically diverse tetrapods, and have undergone 300 million years of adaptive evolution. Within the reptilian tetrapods, geckos possess several interesting features, including the ability to regenerate autotomized tails and to climb on smooth surfaces. Here we sequence the genome of Gekko japonicus (Schlegel's Japanese Gecko) and investigate genetic elements related to its physiology. We obtain a draft G. japonicus genome sequence of 2.55 Gb and annotated 22,487 genes. Comparative genomic analysis reveals specific gene family expansions or reductions that are associated with the formation of adhesive setae, nocturnal vision and tail regeneration, as well as the diversification of olfactory sensation. The obtained genomic data provide robust genetic evidence of adaptive evolution in reptiles. PMID:26598231

Narrow-Host-Range Bacteriophages That Infect Rhizobium etli Associate with Distinct Genomic Types

PubMed Central

Santamaría, Rosa Isela; Bustos, Patricia; Sepúlveda-Robles, Omar; Lozano, Luis; Rodríguez, César; Fernández, José Luis; Juárez, Soledad; Kameyama, Luis; Guarneros, Gabriel; Dávila, Guillermo

2014-01-01

In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ∼45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species. PMID:24185856

Draft genome sequences of 50 MRSA ST5 isolates obtained from a U.S. hospital

USDA-ARS?s Scientific Manuscript database

Methicillin resistant Staphylococcus aureus (MRSA) can be a commensal or pathogen in humans. Pathogenicity and disease are related to the acquisition of mobile genetic elements encoding virulence and antimicrobial resistance genes. Here, we report draft genome sequences for 50 clinical MRSA isolates...

Transcriptome assembly, gene annotation and tissue gene expression atlas of the rainbow trout

USDA-ARS?s Scientific Manuscript database

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complimented by transcriptome information that will enhance genome assembly and annotation. Previously, we reported a transcriptome reference sequence using a 19X coverage of Sanger and 454-pyrosequencing dat...

Draft genome sequence of the silver pomfret fish, Pampus argenteus.

PubMed

AlMomin, Sabah; Kumar, Vinod; Al-Amad, Sami; Al-Hussaini, Mohsen; Dashti, Talal; Al-Enezi, Khaznah; Akbar, Abrar

2016-01-01

Silver pomfret, Pampus argenteus, is a fish species from coastal waters. Despite its high commercial value, this edible fish has not been sequenced. Hence, its genetic and genomic studies have been limited. We report the first draft genome sequence of the silver pomfret obtained using a Next Generation Sequencing (NGS) technology. We assembled 38.7 Gb of nucleotides into scaffolds of 350 Mb with N50 of about 1.5 kb, using high quality paired end reads. These scaffolds represent 63.7% of the estimated silver pomfret genome length. The newly sequenced and assembled genome has 11.06% repetitive DNA regions, and this percentage is comparable to that of the tilapia genome. The genome analysis predicted 16 322 genes. About 91% of these genes showed homology with known proteins. Many gene clusters were annotated to protein and fatty-acid metabolism pathways that may be important in the context of the meat texture and immune system developmental processes. The reference genome can pave the way for the identification of many other genomic features that could improve breeding and population-management strategies, and it can also help characterize the genetic diversity of P. argenteus.

The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation.

PubMed

Mavromatis, Konstantinos; Land, Miriam L; Brettin, Thomas S; Quest, Daniel J; Copeland, Alex; Clum, Alicia; Goodwin, Lynne; Woyke, Tanja; Lapidus, Alla; Klenk, Hans Peter; Cottingham, Robert W; Kyrpides, Nikos C

2012-01-01

The emergence of next generation sequencing (NGS) has provided the means for rapid and high throughput sequencing and data generation at low cost, while concomitantly creating a new set of challenges. The number of available assembled microbial genomes continues to grow rapidly and their quality reflects the quality of the sequencing technology used, but also of the analysis software employed for assembly and annotation. In this work, we have explored the quality of the microbial draft genomes across various sequencing technologies. We have compared the draft and finished assemblies of 133 microbial genomes sequenced at the Department of Energy-Joint Genome Institute and finished at the Los Alamos National Laboratory using a variety of combinations of sequencing technologies, reflecting the transition of the institute from Sanger-based sequencing platforms to NGS platforms. The quality of the public assemblies and of the associated gene annotations was evaluated using various metrics. Results obtained with the different sequencing technologies, as well as their effects on downstream processes, were analyzed. Our results demonstrate that the Illumina HiSeq 2000 sequencing system, the primary sequencing technology currently used for de novo genome sequencing and assembly at JGI, has various advantages in terms of total sequence throughput and cost, but it also introduces challenges for the downstream analyses. In all cases assembly results although on average are of high quality, need to be viewed critically and consider sources of errors in them prior to analysis. These data follow the evolution of microbial sequencing and downstream processing at the JGI from draft genome sequences with large gaps corresponding to missing genes of significant biological role to assemblies with multiple small gaps (Illumina) and finally to assemblies that generate almost complete genomes (Illumina+PacBio).

Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri.

PubMed

Nigg, Jared C; Nouri, Shahideh; Falk, Bryce W

2016-07-28

Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera. Copyright © 2016 Nigg et al.

Complete mitochondrial genome of Eagle Owl (Bubo bubo, Strigiformes; Strigidae) from China.

PubMed

Hengjiu, Tian; Jianwei, Ji; Shi, Yang; Zhiming, Zhang; Laghari, Muhammad Younis; Narejo, Naeem Tariq; Lashari, Punhal

2016-01-01

In the present study, the complete mitochondrial genome sequence of Bubo bubo using PCR amplification, sequencing and assembling has been obtained for the first time. The total length of the mitochondrial genome was 16,250  bp, with the base composition of 29.88% A, 34.16% C, 14.35% G, and 21.58% T. It contained 37 genes (2 ribosomal RNA genes, 13 protein-coding genes and 22 transfer RNA genes) and a major non-coding control region (D-loop region). The complete mitochondrial genome sequence of Bubo bubo provides an important data set for further investigation on the phylogenetic relationships within Strigiformes.

Complete Genome Sequence of Genotype VI Newcastle Disease Viruses Isolated from Pigeons in Pakistan

PubMed Central

Wajid, Abdul; Rehmani, Shafqat Fatima; Sharma, Poonam; Goraichuk, Iryna V.; Dimitrov, Kiril M.

2016-01-01

Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion protein genes and complete genomes classified the isolates as members of NDV class II, genotype VI. PMID:27540069

Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

PubMed

Stepanauskas, Ramunas; Fergusson, Elizabeth A; Brown, Joseph; Poulton, Nicole J; Tupper, Ben; Labonté, Jessica M; Becraft, Eric D; Brown, Julia M; Pachiadaki, Maria G; Povilaitis, Tadas; Thompson, Brian P; Mascena, Corianna J; Bellows, Wendy K; Lubys, Arvydas

2017-07-20

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.

OrthoANI: An improved algorithm and software for calculating average nucleotide identity.

PubMed

Lee, Imchang; Ouk Kim, Yeong; Park, Sang-Cheol; Chun, Jongsik

2016-02-01

Species demarcation in Bacteria and Archaea is mainly based on overall genome relatedness, which serves a framework for modern microbiology. Current practice for obtaining these measures between two strains is shifting from experimentally determined similarity obtained by DNA-DNA hybridization (DDH) to genome-sequence-based similarity. Average nucleotide identity (ANI) is a simple algorithm that mimics DDH. Like DDH, ANI values between two genome sequences may be different from each other when reciprocal calculations are compared. We compared 63 690 pairs of genome sequences and found that the differences in reciprocal ANI values are significantly high, exceeding 1 % in some cases. To resolve this problem of not being symmetrical, a new algorithm, named OrthoANI, was developed to accommodate the concept of orthology for which both genome sequences were fragmented and only orthologous fragment pairs taken into consideration for calculating nucleotide identities. OrthoANI is highly correlated with ANI (using BLASTn) and the former showed approximately 0.1 % higher values than the latter. In conclusion, OrthoANI provides a more robust and faster means of calculating average nucleotide identity for taxonomic purposes. The standalone software tools are freely available at http://www.ezbiocloud.net/sw/oat.

Identification of RAN1 orthologue associated with sex determination through whole genome sequencing analysis in fig (Ficus carica L.).

PubMed

Mori, Kazuki; Shirasawa, Kenta; Nogata, Hitoshi; Hirata, Chiharu; Tashiro, Kosuke; Habu, Tsuyoshi; Kim, Sangwan; Himeno, Shuichi; Kuhara, Satoru; Ikegami, Hidetoshi

2017-01-25

With the aim of identifying sex determinants of fig, we generated the first draft genome sequence of fig and conducted the subsequent analyses. Linkage analysis with a high-density genetic map established by a restriction-site associated sequencing technique, and genome-wide association study followed by whole-genome resequencing analysis identified two missense mutations in RESPONSIVE-TO-ANTAGONIST1 (RAN1) orthologue encoding copper-transporting ATPase completely associated with sex phenotypes of investigated figs. This result suggests that RAN1 is a possible sex determinant candidate in the fig genome. The genomic resources and genetic findings obtained in this study can contribute to general understanding of Ficus species and provide an insight into fig's and plant's sex determination system.

de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer.

PubMed

Istace, Benjamin; Friedrich, Anne; d'Agata, Léo; Faye, Sébastien; Payen, Emilie; Beluche, Odette; Caradec, Claudia; Davidas, Sabrina; Cruaud, Corinne; Liti, Gianni; Lemainque, Arnaud; Engelen, Stefan; Wincker, Patrick; Schacherer, Joseph; Aury, Jean-Marc

2017-02-01

Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds. The Oxford Nanopore technology is truly disruptive; it has the potential to revolutionize genomic applications due to its portability, low cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65 % of the chromosomes. This high contiguity allowed us to accurately detect large structural variations across the 21 studied genomes. Because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy. Our analyses show that the Oxford Nanopore technology is already usable for de novo sequencing and assembly; however, non-random errors in homopolymers require polishing the consensus using an alternate sequencing technology. © The Author 2017. Published by Oxford University Press.

de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer

PubMed Central

Istace, Benjamin; Friedrich, Anne; d'Agata, Léo; Faye, Sébastien; Payen, Emilie; Beluche, Odette; Caradec, Claudia; Davidas, Sabrina; Cruaud, Corinne; Liti, Gianni; Lemainque, Arnaud; Engelen, Stefan; Wincker, Patrick; Schacherer, Joseph

2017-01-01

Abstract Background: Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds. The Oxford Nanopore technology is truly disruptive; it has the potential to revolutionize genomic applications due to its portability, low cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Results: Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65 % of the chromosomes. This high contiguity allowed us to accurately detect large structural variations across the 21 studied genomes. Conclusion: Because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy. Our analyses show that the Oxford Nanopore technology is already usable for de novo sequencing and assembly; however, non-random errors in homopolymers require polishing the consensus using an alternate sequencing technology. PMID:28369459

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

PubMed Central

2011-01-01

Background BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. Results This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed. PMID:21794110

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes.

PubMed

Feltus, Frank A; Saski, Christopher A; Mockaitis, Keithanne; Haiminen, Niina; Parida, Laxmi; Smith, Zachary; Ford, James; Staton, Margaret E; Ficklin, Stephen P; Blackmon, Barbara P; Cheng, Chun-Huai; Schnell, Raymond J; Kuhn, David N; Motamayor, Juan-Carlos

2011-07-27

BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

Complete genome sequence of Metallosphaera cuprina, a metal sulfide-oxidizing archaeon from a hot spring.

PubMed

Liu, Li-Jun; You, Xiao-Yan; Zheng, Huajun; Wang, Shengyue; Jiang, Cheng-Ying; Liu, Shuang-Jiang

2011-07-01

The genome of the metal sulfide-oxidizing, thermoacidophilic strain Metallosphaera cuprina Ar-4 has been completely sequenced and annotated. Originally isolated from a sulfuric hot spring, strain Ar-4 grows optimally at 65°C and a pH of 3.5. The M. cuprina genome has a 1,840,348-bp circular chromosome (2,029 open reading frames [ORFs]) and is 16% smaller than the previously sequenced Metallosphaera sedula genome. Compared to the M. sedula genome, there are no counterpart genes in the M. cuprina genome for about 480 ORFs in the M. sedula genome, of which 243 ORFs are annotated as hypothetical protein genes. Still, there are 233 ORFs uniquely occurring in M. cuprina. Genome annotation supports that M. cuprina lives a facultative life on CO(2) and organics and obtains energy from oxidation of sulfidic ores and reduced inorganic sulfuric compounds.

Sequence-Dependent Persistence Length of Long DNA

NASA Astrophysics Data System (ADS)

Chuang, Hui-Min; Reifenberger, Jeffrey G.; Cao, Han; Dorfman, Kevin D.

2017-12-01

Using a high-throughput genome-mapping approach, we obtained circa 50 million measurements of the extension of internal human DNA segments in a 41 nm ×41 nm nanochannel. The underlying DNA sequences, obtained by mapping to the reference human genome, are 2.5-393 kilobase pairs long and contain percent GC contents between 32.5% and 60%. Using Odijk's theory for a channel-confined wormlike chain, these data reveal that the DNA persistence length increases by almost 20% as the percent GC content increases. The increased persistence length is rationalized by a model, containing no adjustable parameters, that treats the DNA as a statistical terpolymer with a sequence-dependent intrinsic persistence length and a sequence-independent electrostatic persistence length.

Genome sequencing of a single tardigrade Hypsibius dujardini individual

PubMed Central

Arakawa, Kazuharu; Yoshida, Yuki; Tomita, Masaru

2016-01-01

Tardigrades are ubiquitous microscopic animals that play an important role in the study of metazoan phylogeny. Most terrestrial tardigrades can withstand extreme environments by entering an ametabolic desiccated state termed anhydrobiosis. Due to their small size and the non-axenic nature of laboratory cultures, molecular studies of tardigrades are prone to contamination. To minimize the possibility of microbial contaminations and to obtain high-quality genomic information, we have developed an ultra-low input library sequencing protocol to enable the genome sequencing of a single tardigrade Hypsibius dujardini individual. Here, we describe the details of our sequencing data and the ultra-low input library preparation methodologies. PMID:27529330

Genome sequencing of a single tardigrade Hypsibius dujardini individual.

PubMed

Arakawa, Kazuharu; Yoshida, Yuki; Tomita, Masaru

2016-08-16

Tardigrades are ubiquitous microscopic animals that play an important role in the study of metazoan phylogeny. Most terrestrial tardigrades can withstand extreme environments by entering an ametabolic desiccated state termed anhydrobiosis. Due to their small size and the non-axenic nature of laboratory cultures, molecular studies of tardigrades are prone to contamination. To minimize the possibility of microbial contaminations and to obtain high-quality genomic information, we have developed an ultra-low input library sequencing protocol to enable the genome sequencing of a single tardigrade Hypsibius dujardini individual. Here, we describe the details of our sequencing data and the ultra-low input library preparation methodologies.

Draft Genome Sequence, and a Sequence-Defined Genetic Linkage Map of the Legume Crop Species Lupinus angustifolius L

PubMed Central

Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W.; Howieson, John G.; Li, Chengdao

2013-01-01

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species. PMID:23734219

Draft genome sequence, and a sequence-defined genetic linkage map of the legume crop species Lupinus angustifolius L.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W; Howieson, John G; Li, Chengdao

2013-01-01

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species.

Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

PubMed

Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K P; Woo, Patrick C Y

2015-10-22

Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology

PubMed Central

Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K. P.; Woo, Patrick C. Y.

2015-01-01

Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10–49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n = 2), Pichia (Candida) norvegensis (n = 2), Candida tropicalis (n = 1) and Saccharomyces cerevisiae (n = 1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study. PMID:26506340

Ultraaccurate genome sequencing and haplotyping of single human cells.

PubMed

Chu, Wai Keung; Edge, Peter; Lee, Ho Suk; Bansal, Vikas; Bafna, Vineet; Huang, Xiaohua; Zhang, Kun

2017-11-21

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10 -8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs. Copyright © 2017 the Author(s). Published by PNAS.

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

PubMed Central

Staton, Margaret; Best, Teodora; Khodwekar, Sudhir; Owusu, Sandra; Xu, Tao; Xu, Yi; Jennings, Tara; Cronn, Richard; Arumuganathan, A. Kathiravetpilla; Coggeshall, Mark; Gailing, Oliver; Liang, Haiying; Romero-Severson, Jeanne; Schlarbaum, Scott; Carlson, John E.

2015-01-01

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence. PMID:26698853

Scanning the Effects of Ethyl Methanesulfonate on the Whole Genome of Lotus japonicus Using Second-Generation Sequencing Analysis

PubMed Central

Mohd-Yusoff, Nur Fatihah; Ruperao, Pradeep; Tomoyoshi, Nurain Emylia; Edwards, David; Gresshoff, Peter M.; Biswas, Bandana; Batley, Jacqueline

2015-01-01

Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm. PMID:25660167

Genome Sequences of Eight Aspergillus flavus spp. and One A. parasiticus sp., Isolated From Peanut Seeds in Georgia

USDA-ARS?s Scientific Manuscript database

Aspergillus flavus and A. parasiticus fungi, carcinogen-mycotoxins producers, infect peanut seeds, causing considerable impact on both human health and the economy. Here we report 9 genome sequences of Aspergillus spp. isolated from peanut seeds. The information obtained will allow conducting biodiv...

Complete genome sequence of genotype VI Newcastle disease viruses isolated from pigeons in Pakistan

USDA-ARS?s Scientific Manuscript database

Two complete genome sequences of Newcastle disease virus (NDV) are described here. Virulent isolates pigeon/Pakistan/Lahore/21A/2015 and pigeon/Pakistan/Lahore/25A/2015 were obtained from racing pigeons sampled in the Pakistani province of Punjab during 2015. Phylogenetic analysis of the fusion prot...

Genome Sequences of Multidrug-Resistant, Colistin-Susceptible and -Resistant Klebsiella pneumoniae Clinical Isolates from Pakistan

PubMed Central

Crawford, Matthew A.; Timme, Ruth; Lomonaco, Sara; Lascols, Christine; Fisher, Debra J.; Sharma, Shashi K.; Strain, Errol; Allard, Marc W.; Brown, Eric W.; McFarland, Melinda A.; Croley, Tim; Hammack, Thomas S.; Weigel, Linda M.; Anderson, Kevin; Hodge, David R.; Pillai, Segaran P.; Morse, Stephen A.; Khan, Erum

2016-01-01

The emergence and spread of colistin resistance among multidrug-resistant (MDR) Klebsiella pneumoniae represent a critical threat to global health. Here, we report the complete genome sequences of 10 MDR, colistin-susceptible and -resistant K. pneumoniae clinical isolates obtained in Pakistan between 2010 and 2013. PMID:27979956

Draft genome sequences of 1 MSSA and 7 MRSA ST5 isolates obtained from California

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus is a commensal of humans that can cause a spectrum of diseases. An isolate’s capacity to cause disease is partially attributed to the acquisition of novel mobile genetic elements. This report provides the draft genome sequence of one methicillin susceptible and seven methicilli...

Complete Genome Sequence of Kluyveromyces lactis Strain GG799, a Common Yeast Host for Heterologous Protein Expression

PubMed Central

Chuzel, Léa; Ganatra, Mehul B.; Schermerhorn, Kelly M.; Gardner, Andrew F.; Anton, Brian P.

2017-01-01

ABSTRACT We report the genome sequence of the dairy yeast Kluyveromyces lactis strain GG799 obtained using the Pacific Biosciences RS II platform. K. lactis strain GG799 is a common host for the expression of proteins at both laboratory and industrial scales. PMID:28751387

Whole-Genome Sequencing of Lactobacillus salivarius Strains BCRC 14759 and BCRC 12574

PubMed Central

Chiu, Shih-Hau; Wang, Li-Ting; Huang, Lina

2017-01-01

ABSTRACT Lactobacillus salivarius BCRC 14759 has been identified as a high-exopolysaccharide-producing strain with potential as a probiotic or fermented dairy product. Here, we report the genome sequences of L. salivarius BCRC 14759 and the comparable strain BCRC 12574, isolated from human saliva. The PacBio RSII sequencing platform was used to obtain high-quality assemblies for characterization of this probiotic candidate. PMID:29167259

Whole genome sequencing identifies influenza A H3N2 transmission and offers superior resolution to classical typing methods.

PubMed

Meinel, Dominik M; Heinzinger, Susanne; Eberle, Ute; Ackermann, Nikolaus; Schönberger, Katharina; Sing, Andreas

2018-02-01

Influenza with its annual epidemic waves is a major cause of morbidity and mortality worldwide. However, only little whole genome data are available regarding the molecular epidemiology promoting our understanding of viral spread in human populations. We implemented a RT-PCR strategy starting from patient material to generate influenza A whole genome sequences for molecular epidemiological surveillance. Samples were obtained within the Bavarian Influenza Sentinel. The complete influenza virus genome was amplified by a one-tube multiplex RT-PCR and sequenced on an Illumina MiSeq. We report whole genomic sequences for 50 influenza A H3N2 viruses, which was the predominating virus in the season 2014/15, directly from patient specimens. The dataset included random samples from Bavaria (Germany) throughout the influenza season and samples from three suspected transmission clusters. We identified the outbreak samples based on sequence identity. Whole genome sequencing (WGS) was superior in resolution compared to analysis of single segments or partial segment analysis. Additionally, we detected manifestation of substantial amounts of viral quasispecies in several patients, carrying mutations varying from the dominant virus in each patient. Our rapid whole genome sequencing approach for influenza A virus shows that WGS can effectively be used to detect and understand outbreaks in large communities. Additionally, the genomic data provide in-depth details about the circulating virus within one season.

Genome of Drosophila suzukii, the Spotted Wing Drosophila

PubMed Central

Chiu, Joanna C.; Jiang, Xuanting; Zhao, Li; Hamm, Christopher A.; Cridland, Julie M.; Saelao, Perot; Hamby, Kelly A.; Lee, Ernest K.; Kwok, Rosanna S.; Zhang, Guojie; Zalom, Frank G.; Walton, Vaughn M.; Begun, David J.

2013-01-01

Drosophila suzukii Matsumura (spotted wing drosophila) has recently become a serious pest of a wide variety of fruit crops in the United States as well as in Europe, leading to substantial yearly crop losses. To enable basic and applied research of this important pest, we sequenced the D. suzukii genome to obtain a high-quality reference sequence. Here, we discuss the basic properties of the genome and transcriptome and describe patterns of genome evolution in D. suzukii and its close relatives. Our analyses and genome annotations are presented in a web portal, SpottedWingFlyBase, to facilitate public access. PMID:24142924

SMRT sequencing data for Garcinia mangostana L. variety Mesta.

PubMed

Midin, Mohd Razik; Loke, Kok-Keong; Madon, Maria; Nordin, Mohd Shukor; Goh, Hoe-Han; Mohd Noor, Normah

2017-06-01

The "Queen of Fruits" mangosteen ( Garcinia mangostana L.) produces commercially important fruits with desirable taste of flesh and pericarp rich in xanthones with medicinal properties. To date, only limited knowledge is available on the cytogenetics and genome sequences of a common variety of mangosteen (Abu Bakar et al., 2016 [1]). Here, we report the first single-molecule real-time (SMRT) sequencing data from whole genome sequencing of mangosteen of Mesta variety. Raw reads of the SMRT sequencing project can be obtained from SRA database with the accession numbers SRX2718652 until SRX2718659.

Genome Survey Sequencing of Luffa Cylindrica L. and Microsatellite High Resolution Melting (SSR-HRM) Analysis for Genetic Relationship of Luffa Genotypes

PubMed Central

An, Jianyu; Yin, Mengqi; Zhang, Qin; Gong, Dongting; Jia, Xiaowen; Guan, Yajing; Hu, Jin

2017-01-01

Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica, about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (≥5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both “Zheda 23” and “Zheda 83”. Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species. PMID:28891982

Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association

PubMed Central

2012-01-01

Background The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. Results Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. Conclusions Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences. PMID:22409516

Random Amplification and Pyrosequencing for Identification of Novel Viral Genome Sequences

PubMed Central

Hang, Jun; Forshey, Brett M.; Kochel, Tadeusz J.; Li, Tao; Solórzano, Víctor Fiestas; Halsey, Eric S.; Kuschner, Robert A.

2012-01-01

ssRNA viruses have high levels of genomic divergence, which can lead to difficulty in genomic characterization of new viruses using traditional PCR amplification and sequencing methods. In this study, random reverse transcription, anchored random PCR amplification, and high-throughput pyrosequencing were used to identify orthobunyavirus sequences from total RNA extracted from viral cultures of acute febrile illness specimens. Draft genome sequence for the orthobunyavirus L segment was assembled and sequentially extended using de novo assembly contigs from pyrosequencing reads and orthobunyavirus sequences in GenBank as guidance. Accuracy and continuous coverage were achieved by mapping all reads to the L segment draft sequence. Subsequently, RT-PCR and Sanger sequencing were used to complete the genome sequence. The complete L segment was found to be 6936 bases in length, encoding a 2248-aa putative RNA polymerase. The identified L segment was distinct from previously published South American orthobunyaviruses, sharing 63% and 54% identity at the nucleotide and amino acid level, respectively, with the complete Oropouche virus L segment and 73% and 81% identity at the nucleotide and amino acid level, respectively, with a partial Caraparu virus L segment. The result demonstrated the effectiveness of a sequence-independent amplification and next-generation sequencing approach for obtaining complete viral genomes from total nucleic acid extracts and its use in pathogen discovery. PMID:22468136

Protein family clustering for structural genomics.

PubMed

Yan, Yongpan; Moult, John

2005-10-28

A major goal of structural genomics is the provision of a structural template for a large fraction of protein domains. The magnitude of this task depends on the number and nature of protein sequence families. With a large number of bacterial genomes now fully sequenced, it is possible to obtain improved estimates of the number and diversity of families in that kingdom. We have used an automated clustering procedure to group all sequences in a set of genomes into protein families. Bench-marking shows the clustering method is sensitive at detecting remote family members, and has a low level of false positives. This comprehensive protein family set has been used to address the following questions. (1) What is the structure coverage for currently known families? (2) How will the number of known apparent families grow as more genomes are sequenced? (3) What is a practical strategy for maximizing structure coverage in future? Our study indicates that approximately 20% of known families with three or more members currently have a representative structure. The study indicates also that the number of apparent protein families will be considerably larger than previously thought: We estimate that, by the criteria of this work, there will be about 250,000 protein families when 1000 microbial genomes have been sequenced. However, the vast majority of these families will be small, and it will be possible to obtain structural templates for 70-80% of protein domains with an achievable number of representative structures, by systematically sampling the larger families.

Sequences of multiple bacterial genomes and a Chlamydia trachomatis genotype from direct sequencing of DNA derived from a vaginal swab diagnostic specimen.

PubMed

Andersson, P; Klein, M; Lilliebridge, R A; Giffard, P M

2013-09-01

Ultra-deep Illumina sequencing was performed on whole genome amplified DNA derived from a Chlamydia trachomatis-positive vaginal swab. Alignment of reads with reference genomes allowed robust SNP identification from the C. trachomatis chromosome and plasmid. This revealed that the C. trachomatis in the specimen was very closely related to the sequenced urogenital, serovar F, clade T1 isolate F-SW4. In addition, high genome-wide coverage was obtained for Prevotella melaninogenica, Gardnerella vaginalis, Clostridiales genomosp. BVAB3 and Mycoplasma hominis. This illustrates the potential of metagenome data to provide high resolution bacterial typing data from multiple taxa in a diagnostic specimen. ©2013 The Authors Clinical Microbiology and Infection ©2013 European Society of Clinical Microbiology and Infectious Diseases.

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.

PubMed

Wang, Zhiwen; Hobson, Neil; Galindo, Leonardo; Zhu, Shilin; Shi, Daihu; McDill, Joshua; Yang, Linfeng; Hawkins, Simon; Neutelings, Godfrey; Datla, Raju; Lambert, Georgina; Galbraith, David W; Grassa, Christopher J; Geraldes, Armando; Cronk, Quentin C; Cullis, Christopher; Dash, Prasanta K; Kumar, Polumetla A; Cloutier, Sylvie; Sharpe, Andrew G; Wong, Gane K-S; Wang, Jun; Deyholos, Michael K

2012-11-01

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

The DL1 repeats in the genome of Diphyllobothrium latum.

PubMed

Usmanova, Nadezhda M; Kazakov, Vasiliy I

2010-07-01

Diphyllobothrium latum is a widespread intestinal parasite, which has a great clinical relevance, but there are no sequences of its nuclear genome. In this paper, a repetitive element in the D. latum genome is firstly described. The adult D. latum was obtained in the result of expulsion from intestinum of a patient suffering from diphyllobothriasis. Genomic DNA was isolated from several proglottids of this individual. PstI restriction products of D. latum genomic DNA were sequenced. Polymerase chain reaction (PCR) amplification of these products using genomic DNA and selected primers was carried out. Thereby a cluster of a repetitive element, called DL1, was discovered. For precise identification of a beginning and an end of the repeat, a product of PCR amplification of D. latum genomic DNA with one specific primer was sequenced. In discussion, several evidences that DL1 repeat is a member of the SINE family of retroposons were adduced.

Probabilistic topic modeling for the analysis and classification of genomic sequences

PubMed Central

2015-01-01

Background Studies on genomic sequences for classification and taxonomic identification have a leading role in the biomedical field and in the analysis of biodiversity. These studies are focusing on the so-called barcode genes, representing a well defined region of the whole genome. Recently, alignment-free techniques are gaining more importance because they are able to overcome the drawbacks of sequence alignment techniques. In this paper a new alignment-free method for DNA sequences clustering and classification is proposed. The method is based on k-mers representation and text mining techniques. Methods The presented method is based on Probabilistic Topic Modeling, a statistical technique originally proposed for text documents. Probabilistic topic models are able to find in a document corpus the topics (recurrent themes) characterizing classes of documents. This technique, applied on DNA sequences representing the documents, exploits the frequency of fixed-length k-mers and builds a generative model for a training group of sequences. This generative model, obtained through the Latent Dirichlet Allocation (LDA) algorithm, is then used to classify a large set of genomic sequences. Results and conclusions We performed classification of over 7000 16S DNA barcode sequences taken from Ribosomal Database Project (RDP) repository, training probabilistic topic models. The proposed method is compared to the RDP tool and Support Vector Machine (SVM) classification algorithm in a extensive set of trials using both complete sequences and short sequence snippets (from 400 bp to 25 bp). Our method reaches very similar results to RDP classifier and SVM for complete sequences. The most interesting results are obtained when short sequence snippets are considered. In these conditions the proposed method outperforms RDP and SVM with ultra short sequences and it exhibits a smooth decrease of performance, at every taxonomic level, when the sequence length is decreased. PMID:25916734

Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

PubMed

Marcy, Yann; Ishoey, Thomas; Lasken, Roger S; Stockwell, Timothy B; Walenz, Brian P; Halpern, Aaron L; Beeson, Karen Y; Goldberg, Susanne M D; Quake, Stephen R

2007-09-01

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

Sequencing, Analysis, and Annotation of Expressed Sequence Tags for Camelus dromedarius

PubMed Central

Al-Swailem, Abdulaziz M.; Shehata, Maher M.; Abu-Duhier, Faisel M.; Al-Yamani, Essam J.; Al-Busadah, Khalid A.; Al-Arawi, Mohammed S.; Al-Khider, Ali Y.; Al-Muhaimeed, Abdullah N.; Al-Qahtani, Fahad H.; Manee, Manee M.; Al-Shomrani, Badr M.; Al-Qhtani, Saad M.; Al-Harthi, Amer S.; Akdemir, Kadir C.; Otu, Hasan H.

2010-01-01

Despite its economical, cultural, and biological importance, there has not been a large scale sequencing project to date for Camelus dromedarius. With the goal of sequencing complete DNA of the organism, we first established and sequenced camel EST libraries, generating 70,272 reads. Following trimming, chimera check, repeat masking, cluster and assembly, we obtained 23,602 putative gene sequences, out of which over 4,500 potentially novel or fast evolving gene sequences do not carry any homology to other available genomes. Functional annotation of sequences with similarities in nucleotide and protein databases has been obtained using Gene Ontology classification. Comparison to available full length cDNA sequences and Open Reading Frame (ORF) analysis of camel sequences that exhibit homology to known genes show more than 80% of the contigs with an ORF>300 bp and ∼40% hits extending to the start codons of full length cDNAs suggesting successful characterization of camel genes. Similarity analyses are done separately for different organisms including human, mouse, bovine, and rat. Accompanying web portal, CAGBASE (http://camel.kacst.edu.sa/), hosts a relational database containing annotated EST sequences and analysis tools with possibility to add sequences from public domain. We anticipate our results to provide a home base for genomic studies of camel and other comparative studies enabling a starting point for whole genome sequencing of the organism. PMID:20502665

Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses.

PubMed

Ma, Peng-Fei; Guo, Zhen-Hua; Li, De-Zhu

2012-01-01

Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change. We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses. Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects.

Rapid Sequencing of the Bamboo Mitochondrial Genome Using Illumina Technology and Parallel Episodic Evolution of Organelle Genomes in Grasses

PubMed Central

Ma, Peng-Fei; Guo, Zhen-Hua; Li, De-Zhu

2012-01-01

Background Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change. Methodology/Principal Findings We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses. Conclusions/Significance Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects. PMID:22272330

Downsizing genomic medicine: approaching the ethical complexity of whole-genome sequencing by starting small.

PubMed

Sharp, Richard R

2011-03-01

As we look to a time when whole-genome sequencing is integrated into patient care, it is possible to anticipate a number of ethical challenges that will need to be addressed. The most intractable of these concern informed consent and the responsible management of very large amounts of genetic information. Given the range of possible findings, it remains unclear to what extent it will be possible to obtain meaningful patient consent to genomic testing. Equally unclear is how clinicians will disseminate the enormous volume of genetic information produced by whole-genome sequencing. Toward developing practical strategies for managing these ethical challenges, we propose a research agenda that approaches multiplexed forms of clinical genetic testing as natural laboratories in which to develop best practices for managing the ethical complexities of genomic medicine.

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project.

PubMed

Belkorchia, Abdel; Biderre, Corinne; Militon, Cécile; Polonais, Valérie; Wincker, Patrick; Jubin, Claire; Delbac, Frédéric; Peyretaillade, Eric; Peyret, Pierre

2008-03-01

Brachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity.

Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data.

PubMed

Jayakumar, Vasanthan; Sakakibara, Yasubumi

2017-11-03

Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.

Application of representational difference analysis to identify genomic differences between Bradyrhizobium elkanii and B. Japonicum species.

PubMed

Soares, René Arderius; Passaglia, Luciane Maria Pereira

2010-10-01

Bradyrhizobium elkanii is successfully used in the formulation of commercial inoculants and, together with B. japonicum, it fully supplies the plant nitrogen demands. Despite the similarity between B. japonicum and B. elkanii species, several works demonstrated genetic and physiological differences between them. In this work Representational Difference Analysis (RDA) was used for genomic comparison between B. elkanii SEMIA 587, a crop inoculant strain, and B. japonicum USDA 110, a reference strain. Two hundred sequences were obtained. From these, 46 sequences belonged exclusively to the genome of B. elkanii strain, and 154 showed similarity to sequences from B. japonicum genome. From the 46 sequences with no similarity to sequences from B. japonicum, 39 showed no similarity to sequences in public databases and seven showed similarity to sequences of genes coding for known proteins. These seven sequences were divided in three groups: similar to sequences from other Bradyrhizobium strains, similar to sequences from other nitrogen-fixing bacteria, and similar to sequences from non nitrogen-fixing bacteria. These new sequences could be used as DNA markers in order to investigate the rates of genetic material gain and loss in natural Bradyrhizobium strains.

Whole-Genome Sequencing of Lactobacillus salivarius Strains BCRC 14759 and BCRC 12574.

PubMed

Chiu, Shih-Hau; Chen, Chien-Chi; Wang, Li-Ting; Huang, Lina

2017-11-22

Lactobacillus salivarius BCRC 14759 has been identified as a high-exopolysaccharide-producing strain with potential as a probiotic or fermented dairy product. Here, we report the genome sequences of L. salivarius BCRC 14759 and the comparable strain BCRC 12574, isolated from human saliva. The PacBio RSII sequencing platform was used to obtain high-quality assemblies for characterization of this probiotic candidate. Copyright © 2017 Chiu et al.

PepLine: a software pipeline for high-throughput direct mapping of tandem mass spectrometry data on genomic sequences.

PubMed

Ferro, Myriam; Tardif, Marianne; Reguer, Erwan; Cahuzac, Romain; Bruley, Christophe; Vermat, Thierry; Nugues, Estelle; Vigouroux, Marielle; Vandenbrouck, Yves; Garin, Jérôme; Viari, Alain

2008-05-01

PepLine is a fully automated software which maps MS/MS fragmentation spectra of trypsic peptides to genomic DNA sequences. The approach is based on Peptide Sequence Tags (PSTs) obtained from partial interpretation of QTOF MS/MS spectra (first module). PSTs are then mapped on the six-frame translations of genomic sequences (second module) giving hits. Hits are then clustered to detect potential coding regions (third module). Our work aimed at optimizing the algorithms of each component to allow the whole pipeline to proceed in a fully automated manner using raw nucleic acid sequences (i.e., genomes that have not been "reduced" to a database of ORFs or putative exons sequences). The whole pipeline was tested on controlled MS/MS spectra sets from standard proteins and from Arabidopsis thaliana envelope chloroplast samples. Our results demonstrate that PepLine competed with protein database searching softwares and was fast enough to potentially tackle large data sets and/or high size genomes. We also illustrate the potential of this approach for the detection of the intron/exon structure of genes.

Whole-Genome Sequences of Four Strains Closely Related to Members of the Mycobacterium chelonae Group, Isolated from Biofilms in a Drinking Water Distribution System Simulator

EPA Science Inventory

We report the draft genome sequences of four Mycobacterium chelonae group strains from biofilms obtained after a ‘chlorine burn’ in a chloraminated drinking water distribution system simulator. These opportunistic pathogens have been detected in drinking and hospital water distr...

Single nucleotide polymorphisms in common bean: their discovery and genotyping using a multiplex detection system

USDA-ARS?s Scientific Manuscript database

Single-nucleotide Polymorphism (SNP) markers are by far the most common form of DNA polymorphism in a genome. The objectives of this study were to discover SNPs in common bean comparing sequences from coding and non-coding regions obtained from Genbank and genomic DNA and to compare sequencing resu...

Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome

USDA-ARS?s Scientific Manuscript database

Two cell lines, designated MARC.BGCF.2 and MARC.BGCF.1-3, were initiated from skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. These cell lines were submitted to American Type Culture Collection (ATCC, Manassas, VA, USA) and will be made publicly avai...

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits

PubMed Central

2011-01-01

Background Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18) to duodecaploid (12X = 108). Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome. Results A switchgrass BAC library constructed by partial digestion of nuclear DNA with EcoRI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective). Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice OsBRI1 locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy. Conclusions The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy. PMID:21767393

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits.

PubMed

Saski, Christopher A; Li, Zhigang; Feltus, Frank A; Luo, Hong

2011-07-18

Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18) to duodecaploid (12X = 108). Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome. A switchgrass BAC library constructed by partial digestion of nuclear DNA with EcoRI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective). Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice OsBRI1 locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy. The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells.

PubMed

Yajima, Misako; Ikuta, Kazufumi; Kanda, Teru

2018-04-03

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells

PubMed Central

Ikuta, Kazufumi; Kanda, Teru

2018-01-01

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically. PMID:29614006

Clinical sequencing in leukemia with the assistance of artificial intelligence.

PubMed

Tojo, Arinobu

2017-01-01

Next generation sequencing (NGS) of cancer genomes is now becoming a prerequisite for accurate diagnosis and proper treatment in clinical oncology. Because the genomic regions for NGS expand from a certain set of genes to the whole exome or whole genome, the resulting sequence data becomes incredibly enormous and makes it quite laborious to translate the genomic data into medicine, so-called annotation and curation. We organized a clinical sequencing team and established a bidirectional (bed-to-bench and bench-to-bed) system to integrate clinical and genomic data for hematological malignancies. We also started a collaborative research project with IBM Japan to adopt the artificial intelligence Watson for Genomics (WfG) to the pipeline of medical informatics. Genomic DNA was prepared from malignant as well as normal tissues in each patient and subjected to NGS. Sequence data was analyzed using an in-house semi-automated pipeline in combination with WfG, which was used to identify candidate driver mutations and relevant pathways from which applicable drug information was deduced. Currently, we have analyzed more than 150 patients with hematological disorders, including AML and ALL, and obtained many informative findings. In this presentation, I will introduce some of the achievements we have made so far.

Different phylogenomic approaches to resolve the evolutionary relationships among model fish species.

PubMed

Negrisolo, Enrico; Kuhl, Heiner; Forcato, Claudio; Vitulo, Nicola; Reinhardt, Richard; Patarnello, Tomaso; Bargelloni, Luca

2010-12-01

Comparative genomics holds the promise to magnify the information obtained from individual genome sequencing projects, revealing common features conserved across genomes and identifying lineage-specific characteristics. To implement such a comparative approach, a robust phylogenetic framework is required to accurately reconstruct evolution at the genome level. Among vertebrate taxa, teleosts represent the second best characterized group, with high-quality draft genome sequences for five model species (Danio rerio, Gasterosteus aculeatus, Oryzias latipes, Takifugu rubripes, and Tetraodon nigroviridis), and several others are in the finishing lane. However, the relationships among the acanthomorph teleost model fishes remain an unresolved taxonomic issue. Here, a genomic region spanning over 1.2 million base pairs was sequenced in the teleost fish Dicentrarchus labrax. Together with genomic data available for the above fish models, the new sequence was used to identify unique orthologous genomic regions shared across all target taxa. Different strategies were applied to produce robust multiple gene and genomic alignments spanning from 11,802 to 186,474 amino acid/nucleotide positions. Ten data sets were analyzed according to Bayesian inference, maximum likelihood, maximum parsimony, and neighbor joining methods. Extensive analyses were performed to explore the influence of several factors (e.g., alignment methodology, substitution model, data set partitions, and long-branch attraction) on the tree topology. Although a general consensus was observed for a closer relationship between G. aculeatus (Gasterosteidae) and Di. labrax (Moronidae) with the atherinomorph O. latipes (Beloniformes) sister taxon of this clade, with the tetraodontiform group Ta. rubripes and Te. nigroviridis (Tetraodontiformes) representing a more distantly related taxon among acanthomorph model fish species, conflicting results were obtained between data sets and methods, especially with respect to the choice of alignment methodology applied to noncoding parts of the genomic region under study. This may limit the use of intergenic/noncoding sequences in phylogenomics until more robust alignment algorithms are developed.

Discovery and Complete Genome Sequence of a Bacteriophage from an Obligate Intracellular Symbiont of a Cellulolytic Protist in the Termite Gut

PubMed Central

Pramono, Ajeng K.; Kuwahara, Hirokazu; Itoh, Takehiko; Toyoda, Atsushi; Yamada, Akinori; Hongoh, Yuichi

2017-01-01

Termites depend nutritionally on their gut microbes, and protistan, bacterial, and archaeal gut communities have been extensively studied. However, limited information is available on viruses in the termite gut. We herein report the complete genome sequence (99,517 bp) of a phage obtained during a genome analysis of “Candidatus Azobacteroides pseudotrichonymphae” phylotype ProJPt-1, which is an obligate intracellular symbiont of the cellulolytic protist Pseudotrichonympha sp. in the gut of the termite Prorhinotermes japonicus. The genome of the phage, designated ProJPt-Bp1, was circular or circularly permuted, and was not integrated into the two circular chromosomes or five circular plasmids composing the host ProJPt-1 genome. The phage was putatively affiliated with the order Caudovirales based on sequence similarities with several phage-related genes; however, most of the 52 protein-coding sequences had no significant homology to sequences in the databases. The phage genome contained a tRNA-Gln (CAG) gene, which showed the highest sequence similarity to the tRNA-Gln (CAA) gene of the host “Ca. A. pseudotrichonymphae” phylotype ProJPt-1. Since the host genome lacked a tRNA-Gln (CAG) gene, the phage tRNA gene may compensate for differences in codon usage bias between the phage and host genomes. The phage genome also contained a non-coding region with high nucleotide sequence similarity to a region in one of the host plasmids. No other phage-related sequences were found in the host ProJPt-1 genome. To the best of our knowledge, this is the first report of a phage from an obligate, mutualistic endosymbiont permanently associated with eukaryotic cells. PMID:28321010

Sequencing and characterizing the genome of Estrella lausannensis as an undergraduate project: training students and biological insights.

PubMed

Bertelli, Claire; Aeby, Sébastien; Chassot, Bérénice; Clulow, James; Hilfiker, Olivier; Rappo, Samuel; Ritzmann, Sébastien; Schumacher, Paolo; Terrettaz, Céline; Benaglio, Paola; Falquet, Laurent; Farinelli, Laurent; Gharib, Walid H; Goesmann, Alexander; Harshman, Keith; Linke, Burkhard; Miyazaki, Ryo; Rivolta, Carlo; Robinson-Rechavi, Marc; van der Meer, Jan Roelof; Greub, Gilbert

2015-01-01

With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by "embedded bioinformaticians," i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the "Sequence a genome" class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses.

The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants.

PubMed

Fajardo, Diego; Schlautman, Brandon; Steffan, Shawn; Polashock, James; Vorsa, Nicholi; Zalapa, Juan

2014-02-25

This is the first de novo assembly and annotation of a complete mitochondrial genome in the Ericales order from the American cranberry (Vaccinium macrocarpon Ait.). Moreover, only four complete Asterid mitochondrial genomes have been made publicly available. The cranberry mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with relatively little repetitive sequences and DNA of plastid origin. The complete mitochondrial genome of cranberry was annotated obtaining a total of 34 genes classified based on their putative function, plus three ribosomal RNAs, and 17 transfer RNAs. Maternal organellar cranberry inheritance was inferred by analyzing gene variation in the cranberry mitochondria and plastid genomes. The annotation of cranberry mitochondrial genome revealed the presence of two copies of tRNA-Sec and a selenocysteine insertion sequence (SECIS) element which were lost in plants during evolution. This is the first report of a land plant possessing selenocysteine insertion machinery at the sequence level. Published by Elsevier B.V.

Assembly of highly repetitive genomes using short reads: the genome of discrete typing unit III Trypanosoma cruzi strain 231.

PubMed

Baptista, Rodrigo P; Reis-Cunha, Joao Luis; DeBarry, Jeremy D; Chiari, Egler; Kissinger, Jessica C; Bartholomeu, Daniella C; Macedo, Andrea M

2018-02-14

Next-generation sequencing (NGS) methods are low-cost high-throughput technologies that produce thousands to millions of sequence reads. Despite the high number of raw sequence reads, their short length, relative to Sanger, PacBio or Nanopore reads, complicates the assembly of genomic repeats. Many genome tools are available, but the assembly of highly repetitive genome sequences using only NGS short reads remains challenging. Genome assembly of organisms responsible for important neglected diseases such as Trypanosoma cruzi, the aetiological agent of Chagas disease, is known to be challenging because of their repetitive nature. Only three of six recognized discrete typing units (DTUs) of the parasite have their draft genomes published and therefore genome evolution analyses in the taxon are limited. In this study, we developed a computational workflow to assemble highly repetitive genomes via a combination of de novo and reference-based assembly strategies to better overcome the intrinsic limitations of each, based on Illumina reads. The highly repetitive genome of the human-infecting parasite T. cruzi 231 strain was used as a test subject. The combined-assembly approach shown in this study benefits from the reference-based assembly ability to resolve highly repetitive sequences and from the de novo capacity to assemble genome-specific regions, improving the quality of the assembly. The acceptable confidence obtained by analyzing our results showed that our combined approach is an attractive option to assemble highly repetitive genomes with NGS short reads. Phylogenomic analysis including the 231 strain, the first representative of DTU III whose genome was sequenced, was also performed and provides new insights into T. cruzi genome evolution.

Modeling genome coverage in single-cell sequencing

PubMed Central

Daley, Timothy; Smith, Andrew D.

2014-01-01

Motivation: Single-cell DNA sequencing is necessary for examining genetic variation at the cellular level, which remains hidden in bulk sequencing experiments. But because they begin with such small amounts of starting material, the amount of information that is obtained from single-cell sequencing experiment is highly sensitive to the choice of protocol employed and variability in library preparation. In particular, the fraction of the genome represented in single-cell sequencing libraries exhibits extreme variability due to quantitative biases in amplification and loss of genetic material. Results: We propose a method to predict the genome coverage of a deep sequencing experiment using information from an initial shallow sequencing experiment mapped to a reference genome. The observed coverage statistics are used in a non-parametric empirical Bayes Poisson model to estimate the gain in coverage from deeper sequencing. This approach allows researchers to know statistical features of deep sequencing experiments without actually sequencing deeply, providing a basis for optimizing and comparing single-cell sequencing protocols or screening libraries. Availability and implementation: The method is available as part of the preseq software package. Source code is available at http://smithlabresearch.org/preseq. Contact: andrewds@usc.edu Supplementary information: Supplementary material is available at Bioinformatics online. PMID:25107873

BAC end sequencing of Pacific white shrimp Litopenaeus vannamei: a glimpse into the genome of Penaeid shrimp

NASA Astrophysics Data System (ADS)

Zhao, Cui; Zhang, Xiaojun; Liu, Chengzhang; Huan, Pin; Li, Fuhua; Xiang, Jianhai; Huang, Chao

2012-05-01

Little is known about the genome of Pacific white shrimp ( Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 pairedends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.

An integrated semiconductor device enabling non-optical genome sequencing.

PubMed

Rothberg, Jonathan M; Hinz, Wolfgang; Rearick, Todd M; Schultz, Jonathan; Mileski, William; Davey, Mel; Leamon, John H; Johnson, Kim; Milgrew, Mark J; Edwards, Matthew; Hoon, Jeremy; Simons, Jan F; Marran, David; Myers, Jason W; Davidson, John F; Branting, Annika; Nobile, John R; Puc, Bernard P; Light, David; Clark, Travis A; Huber, Martin; Branciforte, Jeffrey T; Stoner, Isaac B; Cawley, Simon E; Lyons, Michael; Fu, Yutao; Homer, Nils; Sedova, Marina; Miao, Xin; Reed, Brian; Sabina, Jeffrey; Feierstein, Erika; Schorn, Michelle; Alanjary, Mohammad; Dimalanta, Eileen; Dressman, Devin; Kasinskas, Rachel; Sokolsky, Tanya; Fidanza, Jacqueline A; Namsaraev, Eugeni; McKernan, Kevin J; Williams, Alan; Roth, G Thomas; Bustillo, James

2011-07-20

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

The Biofuel Feedstock Genomics Resource: a web-based portal and database to enable functional genomics of plant biofuel feedstock species.

PubMed

Childs, Kevin L; Konganti, Kranti; Buell, C Robin

2012-01-01

Major feedstock sources for future biofuel production are likely to be high biomass producing plant species such as poplar, pine, switchgrass, sorghum and maize. One active area of research in these species is genome-enabled improvement of lignocellulosic biofuel feedstock quality and yield. To facilitate genomic-based investigations in these species, we developed the Biofuel Feedstock Genomic Resource (BFGR), a database and web-portal that provides high-quality, uniform and integrated functional annotation of gene and transcript assembly sequences from species of interest to lignocellulosic biofuel feedstock researchers. The BFGR includes sequence data from 54 species and permits researchers to view, analyze and obtain annotation at the gene, transcript, protein and genome level. Annotation of biochemical pathways permits the identification of key genes and transcripts central to the improvement of lignocellulosic properties in these species. The integrated nature of the BFGR in terms of annotation methods, orthologous/paralogous relationships and linkage to seven species with complete genome sequences allows comparative analyses for biofuel feedstock species with limited sequence resources. Database URL: http://bfgr.plantbiology.msu.edu.

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

PubMed

Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

2016-01-01

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism

PubMed Central

Yin, Ling; An, Yunhe; Qu, Junjie; Li, Xinlong; Zhang, Yali; Dry, Ian; Wu, Huijuan; Lu, Jiang

2017-01-01

Plasmopara viticola causes downy mildew disease of grapevine which is one of the most devastating diseases of viticulture worldwide. Here we report a 101.3 Mb whole genome sequence of P. viticola isolate ‘JL-7-2’ obtained by a combination of Illumina and PacBio sequencing technologies. The P. viticola genome contains 17,014 putative protein-coding genes and has ~26% repetitive sequences. A total of 1,301 putative secreted proteins, including 100 putative RXLR effectors and 90 CRN effectors were identified in this genome. In the secretome, 261 potential pathogenicity genes and 95 carbohydrate-active enzymes were predicted. Transcriptional analysis revealed that most of the RXLR effectors, pathogenicity genes and carbohydrate-active enzymes were significantly up-regulated during infection. Comparative genomic analysis revealed that P. viticola evolved independently from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. The availability of the P. viticola genome provides a valuable resource not only for comparative genomic analysis and evolutionary studies among oomycetes, but also enhance our knowledge on the mechanism of interactions between this biotrophic pathogen and its host. PMID:28417959

Complete genome analysis of highly pathogenic bovine ephemeral fever virus isolated in Turkey in 2012.

PubMed

Abayli, Hasan; Tonbak, Sukru; Azkur, Ahmet Kursat; Bulut, Hakan

2017-10-01

Relatively high prevalence and mortality rates of bovine ephemeral fever (BEF) have been reported in recent epidemics in some countries, including Turkey, when compared with previous outbreaks. A limited number of complete genome sequences of BEF virus (BEFV) are available in the GenBank Database. In this study, the complete genome of highly pathogenic BEFV isolated during an outbreak in Turkey in 2012 was analyzed for genetic characterization. The complete genome of the Turkish BEFV isolate was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. It was found that the complete genome of the Turkish BEFV isolate was 14,901 nt in length. The complete genome sequence obtained from the study showed 91-92% identity at nucleotide level to Australian (BB7721) and Chinese (Bovine/China/Henan1/2012) BEFV isolates. Phylogenetic analysis of the glycoprotein gene of the Turkish BEFV isolate also showed that Turkish isolates were closely related to Israeli isolates. Because of the limited number of complete BEFV genome sequences, the results from this study will be useful for understanding the global molecular epidemiology and geodynamics of BEF.

Use of Genome Sequence Information for Meat Quality Trait QTL Mining for Causal Genes and Mutations on Pig Chromosome 17

PubMed Central

Hu, Zhi-Liang; Ramos, Antonio M.; Humphray, Sean J.; Rogers, Jane; Reecy, James M.; Rothschild, Max F.

2011-01-01

The newly available pig genome sequence has provided new information to fine map quantitative trait loci (QTL) in order to eventually identify causal variants. With targeted genomic sequencing efforts, we were able to obtain high quality BAC sequences that cover a region on pig chromosome 17 where a number of meat quality QTL have been previously discovered. Sequences from 70 BAC clones were assembled to form an 8-Mbp contig. Subsequently, we successfully mapped five previously identified QTL, three for meat color and two for lactate related traits, to the contig. With an additional 25 genetic markers that were identified by sequence comparison, we were able to carry out further linkage disequilibrium analysis to narrow down the genomic locations of these QTL, which allowed identification of the chromosomal regions that likely contain the causative variants. This research has provided one practical approach to combine genetic and molecular information for QTL mining. PMID:22303339

A Deep-Coverage Tomato BAC Library and Prospects Toward Development of an STC Framework for Genome Sequencing

PubMed Central

Budiman, Muhammad A.; Mao, Long; Wood, Todd C.; Wing, Rod A.

2000-01-01

Recently a new strategy using BAC end sequences as sequence-tagged connectors (STCs) was proposed for whole-genome sequencing projects. In this study, we present the construction and detailed characterization of a 15.0 haploid genome equivalent BAC library for the cultivated tomato, Lycopersicon esculentum cv. Heinz 1706. The library contains 129,024 clones with an average insert size of 117.5 kb and a chloroplast content of 1.11%. BAC end sequences from 1490 ends were generated and analyzed as a preliminary evaluation for using this library to develop an STC framework to sequence the tomato genome. A total of 1205 BAC end sequences (80.9%) were obtained, with an average length of 360 high-quality bases, and were searched against the GenBank database. Using a cutoff expectation value of <10−6, and combining the results from BLASTN, BLASTX, and TBLASTX searches, 24.3% of the BAC end sequences were similar to known sequences, of which almost half (48.7%) share sequence similarities to retrotransposons and 7% to known genes. Some of the transposable element sequences were the first reported in tomato, such as sequences similar to maize transposon Activator (Ac) ORF and tobacco pararetrovirus-like sequences. Interestingly, there were no BAC end sequences similar to the highly repeated TGRI and TGRII elements. However, the majority (70.3%) of STCs did not share significant sequence similarities to any sequences in GenBank at either the DNA or predicted protein levels, indicating that a large portion of the tomato genome is still unknown. Our data demonstrate that this BAC library is suitable for developing an STC database to sequence the tomato genome. The advantages of developing an STC framework for whole-genome sequencing of tomato are discussed. [The BAC end sequences described in this paper have been deposited in the GenBank data library under accession nos. AQ367111–AQ368361.] PMID:10645957

Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray.

PubMed

Pardo, Belén G; Álvarez-Dios, José Antonio; Cao, Asunción; Ramilo, Andrea; Gómez-Tato, Antonio; Planas, Josep V; Villalba, Antonio; Martínez, Paulino

2016-12-01

The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Complete genome sequence of an attenuated Sparfloxacin resistant Streptococcus agalactiae strain 138spar

USDA-ARS?s Scientific Manuscript database

Through selection of resistance to sparfloxacin, an attenuated Streptococcus agalactiae strain 138spar was obtained from its virulent parent strain S. agalactiae 138P. The full genome of S. agalactiae 138spar is 1,838,126 bp. The availability of this genome will allow comparative genomics to identi...

Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia.

PubMed

Shortt, Jonathan A; Card, Daren C; Schield, Drew R; Liu, Yang; Zhong, Bo; Castoe, Todd A; Carlton, Elizabeth J; Pollock, David D

2017-01-01

In areas where schistosomiasis control programs have been implemented, morbidity and prevalence have been greatly reduced. However, to sustain these reductions and move towards interruption of transmission, new tools for disease surveillance are needed. Genomic methods have the potential to help trace the sources of new infections, and allow us to monitor drug resistance. Large-scale genotyping efforts for schistosome species have been hindered by cost, limited numbers of established target loci, and the small amount of DNA obtained from miracidia, the life stage most readily acquired from humans. Here, we present a method using next generation sequencing to provide high-resolution genomic data from S. japonicum for population-based studies. We applied whole genome amplification followed by double digest restriction site associated DNA sequencing (ddRADseq) to individual S. japonicum miracidia preserved on Whatman FTA cards. We found that we could effectively and consistently survey hundreds of thousands of variants from 10,000 to 30,000 loci from archived miracidia as old as six years. An analysis of variation from eight miracidia obtained from three hosts in two villages in Sichuan showed clear population structuring by village and host even within this limited sample. This high-resolution sequencing approach yields three orders of magnitude more information than microsatellite genotyping methods that have been employed over the last decade, creating the potential to answer detailed questions about the sources of human infections and to monitor drug resistance. Costs per sample range from $50-$200, depending on the amount of sequence information desired, and we expect these costs can be reduced further given continued reductions in sequencing costs, improvement of protocols, and parallelization. This approach provides new promise for using modern genome-scale sampling to S. japonicum surveillance, and could be applied to other schistosome species and other parasitic helminthes.

Noninvasive Online Measurement of Genome Lengths of Mammalian Tissues in Bulk by 14 MeV Neutron Atometry

NASA Astrophysics Data System (ADS)

Maglich, Bogdan; Radovic, Anna; Druey, Christian

2012-10-01

Genome length, L=, no. of DNA nucleotide base pairs in cell of bovine (b) and porcine (p) tissues, closest to human genome, were hitherto measured by genomic sequencing Lb=3, Lp=2.7 Giga base pairs [1,2] (Gbp) errors not given. - We report measurements of Lb/Lp and Lb, Lp without sequencing by atometry [3,4]. No. of O and C atoms, N, in nucleotide molecules, was obtained from prompt γ rate, G, emitted in inel. scatt. 14 MeV neutrons, with nuclei of C, O, in nucleotide molecule. Since G prop. N, Lb/Lp=Gb/Gp. p and b meat was irradiated for 30'. From msd G we obtained Lb /Lp=1.28±0.02 16% greater than [1,2]. We got absolute Lb=1.65/f, Lp=1.28/f Gbp, 0.3

Genome Sequence of Candidatus Nitrososphaera evergladensis from Group I.1b Enriched from Everglades Soil Reveals Novel Genomic Features of the Ammonia-Oxidizing Archaea

PubMed Central

Zhalnina, Kateryna V.; Dias, Raquel; Leonard, Michael T.; Dorr de Quadros, Patricia; Camargo, Flavio A. O.; Drew, Jennifer C.; Farmerie, William G.; Daroub, Samira H.; Triplett, Eric W.

2014-01-01

The activity of ammonia-oxidizing archaea (AOA) leads to the loss of nitrogen from soil, pollution of water sources and elevated emissions of greenhouse gas. To date, eight AOA genomes are available in the public databases, seven are from the group I.1a of the Thaumarchaeota and only one is from the group I.1b, isolated from hot springs. Many soils are dominated by AOA from the group I.1b, but the genomes of soil representatives of this group have not been sequenced and functionally characterized. The lack of knowledge of metabolic pathways of soil AOA presents a critical gap in understanding their role in biogeochemical cycles. Here, we describe the first complete genome of soil archaeon Candidatus Nitrososphaera evergladensis, which has been reconstructed from metagenomic sequencing of a highly enriched culture obtained from an agricultural soil. The AOA enrichment was sequenced with the high throughput next generation sequencing platforms from Pacific Biosciences and Ion Torrent. The de novo assembly of sequences resulted in one 2.95 Mb contig. Annotation of the reconstructed genome revealed many similarities of the basic metabolism with the rest of sequenced AOA. Ca. N. evergladensis belongs to the group I.1b and shares only 40% of whole-genome homology with the closest sequenced relative Ca. N. gargensis. Detailed analysis of the genome revealed coding sequences that were completely absent from the group I.1a. These unique sequences code for proteins involved in control of DNA integrity, transporters, two-component systems and versatile CRISPR defense system. Notably, genomes from the group I.1b have more gene duplications compared to the genomes from the group I.1a. We suggest that the presence of these unique genes and gene duplications may be associated with the environmental versatility of this group. PMID:24999826

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

PubMed

Seoane-Zonjic, Pedro; Cañas, Rafael A; Bautista, Rocío; Gómez-Maldonado, Josefa; Arrillaga, Isabel; Fernández-Pozo, Noé; Claros, M Gonzalo; Cánovas, Francisco M; Ávila, Concepción

2016-02-27

In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a Ruby gem for this class of analyses.

Exploring Pandora's Box: Potential and Pitfalls of Low Coverage Genome Surveys for Evolutionary Biology

PubMed Central

Leese, Florian; Mayer, Christoph; Agrawal, Shobhit; Dambach, Johannes; Dietz, Lars; Doemel, Jana S.; Goodall-Copstake, William P.; Held, Christoph; Jackson, Jennifer A.; Lampert, Kathrin P.; Linse, Katrin; Macher, Jan N.; Nolzen, Jennifer; Raupach, Michael J.; Rivera, Nicole T.; Schubart, Christoph D.; Striewski, Sebastian; Tollrian, Ralph; Sands, Chester J.

2012-01-01

High throughput sequencing technologies are revolutionizing genetic research. With this “rise of the machines”, genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02–25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers. PMID:23185309

UFO: a web server for ultra-fast functional profiling of whole genome protein sequences.

PubMed

Meinicke, Peter

2009-09-02

Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

Genomic epidemiology of a protracted hospital outbreak caused by multidrug-resistant Acinetobacter baumannii in Birmingham, England.

PubMed

Halachev, Mihail R; Chan, Jacqueline Z-M; Constantinidou, Chrystala I; Cumley, Nicola; Bradley, Craig; Smith-Banks, Matthew; Oppenheim, Beryl; Pallen, Mark J

2014-01-01

Multidrug-resistant Acinetobacter baumannii commonly causes hospital outbreaks. However, within an outbreak, it can be difficult to identify the routes of cross-infection rapidly and accurately enough to inform infection control. Here, we describe a protracted hospital outbreak of multidrug-resistant A. baumannii, in which whole-genome sequencing (WGS) was used to obtain a high-resolution view of the relationships between isolates. To delineate and investigate the outbreak, we attempted to genome-sequence 114 isolates that had been assigned to the A. baumannii complex by the Vitek2 system and obtained informative draft genome sequences from 102 of them. Genomes were mapped against an outbreak reference sequence to identify single nucleotide variants (SNVs). We found that the pulsotype 27 outbreak strain was distinct from all other genome-sequenced strains. Seventy-four isolates from 49 patients could be assigned to the pulsotype 27 outbreak on the basis of genomic similarity, while WGS allowed 18 isolates to be ruled out of the outbreak. Among the pulsotype 27 outbreak isolates, we identified 31 SNVs and seven major genotypic clusters. In two patients, we documented within-host diversity, including mixtures of unrelated strains and within-strain clouds of SNV diversity. By combining WGS and epidemiological data, we reconstructed potential transmission events that linked all but 10 of the patients and confirmed links between clinical and environmental isolates. Identification of a contaminated bed and a burns theatre as sources of transmission led to enhanced environmental decontamination procedures. WGS is now poised to make an impact on hospital infection prevention and control, delivering cost-effective identification of routes of infection within a clinically relevant timeframe and allowing infection control teams to track, and even prevent, the spread of drug-resistant hospital pathogens.

High-Throughput Sequencing, a Versatile Weapon to Support Genome-Based Diagnosis in Infectious Diseases: Applications to Clinical Bacteriology

PubMed Central

Caboche, Ségolène; Audebert, Christophe; Hot, David

2014-01-01

The recent progresses of high-throughput sequencing (HTS) technologies enable easy and cost-reduced access to whole genome sequencing (WGS) or re-sequencing. HTS associated with adapted, automatic and fast bioinformatics solutions for sequencing applications promises an accurate and timely identification and characterization of pathogenic agents. Many studies have demonstrated that data obtained from HTS analysis have allowed genome-based diagnosis, which has been consistent with phenotypic observations. These proofs of concept are probably the first steps toward the future of clinical microbiology. From concept to routine use, many parameters need to be considered to promote HTS as a powerful tool to help physicians and clinicians in microbiological investigations. This review highlights the milestones to be completed toward this purpose. PMID:25437800

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

The complete validated mitochondrial genome of the silver gemfish Rexea solandri (Cuvier, 1832) (Perciformes, Gempylidae).

PubMed

Bustamante, Carlos; Ovenden, Jennifer R

2016-01-01

The silver gemfish Rexea solandri is an important economic resource but Vulnerable to overfishing in Australian waters. The complete mitochondrial genome sequence is described from 1.6 million reads obtained via next generation sequencing. The total length of the mitogenome is 16,350 bp comprising 2 rRNA, 13 protein-coding genes, 22 tRNA and 2 non-coding regions. The mitogenome sequence was validated against sequences of PCR fragments and BLAST queries of Genbank. Gene order was equivalent to that found in marine fishes.

Long-PCR based next generation sequencing of the whole mitochondrial genome of the peacock skate Pavoraja nitida (Elasmobranchii: Arhynchobatidae).

PubMed

Yang, Lei; Naylor, Gavin J P

2016-01-01

We determined the complete mitochondrial genome sequence (16,760 bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes.

Data Release: DNA barcodes of plant species collected for the Global Genome Initiative for Gardens Program, National Museum of Natural History, Smithsonian Institution

PubMed Central

Zúñiga, Jose D.; Gostel, Morgan R.; Mulcahy, Daniel G.; Barker, Katharine; Asia Hill; Sedaghatpour, Maryam; Vo, Samantha Q.; Funk, Vicki A.; Coddington, Jonathan A.

2017-01-01

Abstract The Global Genome Initiative has sequenced and released 1961 DNA barcodes for genetic samples obtained as part of the Global Genome Initiative for Gardens Program. The dataset includes barcodes for 29 plant families and 309 genera that did not have sequences flagged as barcodes in GenBank and sequences from officially recognized barcoding genetic markers meet the data standard of the Consortium for the Barcode of Life. The genetic samples were deposited in the Smithsonian Institution’s National Museum of Natural History Biorepository and their records were made public through the Global Genome Biodiversity Network’s portal. The DNA barcodes are now available on GenBank. PMID:29118648

Comparison of Intracellular "Ca. Endomicrobium Trichonymphae" Genomovars Illuminates the Requirement and Decay of Defense Systems against Foreign DNA.

PubMed

Izawa, Kazuki; Kuwahara, Hirokazu; Kihara, Kumiko; Yuki, Masahiro; Lo, Nathan; Itoh, Takehiko; Ohkuma, Moriya; Hongoh, Yuichi

2016-10-13

"Candidatus Endomicrobium trichonymphae" (Bacteria; Elusimicrobia) is an obligate intracellular symbiont of the cellulolytic protist genus Trichonympha in the termite gut. A previous genome analysis of "Ca Endomicrobium trichonymphae" phylotype Rs-D17 (genomovar Ri2008), obtained from a Trichonympha agilis cell in the gut of the termite Reticulitermes speratus, revealed that its genome is small (1.1 Mb) and contains many pseudogenes; it is in the course of reductive genome evolution. Here we report the complete genome sequence of another Rs-D17 genomovar, Ti2015, obtained from a different T. agilis cell present in an R. speratus gut. These two genomovars share most intact protein-coding genes and pseudogenes, showing 98.6% chromosome sequence similarity. However, characteristic differences were found in their defense systems, which comprised restriction-modification and CRISPR/Cas systems. The repertoire of intact restriction-modification systems differed between the genomovars, and two of the three CRISPR/Cas loci in genomovar Ri2008 are pseudogenized or missing in genomovar Ti2015. These results suggest relaxed selection pressure for maintaining these defense systems. Nevertheless, the remaining CRISPR/Cas system in each genomovar appears to be active; none of the "spacer" sequences (112 in Ri2008 and 128 in Ti2015) were shared whereas the "repeat" sequences were identical. Furthermore, we obtained draft genomes of three additional endosymbiotic Endomicrobium phylotypes from different host protist species, and discovered multiple, intact CRISPR/Cas systems in each genome. Collectively, unlike bacteriome endosymbionts in insects, the Endomicrobium endosymbionts of termite-gut protists appear to require defense against foreign DNA, although the required level of defense has likely been reduced during their intracellular lives. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Draft Genome Sequence of the Deinococcus-Thermus Bacterium Meiothermus ruber Strain A

DOE PAGES

Thiel, Vera; Tomsho, Lynn P.; Burhans, Richard; ...

2015-03-26

The draft genome sequence of the Deinococcus-Thermus group bacterium Meiothermus ruber strain A, isolated from a cyanobacterial enrichment culture obtained from Octopus Spring (Yellowstone National Park, WY), comprises 2,968,099 bp in 170 contigs. It is predicted to contain 2,895 protein-coding genes, 44 tRNA-coding genes, and 2 rRNA operons.

Draft genome of the medaka fish: a comprehensive resource for medaka developmental genetics and vertebrate evolutionary biology.

PubMed

Takeda, Hiroyuki

2008-06-01

The medaka Oryzias latipes is a small egg-laying freshwater teleost, and has become an excellent model system for developmental genetics and evolutionary biology. The medaka genome is relatively small in size, approximately 800 Mb, and the genome sequencing project was recently completed by Japanese research groups, providing a high-quality draft genome sequence of the inbred Hd-rR strain of medaka. In this review, I present an overview of the medaka genome project including genome resources, followed by specific findings obtained with the medaka draft genome. In particular, I focus on the analysis that was done by taking advantage of the medaka system, such as the sex chromosome differentiation and the regional history of medaka species using single nucleotide polymorphisms as genomic markers.

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes.

PubMed

Haiminen, Niina; Feltus, F Alex; Parida, Laxmi

2011-04-15

We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies.

Complete genome sequence of Ikoma lyssavirus.

PubMed

Marston, Denise A; Ellis, Richard J; Horton, Daniel L; Kuzmin, Ivan V; Wise, Emma L; McElhinney, Lorraine M; Banyard, Ashley C; Ngeleja, Chanasa; Keyyu, Julius; Cleaveland, Sarah; Lembo, Tiziana; Rupprecht, Charles E; Fooks, Anthony R

2012-09-01

Lyssaviruses (family Rhabdoviridae) constitute one of the most important groups of viral zoonoses globally. All lyssaviruses cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Currently available vaccines are highly protective against the predominantly circulating lyssavirus species. Using next-generation sequencing technologies, we have obtained the whole-genome sequence for a novel lyssavirus, Ikoma lyssavirus (IKOV), isolated from an African civet in Tanzania displaying clinical signs of rabies. Genetically, this virus is the most divergent within the genus Lyssavirus. Characterization of the genome will help to improve our understanding of lyssavirus diversity and enable investigation into vaccine-induced immunity and protection.

The complete mitochondrial genome sequence of the Tibetan red fox (Vulpes vulpes montana).

PubMed

Zhang, Jin; Zhang, Honghai; Zhao, Chao; Chen, Lei; Sha, Weilai; Liu, Guangshuai

2015-01-01

In this study, the complete mitochondrial genome of the Tibetan red fox (Vulpes Vulpes montana) was sequenced for the first time using blood samples obtained from a wild female red fox captured from Lhasa in Tibet, China. Qinghai--Tibet Plateau is the highest plateau in the world with an average elevation above 3500 m. Sequence analysis showed it contains 12S rRNA gene, 16S rRNA gene, 22 tRNA genes, 13 protein-coding genes and 1 control region (CR). The variable tandem repeats in CR is the main reason of the length variability of mitochondrial genome among canide animals.

Toscana Virus Genome Stability: Data from a Meningoencephalitis Case in Mantua, Italy

PubMed Central

Baggieri, Melissa; Gattuso, Gianni; Fortuna, Claudia; Remoli, Maria Elena; Vaccari, Gabriele; Zaccaria, Guendalina; Marchi, Antonella; Bucci, Paola; Benedetti, Eleonora; Fiorentini, Cristiano; Nicoletti, Loredana

2014-01-01

Abstract In July of 2013, samples from a patient with a neurological syndrome were collected from Mantua hospital and sent to the National Reference Laboratory for Arboviruses (National Institute of Health, Rome). On the basis of the symptoms, serological and molecular assays were performed to diagnose either West Nile virus (WNV) or Toscana virus (TOSV) infection. Molecular and serological tests confirmed TOSV infection. Virus isolation was obtained from cerebrospinal fluid. A full genome sequence was determined from this TOSV strain with next-generation sequencing using Ion Torrent technology. Nucleotide and amino acidic sequences grouped phylogenetically with lineage TOSV A and showed a low genome variability. PMID:25514123

Phylogenic study of Lemnoideae (duckweeds) through complete chloroplast genomes for eight accessions.

PubMed

Ding, Yanqiang; Fang, Yang; Guo, Ling; Li, Zhidan; He, Kaize; Zhao, Yun; Zhao, Hai

2017-01-01

Phylogenetic relationship within different genera of Lemnoideae, a kind of small aquatic monocotyledonous plants, was not well resolved, using either morphological characters or traditional markers. Given that rich genetic information in chloroplast genome makes them particularly useful for phylogenetic studies, we used chloroplast genomes to clarify the phylogeny within Lemnoideae. DNAs were sequenced with next-generation sequencing. The duckweeds chloroplast genomes were indirectly filtered from the total DNA data, or directly obtained from chloroplast DNA data. To test the reliability of assembling the chloroplast genome based on the filtration of the total DNA, two methods were used to assemble the chloroplast genome of Landoltia punctata strain ZH0202. A phylogenetic tree was built on the basis of the whole chloroplast genome sequences using MrBayes v.3.2.6 and PhyML 3.0. Eight complete duckweeds chloroplast genomes were assembled, with lengths ranging from 165,775 bp to 171,152 bp, and each contains 80 protein-coding sequences, four rRNAs, 30 tRNAs and two pseudogenes. The identity of L. punctata strain ZH0202 chloroplast genomes assembled through two methods was 100%, and their sequences and lengths were completely identical. The chloroplast genome comparison demonstrated that the differences in chloroplast genome sizes among the Lemnoideae primarily resulted from variation in non-coding regions, especially from repeat sequence variation. The phylogenetic analysis demonstrated that the different genera of Lemnoideae are derived from each other in the following order: Spirodela , Landoltia , Lemna , Wolffiella , and Wolffia . This study demonstrates potential of whole chloroplast genome DNA as an effective option for phylogenetic studies of Lemnoideae. It also showed the possibility of using chloroplast DNA data to elucidate those phylogenies which were not yet solved well by traditional methods even in plants other than duckweeds.

Phylogenic study of Lemnoideae (duckweeds) through complete chloroplast genomes for eight accessions

PubMed Central

Ding, Yanqiang; Fang, Yang; Guo, Ling; Li, Zhidan; He, Kaize

2017-01-01

Background Phylogenetic relationship within different genera of Lemnoideae, a kind of small aquatic monocotyledonous plants, was not well resolved, using either morphological characters or traditional markers. Given that rich genetic information in chloroplast genome makes them particularly useful for phylogenetic studies, we used chloroplast genomes to clarify the phylogeny within Lemnoideae. Methods DNAs were sequenced with next-generation sequencing. The duckweeds chloroplast genomes were indirectly filtered from the total DNA data, or directly obtained from chloroplast DNA data. To test the reliability of assembling the chloroplast genome based on the filtration of the total DNA, two methods were used to assemble the chloroplast genome of Landoltia punctata strain ZH0202. A phylogenetic tree was built on the basis of the whole chloroplast genome sequences using MrBayes v.3.2.6 and PhyML 3.0. Results Eight complete duckweeds chloroplast genomes were assembled, with lengths ranging from 165,775 bp to 171,152 bp, and each contains 80 protein-coding sequences, four rRNAs, 30 tRNAs and two pseudogenes. The identity of L. punctata strain ZH0202 chloroplast genomes assembled through two methods was 100%, and their sequences and lengths were completely identical. The chloroplast genome comparison demonstrated that the differences in chloroplast genome sizes among the Lemnoideae primarily resulted from variation in non-coding regions, especially from repeat sequence variation. The phylogenetic analysis demonstrated that the different genera of Lemnoideae are derived from each other in the following order: Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia. Discussion This study demonstrates potential of whole chloroplast genome DNA as an effective option for phylogenetic studies of Lemnoideae. It also showed the possibility of using chloroplast DNA data to elucidate those phylogenies which were not yet solved well by traditional methods even in plants other than duckweeds. PMID:29302399

Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms.

PubMed

Barony, Gustavo M; Tavares, Guilherme C; Pereira, Felipe L; Carvalho, Alex F; Dorella, Fernanda A; Leal, Carlos A G; Figueiredo, Henrique C P

2017-10-19

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

Generation and analysis of expressed sequence tags from the bone marrow of Chinese Sika deer.

PubMed

Yao, Baojin; Zhao, Yu; Zhang, Mei; Li, Juan

2012-03-01

Sika deer is one of the best-known and highly valued animals of China. Despite its economic, cultural, and biological importance, there has not been a large-scale sequencing project for Sika deer to date. With the ultimate goal of sequencing the complete genome of this organism, we first established a bone marrow cDNA library for Sika deer and generated a total of 2,025 reads. After processing the sequences, 2,017 high-quality expressed sequence tags (ESTs) were obtained. These ESTs were assembled into 1,157 unigenes, including 238 contigs and 919 singletons. Comparative analyses indicated that 888 (76.75%) of the unigenes had significant matches to sequences in the non-redundant protein database, In addition to highly expressed genes, such as stearoyl-CoA desaturase, cytochrome c oxidase, adipocyte-type fatty acid-binding protein, adiponectin and thymosin beta-4, we also obtained vascular endothelial growth factor-A and heparin-binding growth-associated molecule, both of which are of great importance for angiogenesis research. There were 244 (21.09%) unigenes with no significant match to any sequence in current protein or nucleotide databases, and these sequences may represent genes with unknown function in Sika deer. Open reading frame analysis of the sequences was performed using the getorf program. In addition, the sequences were functionally classified using the gene ontology hierarchy, clusters of orthologous groups of proteins and Kyoto encyclopedia of genes and genomes databases. Analysis of ESTs described in this paper provides an important resource for the transcriptome exploration of Sika deer, and will also facilitate further studies on functional genomics, gene discovery and genome annotation of Sika deer.

Genome Sequence of African Swine Fever Virus BA71, the Virulent Parental Strain of the Nonpathogenic and Tissue-Culture Adapted BA71V.

PubMed

Rodríguez, Javier M; Moreno, Leticia Tais; Alejo, Alí; Lacasta, Anna; Rodríguez, Fernando; Salas, María L

2015-01-01

The strain BA71V has played a key role in African swine fever virus (ASFV) research. It was the first genome sequenced, and remains the only genome completely determined. A large part of the studies on the function of ASFV genes, viral transcription, replication, DNA repair and morphogenesis, has been performed using this model. This avirulent strain was obtained by adaptation to grow in Vero cells of the highly virulent BA71 strain. We report here the analysis of the genome sequence of BA71 in comparison with that of BA71V. They possess the smallest genomes for a virulent or an attenuated ASFV, and are essentially identical except for a relatively small number of changes. We discuss the possible contribution of these changes to virulence. Analysis of the BA71 sequence allowed us to identify new similarities among ASFV proteins, and with database proteins including two ASFV proteins that could function as a two-component signaling network.

ChIP-seq.

PubMed

Kim, Tae Hoon; Dekker, Job

2018-05-01

Owing to its digital nature, ChIP-seq has become the standard method for genome-wide ChIP analysis. Using next-generation sequencing platforms (notably the Illumina Genome Analyzer), millions of short sequence reads can be obtained. The densities of recovered ChIP sequence reads along the genome are used to determine the binding sites of the protein. Although a relatively small amount of ChIP DNA is required for ChIP-seq, the current sequencing platforms still require amplification of the ChIP DNA by ligation-mediated PCR (LM-PCR). This protocol, which involves linker ligation followed by size selection, is the standard ChIP-seq protocol using an Illumina Genome Analyzer. The size-selected ChIP DNA is amplified by LM-PCR and size-selected for the second time. The purified ChIP DNA is then loaded into the Genome Analyzer. The ChIP DNA can also be processed in parallel for ChIP-chip results. © 2018 Cold Spring Harbor Laboratory Press.

A Parvovirus B19 synthetic genome: sequence features and functional competence.

PubMed

Manaresi, Elisabetta; Conti, Ilaria; Bua, Gloria; Bonvicini, Francesca; Gallinella, Giorgio

2017-08-01

Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

gEVE: a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes.

PubMed

Nakagawa, So; Takahashi, Mahoko Ueda

2016-01-01

In mammals, approximately 10% of genome sequences correspond to endogenous viral elements (EVEs), which are derived from ancient viral infections of germ cells. Although most EVEs have been inactivated, some open reading frames (ORFs) of EVEs obtained functions in the hosts. However, EVE ORFs usually remain unannotated in the genomes, and no databases are available for EVE ORFs. To investigate the function and evolution of EVEs in mammalian genomes, we developed EVE ORF databases for 20 genomes of 19 mammalian species. A total of 736,771 non-overlapping EVE ORFs were identified and archived in a database named gEVE (http://geve.med.u-tokai.ac.jp). The gEVE database provides nucleotide and amino acid sequences, genomic loci and functional annotations of EVE ORFs for all 20 genomes. In analyzing RNA-seq data with the gEVE database, we successfully identified the expressed EVE genes, suggesting that the gEVE database facilitates studies of the genomic analyses of various mammalian species.Database URL: http://geve.med.u-tokai.ac.jp. © The Author(s) 2016. Published by Oxford University Press.

gEVE: a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes

PubMed Central

Nakagawa, So; Takahashi, Mahoko Ueda

2016-01-01

In mammals, approximately 10% of genome sequences correspond to endogenous viral elements (EVEs), which are derived from ancient viral infections of germ cells. Although most EVEs have been inactivated, some open reading frames (ORFs) of EVEs obtained functions in the hosts. However, EVE ORFs usually remain unannotated in the genomes, and no databases are available for EVE ORFs. To investigate the function and evolution of EVEs in mammalian genomes, we developed EVE ORF databases for 20 genomes of 19 mammalian species. A total of 736,771 non-overlapping EVE ORFs were identified and archived in a database named gEVE (http://geve.med.u-tokai.ac.jp). The gEVE database provides nucleotide and amino acid sequences, genomic loci and functional annotations of EVE ORFs for all 20 genomes. In analyzing RNA-seq data with the gEVE database, we successfully identified the expressed EVE genes, suggesting that the gEVE database facilitates studies of the genomic analyses of various mammalian species. Database URL: http://geve.med.u-tokai.ac.jp PMID:27242033

Comparative scaffolding and gap filling of ancient bacterial genomes applied to two ancient Yersinia pestis genomes

PubMed Central

Doerr, Daniel; Chauve, Cedric

2017-01-01

Yersinia pestis is the causative agent of the bubonic plague, a disease responsible for several dramatic historical pandemics. Progress in ancient DNA (aDNA) sequencing rendered possible the sequencing of whole genomes of important human pathogens, including the ancient Y. pestis strains responsible for outbreaks of the bubonic plague in London in the 14th century and in Marseille in the 18th century, among others. However, aDNA sequencing data are still characterized by short reads and non-uniform coverage, so assembling ancient pathogen genomes remains challenging and often prevents a detailed study of genome rearrangements. It has recently been shown that comparative scaffolding approaches can improve the assembly of ancient Y. pestis genomes at a chromosome level. In the present work, we address the last step of genome assembly, the gap-filling stage. We describe an optimization-based method AGapEs (ancestral gap estimation) to fill in inter-contig gaps using a combination of a template obtained from related extant genomes and aDNA reads. We show how this approach can be used to refine comparative scaffolding by selecting contig adjacencies supported by a mix of unassembled aDNA reads and comparative signal. We applied our method to two Y. pestis data sets from the London and Marseilles outbreaks, for which we obtained highly improved genome assemblies for both genomes, comprised of, respectively, five and six scaffolds with 95 % of the assemblies supported by ancient reads. We analysed the genome evolution between both ancient genomes in terms of genome rearrangements, and observed a high level of synteny conservation between these strains. PMID:29114402

Sequencing and comparative analyses of the genomes of zoysiagrasses

PubMed Central

Tanaka, Hidenori; Hirakawa, Hideki; Kosugi, Shunichi; Nakayama, Shinobu; Ono, Akiko; Watanabe, Akiko; Hashiguchi, Masatsugu; Gondo, Takahiro; Ishigaki, Genki; Muguerza, Melody; Shimizu, Katsuya; Sawamura, Noriko; Inoue, Takayasu; Shigeki, Yuichi; Ohno, Naoki; Tabata, Satoshi; Akashi, Ryo; Sato, Shusei

2016-01-01

Zoysia is a warm-season turfgrass, which comprises 11 allotetraploid species (2n = 4x = 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession ‘Nagirizaki’ (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella ‘Wakaba’ and Z. pacifica ‘Zanpa’ were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica ‘Kyoto’, Z. japonica ‘Miyagi’ and Z. matrella ‘Chiba Fair Green’, were accumulated, and aligned against the reference genome of ‘Nagirizaki’ along with those from ‘Wakaba’ and ‘Zanpa’. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the ‘Zoysia Genome Database’ at http://zoysia.kazusa.or.jp. PMID:26975196

Sequencing and comparative analyses of the genomes of zoysiagrasses.

PubMed

Tanaka, Hidenori; Hirakawa, Hideki; Kosugi, Shunichi; Nakayama, Shinobu; Ono, Akiko; Watanabe, Akiko; Hashiguchi, Masatsugu; Gondo, Takahiro; Ishigaki, Genki; Muguerza, Melody; Shimizu, Katsuya; Sawamura, Noriko; Inoue, Takayasu; Shigeki, Yuichi; Ohno, Naoki; Tabata, Satoshi; Akashi, Ryo; Sato, Shusei

2016-04-01

Zoysiais a warm-season turfgrass, which comprises 11 allotetraploid species (2n= 4x= 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession 'Nagirizaki' (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella 'Wakaba' and Z. pacifica 'Zanpa' were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica'Kyoto', Z. japonica'Miyagi' and Z. matrella'Chiba Fair Green', were accumulated, and aligned against the reference genome of 'Nagirizaki' along with those from 'Wakaba' and 'Zanpa'. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the 'Zoysia Genome Database' at http://zoysia.kazusa.or.jp. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Noninvasive genome sampling in chimpanzees.

PubMed

Kohn, Michael H

2010-12-01

The inevitable has happened: genomic technologies have been added to our noninvasive genetic sampling repertoire. In this issue of Molecular Ecology, Perry et al. (2010) demonstrate how DNA extraction from chimpanzee faeces, followed by a series of steps to enrich for target loci, can be coupled with next-generation sequencing. These authors collected sequence and single-nucleotide polymorphism (SNP) data at more than 600 genomic loci (chromosome 21 and the X) and the complete mitochondrial DNA. By design, each locus was 'deep sequenced' to enable SNP identification. To demonstrate the reliability of their data, the work included samples from six captive chimps, which allowed for a comparison between presumably genuine SNPs obtained from blood and potentially flawed SNPs deduced from faeces. Thus, with this method, anyone with the resources, skills and ambition to do genome sequencing of wild, elusive, or protected mammals can enjoy all of the benefits of noninvasive sampling. © 2010 Blackwell Publishing Ltd.

Draft genome sequence of Bradyrhizobium sp. strain BR 3262, an effective microsymbiont recommended for cowpea inoculation in Brazil.

PubMed

Simões-Araújo, Jean Luiz; Leite, Jakson; Marie Rouws, Luc Felicianus; Passos, Samuel Ribeiro; Xavier, Gustavo Ribeiro; Rumjanek, Norma Gouvêa; Zilli, Jerri Édson

The strain BR 3262 was isolated from nodule of cowpea (Vigna unguiculata L. Walp) growing in soil of the Atlantic Forest area in Brazil and it is reported as an efficient nitrogen fixing bacterium associated to cowpea. Firstly, this strain was assigned as Bradyrhizobium elkanii, however, recently a more detailed genetic and molecular characterization has indicated it could be a Bradyrhizobium pachyrhizi species. We report here the draft genome sequence of B. pachyrhizi strain BR 3262, an elite bacterium used as inoculant for cowpea. The whole genome with 116 scaffolds, 8,965,178bp and 63.8% of C+G content for BR 3262 was obtained using Illumina MiSeq sequencing technology. Annotation was added by the RAST prokaryotic genome annotation service and shown 8369 coding sequences, 52 RNAs genes, classified in 504 subsystems. Published by Elsevier Editora Ltda.

Genome sequence of the model medicinal mushroom Ganoderma lucidum

PubMed Central

Chen, Shilin; Xu, Jiang; Liu, Chang; Zhu, Yingjie; Nelson, David R.; Zhou, Shiguo; Li, Chunfang; Wang, Lizhi; Guo, Xu; Sun, Yongzhen; Luo, Hongmei; Li, Ying; Song, Jingyuan; Henrissat, Bernard; Levasseur, Anthony; Qian, Jun; Li, Jianqin; Luo, Xiang; Shi, Linchun; He, Liu; Xiang, Li; Xu, Xiaolan; Niu, Yunyun; Li, Qiushi; Han, Mira V.; Yan, Haixia; Zhang, Jin; Chen, Haimei; Lv, Aiping; Wang, Zhen; Liu, Mingzhu; Schwartz, David C.; Sun, Chao

2012-01-01

Ganoderma lucidum is a widely used medicinal macrofungus in traditional Chinese medicine that creates a diverse set of bioactive compounds. Here we report its 43.3-Mb genome, encoding 16,113 predicted genes, obtained using next-generation sequencing and optical mapping approaches. The sequence analysis reveals an impressive array of genes encoding cytochrome P450s (CYPs), transporters and regulatory proteins that cooperate in secondary metabolism. The genome also encodes one of the richest sets of wood degradation enzymes among all of the sequenced basidiomycetes. In all, 24 physical CYP gene clusters are identified. Moreover, 78 CYP genes are coexpressed with lanosterol synthase, and 16 of these show high similarity to fungal CYPs that specifically hydroxylate testosterone, suggesting their possible roles in triterpenoid biosynthesis. The elucidation of the G. lucidum genome makes this organism a potential model system for the study of secondary metabolic pathways and their regulation in medicinal fungi. PMID:22735441

Genome sequencing of methanogenic Archaea Methanosarcina mazei TUC01 strain isolated from an Amazonian Flooded Area

NASA Astrophysics Data System (ADS)

Baraúna, R. A.; Graças, D. A.; Ramos, R. T.; Carneiro, A. R.; Lopes, T. S.; Lima, A. R.; Zahlouth, R. L.; Pellizari, V. H.; Silva, A.

2013-05-01

Methanosarcina mazei is a strictly anaerobic methanogen from the Methanosarcinales order. This species is known for its broad catabolic range among methanogens and is widespread throughout diverse environments. The draft genome of a strain cultivated from the sediment of the Tucuruí hydroelectric power station, the fourth largest hydroelectric dam in the world, is described here. Approximately 80% of methane is produced by biogenic sources, such as methanogenic archaea from M. mazei species. Although the methanogenesis pathway is well known, some aspects of the core genome, genome evolution and shared genes are still unclear. A sediment sample from the Tucuruí hydropower station reservoir was inoculated in mineral media supplemented with acetate and methanol. This media was maintained in an H2:CO2 (80:20) atmosphere to enrich and cultivate M. mazei. The enrichment was conducted at 30°C under standard anaerobic conditions. After several molecular and cellular analyses, total DNA was extracted from a non-pure culture of M. mazei, amplified using phi29 DNA polymerase (BioLabs) and finally used as a source template for genome sequencing. The draft genome was obtained after two rounds of sequencing. First, the genome was sequenced using a SOLiD System V3 with a mate-paired library, which yielded 24,405,103 and 24,399,268 reads (50 bp) for the R3 and F3 tags, respectively. The second round of sequencing was performed using the SOLiD 5500 XL platform with a mate-paired library, resulting in a total of 113,588,848 reads (60 bp) for each tag (F3 and R3). All reads obtained by this procedure were filtered using Quality Assessment software, whereby reads with an average quality score below Phred 20 were removed. Velvet and Edena were used to assemble the reads, and Simplifier was used to remove the redundant sequences. After this, a total of 16,811 contigs were obtained. M. mazei GO1 (AE008384) genome was used to map the contigs and generate the scaffolds. We used the Graphical Contig Analyzer for All Sequencing Platforms software (G4ALL; http://g4all.sourceforge.net/) to manually curate and generate the genome scaffold with gaps. The resultant gaps were manually closed using CLC Genomics Workbench software. M. mazei TUC01 genome contained 3,420,400 bp with a GC content of 42.47% distributed over 3 scaffolds that were annotated by RAST. A total of 2,959 coding DNA sequences (CDS) were predicted. The genome of M. mazei TUC01 (accession number: CP003077) will provide valuable information about the ecology of Methanosarcinales order and more accurate information about the methanogenesis pathway observed in the Neotropics. SPONSOR: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Agência Nacional de Energia Elétrica (ANEEL); Centrais Elétricas do Norte do Brasil (Eletronorte).

A comparative analysis of whole genome sequencing of esophageal adenocarcinoma pre- and post-chemotherapy

PubMed Central

Noorani, Ayesha; Lynch, Andy G.; Achilleos, Achilleas; Eldridge, Matthew; Bower, Lawrence; Weaver, Jamie M.J.; Crawte, Jason; Ong, Chin-Ann; Shannon, Nicholas; MacRae, Shona; Grehan, Nicola; Nutzinger, Barbara; O'Donovan, Maria; Hardwick, Richard; Tavaré, Simon; Fitzgerald, Rebecca C.

2017-01-01

The scientific community has avoided using tissue samples from patients that have been exposed to systemic chemotherapy to infer the genomic landscape of a given cancer. Esophageal adenocarcinoma is a heterogeneous, chemoresistant tumor for which the availability and size of pretreatment endoscopic samples are limiting. This study compares whole-genome sequencing data obtained from chemo-naive and chemo-treated samples. The quality of whole-genomic sequencing data is comparable across all samples regardless of chemotherapy status. Inclusion of samples collected post-chemotherapy increased the proportion of late-stage tumors. When comparing matched pre- and post-chemotherapy samples from 10 cases, the mutational signatures, copy number, and SNV mutational profiles reflect the expected heterogeneity in this disease. Analysis of SNVs in relation to allele-specific copy-number changes pinpoints the common ancestor to a point prior to chemotherapy. For cases in which pre- and post-chemotherapy samples do show substantial differences, the timing of the divergence is near-synchronous with endoreduplication. Comparison across a large prospective cohort (62 treatment-naive, 58 chemotherapy-treated samples) reveals no significant differences in the overall mutation rate, mutation signatures, specific recurrent point mutations, or copy-number events in respect to chemotherapy status. In conclusion, whole-genome sequencing of samples obtained following neoadjuvant chemotherapy is representative of the genomic landscape of esophageal adenocarcinoma. Excluding these samples reduces the material available for cataloging and introduces a bias toward the earlier stages of cancer. PMID:28465312

From days to hours: reporting clinically actionable variants from whole genome sequencing.

PubMed

Middha, Sumit; Baheti, Saurabh; Hart, Steven N; Kocher, Jean-Pierre A

2014-01-01

As the cost of whole genome sequencing (WGS) decreases, clinical laboratories will be looking at broadly adopting this technology to screen for variants of clinical significance. To fully leverage this technology in a clinical setting, results need to be reported quickly, as the turnaround rate could potentially impact patient care. The latest sequencers can sequence a whole human genome in about 24 hours. However, depending on the computing infrastructure available, the processing of data can take several days, with the majority of computing time devoted to aligning reads to genomics regions that are to date not clinically interpretable. In an attempt to accelerate the reporting of clinically actionable variants, we have investigated the utility of a multi-step alignment algorithm focused on aligning reads and calling variants in genomic regions of clinical relevance prior to processing the remaining reads on the whole genome. This iterative workflow significantly accelerates the reporting of clinically actionable variants with no loss of accuracy when compared to genotypes obtained with the OMNI SNP platform or to variants detected with a standard workflow that combines Novoalign and GATK.

Complete mitochondrial genome sequence of Indian medium carp, Labeo gonius (Hamilton, 1822) and its comparison with other related carp species.

PubMed

Behera, Bijay Kumar; Kumari, Kavita; Baisvar, Vishwamitra Singh; Rout, Ajaya Kumar; Pakrashi, Sudip; Paria, Prasenjet; Jena, J K

2017-01-01

In the present study, the complete mitochondrial genome sequence of Labeo gonius is reported using PGM sequencer (Ion Torrent). The complete mitogenome of L. gonius is obtained by the de novo sequences assembly of genomic reads using the Torrent Mapping Alignment Program (TMAP) which is 16 614 bp in length. The mitogenome of L. gonius comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNA genes, and D-loop as control region along with gene order and organization, being similar to most of other fish mitogenomes of NCBI databases. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of Labeo fimbriatus, as reported earlier. The phylogenetic analysis of Cypriniformes depicted that their mitogenomes are closely related to each other. The complete mitogenome sequence of L. gonius would be helpful in understanding the population genetics, phylogenetics, and evolution of Indian Carps.

The complete chloroplast genome of a medicinal plant Epimedium koreanum Nakai (Berberidaceae).

PubMed

Lee, Jung-Hoon; Kim, Kyunghee; Kim, Na-Rae; Lee, Sang-Choon; Yang, Tae-Jin; Kim, Young-Dong

2016-11-01

Epimedium koreanum is a perennial medicinal plant distributed in Eastern Asia. The complete chloroplast genome sequences of E. koreanum was obtained by de novo assembly using whole genome next-generation sequences. The chloroplast genome of E. koreanum was 157 218 bp in length and separated into four distinct regions such as large single copy region (89 600 bp), small single copy region (17 222 bp) and a pair of inverted repeat regions (25 198 bp). The genome contained a total of 112 genes including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that E. koreanum is most closely related to Berberis bealei, a traditional medicinal plant in the Berberidaceae family.

The first complete chloroplast genome sequence of a lycophyte,Huperzia lucidula (Lycopodiaceae)

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

Wolf, Paul G.; Karol, Kenneth G.; Mandoli, Dina F.

2005-02-01

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8x depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,671 bp. Gene order is more similarmore » to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperziachloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophytechloroplast genome data also enable a better reconstruction of the basaltracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.« less

Whole-genome sequence of Escherichia coli serotype O157:H7 strain B6914-ARS

USDA-ARS?s Scientific Manuscript database

Escherichia coli serotype O157:H7 strain B6914-MS1 is a Shiga toxin-deficient human fecal isolate obtained by the Centers for Disease Control and Prevention that has been used extensively in applied research studies. Here we report the genome sequence of strain B6914-ARS, a B6914-MS1 clone that has ...

Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry.

PubMed

Asara, John M; Schweitzer, Mary H; Freimark, Lisa M; Phillips, Matthew; Cantley, Lewis C

2007-04-13

Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum) and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to be obtained.

COMPLETE GENOMIC SEQUENCE OF VIRULENT PIGEON PARAMYXOVIRUS IN LAUGHING DOVES (STREPTOPELIA SENEGALENSIS) IN KENYA.

PubMed

Obanda, Vincent; Michuki, George; Jowers, Michael J; Rumberia, Cecilia; Mutinda, Mathew; Lwande, Olivia Wesula; Wangoru, Kihara; Kasiiti-Orengo, Jacquiline; Yongo, Moses; Angelone-Alasaad, Samer

2016-07-01

Following mass deaths of Laughing Doves (Streptopelia senegalensis) in different localities throughout Kenya, internal organs obtained during necropsy of two moribund birds were sampled and analyzed by next generation sequencing. We isolated the virulent strain of pigeon paramyxovirus type-1 (PPMV-1), PPMV1/Laughing Dove/Kenya/Isiolo/B2/2012, which had a characteristic fusion gene motif (110)GGRRQKRF(117). We obtained a partial full genome of 15,114 nucleotides. The phylogenetic relationship based on the fusion gene and genomic sequence grouped our isolate as class II genotype VI, a group of viruses commonly isolated from wild birds but potentially lethal to Chickens ( Gallus gallus domesticus ). The fusion gene isolate clustered with PPMV-I strains from pigeons (Columbidae) in Nigeria. The complete genome showed a basal and highly divergent lineage to American, European, and Asian strains, indicating a divergent evolutionary pathway. The isolated strain is highly virulent and apparently species-specific to Laughing Doves in Kenya. Risk of transmission of such a strain to poultry is potentially high whereas the cyclic epizootic in doves is a threat to conservation of wild Columbidae in Kenya.

Effective de novo assembly of fish genome using haploid larvae.

PubMed

Iwasaki, Yuki; Nishiki, Issei; Nakamura, Yoji; Yasuike, Motoshige; Kai, Wataru; Nomura, Kazuharu; Yoshida, Kazunori; Nomura, Yousuke; Fujiwara, Atushi; Kobayashi, Takanori; Ototake, Mitsuru

2016-02-01

Recent improvements in next-generation sequencing technology have made it possible to do whole genome sequencing, on even non-model eukaryote species with no available reference genomes. However, de novo assembly of diploid genomes is still a big challenge because of allelic variation. The aim of this study was to determine the feasibility of utilizing the genome of haploid fish larvae for de novo assembly of whole-genome sequences. We compared the efficiency of assembly using the haploid genome of yellowtail (Seriola quinqueradiata) with that using the diploid genome obtained from the dam. De novo assembly from the haploid and the diploid sequence reads (100 million reads per each datasets) generated by the Ion Proton sequencer (200 bp) was done under two different assembly algorithms, namely overlap-layout-consensus (OLC) and de Bruijn graph (DBG). This revealed that the assembly of the haploid genome significantly reduced (approximately 22% for OLC, 9% for DBG) the total number of contigs (with longer average and N50 contig lengths) when compared to the diploid genome assembly. The haploid assembly also improved the quality of the scaffolds by reducing the number of regions with unassigned nucleotides (Ns) (total length of Ns; 45,331,916 bp for haploids and 67,724,360 bp for diploids) in OLC-based assemblies. It appears clear that the haploid genome assembly is better because the allelic variation in the diploid genome disrupts the extension of contigs during the assembly process. Our results indicate that utilizing the genome of haploid larvae leads to a significant improvement in the de novo assembly process, thus providing a novel strategy for the construction of reference genomes from non-model diploid organisms such as fish. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of mango (Mangifera indica L.) transcriptome and chloroplast genome.

PubMed

Azim, M Kamran; Khan, Ishtaiq A; Zhang, Yong

2014-05-01

We characterized mango leaf transcriptome and chloroplast genome using next generation DNA sequencing. The RNA-seq output of mango transcriptome generated >12 million reads (total nucleotides sequenced >1 Gb). De novo transcriptome assembly generated 30,509 unigenes with lengths in the range of 300 to ≥3,000 nt and 67× depth of coverage. Blast searching against nonredundant nucleotide databases and several Viridiplantae genomic datasets annotated 24,593 mango unigenes (80% of total) and identified Citrus sinensis as closest neighbor of mango with 9,141 (37%) matched sequences. The annotation with gene ontology and Clusters of Orthologous Group terms categorized unigene sequences into 57 and 25 classes, respectively. More than 13,500 unigenes were assigned to 293 KEGG pathways. Besides major plant biology related pathways, KEGG based gene annotation pointed out active presence of an array of biochemical pathways involved in (a) biosynthesis of bioactive flavonoids, flavones and flavonols, (b) biosynthesis of terpenoids and lignins and (c) plant hormone signal transduction. The mango transcriptome sequences revealed 235 proteases belonging to five catalytic classes of proteolytic enzymes. The draft genome of mango chloroplast (cp) was obtained by a combination of Sanger and next generation sequencing. The draft mango cp genome size is 151,173 bp with a pair of inverted repeats of 27,093 bp separated by small and large single copy regions, respectively. Out of 139 genes in mango cp genome, 91 found to be protein coding. Sequence analysis revealed cp genome of C. sinensis as closest neighbor of mango. We found 51 short repeats in mango cp genome supposed to be associated with extensive rearrangements. This is the first report of transcriptome and chloroplast genome analysis of any Anacardiaceae family member.

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PubMed

Straub, Shannon C K; Parks, Matthew; Weitemier, Kevin; Fishbein, Mark; Cronn, Richard C; Liston, Aaron

2012-02-01

Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

Transcriptome sequence analysis of an ornamental plant, Ananas comosus var. bracteatus, revealed the potential unigenes involved in terpenoid and phenylpropanoid biosynthesis.

PubMed

Ma, Jun; Kanakala, S; He, Yehua; Zhang, Junli; Zhong, Xiaolan

2015-01-01

Ananas comosus var. bracteatus (Red Pineapple) is an important ornamental plant for its colorful leaves and decorative red fruits. Because of its complex genome, it is difficult to understand the molecular mechanisms involved in the growth and development. Thus high-throughput transcriptome sequencing of Ananas comosus var. bracteatus is necessary to generate large quantities of transcript sequences for the purpose of gene discovery and functional genomic studies. The Ananas comosus var. bracteatus transcriptome was sequenced by the Illumina paired-end sequencing technology. We obtained a total of 23.5 million high quality sequencing reads, 1,555,808 contigs and 41,052 unigenes. In total 41,052 unigenes of Ananas comosus var. bracteatus, 23,275 unigenes were annotated in the NCBI non-redundant protein database and 23,134 unigenes were annotated in the Swiss-Port database. Out of these, 17,748 and 8,505 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. Functional annotation against Kyoto Encyclopedia of Genes and Genomes Pathway database identified 5,825 unigenes which were mapped to 117 pathways. The assembly predicted many unigenes that were previously unknown. The annotated unigenes were compared against pineapple, rice, maize, Arabidopsis, and sorghum. Unigenes that did not match any of those five sequence datasets are considered to be Ananas comosus var. bracteatus unique. We predicted unigenes encoding enzymes involved in terpenoid and phenylpropanoid biosynthesis. The sequence data provide the most comprehensive transcriptomic resource currently available for Ananas comosus var. bracteatus. To our knowledge; this is the first report on the de novo transcriptome sequencing of the Ananas comosus var. bracteatus. Unigenes obtained in this study, may help improve future gene expression, genetic and genomics studies in Ananas comosus var. bracteatus.

Transcriptome Sequence Analysis of an Ornamental Plant, Ananas comosus var. bracteatus, Revealed the Potential Unigenes Involved in Terpenoid and Phenylpropanoid Biosynthesis

PubMed Central

Ma, Jun; Kanakala, S.; He, Yehua; Zhang, Junli; Zhong, Xiaolan

2015-01-01

Background Ananas comosus var. bracteatus (Red Pineapple) is an important ornamental plant for its colorful leaves and decorative red fruits. Because of its complex genome, it is difficult to understand the molecular mechanisms involved in the growth and development. Thus high-throughput transcriptome sequencing of Ananas comosus var. bracteatus is necessary to generate large quantities of transcript sequences for the purpose of gene discovery and functional genomic studies. Results The Ananas comosus var. bracteatus transcriptome was sequenced by the Illumina paired-end sequencing technology. We obtained a total of 23.5 million high quality sequencing reads, 1,555,808 contigs and 41,052 unigenes. In total 41,052 unigenes of Ananas comosus var. bracteatus, 23,275 unigenes were annotated in the NCBI non-redundant protein database and 23,134 unigenes were annotated in the Swiss-Port database. Out of these, 17,748 and 8,505 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. Functional annotation against Kyoto Encyclopedia of Genes and Genomes Pathway database identified 5,825 unigenes which were mapped to 117 pathways. The assembly predicted many unigenes that were previously unknown. The annotated unigenes were compared against pineapple, rice, maize, Arabidopsis, and sorghum. Unigenes that did not match any of those five sequence datasets are considered to be Ananas comosus var. bracteatus unique. We predicted unigenes encoding enzymes involved in terpenoid and phenylpropanoid biosynthesis. Conclusion The sequence data provide the most comprehensive transcriptomic resource currently available for Ananas comosus var. bracteatus. To our knowledge; this is the first report on the de novo transcriptome sequencing of the Ananas comosus var. bracteatus. Unigenes obtained in this study, may help improve future gene expression, genetic and genomics studies in Ananas comosus var. bracteatus. PMID:25769053

Enhanced sequencing coverage with digital droplet multiple displacement amplification

PubMed Central

Sidore, Angus M.; Lan, Freeman; Lim, Shaun W.; Abate, Adam R.

2016-01-01

Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing. PMID:26704978

Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae).

PubMed

Walker, Joseph F; Zanis, Michael J; Emery, Nancy C

2014-04-01

Complete chloroplast genome studies can help resolve relationships among large, complex plant lineages such as Asteraceae. We present the first whole plastome from the Madieae tribe and compare its sequence variation to other chloroplast genomes in Asteraceae. We used high throughput sequencing to obtain the Lasthenia burkei chloroplast genome. We compared sequence structure and rates of molecular evolution in the small single copy (SSC), large single copy (LSC), and inverted repeat (IR) regions to those for eight Asteraceae accessions and one Solanaceae accession. The chloroplast sequence of L. burkei is 150 746 bp and contains 81 unique protein coding genes and 4 coding ribosomal RNA sequences. We identified three major inversions in the L. burkei chloroplast, all of which have been found in other Asteraceae lineages, and a previously unreported inversion in Lactuca sativa. Regions flanking inversions contained tRNA sequences, but did not have particularly high G + C content. Substitution rates varied among the SSC, LSC, and IR regions, and rates of evolution within each region varied among species. Some observed differences in rates of molecular evolution may be explained by the relative proportion of coding to noncoding sequence within regions. Rates of molecular evolution vary substantially within and among chloroplast genomes, and major inversion events may be promoted by the presence of tRNAs. Collectively, these results provide insight into different mechanisms that may promote intramolecular recombination and the inversion of large genomic regions in the plastome.

Application of industrial scale genomics to discovery of therapeutic targets in heart failure.

PubMed

Mehraban, F; Tomlinson, J E

2001-12-01

In recent years intense activity in both academic and industrial sectors has provided a wealth of information on the human genome with an associated impressive increase in the number of novel gene sequences deposited in sequence data repositories and patent applications. This genomic industrial revolution has transformed the way in which drug target discovery is now approached. In this article we discuss how various differential gene expression (DGE) technologies are being utilized for cardiovascular disease (CVD) drug target discovery. Other approaches such as sequencing cDNA from cardiovascular derived tissues and cells coupled with bioinformatic sequence analysis are used with the aim of identifying novel gene sequences that may be exploited towards target discovery. Additional leverage from gene sequence information is obtained through identification of polymorphisms that may confer disease susceptibility and/or affect drug responsiveness. Pharmacogenomic studies are described wherein gene expression-based techniques are used to evaluate drug response and/or efficacy. Industrial-scale genomics supports and addresses not only novel target gene discovery but also the burgeoning issues in pharmaceutical and clinical cardiovascular medicine relative to polymorphic gene responses.

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

PubMed Central

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-01-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents. PMID:22735701

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

PubMed

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-08-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Identification of Sinorhizobium (Ensifer) medicae based on a specific genomic sequence unveiled by M13-PCR fingerprinting.

PubMed

Dourado, Ana Catarina; Alves, Paula I L; Tenreiro, Tania; Ferreira, Eugénio M; Tenreiro, Rogério; Fareleira, Paula; Crespo, M Teresa Barreto

2009-12-01

A collection of nodule isolates from Medicago polymorpha obtained from southern and central Portugal was evaluated by M13-PCR fingerprinting and hierarchical cluster analysis. Several genomic clusters were obtained which, by 16S rRNA gene sequencing of selected representatives, were shown to be associated with particular taxonomic groups of rhizobia and other soil bacteria. The method provided a clear separation between rhizobia and co-isolated non-symbiotic soil contaminants. Ten M13-PCR groups were assigned to Sinorhizobium (Ensifer) medicae and included all isolates responsible for the formation of nitrogen-fixing nodules upon re-inoculation of M. polymorpha test-plants. In addition, enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting indicated a high genomic heterogeneity within the major M13- PCR clusters of S. medicae isolates. Based on nucleotide sequence data of an M13-PCR amplicon of ca. 1500 bp, observed only in S. medicae isolates and spanning locus Smed_3707 to Smed_3709 from the pSMED01 plasmid sequence of S. medicae WSM419 genome's sequence, a pair of PCR primers was designed and used for direct PCR amplification of a 1399-bp sequence within this fragment. Additional in silico and in vitro experiments, as well as phylogenetic analysis, confirmed the specificity of this primer combination and therefore the reliability of this approach in the prompt identification of S. medicae isolates and their distinction from other soil bacteria.

From cultured to uncultured genome sequences: metagenomics and modeling microbial ecosystems.

PubMed

Garza, Daniel R; Dutilh, Bas E

2015-11-01

Microorganisms and the viruses that infect them are the most numerous biological entities on Earth and enclose its greatest biodiversity and genetic reservoir. With strength in their numbers, these microscopic organisms are major players in the cycles of energy and matter that sustain all life. Scientists have only scratched the surface of this vast microbial world through culture-dependent methods. Recent developments in generating metagenomes, large random samples of nucleic acid sequences isolated directly from the environment, are providing comprehensive portraits of the composition, structure, and functioning of microbial communities. Moreover, advances in metagenomic analysis have created the possibility of obtaining complete or nearly complete genome sequences from uncultured microorganisms, providing important means to study their biology, ecology, and evolution. Here we review some of the recent developments in the field of metagenomics, focusing on the discovery of genetic novelty and on methods for obtaining uncultured genome sequences, including through the recycling of previously published datasets. Moreover we discuss how metagenomics has become a core scientific tool to characterize eco-evolutionary patterns of microbial ecosystems, thus allowing us to simultaneously discover new microbes and study their natural communities. We conclude by discussing general guidelines and challenges for modeling the interactions between uncultured microorganisms and viruses based on the information contained in their genome sequences. These models will significantly advance our understanding of the functioning of microbial ecosystems and the roles of microbes in the environment.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing.

PubMed

Hribová, Eva; Neumann, Pavel; Matsumoto, Takashi; Roux, Nicolas; Macas, Jirí; Dolezel, Jaroslav

2010-09-16

Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing

PubMed Central

2010-01-01

Background Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. Results In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. Conclusion A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection. PMID:20846365

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes

PubMed Central

2011-01-01

Background We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. Results The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. Conclusions BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies. PMID:21496274

Transcriptome analysis of eyestalk and hemocytes in the ridgetail white prawn Exopalaemon carinicauda: assembly, annotation and marker discovery.

PubMed

Li, Jitao; Li, Jian; Chen, Ping; Liu, Ping; He, Yuying

2015-01-01

The ridgetail white prawn Exopalaemon carinicauda is one of major economic mariculture species in eastern China. The deficiency of genomic and transcriptomic data is becoming the bottleneck of further researches on its good traits. In the present study, 454 pyrosequencing was undertaken to investigate the transcriptome profiles of E. carinicauda. A collection of 1,028,710 sequence reads (459.59 Mb) obtained from cDNA prepared from eyestalk and hemocytes was assembled into 162,056 expressed sequence tags (ESTs). Of these, 29.88 % of 48,428 contigs and 70.12 % of 113,628 singlets possessed high similarities to sequences in the GenBank non-redundant database, with most significant (E value <1e(-10)) unigenes matches occurring with crustacean and insect sequences. KEGG analysis of unigenes identified putative members of biological pathways related to growth and immunity. In addition, we obtained a total of putative 125,112 SNPs and 13,467 microsatellites. These results will contribute to the understanding of the genome makeup and provide useful information for future functional genomic research in E. carinicauda.

A Year of Infection in the Intensive Care Unit: Prospective Whole Genome Sequencing of Bacterial Clinical Isolates Reveals Cryptic Transmissions and Novel Microbiota

PubMed Central

Roach, David J.; Burton, Joshua N.; Lee, Choli; Stackhouse, Bethany; Butler-Wu, Susan M.; Cookson, Brad T.

2015-01-01

Bacterial whole genome sequencing holds promise as a disruptive technology in clinical microbiology, but it has not yet been applied systematically or comprehensively within a clinical context. Here, over the course of one year, we performed prospective collection and whole genome sequencing of nearly all bacterial isolates obtained from a tertiary care hospital’s intensive care units (ICUs). This unbiased collection of 1,229 bacterial genomes from 391 patients enables detailed exploration of several features of clinical pathogens. A sizable fraction of isolates identified as clinically relevant corresponded to previously undescribed species: 12% of isolates assigned a species-level classification by conventional methods actually qualified as distinct, novel genomospecies on the basis of genomic similarity. Pan-genome analysis of the most frequently encountered pathogens in the collection revealed substantial variation in pan-genome size (1,420 to 20,432 genes) and the rate of gene discovery (1 to 152 genes per isolate sequenced). Surprisingly, although potential nosocomial transmission of actively surveilled pathogens was rare, 8.7% of isolates belonged to genomically related clonal lineages that were present among multiple patients, usually with overlapping hospital admissions, and were associated with clinically significant infection in 62% of patients from which they were recovered. Multi-patient clonal lineages were particularly evident in the neonatal care unit, where seven separate Staphylococcus epidermidis clonal lineages were identified, including one lineage associated with bacteremia in 5/9 neonates. Our study highlights key differences in the information made available by conventional microbiological practices versus whole genome sequencing, and motivates the further integration of microbial genome sequencing into routine clinical care. PMID:26230489

Assembling the Marine Metagenome, One Cell at a Time

PubMed Central

Woyke, Tanja; Xie, Gary; Copeland, Alex; González, José M.; Han, Cliff; Kiss, Hajnalka; Saw, Jimmy H.; Senin, Pavel; Yang, Chi; Chatterji, Sourav; Cheng, Jan-Fang; Eisen, Jonathan A.; Sieracki, Michael E.; Stepanauskas, Ramunas

2009-01-01

The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa. PMID:19390573

Single nucleotide polymorphism analysis of Korean native chickens using next generation sequencing data.

PubMed

Seo, Dong-Won; Oh, Jae-Don; Jin, Shil; Song, Ki-Duk; Park, Hee-Bok; Heo, Kang-Nyeong; Shin, Younhee; Jung, Myunghee; Park, Junhyung; Jo, Cheorun; Lee, Hak-Kyo; Lee, Jun-Heon

2015-02-01

There are five native chicken lines in Korea, which are mainly classified by plumage colors (black, white, red, yellow, gray). These five lines are very important genetic resources in the Korean poultry industry. Based on a next generation sequencing technology, whole genome sequence and reference assemblies were performed using Gallus_gallus_4.0 (NCBI) with whole genome sequences from these lines to identify common and novel single nucleotide polymorphisms (SNPs). We obtained 36,660,731,136 ± 1,257,159,120 bp of raw sequence and average 26.6-fold of 25-29 billion reference assembly sequences representing 97.288 % coverage. Also, 4,006,068 ± 97,534 SNPs were observed from 29 autosomes and the Z chromosome and, of these, 752,309 SNPs are the common SNPs across lines. Among the identified SNPs, the number of novel- and known-location assigned SNPs was 1,047,951 ± 14,956 and 2,948,648 ± 81,414, respectively. The number of unassigned known SNPs was 1,181 ± 150 and unassigned novel SNPs was 8,238 ± 1,019. Synonymous SNPs, non-synonymous SNPs, and SNPs having character changes were 26,266 ± 1,456, 11,467 ± 604, 8,180 ± 458, respectively. Overall, 443,048 ± 26,389 SNPs in each bird were identified by comparing with dbSNP in NCBI. The presently obtained genome sequence and SNP information in Korean native chickens have wide applications for further genome studies such as genetic diversity studies to detect causative mutations for economic and disease related traits.

Targeted sequencing for high-resolution evolutionary analyses following genome duplication in salmonid fish: Proof of concept for key components of the insulin-like growth factor axis.

PubMed

Lappin, Fiona M; Shaw, Rebecca L; Macqueen, Daniel J

2016-12-01

High-throughput sequencing has revolutionised comparative and evolutionary genome biology. It has now become relatively commonplace to generate multiple genomes and/or transcriptomes to characterize the evolution of large taxonomic groups of interest. Nevertheless, such efforts may be unsuited to some research questions or remain beyond the scope of some research groups. Here we show that targeted high-throughput sequencing offers a viable alternative to study genome evolution across a vertebrate family of great scientific interest. Specifically, we exploited sequence capture and Illumina sequencing to characterize the evolution of key components from the insulin-like growth (IGF) signalling axis of salmonid fish at unprecedented phylogenetic resolution. The IGF axis represents a central governor of vertebrate growth and its core components were expanded by whole genome duplication in the salmonid ancestor ~95Ma. Using RNA baits synthesised to genes encoding the complete family of IGF binding proteins (IGFBP) and an IGF hormone (IGF2), we captured, sequenced and assembled orthologous and paralogous exons from species representing all ten salmonid genera. This approach generated 299 novel sequences, most as complete or near-complete protein-coding sequences. Phylogenetic analyses confirmed congruent evolutionary histories for all nineteen recognized salmonid IGFBP family members and identified novel salmonid-specific IGF2 paralogues. Moreover, we reconstructed the evolution of duplicated IGF axis paralogues across a replete salmonid phylogeny, revealing complex historic selection regimes - both ancestral to salmonids and lineage-restricted - that frequently involved asymmetric paralogue divergence under positive and/or relaxed purifying selection. Our findings add to an emerging literature highlighting diverse applications for targeted sequencing in comparative-evolutionary genomics. We also set out a viable approach to obtain large sets of nuclear genes for any member of the salmonid family, which should enable insights into the evolutionary role of whole genome duplication before additional nuclear genome sequences become available. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Investigation of the Evolutionary Development of the Genus Bifidobacterium by Comparative Genomics

PubMed Central

Lugli, Gabriele Andrea; Milani, Christian; Turroni, Francesca; Duranti, Sabrina; Ferrario, Chiara; Viappiani, Alice; Mancabelli, Leonardo; Mangifesta, Marta; Taminiau, Bernard; Delcenserie, Véronique; van Sinderen, Douwe

2014-01-01

The Bifidobacterium genus currently encompasses 48 recognized taxa, which have been isolated from different ecosystems. However, the current phylogeny of bifidobacteria is hampered by the relative paucity of genotypic data. Here, we reassessed the taxonomy of this bacterial genus using genome-based approaches, which demonstrated that the previous taxonomic view of bifidobacteria contained several inconsistencies. In particular, high levels of genetic relatedness were shown to exist between particular Bifidobacterium taxa which would not justify their status as separate species. The results presented are here based on average nucleotide identity analysis involving the genome sequences for each type strain of the 48 bifidobacterial taxa, as well as phylogenetic comparative analysis of the predicted core genome of the Bifidobacterium genus. The results of this study demonstrate that the availability of complete genome sequences allows the reconstruction of a more robust bifidobacterial phylogeny than that obtained from a single gene-based sequence comparison, thus discouraging the assignment of a new or separate bifidobacterial taxon without such a genome-based validation. PMID:25107967

Read clouds uncover variation in complex regions of the human genome

PubMed Central

Bishara, Alex; Liu, Yuling; Weng, Ziming; Kashef-Haghighi, Dorna; Newburger, Daniel E.; West, Robert; Sidow, Arend; Batzoglou, Serafim

2015-01-01

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies. PMID:26286554

Survey of genome sequences in a wild sweet potato, Ipomoea trifida (H. B. K.) G. Don

PubMed Central

Hirakawa, Hideki; Okada, Yoshihiro; Tabuchi, Hiroaki; Shirasawa, Kenta; Watanabe, Akiko; Tsuruoka, Hisano; Minami, Chiharu; Nakayama, Shinobu; Sasamoto, Shigemi; Kohara, Mitsuyo; Kishida, Yoshie; Fujishiro, Tsunakazu; Kato, Midori; Nanri, Keiko; Komaki, Akiko; Yoshinaga, Masaru; Takahata, Yasuhiro; Tanaka, Masaru; Tabata, Satoshi; Isobe, Sachiko N.

2015-01-01

Ipomoea trifida (H. B. K.) G. Don. is the most likely diploid ancestor of the hexaploid sweet potato, I. batatas (L.) Lam. To assist in analysis of the sweet potato genome, de novo whole-genome sequencing was performed with two lines of I. trifida, namely the selfed line Mx23Hm and the highly heterozygous line 0431-1, using the Illumina HiSeq platform. We classified the sequences thus obtained as either ‘core candidates’ (common to the two lines) or ‘line specific’. The total lengths of the assembled sequences of Mx23Hm (ITR_r1.0) was 513 Mb, while that of 0431-1 (ITRk_r1.0) was 712 Mb. Of the assembled sequences, 240 Mb (Mx23Hm) and 353 Mb (0431-1) were classified into core candidate sequences. A total of 62,407 (62.4 Mb) and 109,449 (87.2 Mb) putative genes were identified, respectively, in the genomes of Mx23Hm and 0431-1, of which 11,823 were derived from core sequences of Mx23Hm, while 28,831 were from the core candidate sequence of 0431-1. There were a total of 1,464,173 single-nucleotide polymorphisms and 16,682 copy number variations (CNVs) in the two assembled genomic sequences (under the condition of log2 ratio of >1 and CNV size >1,000 bases). The results presented here are expected to contribute to the progress of genomic and genetic studies of I. trifida, as well as studies of the sweet potato and the genus Ipomoea in general. PMID:25805887

Professionally Responsible Disclosure of Genomic Sequencing Results in Pediatric Practice

PubMed Central

Brothers, Kyle B.; Chung, Wendy K.; Joffe, Steven; Koenig, Barbara A.; Wilfond, Benjamin; Yu, Joon-Ho

2015-01-01

Genomic sequencing is being rapidly introduced into pediatric clinical practice. The results of sequencing are distinctive for their complexity and subsequent challenges of interpretation for generalist and specialist pediatricians, parents, and patients. Pediatricians therefore need to prepare for the professionally responsible disclosure of sequencing results to parents and patients and guidance of parents and patients in the interpretation and use of these results, including managing uncertain data. This article provides an ethical framework to guide and evaluate the professionally responsible disclosure of the results of genomic sequencing in pediatric practice. The ethical framework comprises 3 core concepts of pediatric ethics: the best interests of the child standard, parental surrogate decision-making, and pediatric assent. When recommending sequencing, pediatricians should explain the nature of the proposed test, its scope and complexity, the categories of results, and the concept of a secondary or incidental finding. Pediatricians should obtain the informed permission of parents and the assent of mature adolescents about the scope of sequencing to be performed and the return of results. PMID:26371191

Complete Genome Sequence of a New Firmicutes Species Isolated from Anaerobic Biomass Hydrolysis.

PubMed

Abendroth, Christian; Hahnke, Sarah; Codoñer, Francisco M; Klocke, Michael; Luschnig, Olaf; Porcar, Manuel

2017-10-05

A new Firmicutes isolate, strain HV4-6-A5C, was obtained from the hydrolysis stage of a mesophilic and anaerobic two-stage lab-scale leach-bed system for biomethanation of fresh grass. It is assumed that the bacterial isolate contributes to plant biomass degradation. Here, we report a draft annotated genome sequence of this organism. Copyright © 2017 Abendroth et al.

Genome data from a sixteenth century pig illuminate modern breed relationships

PubMed Central

Ramírez, O; Burgos-Paz, W; Casas, E; Ballester, M; Bianco, E; Olalde, I; Santpere, G; Novella, V; Gut, M; Lalueza-Fox, C; Saña, M; Pérez-Enciso, M

2015-01-01

Ancient DNA (aDNA) provides direct evidence of historical events that have modeled the genome of modern individuals. In livestock, resolving the differences between the effects of initial domestication and of subsequent modern breeding is not straight forward without aDNA data. Here, we have obtained shotgun genome sequence data from a sixteenth century pig from Northeastern Spain (Montsoriu castle), the ancient pig was obtained from an extremely well-preserved and diverse assemblage. In addition, we provide the sequence of three new modern genomes from an Iberian pig, Spanish wild boar and a Guatemalan Creole pig. Comparison with both mitochondrial and autosomal genome data shows that the ancient pig is closely related to extant Iberian pigs and to European wild boar. Although the ancient sample was clearly domestic, admixture with wild boar also occurred, according to the D-statistics. The close relationship between Iberian, European wild boar and the ancient pig confirms that Asian introgression in modern Iberian pigs has not existed or has been negligible. In contrast, the Guatemalan Creole pig clusters apart from the Iberian pig genome, likely due to introgression from international breeds. PMID:25204303

Increased genomic prediction accuracy in wheat breeding through spatial adjustment of field trial data.

PubMed

Lado, Bettina; Matus, Ivan; Rodríguez, Alejandra; Inostroza, Luis; Poland, Jesse; Belzile, François; del Pozo, Alejandro; Quincke, Martín; Castro, Marina; von Zitzewitz, Jarislav

2013-12-09

In crop breeding, the interest of predicting the performance of candidate cultivars in the field has increased due to recent advances in molecular breeding technologies. However, the complexity of the wheat genome presents some challenges for applying new technologies in molecular marker identification with next-generation sequencing. We applied genotyping-by-sequencing, a recently developed method to identify single-nucleotide polymorphisms, in the genomes of 384 wheat (Triticum aestivum) genotypes that were field tested under three different water regimes in Mediterranean climatic conditions: rain-fed only, mild water stress, and fully irrigated. We identified 102,324 single-nucleotide polymorphisms in these genotypes, and the phenotypic data were used to train and test genomic selection models intended to predict yield, thousand-kernel weight, number of kernels per spike, and heading date. Phenotypic data showed marked spatial variation. Therefore, different models were tested to correct the trends observed in the field. A mixed-model using moving-means as a covariate was found to best fit the data. When we applied the genomic selection models, the accuracy of predicted traits increased with spatial adjustment. Multiple genomic selection models were tested, and a Gaussian kernel model was determined to give the highest accuracy. The best predictions between environments were obtained when data from different years were used to train the model. Our results confirm that genotyping-by-sequencing is an effective tool to obtain genome-wide information for crops with complex genomes, that these data are efficient for predicting traits, and that correction of spatial variation is a crucial ingredient to increase prediction accuracy in genomic selection models.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

PubMed Central

Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

2004-01-01

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species

PubMed Central

Legault, Boris A; Lopez-Lopez, Arantxa; Alba-Casado, Jose Carlos; Doolittle, W Ford; Bolhuis, Henk; Rodriguez-Valera, Francisco; Papke, R Thane

2006-01-01

Background Mature saturated brine (crystallizers) communities are largely dominated (>80% of cells) by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically simplified environment. Similar studies carried out in other extreme environments have revealed very little diversity in gene content among the cell lineages present. Results The metagenome of the microbial community of a crystallizer pond has been analyzed by end sequencing a 2000 clone fosmid library and comparing the sequences obtained with the genome sequence of "Haloquadratum walsbyi". The genome of the sequenced strain was retrieved nearly complete within this environmental DNA library. However, many ORF's that could be ascribed to the "Haloquadratum" metapopulation by common genome characteristics or scaffolding to the strain genome were not present in the specific sequenced isolate. Particularly, three regions of the sequenced genome were associated with multiple rearrangements and the presence of different genes from the metapopulation. Many transposition and phage related genes were found within this pool which, together with the associated atypical GC content in these areas, supports lateral gene transfer mediated by these elements as the most probable genetic cause of this variability. Additionally, these sequences were highly enriched in putative regulatory and signal transduction functions. Conclusion These results point to a large pan-genome (total gene repertoire of the genus/species) even in this highly specialized extremophile and at a single geographic location. The extensive gene repertoire is what might be expected of a population that exploits a diverse nutrient pool, resulting from the degradation of biomass produced at lower salinities. PMID:16820057

Next-generation sequencing of mixed genomic DNA allows efficient assembly of rearranged mitochondrial genomes in Amolops chunganensis and Quasipaa boulengeri

PubMed Central

Yuan, Siqi; Zheng, Yuchi; Zeng, Xiaomao

2016-01-01

Recent improvements in next-generation sequencing (NGS) technologies can facilitate the obtainment of mitochondrial genomes. However, it is not clear whether NGS could be effectively used to reconstruct the mitogenome with high gene rearrangement. These high rearrangements would cause amplification failure, and/or assembly and alignment errors. Here, we choose two frogs with rearranged gene order, Amolops chunganensis and Quasipaa boulengeri, to test whether gene rearrangements affect the mitogenome assembly and alignment by using NGS. The mitogenomes with gene rearrangements are sequenced through Illumina MiSeq genomic sequencing and assembled effectively by Trinity v2.1.0 and SOAPdenovo2. Gene order and contents in the mitogenome of A. chunganensis and Q. boulengeri are typical neobatrachian pattern except for rearrangements at the position of “WANCY” tRNA genes cluster. Further, the mitogenome of Q. boulengeri is characterized with a tandem duplication of trnM. Moreover, we utilize 13 protein-coding genes of A. chunganensis, Q. boulengeri and other neobatrachians to reconstruct the phylogenetic tree for evaluating mitochondrial sequence authenticity of A. chunganensis and Q. boulengeri. In this work, we provide nearly complete mitochondrial genomes of A. chunganensis and Q. boulengeri. PMID:27994980

The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4

PubMed Central

Duan, Jin; Jiang, Wei; Cheng, Zhenyu; Heikkila, John J.; Glick, Bernard R.

2013-01-01

The plant growth-promoting bacterium (PGPB) Pseudomonas sp. UW4, previously isolated from the rhizosphere of common reeds growing on the campus of the University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentrations of salt, cold, heavy metals, drought and phytopathogens. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. sp. UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,423 predicted protein-coding sequences that occupy 87.2% of the genome. Nineteen genomic islands (GIs) were predicted and thirty one complete putative insertion sequences were identified. Genes potentially involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, trehalose production, siderophore production, acetoin synthesis, and phosphate solubilization were determined. Moreover, genes that contribute to the environmental fitness of UW4 were also observed including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Whole-genome comparison with other completely sequenced Pseudomonas strains and phylogeny of four concatenated “housekeeping” genes (16S rRNA, gyrB, rpoB and rpoD) of 128 Pseudomonas strains revealed that UW4 belongs to the fluorescens group, jessenii subgroup. PMID:23516524

dbWGFP: a database and web server of human whole-genome single nucleotide variants and their functional predictions.

PubMed

Wu, Jiaxin; Wu, Mengmeng; Li, Lianshuo; Liu, Zhuo; Zeng, Wanwen; Jiang, Rui

2016-01-01

The recent advancement of the next generation sequencing technology has enabled the fast and low-cost detection of all genetic variants spreading across the entire human genome, making the application of whole-genome sequencing a tendency in the study of disease-causing genetic variants. Nevertheless, there still lacks a repository that collects predictions of functionally damaging effects of human genetic variants, though it has been well recognized that such predictions play a central role in the analysis of whole-genome sequencing data. To fill this gap, we developed a database named dbWGFP (a database and web server of human whole-genome single nucleotide variants and their functional predictions) that contains functional predictions and annotations of nearly 8.58 billion possible human whole-genome single nucleotide variants. Specifically, this database integrates 48 functional predictions calculated by 17 popular computational methods and 44 valuable annotations obtained from various data sources. Standalone software, user-friendly query services and free downloads of this database are available at http://bioinfo.au.tsinghua.edu.cn/dbwgfp. dbWGFP provides a valuable resource for the analysis of whole-genome sequencing, exome sequencing and SNP array data, thereby complementing existing data sources and computational resources in deciphering genetic bases of human inherited diseases. © The Author(s) 2016. Published by Oxford University Press.

A post-assembly genome-improvement toolkit (PAGIT) to obtain annotated genomes from contigs.

PubMed

Swain, Martin T; Tsai, Isheng J; Assefa, Samual A; Newbold, Chris; Berriman, Matthew; Otto, Thomas D

2012-06-07

Genome projects now produce draft assemblies within weeks owing to advanced high-throughput sequencing technologies. For milestone projects such as Escherichia coli or Homo sapiens, teams of scientists were employed to manually curate and finish these genomes to a high standard. Nowadays, this is not feasible for most projects, and the quality of genomes is generally of a much lower standard. This protocol describes software (PAGIT) that is used to improve the quality of draft genomes. It offers flexible functionality to close gaps in scaffolds, correct base errors in the consensus sequence and exploit reference genomes (if available) in order to improve scaffolding and generating annotations. The protocol is most accessible for bacterial and small eukaryotic genomes (up to 300 Mb), such as pathogenic bacteria, malaria and parasitic worms. Applying PAGIT to an E. coli assembly takes ∼24 h: it doubles the average contig size and annotates over 4,300 gene models.

[Phylogenetic analysis of genomes of Vibrio cholerae strains isolated on the territory of Rostov region].

PubMed

Kuleshov, K V; Markelov, M L; Dedkov, V G; Vodop'ianov, A S; Kermanov, A V; Pisanov, R V; Kruglikov, V D; Mazrukho, A B; Maleev, V V; Shipulin, G A

2013-01-01

Determination of origin of 2 Vibrio cholerae strains isolated on the territory of Rostov region by using full genome sequencing data. Toxigenic strain 2011 EL- 301 V. cholerae 01 El Tor Inaba No. 301 (ctxAB+, tcpA+) and nontoxigenic strain V. cholerae O1 Ogawa P- 18785 (ctxAB-, tcpA+) were studied. Sequencing was carried out on the MiSeq platform. Phylogenetic analysis of the genomes obtained was carried out based on comparison of conservative part of the studied and 54 previously sequenced genomes. 2011EL-301 strain genome was presented by 164 contigs with an average coverage of 100, N50 parameter was 132 kb, for strain P- 18785 - 159 contigs with a coverage of69, N50 - 83 kb. The contigs obtained for strain 2011 EL-301 were deposited in DDBJ/EMBL/GenBank databases with access code AJFN02000000, for strain P-18785 - ANHS00000000. 716 protein-coding orthologous genes were detected. Based on phylogenetic analysis strain P- 18785 belongs to PG-1 subgroup (a group of predecessor strains of the 7th pandemic). Strain 2011EL-301 belongs to groups of strains of the 7th pandemic and is included into the cluster with later isolates that are associated with cases of cholera in South Africa and cases of import of cholera to the USA from Pakistan. The data obtained allows to establish phylogenetic connections with V cholerae strains isolated earlier.

The Genome Sequence of the Leaf-Cutter Ant Atta cephalotes Reveals Insights into Its Obligate Symbiotic Lifestyle

PubMed Central

Suen, Garret; Holt, Carson; Abouheif, Ehab; Bornberg-Bauer, Erich; Bouffard, Pascal; Caldera, Eric J.; Cash, Elizabeth; Cavanaugh, Amy; Denas, Olgert; Elhaik, Eran; Favé, Marie-Julie; Gadau, Jürgen; Gibson, Joshua D.; Graur, Dan; Grubbs, Kirk J.; Hagen, Darren E.; Harkins, Timothy T.; Helmkampf, Martin; Hu, Hao; Johnson, Brian R.; Kim, Jay; Marsh, Sarah E.; Moeller, Joseph A.; Muñoz-Torres, Mónica C.; Murphy, Marguerite C.; Naughton, Meredith C.; Nigam, Surabhi; Overson, Rick; Rajakumar, Rajendhran; Reese, Justin T.; Scott, Jarrod J.; Smith, Chris R.; Tao, Shu; Tsutsui, Neil D.; Viljakainen, Lumi; Wissler, Lothar; Yandell, Mark D.; Zimmer, Fabian; Taylor, James; Slater, Steven C.; Clifton, Sandra W.; Warren, Wesley C.; Elsik, Christine G.; Smith, Christopher D.; Weinstock, George M.; Gerardo, Nicole M.; Currie, Cameron R.

2011-01-01

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host–microbe symbioses. PMID:21347285

Genome-wide comparison of medieval and modern Mycobacterium leprae.

PubMed

Schuenemann, Verena J; Singh, Pushpendra; Mendum, Thomas A; Krause-Kyora, Ben; Jäger, Günter; Bos, Kirsten I; Herbig, Alexander; Economou, Christos; Benjak, Andrej; Busso, Philippe; Nebel, Almut; Boldsen, Jesper L; Kjellström, Anna; Wu, Huihai; Stewart, Graham R; Taylor, G Michael; Bauer, Peter; Lee, Oona Y-C; Wu, Houdini H T; Minnikin, David E; Besra, Gurdyal S; Tucker, Katie; Roffey, Simon; Sow, Samba O; Cole, Stewart T; Nieselt, Kay; Krause, Johannes

2013-07-12

Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.

Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia

PubMed Central

Shortt, Jonathan A.; Card, Daren C.; Schield, Drew R.; Liu, Yang; Zhong, Bo; Castoe, Todd A.

2017-01-01

Background In areas where schistosomiasis control programs have been implemented, morbidity and prevalence have been greatly reduced. However, to sustain these reductions and move towards interruption of transmission, new tools for disease surveillance are needed. Genomic methods have the potential to help trace the sources of new infections, and allow us to monitor drug resistance. Large-scale genotyping efforts for schistosome species have been hindered by cost, limited numbers of established target loci, and the small amount of DNA obtained from miracidia, the life stage most readily acquired from humans. Here, we present a method using next generation sequencing to provide high-resolution genomic data from S. japonicum for population-based studies. Methodology/Principal Findings We applied whole genome amplification followed by double digest restriction site associated DNA sequencing (ddRADseq) to individual S. japonicum miracidia preserved on Whatman FTA cards. We found that we could effectively and consistently survey hundreds of thousands of variants from 10,000 to 30,000 loci from archived miracidia as old as six years. An analysis of variation from eight miracidia obtained from three hosts in two villages in Sichuan showed clear population structuring by village and host even within this limited sample. Conclusions/Significance This high-resolution sequencing approach yields three orders of magnitude more information than microsatellite genotyping methods that have been employed over the last decade, creating the potential to answer detailed questions about the sources of human infections and to monitor drug resistance. Costs per sample range from $50-$200, depending on the amount of sequence information desired, and we expect these costs can be reduced further given continued reductions in sequencing costs, improvement of protocols, and parallelization. This approach provides new promise for using modern genome-scale sampling to S. japonicum surveillance, and could be applied to other schistosome species and other parasitic helminthes. PMID:28107347

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

PubMed

Quail, Michael A; Smith, Miriam; Coupland, Paul; Otto, Thomas D; Harris, Simon R; Connor, Thomas R; Bertoni, Anna; Swerdlow, Harold P; Gu, Yong

2012-07-24

Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent's PGM, Pacific Biosciences' RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys

PubMed Central

2014-01-01

Background Clinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo). Methods Six single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome. Results Complete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens. Conclusions Mitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology. PMID:25034633

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys.

PubMed

Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Barta, John R

2014-07-17

Clinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo). Six single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome. Complete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens. Mitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology.

Comprehensive Survey of Genetic Diversity in Chloroplast Genomes and 45S nrDNAs within Panax ginseng Species

PubMed Central

Kim, Kyunghee; Lee, Sang-Choon; Lee, Junki; Lee, Hyun Oh; Joh, Ho Jun; Kim, Nam-Hoon; Park, Hyun-Seung; Yang, Tae-Jin

2015-01-01

We report complete sequences of chloroplast (cp) genome and 45S nuclear ribosomal DNA (45S nrDNA) for 11 Panax ginseng cultivars. We have obtained complete sequences of cp and 45S nrDNA, the representative barcoding target sequences for cytoplasm and nuclear genome, respectively, based on low coverage NGS sequence of each cultivar. The cp genomes sizes ranged from 156,241 to 156,425 bp and the major size variation was derived from differences in copy number of tandem repeats in the ycf1 gene and in the intergenic regions of rps16-trnUUG and rpl32-trnUAG. The complete 45S nrDNA unit sequences were 11,091 bp, representing a consensus single transcriptional unit with an intergenic spacer region. Comparative analysis of these sequences as well as those previously reported for three Chinese accessions identified very rare but unique polymorphism in the cp genome within P. ginseng cultivars. There were 12 intra-species polymorphisms (six SNPs and six InDels) among 14 cultivars. We also identified five SNPs from 45S nrDNA of 11 Korean ginseng cultivars. From the 17 unique informative polymorphic sites, we developed six reliable markers for analysis of ginseng diversity and cultivar authentication. PMID:26061692

A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi.

PubMed

Tan, Qian-Qian; Zhu, Li; Li, Yi; Liu, Wen; Ma, Wei-Hua; Lei, Chao-Liang; Wang, Xiao-Ping

2015-01-01

The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species. Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes. The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.

Development of Novel SSR Markers for Flax (Linum usitatissimum L.) Using Reduced-Representation Genome Sequencing.

PubMed

Wu, Jianzhong; Zhao, Qian; Wu, Guangwen; Zhang, Shuquan; Jiang, Tingbo

2016-01-01

Flax ( Linum usitatissimum L.) is a major fiber and oil yielding crop grown in northeastern China. Identification of flax molecular markers is a key step toward improving flax yield and quality via marker-assisted breeding. Simple sequence repeat (SSR) markers, which are based on genomic structural variation, are considered the most valuable type of genetic marker for this purpose. In this study, we screened 1574 microsatellites from Linum usitatissimum L. obtained using reduced representation genome sequencing (RRGS) to systematically identify SSR markers. The resulting set of microsatellites consisted mainly of trinucleotide (56.10%) and dinucleotide (35.23%) repeats, with each motif consisting of 5-8 repeats. We then evaluated marker sensitivity and specificity based on samples of 48 flax isolates obtained from northeastern China. Using the new SSR panel, the results demonstrated that fiber flax and oilseed flax varieties clustered into two well separated groups. The novel SSR markers developed in this study show potential value for selection of varieties for use in flax breeding programs.

Ultrafast Comparison of Personal Genomes via Precomputed Genome Fingerprints.

PubMed

Glusman, Gustavo; Mauldin, Denise E; Hood, Leroy E; Robinson, Max

2017-01-01

We present an ultrafast method for comparing personal genomes. We transform the standard genome representation (lists of variants relative to a reference) into "genome fingerprints" via locality sensitive hashing. The resulting genome fingerprints can be meaningfully compared even when the input data were obtained using different sequencing technologies, processed using different pipelines, represented in different data formats and relative to different reference versions. Furthermore, genome fingerprints are robust to up to 30% missing data. Because of their reduced size, computation on the genome fingerprints is fast and requires little memory. For example, we could compute all-against-all pairwise comparisons among the 2504 genomes in the 1000 Genomes data set in 67 s at high quality (21 μs per comparison, on a single processor), and achieved a lower quality approximation in just 11 s. Efficient computation enables scaling up a variety of important genome analyses, including quantifying relatedness, recognizing duplicative sequenced genomes in a set, population reconstruction, and many others. The original genome representation cannot be reconstructed from its fingerprint, effectively decoupling genome comparison from genome interpretation; the method thus has significant implications for privacy-preserving genome analytics.

Complete genome sequence of maize yellow striate virus, a new cytorhabdovirus infecting maize and wheat crops in Argentina.

PubMed

Maurino, Fernanda; Dumón, Analía D; Llauger, Gabriela; Alemandri, Vanina; de Haro, Luis A; Mattio, M Fernanda; Del Vas, Mariana; Laguna, Irma Graciela; Giménez Pecci, María de la Paz

2018-01-01

A rhabdovirus infecting maize and wheat crops in Argentina was molecularly characterized. Through next-generation sequencing (NGS) of symptomatic leaf samples, the complete genome was obtained of two isolates of maize yellow striate virus (MYSV), a putative new rhabdovirus, differing by only 0.4% at the nucleotide level. The MYSV genome consists of 12,654 nucleotides for maize and wheat virus isolates, and shares 71% nucleotide sequence identity with the complete genome of barley yellow striate mosaic virus (BYSMV, NC028244). Ten open reading frames (ORFs) were predicted in the MYSV genome from the antigenomic strand and were compared with their BYSMV counterparts. The highest amino acid sequence identity of the MYSV and BYSMV proteins was 80% between the L proteins, and the lowest was 37% between the proteins 4. Phylogenetic analysis suggested that the MYSV isolates are new members of the genus Cytorhabdovirus, family Rhabdoviridae. Yellow striate, affecting maize and wheat crops in Argentina, is an emergent disease that presents a potential economic risk for these widely distributed crops.

Genome Sequence of African Swine Fever Virus BA71, the Virulent Parental Strain of the Nonpathogenic and Tissue-Culture Adapted BA71V

PubMed Central

Rodríguez, Javier M.; Moreno, Leticia Tais; Alejo, Alí; Lacasta, Anna; Rodríguez, Fernando; Salas, María L.

2015-01-01

The strain BA71V has played a key role in African swine fever virus (ASFV) research. It was the first genome sequenced, and remains the only genome completely determined. A large part of the studies on the function of ASFV genes, viral transcription, replication, DNA repair and morphogenesis, has been performed using this model. This avirulent strain was obtained by adaptation to grow in Vero cells of the highly virulent BA71 strain. We report here the analysis of the genome sequence of BA71 in comparison with that of BA71V. They possess the smallest genomes for a virulent or an attenuated ASFV, and are essentially identical except for a relatively small number of changes. We discuss the possible contribution of these changes to virulence. Analysis of the BA71 sequence allowed us to identify new similarities among ASFV proteins, and with database proteins including two ASFV proteins that could function as a two-component signaling network. PMID:26618713

Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia.

PubMed

Khaw, Yam Sim; Chan, Yoke Fun; Jafar, Faizatul Lela; Othman, Norlijah; Chee, Hui Yee

2016-01-01

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5' and 3' non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63-81% among themselves and 63-96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection.

Comparative Genetic Analyses of Human Rhinovirus C (HRV-C) Complete Genome from Malaysia

PubMed Central

Khaw, Yam Sim; Chan, Yoke Fun; Jafar, Faizatul Lela; Othman, Norlijah; Chee, Hui Yee

2016-01-01

Human rhinovirus-C (HRV-C) has been implicated in more severe illnesses than HRV-A and HRV-B, however, the limited number of HRV-C complete genomes (complete 5′ and 3′ non-coding region and open reading frame sequences) has hindered the in-depth genetic study of this virus. This study aimed to sequence seven complete HRV-C genomes from Malaysia and compare their genetic characteristics with the 18 published HRV-Cs. Seven Malaysian HRV-C complete genomes were obtained with newly redesigned primers. The seven genomes were classified as HRV-C6, C12, C22, C23, C26, C42, and pat16 based on the VP4/VP2 and VP1 pairwise distance threshold classification. Five of the seven Malaysian isolates, namely, 3430-MY-10/C22, 8713-MY-10/C23, 8097-MY-11/C26, 1570-MY-10/C42, and 7383-MY-10/pat16 are the first newly sequenced complete HRV-C genomes. All seven Malaysian isolates genomes displayed nucleotide similarity of 63–81% among themselves and 63–96% with other HRV-Cs. Malaysian HRV-Cs had similar putative immunogenic sites, putative receptor utilization and potential antiviral sites as other HRV-Cs. The genomic features of Malaysian isolates were similar to those of other HRV-Cs. Negative selections were frequently detected in HRV-Cs complete coding sequences indicating that these sequences were under functional constraint. The present study showed that HRV-Cs from Malaysia have diverse genetic sequences but share conserved genomic features with other HRV-Cs. This genetic information could provide further aid in the understanding of HRV-C infection. PMID:27199901

Next-generation sequencing of the Trichinella murrelli mitochondrial genome allows comprehensive comparison of its divergence from the principal agent of human trichinellosis, Trichinella spiralis.

PubMed

Webb, Kristen M; Rosenthal, Benjamin M

2011-01-01

The mitochondrial genome's non-recombinant mode of inheritance and relatively rapid rate of evolution has promoted its use as a marker for studying the biogeographic history and evolutionary interrelationships among many metazoan species. A modest portion of the mitochondrial genome has been defined for 12 species and genotypes of parasites in the genus Trichinella, but its adequacy in representing the mitochondrial genome as a whole remains unclear, as the complete coding sequence has been characterized only for Trichinella spiralis. Here, we sought to comprehensively describe the extent and nature of divergence between the mitochondrial genomes of T. spiralis (which poses the most appreciable zoonotic risk owing to its capacity to establish persistent infections in domestic pigs) and Trichinella murrelli (which is the most prevalent species in North American wildlife hosts, but which poses relatively little risk to the safety of pork). Next generation sequencing methodologies and scaffold and de novo assembly strategies were employed. The entire protein-coding region was sequenced (13,917 bp), along with a portion of the highly repetitive non-coding region (1524 bp) of the mitochondrial genome of T. murrelli with a combined average read depth of 250 reads. The accuracy of base calling, estimated from coding region sequence was found to exceed 99.3%. Genome content and gene order was not found to be significantly different from that of T. spiralis. An overall inter-species sequence divergence of 9.5% was estimated. Significant variation was identified when the amount of variation between species at each gene is compared to the average amount of variation between species across the coding region. Next generation sequencing is a highly effective means to obtain previously unknown mitochondrial genome sequence. Particular to parasites, the extremely deep coverage achieved through this method allows for the detection of sequence heterogeneity between the multiple individuals that necessarily comprise such templates. Copyright © 2010 Elsevier B.V. All rights reserved.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

PubMed

Guérin, Frédéric; Arnaiz, Olivier; Boggetto, Nicole; Denby Wilkes, Cyril; Meyer, Eric; Sperling, Linda; Duharcourt, Sandra

2017-04-26

DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.

Novel Approach to Analyzing MFE of Noncoding RNA Sequences

PubMed Central

George, Tina P.; Thomas, Tessamma

2016-01-01

Genomic studies have become noncoding RNA (ncRNA) centric after the study of different genomes provided enormous information on ncRNA over the past decades. The function of ncRNA is decided by its secondary structure, and across organisms, the secondary structure is more conserved than the sequence itself. In this study, the optimal secondary structure or the minimum free energy (MFE) structure of ncRNA was found based on the thermodynamic nearest neighbor model. MFE of over 2600 ncRNA sequences was analyzed in view of its signal properties. Mathematical models linking MFE to the signal properties were found for each of the four classes of ncRNA analyzed. MFE values computed with the proposed models were in concordance with those obtained with the standard web servers. A total of 95% of the sequences analyzed had deviation of MFE values within ±15% relative to those obtained from standard web servers. PMID:27695341

Novel Approach to Analyzing MFE of Noncoding RNA Sequences.

PubMed

George, Tina P; Thomas, Tessamma

2016-01-01

Genomic studies have become noncoding RNA (ncRNA) centric after the study of different genomes provided enormous information on ncRNA over the past decades. The function of ncRNA is decided by its secondary structure, and across organisms, the secondary structure is more conserved than the sequence itself. In this study, the optimal secondary structure or the minimum free energy (MFE) structure of ncRNA was found based on the thermodynamic nearest neighbor model. MFE of over 2600 ncRNA sequences was analyzed in view of its signal properties. Mathematical models linking MFE to the signal properties were found for each of the four classes of ncRNA analyzed. MFE values computed with the proposed models were in concordance with those obtained with the standard web servers. A total of 95% of the sequences analyzed had deviation of MFE values within ±15% relative to those obtained from standard web servers.

Effects of sample treatments on genome recovery via single-cell genomics

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

Clingenpeel, Scott; Schwientek, Patrick; Hugenholtz, Philip

2014-06-13

It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

Development of self-compressing BLSOM for comprehensive analysis of big sequence data.

PubMed

Kikuchi, Akihito; Ikemura, Toshimichi; Abe, Takashi

2015-01-01

With the remarkable increase in genomic sequence data from various organisms, novel tools are needed for comprehensive analyses of available big sequence data. We previously developed a Batch-Learning Self-Organizing Map (BLSOM), which can cluster genomic fragment sequences according to phylotype solely dependent on oligonucleotide composition and applied to genome and metagenomic studies. BLSOM is suitable for high-performance parallel-computing and can analyze big data simultaneously, but a large-scale BLSOM needs a large computational resource. We have developed Self-Compressing BLSOM (SC-BLSOM) for reduction of computation time, which allows us to carry out comprehensive analysis of big sequence data without the use of high-performance supercomputers. The strategy of SC-BLSOM is to hierarchically construct BLSOMs according to data class, such as phylotype. The first-layer BLSOM was constructed with each of the divided input data pieces that represents the data subclass, such as phylotype division, resulting in compression of the number of data pieces. The second BLSOM was constructed with a total of weight vectors obtained in the first-layer BLSOMs. We compared SC-BLSOM with the conventional BLSOM by analyzing bacterial genome sequences. SC-BLSOM could be constructed faster than BLSOM and cluster the sequences according to phylotype with high accuracy, showing the method's suitability for efficient knowledge discovery from big sequence data.

A Hybrid Approach for the Automated Finishing of Bacterial Genomes

PubMed Central

Robins, William P.; Chin, Chen-Shan; Webster, Dale; Paxinos, Ellen; Hsu, David; Ashby, Meredith; Wang, Susana; Peluso, Paul; Sebra, Robert; Sorenson, Jon; Bullard, James; Yen, Jackie; Valdovino, Marie; Mollova, Emilia; Luong, Khai; Lin, Steven; LaMay, Brianna; Joshi, Amruta; Rowe, Lori; Frace, Michael; Tarr, Cheryl L.; Turnsek, Maryann; Davis, Brigid M; Kasarskis, Andrew; Mekalanos, John J.; Waldor, Matthew K.; Schadt, Eric E.

2013-01-01

Dramatic improvements in DNA sequencing technology have revolutionized our ability to characterize most genomic diversity. However, accurate resolution of large structural events has remained challenging due to the comparatively shorter read lengths of second-generation technologies. Emerging third-generation sequencing technologies, which yield markedly increased read length on rapid time scales and for low cost, have the potential to address assembly limitations. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at > 99.9% accuracy. Complex regions with clinically significant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 reference we obtain 14 and 8 scaffolds greater than 1kb, respectively, correcting several errors in the underlying source data. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly. PMID:22750883

Complete chloroplast and ribosomal sequences for 30 accessions elucidate evolution of Oryza AA genome species

PubMed Central

Kim, Kyunghee; Lee, Sang-Choon; Lee, Junki; Yu, Yeisoo; Yang, Kiwoung; Choi, Beom-Soon; Koh, Hee-Jong; Waminal, Nomar Espinosa; Choi, Hong-Il; Kim, Nam-Hoon; Jang, Woojong; Park, Hyun-Seung; Lee, Jonghoon; Lee, Hyun Oh; Joh, Ho Jun; Lee, Hyeon Ju; Park, Jee Young; Perumal, Sampath; Jayakodi, Murukarthick; Lee, Yun Sun; Kim, Backki; Copetti, Dario; Kim, Soonok; Kim, Sunggil; Lim, Ki-Byung; Kim, Young-Dong; Lee, Jungho; Cho, Kwang-Su; Park, Beom-Seok; Wing, Rod A.; Yang, Tae-Jin

2015-01-01

Cytoplasmic chloroplast (cp) genomes and nuclear ribosomal DNA (nR) are the primary sequences used to understand plant diversity and evolution. We introduce a high-throughput method to simultaneously obtain complete cp and nR sequences using Illumina platform whole-genome sequence. We applied the method to 30 rice specimens belonging to nine Oryza species. Concurrent phylogenomic analysis using cp and nR of several of specimens of the same Oryza AA genome species provides insight into the evolution and domestication of cultivated rice, clarifying three ambiguous but important issues in the evolution of wild Oryza species. First, cp-based trees clearly classify each lineage but can be biased by inter-subspecies cross-hybridization events during speciation. Second, O. glumaepatula, a South American wild rice, includes two cytoplasm types, one of which is derived from a recent interspecies hybridization with O. longistminata. Third, the Australian O. rufipogan-type rice is a perennial form of O. meridionalis. PMID:26506948

Complete genome sequencing of the luminescent bacterium, Vibrio qinghaiensis sp. Q67 using PacBio technology

NASA Astrophysics Data System (ADS)

Gong, Liang; Wu, Yu; Jian, Qijie; Yin, Chunxiao; Li, Taotao; Gupta, Vijai Kumar; Duan, Xuewu; Jiang, Yueming

2018-01-01

Vibrio qinghaiensis sp.-Q67 (Vqin-Q67) is a freshwater luminescent bacterium that continuously emits blue-green light (485 nm). The bacterium has been widely used for detecting toxic contaminants. Here, we report the complete genome sequence of Vqin-Q67, obtained using third-generation PacBio sequencing technology. Continuous long reads were attained from three PacBio sequencing runs and reads >500 bp with a quality value of >0.75 were merged together into a single dataset. This resultant highly-contiguous de novo assembly has no genome gaps, and comprises two chromosomes with substantial genetic information, including protein-coding genes, non-coding RNA, transposon and gene islands. Our dataset can be useful as a comparative genome for evolution and speciation studies, as well as for the analysis of protein-coding gene families, the pathogenicity of different Vibrio species in fish, the evolution of non-coding RNA and transposon, and the regulation of gene expression in relation to the bioluminescence of Vqin-Q67.

Complete plastid genome sequence of goosegrass (Eleusine indica) and comparison with other Poaceae.

PubMed

Zhang, Hui; Hall, Nathan; McElroy, J Scott; Lowe, Elijah K; Goertzen, Leslie R

2017-02-05

Eleusine indica, also known as goosegrass, is a serious weed in at least 42 countries. In this paper we report the complete plastid genome sequence of goosegrass obtained by de novo assembly of paired-end and mate-paired reads generated by Illumina sequencing of total genomic DNA. The goosegrass plastome is a circular molecule of 135,151bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 20,919 bases. The large (LSC) and the small (SSC) single-copy regions span 80,667 bases and 12,646 bases, respectively. The plastome of goosegrass has 38.19% GC content and includes 108 unique genes, of which 76 are protein-coding, 28 are transfer RNA, and 4 are ribosomal RNA. The goosegrass plastome sequence was compared to eight other species of Poaceae. Although generally conserved with respect to Poaceae, this genomic resource will be useful for evolutionary studies within this weed species and the genus Eleusine. Copyright © 2016. Published by Elsevier B.V.

Ancient genomics

PubMed Central

Der Sarkissian, Clio; Allentoft, Morten E.; Ávila-Arcos, María C.; Barnett, Ross; Campos, Paula F.; Cappellini, Enrico; Ermini, Luca; Fernández, Ruth; da Fonseca, Rute; Ginolhac, Aurélien; Hansen, Anders J.; Jónsson, Hákon; Korneliussen, Thorfinn; Margaryan, Ashot; Martin, Michael D.; Moreno-Mayar, J. Víctor; Raghavan, Maanasa; Rasmussen, Morten; Velasco, Marcela Sandoval; Schroeder, Hannes; Schubert, Mikkel; Seguin-Orlando, Andaine; Wales, Nathan; Gilbert, M. Thomas P.; Willerslev, Eske; Orlando, Ludovic

2015-01-01

The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field's focus was previously limited to mitochondrial DNA and a few nuclear markers, whole genome sequences from the deep past can now be retrieved. This breakthrough is tightly connected to the massive sequence throughput of next generation sequencing platforms and the ability to target short and degraded DNA molecules. Many ancient specimens previously unsuitable for DNA analyses because of extensive degradation can now successfully be used as source materials. Additionally, the analytical power obtained by increasing the number of sequence reads to billions effectively means that contamination issues that have haunted aDNA research for decades, particularly in human studies, can now be efficiently and confidently quantified. At present, whole genomes have been sequenced from ancient anatomically modern humans, archaic hominins, ancient pathogens and megafaunal species. Those have revealed important functional and phenotypic information, as well as unexpected adaptation, migration and admixture patterns. As such, the field of aDNA has entered the new era of genomics and has provided valuable information when testing specific hypotheses related to the past. PMID:25487338

Y and W Chromosome Assemblies: Approaches and Discoveries.

PubMed

Tomaszkiewicz, Marta; Medvedev, Paul; Makova, Kateryna D

2017-04-01

Hundreds of vertebrate genomes have been sequenced and assembled to date. However, most sequencing projects have ignored the sex chromosomes unique to the heterogametic sex - Y and W - that are known as sex-limited chromosomes (SLCs). Indeed, haploid and repetitive Y chromosomes in species with male heterogamety (XY), and W chromosomes in species with female heterogamety (ZW), are difficult to sequence and assemble. Nevertheless, obtaining their sequences is important for understanding the intricacies of vertebrate genome function and evolution. Recent progress has been made towards the adaptation of next-generation sequencing (NGS) techniques to deciphering SLC sequences. We review here currently available methodology and results with regard to SLC sequencing and assembly. We focus on vertebrates, but bring in some examples from other taxa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Life in the fast lane for protein crystallization and X-ray crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2005-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

Life in the Fast Lane for Protein Crystallization and X-Ray Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2004-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today s high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

Complete Chloroplast Genome of the Multifunctional Crop Globe Artichoke and Comparison with Other Asteraceae

PubMed Central

Curci, Pasquale L.; De Paola, Domenico; Danzi, Donatella; Vendramin, Giovanni G.; Sonnante, Gabriella

2015-01-01

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for “specific barcode” purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants. PMID:25774672

Complete chloroplast genome of the multifunctional crop globe artichoke and comparison with other Asteraceae.

PubMed

Curci, Pasquale L; De Paola, Domenico; Danzi, Donatella; Vendramin, Giovanni G; Sonnante, Gabriella

2015-01-01

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for "specific barcode" purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants.

Unexpected effects of different genetic backgrounds on identification of genomic rearrangements via whole-genome next generation sequencing.

PubMed

Chen, Zhangguo; Gowan, Katherine; Leach, Sonia M; Viboolsittiseri, Sawanee S; Mishra, Ameet K; Kadoishi, Tanya; Diener, Katrina; Gao, Bifeng; Jones, Kenneth; Wang, Jing H

2016-10-21

Whole genome next generation sequencing (NGS) is increasingly employed to detect genomic rearrangements in cancer genomes, especially in lymphoid malignancies. We recently established a unique mouse model by specifically deleting a key non-homologous end-joining DNA repair gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in germinal center B cells. This mouse model spontaneously develops mature B cell lymphomas (termed G1XP lymphomas). Here, we attempt to employ whole genome NGS to identify novel structural rearrangements, in particular inter-chromosomal translocations (CTXs), in these G1XP lymphomas. We sequenced six lymphoma samples, aligned our NGS data with mouse reference genome (in C57BL/6J (B6) background) and identified CTXs using CREST algorithm. Surprisingly, we detected widespread CTXs in both lymphomas and wildtype control samples, majority of which were false positive and attributable to different genetic backgrounds. In addition, we validated our NGS pipeline by sequencing multiple control samples from distinct tissues of different genetic backgrounds of mouse (B6 vs non-B6). Lastly, our studies showed that widespread false positive CTXs can be generated by simply aligning sequences from different genetic backgrounds of mouse. We conclude that mapping and alignment with reference genome might not be a preferred method for analyzing whole-genome NGS data obtained from a genetic background different from reference genome. Given the complex genetic background of different mouse strains or the heterogeneity of cancer genomes in human patients, in order to minimize such systematic artifacts and uncover novel CTXs, a preferred method might be de novo assembly of personalized normal control genome and cancer cell genome, instead of mapping and aligning NGS data to mouse or human reference genome. Thus, our studies have critical impact on the manner of data analysis for cancer genomics.

Draft genome sequence of Bradyrhizobium manausense strain BR 3351T, an effective symbiont isolated from Amazon rainforest.

PubMed

Simões-Araújo, Jean Luiz; Rumjanek, Norma Gouvêa; Xavier, Gustavo Ribeiro; Zilli, Jerri Édson

The strain BR 3351 T (Bradyrhizobium manausense) was obtained from nodules of cowpea (Vigna unguiculata L. Walp) growing in soil collected from Amazon rainforest. Furthermore, it was observed that the strain has high capacity to fix nitrogen symbiotically in symbioses with cowpea. We report here the draft genome sequence of strain BR 3351 T . The information presented will be important for comparative analysis of nodulation and nitrogen fixation for diazotrophic bacteria. A draft genome with 9,145,311bp and 62.9% of GC content was assembled in 127 scaffolds using 100bp pair-end Illumina MiSeq system. The RAST annotation identified 8603 coding sequences, 51 RNAs genes, classified in 504 subsystems. Published by Elsevier Editora Ltda.

Genomic Analysis of the Chicken Infectious Anemia Virus in a Specific Pathogen-Free Chicken Population in China

PubMed Central

Li, Yang; Wang, Yixin; Fang, Lichun; Fu, Jiayuan; Cui, Shuai; Zhao, Yingjie; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

2016-01-01

The antibody to chicken infectious anemia virus (CIAV) was positive in a specific pathogen-free (SPF) chicken population by ELISA test in our previous inspection, indicating a possible infection with CIAV. In this study, blood samples collected from the SPF chickens were used to isolate CIAV by inoculating into MSB1 cells and PCR amplification. A CIAV strain (SD1403) was isolated and successfully identified. Three overlapping genomic fragments were obtained by PCR amplification and sequencing. The full genome sequence of the SD1403 strain was obtained by aligning the sequences. The genome of the SD1403 strain was 2293 bp with a nucleotide identity of 94.8% to 98.5% when compared with 30 referred CIAV strains. The viral proteins VP2 and VP3 were highly conserved, but VP1 was not relatively conserved. Both amino acids 139 and 144 of VP1 were glutamine, which was in accord with the low pathogenic characteristics. In this study, we first reported that CIAV exists in Chinese SPF chicken populations and may be an important reason why attenuated vaccine can be contaminated with CIAV. PMID:27298822

Genomic Analysis of the Chicken Infectious Anemia Virus in a Specific Pathogen-Free Chicken Population in China.

PubMed

Li, Yang; Wang, Yixin; Fang, Lichun; Fu, Jiayuan; Cui, Shuai; Zhao, Yingjie; Cui, Zhizhong; Chang, Shuang; Zhao, Peng

2016-01-01

The antibody to chicken infectious anemia virus (CIAV) was positive in a specific pathogen-free (SPF) chicken population by ELISA test in our previous inspection, indicating a possible infection with CIAV. In this study, blood samples collected from the SPF chickens were used to isolate CIAV by inoculating into MSB1 cells and PCR amplification. A CIAV strain (SD1403) was isolated and successfully identified. Three overlapping genomic fragments were obtained by PCR amplification and sequencing. The full genome sequence of the SD1403 strain was obtained by aligning the sequences. The genome of the SD1403 strain was 2293 bp with a nucleotide identity of 94.8% to 98.5% when compared with 30 referred CIAV strains. The viral proteins VP2 and VP3 were highly conserved, but VP1 was not relatively conserved. Both amino acids 139 and 144 of VP1 were glutamine, which was in accord with the low pathogenic characteristics. In this study, we first reported that CIAV exists in Chinese SPF chicken populations and may be an important reason why attenuated vaccine can be contaminated with CIAV.

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing

PubMed Central

2011-01-01

Background Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models. PMID:21542930

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

PubMed

Straub, Shannon C K; Fishbein, Mark; Livshultz, Tatyana; Foster, Zachary; Parks, Matthew; Weitemier, Kevin; Cronn, Richard C; Liston, Aaron

2011-05-04

Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.

Biocomputational identification and validation of novel microRNAs predicted from bubaline whole genome shotgun sequences.

PubMed

Manku, H K; Dhanoa, J K; Kaur, S; Arora, J S; Mukhopadhyay, C S

2017-10-01

MicroRNAs (miRNAs) are small (19-25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo. Copyright © 2017 Elsevier Ltd. All rights reserved.

Detection of integrated papillomavirus sequences by ligation-mediated PCR (DIPS-PCR) and molecular characterization in cervical cancer cells.

PubMed

Luft, F; Klaes, R; Nees, M; Dürst, M; Heilmann, V; Melsheimer, P; von Knebel Doeberitz, M

2001-04-01

Human papillomavirus (HPV) genomes usually persist as episomal molecules in HPV associated preneoplastic lesions whereas they are frequently integrated into the host cell genome in HPV-related cancers cells. This suggests that malignant conversion of HPV-infected epithelia is linked to recombination of cellular and viral sequences. Due to technical limitations, precise sequence information on viral-cellular junctions were obtained only for few cell lines and primary lesions. In order to facilitate the molecular analysis of genomic HPV integration, we established a ligation-mediated PCR assay for the detection of integrated papillomavirus sequences (DIPS-PCR). DIPS-PCR was initially used to amplify genomic viral-cellular junctions from HPV-associated cervical cancer cell lines (C4-I, C4-II, SW756, and HeLa) and HPV-immortalized keratinocyte lines (HPKIA, HPKII). In addition to junctions already reported in public data bases, various new fusion fragments were identified. Subsequently, 22 different viral-cellular junctions were amplified from 17 cervical carcinomas and 1 vulval intraepithelial neoplasia (VIN III). Sequence analysis of each junction revealed that the viral E1 open reading frame (ORF) was fused to cellular sequences in 20 of 22 (91%) cases. Chromosomal integration loci mapped to chromosomes 1 (2n), 2 (3n), 7 (2n), 8 (3n), 10 (1n), 14 (5n), 16 (1n), 17 (2n), and mitochondrial DNA (1n), suggesting random distribution of chromosomal integration sites. Precise sequence information obtained by DIPS-PCR was further used to monitor the monoclonal origin of 4 cervical cancers, 1 case of recurrent premalignant lesions and 1 lymph node metastasis. Therefore, DIPS-PCR might allow efficient therapy control and prediction of relapse in patients with HPV-associated anogenital cancers. Copyright 2001 Wiley-Liss, Inc.

Analysis for complete genomic sequence of HLA-B and HLA-C alleles in the Chinese Han population.

PubMed

Zhu, F; He, Y; Zhang, W; He, J; He, J; Xu, X; Lv, H; Yan, L

2011-08-01

In the present study, we have determined the complete genomic sequence and analysed the intron polymorphism of partial HLA-B and HLA-C alleles in the Chinese Han population. Over 3.0 kb DNA fragments of HLA-B and HLA-C loci were amplified by polymerase chain reaction from partial 5' untranslated region to 3' noncoding region respectively, and then the amplified products were sequenced. Full-length nucleotide sequences of 14 HLA-B alleles and 10 HLA-C alleles were obtained and have been submitted to GenBank and IMGT/HLA database. Two novel alleles of HLA-B*52:01:01:02 and HLA-B*59:01:01:02 were identified, and the complete genomic sequence of HLA-B*52:01:01:01 was firstly reported. Totally 157 and 167 polymorphism positions were found in the full-length genomic sequence of HLA-B and HLA-C loci respectively. Our results suggested that many single nucleotide polymorphisms existed in the exon and intron regions, and the data can provide useful information for understanding the evolution of HLA-B and HLA-C alleles. © 2011 Blackwell Publishing Ltd.

Whole-Genome Sequencing and Variant Analysis of Human Papillomavirus 16 Infections.

PubMed

van der Weele, Pascal; Meijer, Chris J L M; King, Audrey J

2017-10-01

Human papillomavirus (HPV) is a strongly conserved DNA virus, high-risk types of which can cause cervical cancer in persistent infections. The most common type found in HPV-attributable cancer is HPV16, which can be subdivided into four lineages (A to D) with different carcinogenic properties. Studies have shown HPV16 sequence diversity in different geographical areas, but only limited information is available regarding HPV16 diversity within a population, especially at the whole-genome level. We analyzed HPV16 major variant diversity and conservation in persistent infections and performed a single nucleotide polymorphism (SNP) comparison between persistent and clearing infections. Materials were obtained in the Netherlands from a cohort study with longitudinal follow-up for up to 3 years. Our analysis shows a remarkably large variant diversity in the population. Whole-genome sequences were obtained for 57 persistent and 59 clearing HPV16 infections, resulting in 109 unique variants. Interestingly, persistent infections were completely conserved through time. One reinfection event was identified where the initial and follow-up samples clustered differently. Non-A1/A2 variants seemed to clear preferentially ( P = 0.02). Our analysis shows that population-wide HPV16 sequence diversity is very large. In persistent infections, the HPV16 sequence was fully conserved. Sequencing can identify HPV16 reinfections, although occurrence is rare. SNP comparison identified no strongly acting effect of the viral genome affecting HPV16 infection clearance or persistence in up to 3 years of follow-up. These findings suggest the progression of an early HPV16 infection could be host related. IMPORTANCE Human papillomavirus 16 (HPV16) is the predominant type found in cervical cancer. Progression of initial infection to cervical cancer has been linked to sequence properties; however, knowledge of variants circulating in European populations, especially with longitudinal follow-up, is limited. By sequencing a number of infections with known follow-up for up to 3 years, we gained initial insights into the genetic diversity of HPV16 and the effects of the viral genome on the persistence of infections. A SNP comparison between sequences obtained from clearing and persistent infections did not identify strongly acting DNA variations responsible for these infection outcomes. In addition, we identified an HPV16 reinfection event where sequencing of initial and follow-up samples showed different HPV16 variants. Based on conventional genotyping, this infection would incorrectly be considered a persistent HPV16 infection. In the context of vaccine efficacy and monitoring studies, such infections could potentially cause reduced reported efficacy or efficiency. Copyright © 2017 van der Weele et al.

Read clouds uncover variation in complex regions of the human genome.

PubMed

Bishara, Alex; Liu, Yuling; Weng, Ziming; Kashef-Haghighi, Dorna; Newburger, Daniel E; West, Robert; Sidow, Arend; Batzoglou, Serafim

2015-10-01

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies. © 2015 Bishara et al.; Published by Cold Spring Harbor Laboratory Press.

Bivalve-specific gene expansion in the pearl oyster genome: implications of adaptation to a sessile lifestyle.

PubMed

Takeuchi, Takeshi; Koyanagi, Ryo; Gyoja, Fuki; Kanda, Miyuki; Hisata, Kanako; Fujie, Manabu; Goto, Hiroki; Yamasaki, Shinichi; Nagai, Kiyohito; Morino, Yoshiaki; Miyamoto, Hiroshi; Endo, Kazuyoshi; Endo, Hirotoshi; Nagasawa, Hiromichi; Kinoshita, Shigeharu; Asakawa, Shuichi; Watabe, Shugo; Satoh, Noriyuki; Kawashima, Takeshi

2016-01-01

Bivalve molluscs have flourished in marine environments, and many species constitute important aquatic resources. Recently, whole genome sequences from two bivalves, the pearl oyster, Pinctada fucata, and the Pacific oyster, Crassostrea gigas, have been decoded, making it possible to compare genomic sequences among molluscs, and to explore general and lineage-specific genetic features and trends in bivalves. In order to improve the quality of sequence data for these purposes, we have updated the entire P. fucata genome assembly. We present a new genome assembly of the pearl oyster, Pinctada fucata (version 2.0). To update the assembly, we conducted additional sequencing, obtaining accumulated sequence data amounting to 193× the P. fucata genome. Sequence redundancy in contigs that was caused by heterozygosity was removed in silico, which significantly improved subsequent scaffolding. Gene model version 2.0 was generated with the aid of manual gene annotations supplied by the P. fucata research community. Comparison of mollusc and other bilaterian genomes shows that gene arrangements of Hox, ParaHox, and Wnt clusters in the P. fucata genome are similar to those of other molluscs. Like the Pacific oyster, P. fucata possesses many genes involved in environmental responses and in immune defense. Phylogenetic analyses of heat shock protein70 and C1q domain-containing protein families indicate that extensive expansion of genes occurred independently in each lineage. Several gene duplication events prior to the split between the pearl oyster and the Pacific oyster are also evident. In addition, a number of tandem duplications of genes that encode shell matrix proteins are also well characterized in the P. fucata genome. Both the Pinctada and Crassostrea lineages have expanded specific gene families in a lineage-specific manner. Frequent duplication of genes responsible for shell formation in the P. fucata genome explains the diversity of mollusc shell structures. These duplications reveal dynamic genome evolution to forge the complex physiology that enables bivalves to employ a sessile lifestyle in the intertidal zone.

Increased Genomic Prediction Accuracy in Wheat Breeding Through Spatial Adjustment of Field Trial Data

PubMed Central

Lado, Bettina; Matus, Ivan; Rodríguez, Alejandra; Inostroza, Luis; Poland, Jesse; Belzile, François; del Pozo, Alejandro; Quincke, Martín; Castro, Marina; von Zitzewitz, Jarislav

2013-01-01

In crop breeding, the interest of predicting the performance of candidate cultivars in the field has increased due to recent advances in molecular breeding technologies. However, the complexity of the wheat genome presents some challenges for applying new technologies in molecular marker identification with next-generation sequencing. We applied genotyping-by-sequencing, a recently developed method to identify single-nucleotide polymorphisms, in the genomes of 384 wheat (Triticum aestivum) genotypes that were field tested under three different water regimes in Mediterranean climatic conditions: rain-fed only, mild water stress, and fully irrigated. We identified 102,324 single-nucleotide polymorphisms in these genotypes, and the phenotypic data were used to train and test genomic selection models intended to predict yield, thousand-kernel weight, number of kernels per spike, and heading date. Phenotypic data showed marked spatial variation. Therefore, different models were tested to correct the trends observed in the field. A mixed-model using moving-means as a covariate was found to best fit the data. When we applied the genomic selection models, the accuracy of predicted traits increased with spatial adjustment. Multiple genomic selection models were tested, and a Gaussian kernel model was determined to give the highest accuracy. The best predictions between environments were obtained when data from different years were used to train the model. Our results confirm that genotyping-by-sequencing is an effective tool to obtain genome-wide information for crops with complex genomes, that these data are efficient for predicting traits, and that correction of spatial variation is a crucial ingredient to increase prediction accuracy in genomic selection models. PMID:24082033

Extensive structural variations between mitochondrial genomes of CMS and normal peppers (Capsicum annuum L.) revealed by complete nucleotide sequencing.

PubMed

Jo, Yeong Deuk; Choi, Yoomi; Kim, Dong-Hwan; Kim, Byung-Dong; Kang, Byoung-Cheorl

2014-07-04

Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp. We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (ψatp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes. Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.

The Génolevures database.

PubMed

Martin, Tiphaine; Sherman, David J; Durrens, Pascal

2011-01-01

The Génolevures online database (URL: http://www.genolevures.org) stores and provides the data and results obtained by the Génolevures Consortium through several campaigns of genome annotation of the yeasts in the Saccharomycotina subphylum (hemiascomycetes). This database is dedicated to large-scale comparison of these genomes, storing not only the different chromosomal elements detected in the sequences, but also the logical relations between them. The database is divided into a public part, accessible to anyone through Internet, and a private part where the Consortium members make genome annotations with our Magus annotation system; this system is used to annotate several related genomes in parallel. The public database is widely consulted and offers structured data, organized using a REST web site architecture that allows for automated requests. The implementation of the database, as well as its associated tools and methods, is evolving to cope with the influx of genome sequences produced by Next Generation Sequencing (NGS). Copyright © 2011 Académie des sciences. Published by Elsevier SAS. All rights reserved.

SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing

PubMed Central

Bankevich, Anton; Nurk, Sergey; Antipov, Dmitry; Gurevich, Alexey A.; Dvorkin, Mikhail; Kulikov, Alexander S.; Lesin, Valery M.; Nikolenko, Sergey I.; Pham, Son; Prjibelski, Andrey D.; Pyshkin, Alexey V.; Sirotkin, Alexander V.; Vyahhi, Nikolay; Tesler, Glenn; Pevzner, Pavel A.

2012-01-01

Abstract The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V−SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online (http://bioinf.spbau.ru/spades). It is distributed as open source software. PMID:22506599

A complete mitochondrial genome sequence of Asian black bear Sichuan subspecies (Ursus thibetanus mupinensis)

PubMed Central

Hou, Wan-ru; Chen, Yu; Wu, Xia; Hu, Jin-chu; Peng, Zheng-song; Yang, Jung; Tang, Zong-xiang; Zhou, Cai-Quan; Li, Yu-ming; Yang, Shi-kui; Du, Yu-jie; Kong, Ling-lu; Ren, Zheng-long; Zhang, Huai-yu; Shuai, Su-rong

2007-01-01

We obtained the complete mitochondrial genome of U.thibetanus mupinensis by DNA sequencing based on the PCR fragments of 18 primers we designed. The results indicate that the mtDNA is 16 868 bp in size, encodes 13 protein genes, 22 tRNA genes, and 2 rRNA genes, with an overall H-strand base composition of 31.2% A, 25.4% C, 15.5% G and 27.9% T. The sequence of the control region (CR) located between tRNA-Pro and tRNA-Phe is 1422 bp in size, consists of 8.43% of the whole genome, GC content is 51.9% and has a 6bp tandem repeat and two 10bp tandem repeats identified by using the Tandem Repeats Finder. U. thibetanus mupinensis mitochondrial genome shares high similarity with those of three other Ursidae: U. americanus (91.46%), U. arctos (89.25%) and U. maritimus (87.66%). PMID:17205108

Comparative genomics of Vibrio cholerae from Haiti, Asia, and Africa.

PubMed

Reimer, Aleisha R; Van Domselaar, Gary; Stroika, Steven; Walker, Matthew; Kent, Heather; Tarr, Cheryl; Talkington, Deborah; Rowe, Lori; Olsen-Rasmussen, Melissa; Frace, Michael; Sammons, Scott; Dahourou, Georges Anicet; Boncy, Jacques; Smith, Anthony M; Mabon, Philip; Petkau, Aaron; Graham, Morag; Gilmour, Matthew W; Gerner-Smidt, Peter

2011-11-01

Cholera was absent from the island of Hispaniola at least a century before an outbreak that began in Haiti in the fall of 2010. Pulsed-field gel electrophoresis (PFGE) analysis of clinical isolates from the Haiti outbreak and recent global travelers returning to the United States showed indistinguishable PFGE fingerprints. To better explore the genetic ancestry of the Haiti outbreak strain, we acquired 23 whole-genome Vibrio cholerae sequences: 9 isolates obtained in Haiti or the Dominican Republic, 12 PFGE pattern-matched isolates linked to Asia or Africa, and 2 nonmatched outliers from the Western Hemisphere. Phylogenies for whole-genome sequences and core genome single-nucleotide polymorphisms showed that the Haiti outbreak strain is genetically related to strains originating in India and Cameroon. However, because no identical genetic match was found among sequenced contemporary isolates, a definitive genetic origin for the outbreak in Haiti remains speculative.

Comparative analyses of putative toxin gene homologs from an Old World viper, Daboia russelii

PubMed Central

Krishnan, Neeraja M.

2017-01-01

Availability of snake genome sequences has opened up exciting areas of research on comparative genomics and gene diversity. One of the challenges in studying snake genomes is the acquisition of biological material from live animals, especially from the venomous ones, making the process cumbersome and time-consuming. Here, we report comparative sequence analyses of putative toxin gene homologs from Russell’s viper (Daboia russelii) using whole-genome sequencing data obtained from shed skin. When compared with the major venom proteins in Russell’s viper studied previously, we found 45–100% sequence similarity between the venom proteins and their putative homologs in the skin. Additionally, comparative analyses of 20 putative toxin gene family homologs provided evidence of unique sequence motifs in nerve growth factor (NGF), platelet derived growth factor (PDGF), Kunitz/Bovine pancreatic trypsin inhibitor (Kunitz BPTI), cysteine-rich secretory proteins, antigen 5, andpathogenesis-related1 proteins (CAP) and cysteine-rich secretory protein (CRISP). In those derived proteins, we identified V11 and T35 in the NGF domain; F23 and A29 in the PDGF domain; N69, K2 and A5 in the CAP domain; and Q17 in the CRISP domain to be responsible for differences in the largest pockets across the protein domain structures in crotalines, viperines and elapids from the in silico structure-based analysis. Similarly, residues F10, Y11 and E20 appear to play an important role in the protein structures across the kunitz protein domain of viperids and elapids. Our study highlights the usefulness of shed skin in obtaining good quality high-molecular weight DNA for comparative genomic studies, and provides evidence towards the unique features and evolution of putative venom gene homologs in vipers. PMID:29230357

Mapping the Space of Genomic Signatures

PubMed Central

Kari, Lila; Hill, Kathleen A.; Sayem, Abu S.; Karamichalis, Rallis; Bryans, Nathaniel; Davis, Katelyn; Dattani, Nikesh S.

2015-01-01

We propose a computational method to measure and visualize interrelationships among any number of DNA sequences allowing, for example, the examination of hundreds or thousands of complete mitochondrial genomes. An "image distance" is computed for each pair of graphical representations of DNA sequences, and the distances are visualized as a Molecular Distance Map: Each point on the map represents a DNA sequence, and the spatial proximity between any two points reflects the degree of structural similarity between the corresponding sequences. The graphical representation of DNA sequences utilized, Chaos Game Representation (CGR), is genome- and species-specific and can thus act as a genomic signature. Consequently, Molecular Distance Maps could inform species identification, taxonomic classifications and, to a certain extent, evolutionary history. The image distance employed, Structural Dissimilarity Index (DSSIM), implicitly compares the occurrences of oligomers of length up to k (herein k = 9) in DNA sequences. We computed DSSIM distances for more than 5 million pairs of complete mitochondrial genomes, and used Multi-Dimensional Scaling (MDS) to obtain Molecular Distance Maps that visually display the sequence relatedness in various subsets, at different taxonomic levels. This general-purpose method does not require DNA sequence alignment and can thus be used to compare similar or vastly different DNA sequences, genomic or computer-generated, of the same or different lengths. We illustrate potential uses of this approach by applying it to several taxonomic subsets: phylum Vertebrata, (super)kingdom Protista, classes Amphibia-Insecta-Mammalia, class Amphibia, and order Primates. This analysis of an extensive dataset confirms that the oligomer composition of full mtDNA sequences can be a source of taxonomic information. This method also correctly finds the mtDNA sequences most closely related to that of the anatomically modern human (the Neanderthal, the Denisovan, and the chimp), and that the sequence most different from it in this dataset belongs to a cucumber. PMID:26000734

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes

PubMed Central

Yasui, Yasuo; Hirakawa, Hideki; Ueno, Mariko; Matsui, Katsuhiro; Katsube-Tanaka, Tomoyuki; Yang, Soo Jung; Aii, Jotaro; Sato, Shingo; Mori, Masashi

2016-01-01

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits. PMID:27037832

BAUM: improving genome assembly by adaptive unique mapping and local overlap-layout-consensus approach.

PubMed

Wang, Anqi; Wang, Zhanyu; Li, Zheng; Li, Lei M

2018-06-15

It is highly desirable to assemble genomes of high continuity and consistency at low cost. The current bottleneck of draft genome continuity using the second generation sequencing (SGS) reads is primarily caused by uncertainty among repetitive sequences. Even though the single-molecule real-time sequencing technology is very promising to overcome the uncertainty issue, its relatively high cost and error rate add burden on budget or computation. Many long-read assemblers take the overlap-layout-consensus (OLC) paradigm, which is less sensitive to sequencing errors, heterozygosity and variability of coverage. However, current assemblers of SGS data do not sufficiently take advantage of the OLC approach. Aiming at minimizing uncertainty, the proposed method BAUM, breaks the whole genome into regions by adaptive unique mapping; then the local OLC is used to assemble each region in parallel. BAUM can (i) perform reference-assisted assembly based on the genome of a close species (ii) or improve the results of existing assemblies that are obtained based on short or long sequencing reads. The tests on two eukaryote genomes, a wild rice Oryza longistaminata and a parrot Melopsittacus undulatus, show that BAUM achieved substantial improvement on genome size and continuity. Besides, BAUM reconstructed a considerable amount of repetitive regions that failed to be assembled by existing short read assemblers. We also propose statistical approaches to control the uncertainty in different steps of BAUM. http://www.zhanyuwang.xin/wordpress/index.php/2017/07/21/baum. Supplementary data are available at Bioinformatics online.

Trypsin-like Proteins of the Fungi as Possible Markers of Phytopathogenicity

USDA-ARS?s Scientific Manuscript database

Sequences of peptidases with conserved motifs around the active site residues that are characteristic of trypsins (similar to trypsin peptidases, STP) were obtained from publicly available fungal genomes and related databases. Among the 74 fungal genomes, 30 species of parasitic Ascomycota contained...

GFam: a platform for automatic annotation of gene families.

PubMed

Sasidharan, Rajkumar; Nepusz, Tamás; Swarbreck, David; Huala, Eva; Paccanaro, Alberto

2012-10-01

We have developed GFam, a platform for automatic annotation of gene/protein families. GFam provides a framework for genome initiatives and model organism resources to build domain-based families, derive meaningful functional labels and offers a seamless approach to propagate functional annotation across periodic genome updates. GFam is a hybrid approach that uses a greedy algorithm to chain component domains from InterPro annotation provided by its 12 member resources followed by a sequence-based connected component analysis of un-annotated sequence regions to derive consensus domain architecture for each sequence and subsequently generate families based on common architectures. Our integrated approach increases sequence coverage by 7.2 percentage points and residue coverage by 14.6 percentage points higher than the coverage relative to the best single-constituent database within InterPro for the proteome of Arabidopsis. The true power of GFam lies in maximizing annotation provided by the different InterPro data sources that offer resource-specific coverage for different regions of a sequence. GFam's capability to capture higher sequence and residue coverage can be useful for genome annotation, comparative genomics and functional studies. GFam is a general-purpose software and can be used for any collection of protein sequences. The software is open source and can be obtained from http://www.paccanarolab.org/software/gfam/.

A Pan-HIV Strategy for Complete Genome Sequencing

PubMed Central

Yamaguchi, Julie; Alessandri-Gradt, Elodie; Tell, Robert W.; Brennan, Catherine A.

2015-01-01

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5′ end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance. PMID:26699702

Complete mitochondrial genome sequence of the common bean anthracnose pathogen Colletotrichum lindemuthianum.

PubMed

Gutiérrez, Pablo; Alzate, Juan; Yepes, Mauricio Salazar; Marín, Mauricio

2016-01-01

Colletotrichum lindemuthianum is the causal agent of anthracnose in common bean (Phaseolus vulgaris), one of the most limiting factors for this crop in South and Central America. In this work, the mitochondrial sequence of a Colombian isolate of C. lindemuthianum obtained from a common bean plant (var. Cargamanto) with anthracnose symptoms is presented. The mtDNA codes for 13 proteins of the respiratory chain, 1 ribosomal protein, 2 homing endonucleases, 2 ribosomal RNAs and 28 tRNAs. This is the first report of a complete mtDNA genome sequence from C. lindemuthianum.

Applying Agrep to r-NSA to solve multiple sequences approximate matching.

PubMed

Ni, Bing; Wong, Man-Hon; Lam, Chi-Fai David; Leung, Kwong-Sak

2014-01-01

This paper addresses the approximate matching problem in a database consisting of multiple DNA sequences, where the proposed approach applies Agrep to a new truncated suffix array, r-NSA. The construction time of the structure is linear to the database size, and the computations of indexing a substring in the structure are constant. The number of characters processed in applying Agrep is analysed theoretically, and the theoretical upper-bound can approximate closely the empirical number of characters, which is obtained through enumerating the characters in the actual structure built. Experiments are carried out using (synthetic) random DNA sequences, as well as (real) genome sequences including Hepatitis-B Virus and X-chromosome. Experimental results show that, compared to the straight-forward approach that applies Agrep to multiple sequences individually, the proposed approach solves the matching problem in much shorter time. The speed-up of our approach depends on the sequence patterns, and for highly similar homologous genome sequences, which are the common cases in real-life genomes, it can be up to several orders of magnitude.

A Partial Least Squares Based Procedure for Upstream Sequence Classification in Prokaryotes.

PubMed

Mehmood, Tahir; Bohlin, Jon; Snipen, Lars

2015-01-01

The upstream region of coding genes is important for several reasons, for instance locating transcription factor, binding sites, and start site initiation in genomic DNA. Motivated by a recently conducted study, where multivariate approach was successfully applied to coding sequence modeling, we have introduced a partial least squares (PLS) based procedure for the classification of true upstream prokaryotic sequence from background upstream sequence. The upstream sequences of conserved coding genes over genomes were considered in analysis, where conserved coding genes were found by using pan-genomics concept for each considered prokaryotic species. PLS uses position specific scoring matrix (PSSM) to study the characteristics of upstream region. Results obtained by PLS based method were compared with Gini importance of random forest (RF) and support vector machine (SVM), which is much used method for sequence classification. The upstream sequence classification performance was evaluated by using cross validation, and suggested approach identifies prokaryotic upstream region significantly better to RF (p-value < 0.01) and SVM (p-value < 0.01). Further, the proposed method also produced results that concurred with known biological characteristics of the upstream region.

Identification by whole-genome resequencing of gene defect responsible for severe hypercholesterolemia

PubMed Central

Rios, Jonathan; Stein, Evan; Shendure, Jay; Hobbs, Helen H.; Cohen, Jonathan C.

2010-01-01

Whole-genome sequencing is a potentially powerful tool for the diagnosis of genetic diseases. Here, we used sequencing-by-ligation to sequence the genome of an 11-month-old breast-fed girl with xanthomas and very high plasma cholesterol levels (1023 mg/dl). Her parents had normal plasma cholesterol levels and reported no family history of hypercholesterolemia, suggesting either an autosomal recessive disorder or a de novo mutation. Known genetic causes of severe hypercholesterolemia were ruled out by sequencing the responsible genes (LDLRAP, LDLR, PCSK9, APOE and APOB), and sitosterolemia was ruled out by documenting a normal plasma sitosterol:cholesterol ratio. Sequencing revealed 3 797 207 deviations from the reference sequence, of which 9726 were nonsynonymous single-nucleotide substitutions. A total of 9027 of the nonsynonymous substitutions were present in dbSNP or in 21 additional individuals from whom complete exonic sequences were available. The 699 novel nonsynonymous substitutions were distributed among 604 genes, 23 of which were single-copy genes that each contained 2 nonsynonymous substitutions consistent with an autosomal recessive model. One gene, ABCG5, had two nonsense mutations (Q16X and R446X). This finding indicated that the infant has sitosterolemia. Thus, whole-genome sequencing led to the diagnosis of a known disease with an atypical presentation. Diagnosis was confirmed by the finding of severe sitosterolemia in a blood sample obtained after the infant had been weaned. These findings demonstrate that whole-genome (or exome) sequencing can be a valuable aid to diagnose genetic diseases, even in individual patients. PMID:20719861

Genome sequencing and comparative genomics reveal a repertoire of putative pathogenicity genes in chilli anthracnose fungus Colletotrichum truncatum.

PubMed

Rao, Soumya; Nandineni, Madhusudan R

2017-01-01

Colletotrichum truncatum, a major fungal phytopathogen, causes the anthracnose disease on an economically important spice crop chilli (Capsicum annuum), resulting in huge economic losses in tropical and sub-tropical countries. It follows a subcuticular intramural infection strategy on chilli with a short, asymptomatic, endophytic phase, which contrasts with the intracellular hemibiotrophic lifestyle adopted by most of the Colletotrichum species. However, little is known about the molecular determinants and the mechanism of pathogenicity in this fungus. A high quality whole genome sequence and gene annotation based on transcriptome data of an Indian isolate of C. truncatum from chilli has been obtained. Analysis of the genome sequence revealed a rich repertoire of pathogenicity genes in C. truncatum encoding secreted proteins, effectors, plant cell wall degrading enzymes, secondary metabolism associated proteins, with potential roles in the host-specific infection strategy, placing it next only to the Fusarium species. The size of genome assembly, number of predicted genes and some of the functional categories were similar to other sequenced Colletotrichum species. The comparative genomic analyses with other species and related fungi identified some unique genes and certain highly expanded gene families of CAZymes, proteases and secondary metabolism associated genes in the genome of C. truncatum. The draft genome assembly and functional annotation of potential pathogenicity genes of C. truncatum provide an important genomic resource for understanding the biology and lifestyle of this important phytopathogen and will pave the way for designing efficient disease control regimens.

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

PubMed

de Vries, Ronald P; Riley, Robert; Wiebenga, Ad; Aguilar-Osorio, Guillermo; Amillis, Sotiris; Uchima, Cristiane Akemi; Anderluh, Gregor; Asadollahi, Mojtaba; Askin, Marion; Barry, Kerrie; Battaglia, Evy; Bayram, Özgür; Benocci, Tiziano; Braus-Stromeyer, Susanna A; Caldana, Camila; Cánovas, David; Cerqueira, Gustavo C; Chen, Fusheng; Chen, Wanping; Choi, Cindy; Clum, Alicia; Dos Santos, Renato Augusto Corrêa; Damásio, André Ricardo de Lima; Diallinas, George; Emri, Tamás; Fekete, Erzsébet; Flipphi, Michel; Freyberg, Susanne; Gallo, Antonia; Gournas, Christos; Habgood, Rob; Hainaut, Matthieu; Harispe, María Laura; Henrissat, Bernard; Hildén, Kristiina S; Hope, Ryan; Hossain, Abeer; Karabika, Eugenia; Karaffa, Levente; Karányi, Zsolt; Kraševec, Nada; Kuo, Alan; Kusch, Harald; LaButti, Kurt; Lagendijk, Ellen L; Lapidus, Alla; Levasseur, Anthony; Lindquist, Erika; Lipzen, Anna; Logrieco, Antonio F; MacCabe, Andrew; Mäkelä, Miia R; Malavazi, Iran; Melin, Petter; Meyer, Vera; Mielnichuk, Natalia; Miskei, Márton; Molnár, Ákos P; Mulé, Giuseppina; Ngan, Chew Yee; Orejas, Margarita; Orosz, Erzsébet; Ouedraogo, Jean Paul; Overkamp, Karin M; Park, Hee-Soo; Perrone, Giancarlo; Piumi, Francois; Punt, Peter J; Ram, Arthur F J; Ramón, Ana; Rauscher, Stefan; Record, Eric; Riaño-Pachón, Diego Mauricio; Robert, Vincent; Röhrig, Julian; Ruller, Roberto; Salamov, Asaf; Salih, Nadhira S; Samson, Rob A; Sándor, Erzsébet; Sanguinetti, Manuel; Schütze, Tabea; Sepčić, Kristina; Shelest, Ekaterina; Sherlock, Gavin; Sophianopoulou, Vicky; Squina, Fabio M; Sun, Hui; Susca, Antonia; Todd, Richard B; Tsang, Adrian; Unkles, Shiela E; van de Wiele, Nathalie; van Rossen-Uffink, Diana; Oliveira, Juliana Velasco de Castro; Vesth, Tammi C; Visser, Jaap; Yu, Jae-Hyuk; Zhou, Miaomiao; Andersen, Mikael R; Archer, David B; Baker, Scott E; Benoit, Isabelle; Brakhage, Axel A; Braus, Gerhard H; Fischer, Reinhard; Frisvad, Jens C; Goldman, Gustavo H; Houbraken, Jos; Oakley, Berl; Pócsi, István; Scazzocchio, Claudio; Seiboth, Bernhard; vanKuyk, Patricia A; Wortman, Jennifer; Dyer, Paul S; Grigoriev, Igor V

2017-02-14

The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Genome sequencing and comparative genomics reveal a repertoire of putative pathogenicity genes in chilli anthracnose fungus Colletotrichum truncatum

PubMed Central

Rao, Soumya

2017-01-01

Colletotrichum truncatum, a major fungal phytopathogen, causes the anthracnose disease on an economically important spice crop chilli (Capsicum annuum), resulting in huge economic losses in tropical and sub-tropical countries. It follows a subcuticular intramural infection strategy on chilli with a short, asymptomatic, endophytic phase, which contrasts with the intracellular hemibiotrophic lifestyle adopted by most of the Colletotrichum species. However, little is known about the molecular determinants and the mechanism of pathogenicity in this fungus. A high quality whole genome sequence and gene annotation based on transcriptome data of an Indian isolate of C. truncatum from chilli has been obtained. Analysis of the genome sequence revealed a rich repertoire of pathogenicity genes in C. truncatum encoding secreted proteins, effectors, plant cell wall degrading enzymes, secondary metabolism associated proteins, with potential roles in the host-specific infection strategy, placing it next only to the Fusarium species. The size of genome assembly, number of predicted genes and some of the functional categories were similar to other sequenced Colletotrichum species. The comparative genomic analyses with other species and related fungi identified some unique genes and certain highly expanded gene families of CAZymes, proteases and secondary metabolism associated genes in the genome of C. truncatum. The draft genome assembly and functional annotation of potential pathogenicity genes of C. truncatum provide an important genomic resource for understanding the biology and lifestyle of this important phytopathogen and will pave the way for designing efficient disease control regimens. PMID:28846714

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis.

PubMed

Yao, Xiaohong; Tang, Ping; Li, Zuozhou; Li, Dawei; Liu, Yifei; Huang, Hongwen

2015-01-01

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5' portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.

Multiplex PCR-Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats.

PubMed

Kim, Won-Keun; No, Jin Sun; Lee, Seung-Ho; Song, Dong Hyun; Lee, Daesang; Kim, Jeong-Ah; Gu, Se Hun; Park, Sunhye; Jeong, Seong Tae; Kim, Heung-Chul; Klein, Terry A; Wiley, Michael R; Palacios, Gustavo; Song, Jin-Won

2018-02-01

Seoul virus (SEOV) poses a worldwide public health threat. This virus, which is harbored by Rattus norvegicus and R. rattus rats, is the causative agent of hemorrhagic fever with renal syndrome (HFRS) in humans, which has been reported in Asia, Europe, the Americas, and Africa. Defining SEOV genome sequences plays a critical role in development of preventive and therapeutic strategies against the unique worldwide hantavirus. We applied multiplex PCR-based next-generation sequencing to obtain SEOV genome sequences from clinical and reservoir host specimens. Epidemiologic surveillance of R. norvegicus rats in South Korea during 2000-2016 demonstrated that the serologic prevalence of enzootic SEOV infections was not significant on the basis of sex, weight (age), and season. Viral loads of SEOV in rats showed wide dissemination in tissues and dynamic circulation among populations. Phylogenetic analyses showed the global diversity of SEOV and possible genomic configuration of genetic exchanges.

Genomic Characterization of a Novel Phage Found in Black Abalone (Haliotis cracherodii) Infected with Withering Syndrome

NASA Astrophysics Data System (ADS)

Closek, C. J.; Langevin, S.; Burge, C. A.; Crosson, L.; White, S.; Friedman, C. S.

2016-02-01

Withering syndrome (WS), caused by the bacterium Candidatus Xenohaliotis californiensis, a Rickettsia-like organism (RLO), infects many species of abalone. Black abalone (Haliotis cracherodii), one of two endangered species of abalone, has experienced high population losses along the California coast due to WS. Recently, we observed reduced pathogenicity and mortality events in RLO-infected abalone when a novel bacteriophage (phage) was also present. To better understand phage-bacterium dynamics and develop more informative diagnostic tools, we sequenced the genome of the novel phage associated with the RLO responsible for WS. Metagenomic sequencing libraries were prepared with extracted genomic DNA from two experimentally infected H. cracherodii and phage sequences were enriched using hydroxyapatite chromatography normalization. Normalized libraries were individually barcoded and sequenced with Illumina MiSeq. Raw sequence reads were processed using VIrominer and de novo assembly produced one single phage-like contig (35.7Kb) from the experimentally infected abalone. This highly divergent genome had closest homology with a virus associated with abalone shriveling syndrome (SS). Of the 34 predicted ORFs, overlapping homology with the SS virus ranged from 20-72%, demonstrating the phage sequenced is genetically distinct from any known phage. The phage-like sequences represented a significant portion of the total reads sequenced ( 2 million of the 12 million paired-end reads; 17%) and we obtained 94,000X coverage across the novel phage genome. Beyond characterization of this novel phage, which appears to reduce pathogenicity of the RLO, the genome enabled us to develop quantitative PCR and in situ hybridization assays as diagnostic tools. These tools allow us to detect and quantify this phage in the endangered H. cracherodii.

First de novo whole genome sequencing and assembly of the pink-footed goose.

PubMed

Pujolar, J M; Dalén, L; Olsen, R A; Hansen, M M; Madsen, J

2018-03-01

Annotated genomes can provide new perspectives on the biology of species. We present the first de novo whole genome sequencing for the pink-footed goose. In order to obtain a high-quality de novo assembly the strategy used was to combine one short insert paired-end library with two mate-pair libraries. The pink-footed goose genome was assembled de novo using three different assemblers and an assembly evaluation was subsequently performed in order to choose the best assembler. For our data, ALLPATHS-LG performed the best, since the assembly produced covers most of the genome, while introducing the fewest errors. A total of 26,134 genes were annotated, with bird species accounting for virtually all BLAST hits. We also estimated the substitution rate in the pink-footed goose, which can be of use in future demographic studies, by using a comparative approach with the genome of the chicken, the mallard and the swan goose. A substitution rate of 1.38×10 -7 per nucleotide per generation was obtained when comparing the genomes of the two closely-related goose species (the pink-footed and the swan goose). Altogether, we provide a valuable tool for future genomic studies aiming at particular genes and regions of the pink-footed goose genome as well as other bird species. Copyright © 2017 Elsevier Inc. All rights reserved.

Complete Chloroplast Genome Sequence of Tartary Buckwheat (Fagopyrum tataricum) and Comparative Analysis with Common Buckwheat (F. esculentum)

PubMed Central

Cho, Kwang-Soo; Yun, Bong-Kyoung; Yoon, Young-Ho; Hong, Su-Young; Mekapogu, Manjulatha; Kim, Kyung-Hee; Yang, Tae-Jin

2015-01-01

We report the chloroplast (cp) genome sequence of tartary buckwheat (Fagopyrum tataricum) obtained by next-generation sequencing technology and compared this with the previously reported common buckwheat (F. esculentum ssp. ancestrale) cp genome. The cp genome of F. tataricum has a total sequence length of 159,272 bp, which is 327 bp shorter than the common buckwheat cp genome. The cp gene content, order, and orientation are similar to those of common buckwheat, but with some structural variation at tandem and palindromic repeat frequencies and junction areas. A total of seven InDels (around 100 bp) were found within the intergenic sequences and the ycf1 gene. Copy number variation of the 21-bp tandem repeat varied in F. tataricum (four repeats) and F. esculentum (one repeat), and the InDel of the ycf1 gene was 63 bp long. Nucleotide and amino acid have highly conserved coding sequence with about 98% homology and four genes—rpoC2, ycf3, accD, and clpP—have high synonymous (Ks) value. PCR based InDel markers were applied to diverse genetic resources of F. tataricum and F. esculentum, and the amplicon size was identical to that expected in silico. Therefore, these InDel markers are informative biomarkers to practically distinguish raw or processed buckwheat products derived from F. tataricum and F. esculentum. PMID:25966355

Privacy Challenges of Genomic Big Data.

PubMed

Shen, Hong; Ma, Jian

2017-01-01

With the rapid advancement of high-throughput DNA sequencing technologies, genomics has become a big data discipline where large-scale genetic information of human individuals can be obtained efficiently with low cost. However, such massive amount of personal genomic data creates tremendous challenge for privacy, especially given the emergence of direct-to-consumer (DTC) industry that provides genetic testing services. Here we review the recent development in genomic big data and its implications on privacy. We also discuss the current dilemmas and future challenges of genomic privacy.

Construction of a plant-transformation-competent BIBAC library and genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.)

PubMed Central

2013-01-01

Background Cotton, one of the world’s leading crops, is important to the world’s textile and energy industries, and is a model species for studies of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. Here, we report the construction of a plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library and comparative genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.) with one of its diploid putative progenitor species, G. raimondii Ulbr. Results We constructed the cotton BIBAC library in a vector competent for high-molecular-weight DNA transformation in different plant species through either Agrobacterium or particle bombardment. The library contains 76,800 clones with an average insert size of 135 kb, providing an approximate 99% probability of obtaining at least one positive clone from the library using a single-copy probe. The quality and utility of the library were verified by identifying BIBACs containing genes important for fiber development, fiber cellulose biosynthesis, seed fatty acid metabolism, cotton-nematode interaction, and bacterial blight resistance. In order to gain an insight into the Upland cotton genome and its relationship with G. raimondii, we sequenced nearly 10,000 BIBAC ends (BESs) randomly selected from the library, generating approximately one BES for every 250 kb along the Upland cotton genome. The retroelement Gypsy/DIRS1 family predominates in the Upland cotton genome, accounting for over 77% of all transposable elements. From the BESs, we identified 1,269 simple sequence repeats (SSRs), of which 1,006 were new, thus providing additional markers for cotton genome research. Surprisingly, comparative sequence analysis showed that Upland cotton is much more diverged from G. raimondii at the genomic sequence level than expected. There seems to be no significant difference between the relationships of the Upland cotton D- and A-subgenomes with the G. raimondii genome, even though G. raimondii contains a D genome (D5). Conclusions The library represents the first BIBAC library in cotton and related species, thus providing tools useful for integrative physical mapping, large-scale genome sequencing and large-scale functional analysis of the Upland cotton genome. Comparative sequence analysis provides insights into the Upland cotton genome, and a possible mechanism underlying the divergence and evolution of polyploid Upland cotton from its diploid putative progenitor species, G. raimondii. PMID:23537070

Complete mitochondrial genome of a wild Siberian tiger.

PubMed

Sun, Yujiao; Lu, Taofeng; Sun, Zhaohui; Guan, Weijun; Liu, Zhensheng; Teng, Liwei; Wang, Shuo; Ma, Yuehui

2015-01-01

In this study, the complete mitochondrial genome of Siberian tiger (Panthera tigris altaica) was sequenced, using muscle tissue obtained from a male wild tiger. The total length of the mitochondrial genome is 16,996 bp. The genome structure of this tiger is in accordance with other Siberian tigers and it contains 12S rRNA gene, 16S rRNA gene, 22 tRNA genes, 13 protein-coding genes, and 1 control region.

In silico comparison of genomic regions containing genes coding for enzymes and transcription factors for the phenylpropanoid pathway in Phaseolus vulgaris L. and Glycine max L. Merr

PubMed Central

Reinprecht, Yarmilla; Yadegari, Zeinab; Perry, Gregory E.; Siddiqua, Mahbuba; Wright, Lori C.; McClean, Phillip E.; Pauls, K. Peter

2013-01-01

Legumes contain a variety of phytochemicals derived from the phenylpropanoid pathway that have important effects on human health as well as seed coat color, plant disease resistance and nodulation. However, the information about the genes involved in this important pathway is fragmentary in common bean (Phaseolus vulgaris L.). The objectives of this research were to isolate genes that function in and control the phenylpropanoid pathway in common bean, determine their genomic locations in silico in common bean and soybean, and analyze sequences of the 4CL gene family in two common bean genotypes. Sequences of phenylpropanoid pathway genes available for common bean or other plant species were aligned, and the conserved regions were used to design sequence-specific primers. The PCR products were cloned and sequenced and the gene sequences along with common bean gene-based (g) markers were BLASTed against the Glycine max v.1.0 genome and the P. vulgaris v.1.0 (Andean) early release genome. In addition, gene sequences were BLASTed against the OAC Rex (Mesoamerican) genome sequence assembly. In total, fragments of 46 structural and regulatory phenylpropanoid pathway genes were characterized in this way and placed in silico on common bean and soybean sequence maps. The maps contain over 250 common bean g and SSR (simple sequence repeat) markers and identify the positions of more than 60 additional phenylpropanoid pathway gene sequences, plus the putative locations of seed coat color genes. The majority of cloned phenylpropanoid pathway gene sequences were mapped to one location in the common bean genome but had two positions in soybean. The comparison of the genomic maps confirmed previous studies, which show that common bean and soybean share genomic regions, including those containing phenylpropanoid pathway gene sequences, with conserved synteny. Indels identified in the comparison of Andean and Mesoamerican common bean 4CL gene sequences might be used to develop inter-pool phenylpropanoid pathway gene-based markers. We anticipate that the information obtained by this study will simplify and accelerate selections of common bean with specific phenylpropanoid pathway alleles to increase the contents of beneficial phenylpropanoids in common bean and other legumes. PMID:24046770

Ultrafast Comparison of Personal Genomes via Precomputed Genome Fingerprints

PubMed Central

Glusman, Gustavo; Mauldin, Denise E.; Hood, Leroy E.; Robinson, Max

2017-01-01

We present an ultrafast method for comparing personal genomes. We transform the standard genome representation (lists of variants relative to a reference) into “genome fingerprints” via locality sensitive hashing. The resulting genome fingerprints can be meaningfully compared even when the input data were obtained using different sequencing technologies, processed using different pipelines, represented in different data formats and relative to different reference versions. Furthermore, genome fingerprints are robust to up to 30% missing data. Because of their reduced size, computation on the genome fingerprints is fast and requires little memory. For example, we could compute all-against-all pairwise comparisons among the 2504 genomes in the 1000 Genomes data set in 67 s at high quality (21 μs per comparison, on a single processor), and achieved a lower quality approximation in just 11 s. Efficient computation enables scaling up a variety of important genome analyses, including quantifying relatedness, recognizing duplicative sequenced genomes in a set, population reconstruction, and many others. The original genome representation cannot be reconstructed from its fingerprint, effectively decoupling genome comparison from genome interpretation; the method thus has significant implications for privacy-preserving genome analytics. PMID:29018478

Sensitive Next-Generation Sequencing Method Reveals Deep Genetic Diversity of HIV-1 in the Democratic Republic of the Congo.

PubMed

Rodgers, Mary A; Wilkinson, Eduan; Vallari, Ana; McArthur, Carole; Sthreshley, Larry; Brennan, Catherine A; Cloherty, Gavin; de Oliveira, Tulio

2017-03-15

As the epidemiological epicenter of the human immunodeficiency virus (HIV) pandemic, the Democratic Republic of the Congo (DRC) is a reservoir of circulating HIV strains exhibiting high levels of diversity and recombination. In this study, we characterized HIV specimens collected in two rural areas of the DRC between 2001 and 2003 to identify rare strains of HIV. The env gp41 region was sequenced and characterized for 172 HIV-positive specimens. The env sequences were predominantly subtype A (43.02%), but 7 other subtypes (33.14%), 20 circulating recombinant forms (CRFs; 11.63%), and 20 unclassified (11.63%) sequences were also found. Of the rare and unclassified subtypes, 18 specimens were selected for next-generation sequencing (NGS) by a modified HIV-switching mechanism at the 5' end of the RNA template (SMART) method to obtain full-genome sequences. NGS produced 14 new complete genomes, which included pure subtype C ( n = 2), D ( n = 1), F1 ( n = 1), H ( n = 3), and J ( n = 1) genomes. The two subtype C genomes and one of the subtype H genomes branched basal to their respective subtype branches but had no evidence of recombination. The remaining 6 genomes were complex recombinants of 2 or more subtypes, including subtypes A1, F, G, H, J, and K and unclassified fragments, including one subtype CRF25 isolate, which branched basal to all CRF25 references. Notably, all recombinant subtype H fragments branched basal to the H clade. Spatial-geographical analysis indicated that the diverse sequences identified here did not expand globally. The full-genome and subgenomic sequences identified in our study population significantly increase the documented diversity of the strains involved in the continually evolving HIV-1 pandemic. IMPORTANCE Very little is known about the ancestral HIV-1 strains that founded the global pandemic, and very few complete genome sequences are available from patients in the Congo Basin, where HIV-1 expanded early in the global pandemic. By sequencing a subgenomic fragment of the HIV-1 envelope from study participants in the DRC, we identified rare variants for complete genome sequencing. The basal branching of some of the complete genome sequences that we recovered suggests that these strains are more closely related to ancestral HIV-1 strains than to previously reported strains and is evidence that the local diversification of HIV in the DRC continues to outpace the diversity of global strains decades after the emergence of the pandemic. Copyright © 2017 Rodgers et al.

The Mitochondrial Genomes of the Zoonotic Canine Filarial Parasites Dirofilaria (Nochtiella) repens and Candidatus Dirofilaria (Nochtiella) Honkongensis Provide Evidence for Presence of Cryptic Species

PubMed Central

Yilmaz, Esra; Fritzenwanker, Moritz; Pantchev, Nikola; Lendner, Mathias; Wongkamchai, Sirichit; Otranto, Domenico; Kroidl, Inge; Dennebaum, Martin; Le, Thanh Hoa; Anh Le, Tran; Ramünke, Sabrina; Schaper, Roland; von Samson-Himmelstjerna, Georg; Poppert, Sven; Krücken, Jürgen

2016-01-01

Background Cutaneous dirofilariosis is a canine mosquito-borne zoonosis that can cause larva migrans disease in humans. Dirofilaria repens is considered an emerging pathogen occurring with high prevalence in Mediterranean areas and many parts of tropical Asia. In Hong Kong, a second species, Candidatus Dirofilaria hongkongensis, has been reported. The present study aimed to compare mitochondrial genomes from these parasites and to obtain population genetic information. Methods and Findings Complete mitochondrial genomes were obtained by PCR and Sanger sequencing or ILLUMINA sequencing for four worms. Cytochrome oxidase subunit 1 sequences identified three as D. repens (all from Europe) and one as C. D. hongkongensis (from India). Mitochondrial genomes have the same organization as in other spirurid nematodes but a higher preference for thymine in the coding strand. Phylogenetic analysis was in contradiction to current taxonomy of the Onchocercidae but in agreement with a recent multi-locus phylogenetic analysis using both mitochondrial and nuclear markers. D. repens and C. D. hongkongensis sequences clustered together and were the common sister group to Dirofilaria immitis. Analysis of a 2.5 kb mitochondrial genome fragment from macrofilaria or canine blood samples from Europe (42), Thailand (2), India (1) and Vietnam (1) revealed only small genetic differences in the D. repens samples including all European and the Vietnam sample. The Indian C. D. hongkongensis and the two Thai samples formed separate clusters and differences were comparatively large. Conclusion Genetic differences between Dirofilaria spp. causing cutaneous disease can be considerable whereas D. repens itself was genetically quite homogenous. C. D. hongkongensis was identified for the first time from the Indian subcontinent. The full mitochondrial genome sequence strengthens the hypothesis that it represents an independent species and the Thai samples might represent another cryptic species, Candidatus Dirofilaria sp. ‘Thailand II’, or a quite divergent population of C. D. hongkongensis. PMID:27727270

Genome-based insights into the resistome and mobilome of multidrug-resistant Aeromonas sp. ARM81 isolated from wastewater.

PubMed

Adamczuk, Marcin; Dziewit, Lukasz

2017-01-01

The draft genome of multidrug-resistant Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, β-lactams or tetracycline. Three different β-lactamase genes were identified, including an extended-spectrum β-lactamase gene bla PER-1 . The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules (i.e., genes encoding a zinc transporter ZitB and a putative glucosyltransferase), and 28 putative transposase genes were identified. The mobility of three insertion sequences (isoforms of previously identified elements ISAs12, ISKpn9 and ISAs26) was confirmed using trap plasmids.

Whole Genome Sequence Analysis of an Alachlor and Endosulfan Degrading Micrococcus sp. strain 2385 Isolated from Ochlockonee River, Florida

PubMed Central

Pathak, Ashish; Chauhan, Ashvini; Ewida, Ayman Y.I.; Stothard, Paul

2016-01-01

We recently isolated Micrococcus sp. strain 2385 from Ochlockonee River, Florida and demonstrated potent biodegradative activity against two commonly used pesticides- alachlor [(2-chloro-2`,6`-diethylphenyl-N (methoxymethyl)acetanilide)] and endosulfan [(6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9methano-2,3,4-benzo(e)di-oxathiepin-3-oxide], respectively. To further identify the repertoire of metabolic functions possessed by strain 2385, a draft genome sequence was obtained, assembled, annotated and analyzed. The genome sequence of Micrococcus sp. strain 2385 consisted of 1,460,461,440 bases which assembled into 175 contigs with an N50 contig length of 50,109 bases and a coverage of 600x. The genome size of this strain was estimated at 2,431,226 base pairs with a G+C content of 72.8 and a total number of 2,268 putative genes. RAST annotated a total of 340 subsystems in the genome of strain 2385 along with the presence of 2,177 coding sequences. A genome wide survey indicated that that strain 2385 harbors a plethora of genes to degrade other pollutants including caprolactam, PAHs (such as naphthalene), styrene, toluene and several chloroaromatic compounds. PMID:27672405

Whole Genome Sequence Analysis of an Alachlor and Endosulfan Degrading Micrococcus sp. strain 2385 Isolated from Ochlockonee River, Florida.

PubMed

Pathak, Ashish; Chauhan, Ashvini; Ewida, Ayman Y I; Stothard, Paul

2016-01-01

We recently isolated Micrococcus sp. strain 2385 from Ochlockonee River, Florida and demonstrated potent biodegradative activity against two commonly used pesticides- alachlor [(2-chloro-2`,6`-diethylphenyl-N (methoxymethyl)acetanilide)] and endosulfan [(6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9methano-2,3,4-benzo(e)di-oxathiepin-3-oxide], respectively. To further identify the repertoire of metabolic functions possessed by strain 2385, a draft genome sequence was obtained, assembled, annotated and analyzed. The genome sequence of Micrococcus sp. strain 2385 consisted of 1,460,461,440 bases which assembled into 175 contigs with an N50 contig length of 50,109 bases and a coverage of 600x. The genome size of this strain was estimated at 2,431,226 base pairs with a G+C content of 72.8 and a total number of 2,268 putative genes. RAST annotated a total of 340 subsystems in the genome of strain 2385 along with the presence of 2,177 coding sequences. A genome wide survey indicated that that strain 2385 harbors a plethora of genes to degrade other pollutants including caprolactam, PAHs (such as naphthalene), styrene, toluene and several chloroaromatic compounds.

GenomeRNAi: a database for cell-based RNAi phenotypes.

PubMed

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

GenomeRNAi: a database for cell-based RNAi phenotypes

PubMed Central

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at PMID:17135194

Using RSAT oligo-analysis and dyad-analysis tools to discover regulatory signals in nucleic sequences.

PubMed

Defrance, Matthieu; Janky, Rekin's; Sand, Olivier; van Helden, Jacques

2008-01-01

This protocol explains how to discover functional signals in genomic sequences by detecting over- or under-represented oligonucleotides (words) or spaced pairs thereof (dyads) with the Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/). Two typical applications are presented: (i) predicting transcription factor-binding motifs in promoters of coregulated genes and (ii) discovering phylogenetic footprints in promoters of orthologous genes. The steps of this protocol include purging genomic sequences to discard redundant fragments, discovering over-represented patterns and assembling them to obtain degenerate motifs, scanning sequences and drawing feature maps. The main strength of the method is its statistical ground: the binomial significance provides an efficient control on the rate of false positives. In contrast with optimization-based pattern discovery algorithms, the method supports the detection of under- as well as over-represented motifs. Computation times vary from seconds (gene clusters) to minutes (whole genomes). The execution of the whole protocol should take approximately 1 h.

Component identification of electron transport chains in curdlan-producing Agrobacterium sp. ATCC 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis.

PubMed

Zhang, Hongtao; Setubal, Joao Carlos; Zhan, Xiaobei; Zheng, Zhiyong; Yu, Lijun; Wu, Jianrong; Chen, Dingqiang

2011-06-01

Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.

Complete mitochondrial genome of the monogonont rotifer, Brachionus koreanus (Rotifera, Brachionidae).

PubMed

Hwang, Dae-Sik; Suga, Koushirou; Sakakura, Yoshitaka; Park, Heum Gi; Hagiwara, Atsushi; Rhee, Jae-Sung; Lee, Jae-Seong

2014-02-01

The complete mitochondrial genome was obtained from the assembled genome data sequenced by next generation sequencing (NGS) technology from the monogonont rotifer Brachionus koreanus. The mitochondrial genome of B. koreanus was composed of two circular chromosomes designated as mtDNA-I (10,421 bp) and mtDNA-II (11,923 bp). The gene contents of B. koreanus were identical with previously reported B. plicatilis mitochondrial genomes. However, gene orders of B. koreanus showed one rearrangement between the two species. Of 12 protein-coding genes (PCGs), 3 genes (ATP6, ND1, and ND3) had an incomplete stop codon. The A + T base composition of B. koreanus mitochondrial genome was high (68.81%). They also showed anti-G bias (12.03% and 10.97%) on the second and third position of PCGs as well as slight anti-C bias (15.96% and 14.31%) on the first and third position of PCGs.

Estimating the Population Mutation Rate from a de novo Assembled Bactrian Camel Genome and Cross-Species Comparison with Dromedary ESTs

PubMed Central

2014-01-01

The Bactrian camel (Camelus bactrianus) and the dromedary (Camelus dromedarius) are among the last species that have been domesticated around 3000–6000 years ago. During domestication, strong artificial (anthropogenic) selection has shaped the livestock, creating a huge amount of phenotypes and breeds. Hence, domestic animals represent a unique resource to understand the genetic basis of phenotypic variation and adaptation. Similar to its late domestication history, the Bactrian camel is also among the last livestock animals to have its genome sequenced and deciphered. As no genomic data have been available until recently, we generated a de novo assembly by shotgun sequencing of a single male Bactrian camel. We obtained 1.6 Gb genomic sequences, which correspond to more than half of the Bactrian camel’s genome. The aim of this study was to identify heterozygous single-nucleotide polymorphisms (SNPs) and to estimate population parameters and nucleotide diversity based on an individual camel. With an average 6.6-fold coverage, we detected over 116 000 heterozygous SNPs and recorded a genome-wide nucleotide diversity similar to that of other domesticated ungulates. More than 20 000 (85%) dromedary expressed sequence tags successfully aligned to our genomic draft. Our results provide a template for future association studies targeting economically relevant traits and to identify changes underlying the process of camel domestication and environmental adaptation. PMID:23454912

Genome sequence of the Japanese oak silk moth, Antheraea yamamai: the first draft genome in the family Saturniidae

PubMed Central

Kim, Seong-Ryul; Kwak, Woori; Kim, Hyaekang; Kim, Kee-Young; Kim, Su-Bae; Choi, Kwang-Ho; Kim, Seong-Wan; Hwang, Jae-Sam; Kim, Minjee; Kim, Iksoo; Goo, Tae-Won

2018-01-01

Abstract Background Antheraea yamamai, also known as the Japanese oak silk moth, is a wild species of silk moth. Silk produced by A. yamamai, referred to as tensan silk, shows different characteristics such as thickness, compressive elasticity, and chemical resistance compared with common silk produced from the domesticated silkworm, Bombyx mori. Its unique characteristics have led to its use in many research fields including biotechnology and medical science, and the scientific as well as economic importance of the wild silk moth continues to gradually increase. However, no genomic information for the wild silk moth, including A. yamamai, is currently available. Findings In order to construct the A. yamamai genome, a total of 147G base pairs using Illumina and Pacbio sequencing platforms were generated, providing 210-fold coverage based on the 700-Mb estimated genome size of A. yamamai. The assembled genome of A. yamamai was 656 Mb (>2 kb) with 3675 scaffolds, and the N50 length of assembly was 739 Kb with a 34.07% GC ratio. Identified repeat elements covered 37.33% of the total genome, and the completeness of the constructed genome assembly was estimated to be 96.7% by Benchmarking Universal Single-Copy Orthologs v2 analysis. A total of 15 481 genes were identified using Evidence Modeler based on the gene prediction results obtained from 3 different methods (ab initio, RNA-seq-based, known-gene-based) and manual curation. Conclusions Here we present the genome sequence of A. yamamai, the first genome sequence of the wild silk moth. These results provide valuable genomic information, which will help enrich our understanding of the molecular mechanisms relating to not only specific phenotypes such as wild silk itself but also the genomic evolution of Saturniidae. PMID:29186418

Complete Genome Sequences of 12 Isolates of Listeria monocytogenes Belonging to Serotypes 1/2a, 1/2b, and 4b Obtained from Food Products and Food-Processing Environments in Canada.

PubMed

Mottawea, Walid; Chen, Shu; Saleh-Lakha, Saleema; Belanger, Sebastien; Ogunremi, Dele

2017-05-11

Listeria monocytogenes is the etiological agent for an often fatal foodborne illness known as listeriosis. Here, we present the complete genome sequences of 12 L. monocytogenes isolates representing the three most common serotypes of this pathogen (1/2a, 1/2b, and 4b), collected in Canada from different food products and environmental sources. © Crown copyright 2017.

Draft Genome Sequence of Enterococcus casseliflavus PAVET15 Obtained from the Oviduct Infection of the Cattle Tick (Rhipicephalus microplus) in Jiutepec, Morelos, Mexico

PubMed Central

Cossío-Bayúgar, R.; Miranda-Miranda, E.; Arreguín-Pérez, C. A.; Lozano, L.; Peréz de la Rosa, D.; Rocha-Martínez, M. K.; Bravo-Díaz, M. A.

2017-01-01

ABSTRACT Enterococcus spp. are Gram-positive lactic acid-producing bacteria found in the intestinal tracts of animals, like mammals, birds, and arthropods. Enterococcus spp. may cause oportunistic infections in vertebrate and invertebrate hosts. We report here the draft genome sequence of Enterococcus casseliflavus PAVET15 containing 3,722,480 bp, with 80 contigs, an N50 of 179,476 bp, and 41.93% G+C content. PMID:28428300

Sequence-Based Genotyping for Marker Discovery and Co-Dominant Scoring in Germplasm and Populations

PubMed Central

Truong, Hoa T.; Ramos, A. Marcos; Yalcin, Feyruz; de Ruiter, Marjo; van der Poel, Hein J. A.; Huvenaars, Koen H. J.; Hogers, René C. J.; van Enckevort, Leonora. J. G.; Janssen, Antoine; van Orsouw, Nathalie J.; van Eijk, Michiel J. T.

2012-01-01

Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n = 222 samples) and lettuce (n = 87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike. PMID:22662172

Application of Genomic Technologies to the Breeding of Trees

PubMed Central

Badenes, Maria L.; Fernández i Martí, Angel; Ríos, Gabino; Rubio-Cabetas, María J.

2016-01-01

The recent introduction of next generation sequencing (NGS) technologies represents a major revolution in providing new tools for identifying the genes and/or genomic intervals controlling important traits for selection in breeding programs. In perennial fruit trees with long generation times and large sizes of adult plants, the impact of these techniques is even more important. High-throughput DNA sequencing technologies have provided complete annotated sequences in many important tree species. Most of the high-throughput genotyping platforms described are being used for studies of genetic diversity and population structure. Dissection of complex traits became possible through the availability of genome sequences along with phenotypic variation data, which allow to elucidate the causative genetic differences that give rise to observed phenotypic variation. Association mapping facilitates the association between genetic markers and phenotype in unstructured and complex populations, identifying molecular markers for assisted selection and breeding. Also, genomic data provide in silico identification and characterization of genes and gene families related to important traits, enabling new tools for molecular marker assisted selection in tree breeding. Deep sequencing of transcriptomes is also a powerful tool for the analysis of precise expression levels of each gene in a sample. It consists in quantifying short cDNA reads, obtained by NGS technologies, in order to compare the entire transcriptomes between genotypes and environmental conditions. The miRNAs are non-coding short RNAs involved in the regulation of different physiological processes, which can be identified by high-throughput sequencing of RNA libraries obtained by reverse transcription of purified short RNAs, and by in silico comparison with known miRNAs from other species. All together, NGS techniques and their applications have increased the resources for plant breeding in tree species, closing the former gap of genetic tools between trees and annual species. PMID:27895664

Amalga-like virus infecting Antonospora locustae, a microsporidian pathogen of grasshoppers, plus related viruses associated with other arthropods.

PubMed

Pyle, Jesse D; Keeling, Patrick J; Nibert, Max L

2017-04-02

A previously reported Expressed Sequence Tag (EST) library from spores of microsporidian Antonospora locustae includes a number of clones with sequence similarities to plant amalgaviruses. Reexamining the sequence accessions from that library, we found additional such clones, contributing to a 3247-nt contig that approximates the length of an amalga-like virus genome. Using A. locustae spores stored from that previous study, and new ones obtained from the same source, we newly visualized the putative dsRNA genome of this virus and obtained amplicons yielding a 3387-nt complete genome sequence. Phylogenetic analyses suggested it as prototype strain of a new genus in family Amalgaviridae. The genome contains two partially overlapping long ORFs, with downstream ORF2 in the +1 frame relative to ORF1 and a proposed motif for +1 ribosomal frameshifting in the region of overlap. Subsequent database searches using the predicted fusion protein sequence of this new amalga-like virus identified related sequences in the transcriptome of a basal hexapod, the springtail species Tetrodontophora bielanensis. We speculate that this second new amalga-like virus (contig length, 3475 nt) likely also derived from a microsporidian, or related organism, which was associated with the springtail specimens at the time of sampling for transcriptome analysis. Other findings of interest include evidence that the ORF1 translation products of these two new amalga-like viruses contain a central region of predicted α-helical coiled coil, as recently reported for plant amalgaviruses, and transcriptome-based evidence for another new amalga-like virus in the transcriptome of another basal hexapod, the two-pronged bristletail species Campodea augens. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of Genomic Technologies to the Breeding of Trees.

PubMed

Badenes, Maria L; Fernández I Martí, Angel; Ríos, Gabino; Rubio-Cabetas, María J

2016-01-01

The recent introduction of next generation sequencing (NGS) technologies represents a major revolution in providing new tools for identifying the genes and/or genomic intervals controlling important traits for selection in breeding programs. In perennial fruit trees with long generation times and large sizes of adult plants, the impact of these techniques is even more important. High-throughput DNA sequencing technologies have provided complete annotated sequences in many important tree species. Most of the high-throughput genotyping platforms described are being used for studies of genetic diversity and population structure. Dissection of complex traits became possible through the availability of genome sequences along with phenotypic variation data, which allow to elucidate the causative genetic differences that give rise to observed phenotypic variation. Association mapping facilitates the association between genetic markers and phenotype in unstructured and complex populations, identifying molecular markers for assisted selection and breeding. Also, genomic data provide in silico identification and characterization of genes and gene families related to important traits, enabling new tools for molecular marker assisted selection in tree breeding. Deep sequencing of transcriptomes is also a powerful tool for the analysis of precise expression levels of each gene in a sample. It consists in quantifying short cDNA reads, obtained by NGS technologies, in order to compare the entire transcriptomes between genotypes and environmental conditions. The miRNAs are non-coding short RNAs involved in the regulation of different physiological processes, which can be identified by high-throughput sequencing of RNA libraries obtained by reverse transcription of purified short RNAs, and by in silico comparison with known miRNAs from other species. All together, NGS techniques and their applications have increased the resources for plant breeding in tree species, closing the former gap of genetic tools between trees and annual species.

Genome sequencing identifies Listeria fleischmannii subsp. coloradonensis subsp. nov., isolated from a ranch.

PubMed

den Bakker, Henk C; Manuel, Clyde S; Fortes, Esther D; Wiedmann, Martin; Nightingale, Kendra K

2013-09-01

Twenty Listeria-like isolates were obtained from environmental samples collected on a cattle ranch in northern Colorado; all of these isolates were found to share an identical partial sigB sequence, suggesting close relatedness. The isolates were similar to members of the genus Listeria in that they were Gram-stain-positive, short rods, oxidase-negative and catalase-positive; the isolates were similar to Listeria fleischmannii because they were non-motile at 25 °C. 16S rRNA gene sequencing for representative isolates and whole genome sequencing for one isolate was performed. The genome of the type strain of Listeria fleischmannii (strain LU2006-1(T)) was also sequenced. The draft genomes were very similar in size and the average MUMmer nucleotide identity across 91% of the genomes was 95.16%. Genome sequence data were used to design primers for a six-gene multi-locus sequence analysis (MLSA) scheme. Phylogenies based on (i) the near-complete 16S rRNA gene, (ii) 31 core genes and (iii) six housekeeping genes illustrated the close relationship of these Listeria-like isolates to Listeria fleischmannii LU2006-1(T). Sufficient genetic divergence of the Listeria-like isolates from the type strain of Listeria fleischmannii and differing phenotypic characteristics warrant these isolates to be classified as members of a distinct infraspecific taxon, for which the name Listeria fleischmannii subsp. coloradonensis subsp. nov. is proposed. The type strain is TTU M1-001(T) ( =BAA-2414(T) =DSM 25391(T)). The isolates of Listeria fleischmannii subsp. coloradonensis subsp. nov. differ from the nominate subspecies by the inability to utilize melezitose, turanose and sucrose, and the ability to utilize inositol. The results also demonstrate the utility of whole genome sequencing to facilitate identification of novel taxa within a well-described genus. The genomes of both subspecies of Listeria fleischmannii contained putative enhancin genes; the Listeria fleischmannii subsp. coloradonensis subsp. nov. genome also encoded a putative mosquitocidal toxin. The presence of these genes suggests possible adaptation to an insect host, and further studies are needed to probe niche adaptation of Listeria fleischmannii.

Transcriptome Assembly, Gene Annotation and Tissue Gene Expression Atlas of the Rainbow Trout

PubMed Central

Salem, Mohamed; Paneru, Bam; Al-Tobasei, Rafet; Abdouni, Fatima; Thorgaard, Gary H.; Rexroad, Caird E.; Yao, Jianbo

2015-01-01

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complemented by transcriptome information that will enhance genome assembly and annotation. Previously, transcriptome reference sequences were reported using data from different sources. Although the previous work added a great wealth of sequences, a complete and well-annotated transcriptome is still needed. In addition, gene expression in different tissues was not completely addressed in the previous studies. In this study, non-normalized cDNA libraries were sequenced from 13 different tissues of a single doubled haploid rainbow trout from the same source used for the rainbow trout genome sequence. A total of ~1.167 billion paired-end reads were de novo assembled using the Trinity RNA-Seq assembler yielding 474,524 contigs > 500 base-pairs. Of them, 287,593 had homologies to the NCBI non-redundant protein database. The longest contig of each cluster was selected as a reference, yielding 44,990 representative contigs. A total of 4,146 contigs (9.2%), including 710 full-length sequences, did not match any mRNA sequences in the current rainbow trout genome reference. Mapping reads to the reference genome identified an additional 11,843 transcripts not annotated in the genome. A digital gene expression atlas revealed 7,678 housekeeping and 4,021 tissue-specific genes. Expression of about 16,000–32,000 genes (35–71% of the identified genes) accounted for basic and specialized functions of each tissue. White muscle and stomach had the least complex transcriptomes, with high percentages of their total mRNA contributed by a small number of genes. Brain, testis and intestine, in contrast, had complex transcriptomes, with a large numbers of genes involved in their expression patterns. This study provides comprehensive de novo transcriptome information that is suitable for functional and comparative genomics studies in rainbow trout, including annotation of the genome. PMID:25793877

Development of Novel SSR Markers for Flax (Linum usitatissimum L.) Using Reduced-Representation Genome Sequencing

PubMed Central

Wu, Jianzhong; Zhao, Qian; Wu, Guangwen; Zhang, Shuquan; Jiang, Tingbo

2017-01-01

Flax (Linum usitatissimum L.) is a major fiber and oil yielding crop grown in northeastern China. Identification of flax molecular markers is a key step toward improving flax yield and quality via marker-assisted breeding. Simple sequence repeat (SSR) markers, which are based on genomic structural variation, are considered the most valuable type of genetic marker for this purpose. In this study, we screened 1574 microsatellites from Linum usitatissimum L. obtained using reduced representation genome sequencing (RRGS) to systematically identify SSR markers. The resulting set of microsatellites consisted mainly of trinucleotide (56.10%) and dinucleotide (35.23%) repeats, with each motif consisting of 5–8 repeats. We then evaluated marker sensitivity and specificity based on samples of 48 flax isolates obtained from northeastern China. Using the new SSR panel, the results demonstrated that fiber flax and oilseed flax varieties clustered into two well separated groups. The novel SSR markers developed in this study show potential value for selection of varieties for use in flax breeding programs. PMID:28133461

Application of whole genome re-sequencing data in the development of diagnostic DNA markers tightly linked to a disease-resistance locus for marker-assisted selection in lupin (Lupinus angustifolius).

PubMed

Yang, Huaan; Jian, Jianbo; Li, Xuan; Renshaw, Daniel; Clements, Jonathan; Sweetingham, Mark W; Tan, Cong; Li, Chengdao

2015-09-02

Molecular marker-assisted breeding provides an efficient tool to develop improved crop varieties. A major challenge for the broad application of markers in marker-assisted selection is that the marker phenotypes must match plant phenotypes in a wide range of breeding germplasm. In this study, we used the legume crop species Lupinus angustifolius (lupin) to demonstrate the utility of whole genome sequencing and re-sequencing on the development of diagnostic markers for molecular plant breeding. Nine lupin cultivars released in Australia from 1973 to 2007 were subjected to whole genome re-sequencing. The re-sequencing data together with the reference genome sequence data were used in marker development, which revealed 180,596 to 795,735 SNP markers from pairwise comparisons among the cultivars. A total of 207,887 markers were anchored on the lupin genetic linkage map. Marker mining obtained an average of 387 SNP markers and 87 InDel markers for each of the 24 genome sequence assembly scaffolds bearing markers linked to 11 genes of agronomic interest. Using the R gene PhtjR conferring resistance to phomopsis stem blight disease as a test case, we discovered 17 candidate diagnostic markers by genotyping and selecting markers on a genetic linkage map. A further 243 candidate diagnostic markers were discovered by marker mining on a scaffold bearing non-diagnostic markers linked to the PhtjR gene. Nine out from the ten tested candidate diagnostic markers were confirmed as truly diagnostic on a broad range of commercial cultivars. Markers developed using these strategies meet the requirements for broad application in molecular plant breeding. We demonstrated that low-cost genome sequencing and re-sequencing data were sufficient and very effective in the development of diagnostic markers for marker-assisted selection. The strategies used in this study may be applied to any trait or plant species. Whole genome sequencing and re-sequencing provides a powerful tool to overcome current limitations in molecular plant breeding, which will enable plant breeders to precisely pyramid favourable genes to develop super crop varieties to meet future food demands.

tRNADB-CE: tRNA gene database well-timed in the era of big sequence data.

PubMed

Abe, Takashi; Inokuchi, Hachiro; Yamada, Yuko; Muto, Akira; Iwasaki, Yuki; Ikemura, Toshimichi

2014-01-01

The tRNA gene data base curated by experts "tRNADB-CE" (http://trna.ie.niigata-u.ac.jp) was constructed by analyzing 1,966 complete and 5,272 draft genomes of prokaryotes, 171 viruses', 121 chloroplasts', and 12 eukaryotes' genomes plus fragment sequences obtained by metagenome studies of environmental samples. 595,115 tRNA genes in total, and thus two times of genes compiled previously, have been registered, for which sequence, clover-leaf structure, and results of sequence-similarity and oligonucleotide-pattern searches can be browsed. To provide collective knowledge with help from experts in tRNA researches, we added a column for enregistering comments to each tRNA. By grouping bacterial tRNAs with an identical sequence, we have found high phylogenetic preservation of tRNA sequences, especially at the phylum level. Since many species-unknown tRNAs from metagenomic sequences have sequences identical to those found in species-known prokaryotes, the identical sequence group (ISG) can provide phylogenetic markers to investigate the microbial community in an environmental ecosystem. This strategy can be applied to a huge amount of short sequences obtained from next-generation sequencers, as showing that tRNADB-CE is a well-timed database in the era of big sequence data. It is also discussed that batch-learning self-organizing-map with oligonucleotide composition is useful for efficient knowledge discovery from big sequence data.

Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

PubMed Central

Be, Nicholas A.; Thissen, James B.; Gardner, Shea N.; McLoughlin, Kevin S.; Fofanov, Viacheslav Y.; Koshinsky, Heather; Ellingson, Sally R.; Brettin, Thomas S.; Jackson, Paul J.; Jaing, Crystal J.

2013-01-01

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy. PMID:24039948

PolyA_DB 3 catalogs cleavage and polyadenylation sites identified by deep sequencing in multiple genomes

PubMed Central

Wang, Ruijia; Nambiar, Ram; Zheng, Dinghai

2018-01-01

Abstract PolyA_DB is a database cataloging cleavage and polyadenylation sites (PASs) in several genomes. Previous versions were based mainly on expressed sequence tags (ESTs), which had a limited amount and could lead to inaccurate PAS identification due to the presence of internal A-rich sequences in transcripts. Here, we present an updated version of the database based solely on deep sequencing data. First, PASs are mapped by the 3′ region extraction and deep sequencing (3′READS) method, ensuring unequivocal PAS identification. Second, a large volume of data based on diverse biological samples increases PAS coverage by 3.5-fold over the EST-based version and provides PAS usage information. Third, strand-specific RNA-seq data are used to extend annotated 3′ ends of genes to obtain more thorough annotations of alternative polyadenylation (APA) sites. Fourth, conservation information of PAS across mammals sheds light on significance of APA sites. The database (URL: http://www.polya-db.org/v3) currently holds PASs in human, mouse, rat and chicken, and has links to the UCSC genome browser for further visualization and for integration with other genomic data. PMID:29069441

Insights from the genome of a high alkaline cellulase producing Aspergillus fumigatus strain obtained from Peruvian Amazon rainforest.

PubMed

Paul, Sujay; Zhang, Angel; Ludeña, Yvette; Villena, Gretty K; Yu, Fengan; Sherman, David H; Gutiérrez-Correa, Marcel

2017-06-10

Here, we report the complete genome sequence of a high alkaline cellulase producing Aspergillus fumigatus strain LMB-35Aa isolated from soil of Peruvian Amazon rainforest. The genome is ∼27.5mb in size, comprises of 228 scaffolds with an average GC content of 50%, and is predicted to contain a total of 8660 protein-coding genes. Of which, 6156 are with known function; it codes for 607 putative CAZymes families potentially involved in carbohydrate metabolism. Several important cellulose degrading genes, such as endoglucanase A, endoglucanase B, endoglucanase D and beta-glucosidase, are also identified. The genome of A. fumigatus strain LMB-35Aa represents the first whole sequenced genome of non-clinical, high cellulase producing A. fumigatus strain isolated from forest soil. Copyright © 2017 Elsevier B.V. All rights reserved.

A High-Density Genetic Map with Array-Based Markers Facilitates Structural and Quantitative Trait Locus Analyses of the Common Wheat Genome

PubMed Central

Iehisa, Julio Cesar Masaru; Ohno, Ryoko; Kimura, Tatsuro; Enoki, Hiroyuki; Nishimura, Satoru; Okamoto, Yuki; Nasuda, Shuhei; Takumi, Shigeo

2014-01-01

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map. PMID:24972598

A high-density genetic map with array-based markers facilitates structural and quantitative trait locus analyses of the common wheat genome.

PubMed

Iehisa, Julio Cesar Masaru; Ohno, Ryoko; Kimura, Tatsuro; Enoki, Hiroyuki; Nishimura, Satoru; Okamoto, Yuki; Nasuda, Shuhei; Takumi, Shigeo

2014-10-01

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Gene discovery in an invasive tephritid model pest species, the Mediterranean fruit fly, Ceratitis capitata

PubMed Central

Gomulski, Ludvik M; Dimopoulos, George; Xi, Zhiyong; Soares, Marcelo B; Bonaldo, Maria F; Malacrida, Anna R; Gasperi, Giuliano

2008-01-01

Background The medfly, Ceratitis capitata, is a highly invasive agricultural pest that has become a model insect for the development of biological control programs. Despite research into the behavior and classical and population genetics of this organism, the quantity of sequence data available is limited. We have utilized an expressed sequence tag (EST) approach to obtain detailed information on transcriptome signatures that relate to a variety of physiological systems in the medfly; this information emphasizes on reproduction, sex determination, and chemosensory perception, since the study was based on normalized cDNA libraries from embryos and adult heads. Results A total of 21,253 high-quality ESTs were obtained from the embryo and head libraries. Clustering analyses performed separately for each library resulted in 5201 embryo and 6684 head transcripts. Considering an estimated 19% overlap in the transcriptomes of the two libraries, they represent about 9614 unique transcripts involved in a wide range of biological processes and molecular functions. Of particular interest are the sequences that share homology with Drosophila genes involved in sex determination, olfaction, and reproductive behavior. The medfly transformer2 (tra2) homolog was identified among the embryonic sequences, and its genomic organization and expression were characterized. Conclusion The sequences obtained in this study represent the first major dataset of expressed genes in a tephritid species of agricultural importance. This resource provides essential information to support the investigation of numerous questions regarding the biology of the medfly and other related species and also constitutes an invaluable tool for the annotation of complete genome sequences. Our study has revealed intriguing findings regarding the transcript regulation of tra2 and other sex determination genes, as well as insights into the comparative genomics of genes implicated in chemosensory reception and reproduction. PMID:18500975

Draft Genome Sequence of a Dictyoglomus sp. from an Enrichment Culture of a New Zealand Geothermal Spring

DOE PAGES

Reysenbach, Anna-Louise; Donaho, John; Kelley, John; ...

2018-03-15

A draft genome of a novelDictyoglomussp., NZ13-RE01, was obtained from a New Zealand hot spring enrichment culture. The 1,927,012-bp genome is similar in both size and G+C content to otherDictyoglomusspp. Like its relatives,Dictyoglomussp. NZ13-RE01 encodes many genes involved in complex carbohydrate metabolism.

Draft Genome Sequence of a Dictyoglomus sp. from an Enrichment Culture of a New Zealand Geothermal Spring

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

Reysenbach, Anna-Louise; Donaho, John; Kelley, John

A draft genome of a novelDictyoglomussp., NZ13-RE01, was obtained from a New Zealand hot spring enrichment culture. The 1,927,012-bp genome is similar in both size and G+C content to otherDictyoglomusspp. Like its relatives,Dictyoglomussp. NZ13-RE01 encodes many genes involved in complex carbohydrate metabolism.

Low-pass sequencing for microbial comparative genomics

PubMed Central

Goo, Young Ah; Roach, Jared; Glusman, Gustavo; Baliga, Nitin S; Deutsch, Kerry; Pan, Min; Kennedy, Sean; DasSarma, Shiladitya; Victor Ng, Wailap; Hood, Leroy

2004-01-01

Background We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed Halobacterium sp. NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe. Results As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits. All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins. Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in H. lacusprofundi and H. marismortui. The core biological functions that govern cellular and genetic mechanisms of H. sp. NRC-1 appear to be conserved in these four other halophiles. Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles. The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between H. sp. NRC-1 and H. lacusprofundi. Conclusion Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively. Identification of multiple IS elements in the genome of H. lacusprofundi and H. marismortui suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich genome of H. sp. NRC-1. Identification of multiple TBP and TFB homologs in these four halophiles are consistent with the hypothesis that different types of complex transcriptional regulation may occur through multiple TBP-TFB combinations in response to rapidly changing environmental conditions. Low-pass shotgun sequence analyses of genomes permit extensive and diverse analyses, and should be generally useful for comparative microbial genomics. PMID:14718067

Using Maximum Entropy to Find Patterns in Genomes

NASA Astrophysics Data System (ADS)

Liu, Sophia; Hockenberry, Adam; Lancichinetti, Andrea; Jewett, Michael; Amaral, Luis

The existence of over- and under-represented sequence motifs in genomes provides evidence of selective evolutionary pressures on biological mechanisms such as transcription, translation, ligand-substrate binding, and host immunity. To accurately identify motifs and other genome-scale patterns of interest, it is essential to be able to generate accurate null models that are appropriate for the sequences under study. There are currently no tools available that allow users to create random coding sequences with specified amino acid composition and GC content. Using the principle of maximum entropy, we developed a method that generates unbiased random sequences with pre-specified amino acid and GC content. Our method is the simplest way to obtain maximally unbiased random sequences that are subject to GC usage and primary amino acid sequence constraints. This approach can also be easily be expanded to create unbiased random sequences that incorporate more complicated constraints such as individual nucleotide usage or even di-nucleotide frequencies. The ability to generate correctly specified null models will allow researchers to accurately identify sequence motifs which will lead to a better understanding of biological processes. National Institute of General Medical Science, Northwestern University Presidential Fellowship, National Science Foundation, David and Lucile Packard Foundation, Camille Dreyfus Teacher Scholar Award.

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.

PubMed

Logan, Grace; Freimanis, Graham L; King, David J; Valdazo-González, Begoña; Bachanek-Bankowska, Katarzyna; Sanderson, Nicholas D; Knowles, Nick J; King, Donald P; Cottam, Eleanor M

2014-09-30

Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5' genomic termini and area immediately flanking the poly(C) region. We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Quantifying the Number of Independent Organelle DNA Insertions in Genome Evolution and Human Health

PubMed Central

Martin, William F.

2017-01-01

Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy. Because numts and nupts are typically identified through sequence comparison tools such as BLAST, disruption of insertions into smaller fragments can lead to systematic overestimation of numt and nupt frequencies. Accurate identification of numts and nupts is important, however, both for better understanding of their role during evolution, and for monitoring their increasingly evident role in human disease. Human populations are polymorphic for 141 numt loci, five numts are causal to genetic disease, and cancer genomic studies are revealing an abundance of numts associated with tumor progression. Here, we report investigation of salient parameters involved in obtaining accurate estimates of numt and nupt numbers in genome sequence data. Numts and nupts from 44 sequenced eukaryotic genomes reveal lineage-specific differences in the number, relative age and frequency of insertional events as well as lineage-specific dynamics of their postinsertional fragmentation. Our findings outline the main technical parameters influencing accurate identification and frequency estimation of numts in genomic studies pertinent to both evolution and human health. PMID:28444372

The complete chloroplast genome sequence of Aconitum coreanum and Aconitum carmichaelii and comparative analysis with other Aconitum species

PubMed Central

Park, Inkyu; Kim, Wook-jin; Yang, Sungyu; Yeo, Sang-Min; Li, Hulin

2017-01-01

Aconitum species (belonging to the Ranunculaceae) are well known herbaceous medicinal ingredients and have great economic value in Asian countries. However, there are still limited genomic resources available for Aconitum species. In this study, we sequenced the chloroplast (cp) genomes of two Aconitum species, A. coreanum and A. carmichaelii, using the MiSeq platform. The two Aconitum chloroplast genomes were 155,880 and 157,040 bp in length, respectively, and exhibited LSC and SSC regions separated by a pair of inverted repeat regions. Both cp genomes had 38% GC content and contained 131 unique functional genes including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene order, content, and orientation of the two Aconitum cp genomes exhibited the general structure of angiosperms, and were similar to those of other Aconitum species. Comparison of the cp genome structure and gene order with that of other Aconitum species revealed general contraction and expansion of the inverted repeat regions and single copy boundary regions. Divergent regions were also identified. In phylogenetic analysis, Aconitum species positon among the Ranunculaceae was determined with other family cp genomes in the Ranunculales. We obtained a barcoding target sequence in a divergent region, ndhC–trnV, and successfully developed a SCAR (sequence characterized amplified region) marker for discrimination of A. coreanum. Our results provide useful genetic information and a specific barcode for discrimination of Aconitum species. PMID:28863163

The complete chloroplast genome sequence of Aconitum coreanum and Aconitum carmichaelii and comparative analysis with other Aconitum species.

PubMed

Park, Inkyu; Kim, Wook-Jin; Yang, Sungyu; Yeo, Sang-Min; Li, Hulin; Moon, Byeong Cheol

2017-01-01

Aconitum species (belonging to the Ranunculaceae) are well known herbaceous medicinal ingredients and have great economic value in Asian countries. However, there are still limited genomic resources available for Aconitum species. In this study, we sequenced the chloroplast (cp) genomes of two Aconitum species, A. coreanum and A. carmichaelii, using the MiSeq platform. The two Aconitum chloroplast genomes were 155,880 and 157,040 bp in length, respectively, and exhibited LSC and SSC regions separated by a pair of inverted repeat regions. Both cp genomes had 38% GC content and contained 131 unique functional genes including 86 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene order, content, and orientation of the two Aconitum cp genomes exhibited the general structure of angiosperms, and were similar to those of other Aconitum species. Comparison of the cp genome structure and gene order with that of other Aconitum species revealed general contraction and expansion of the inverted repeat regions and single copy boundary regions. Divergent regions were also identified. In phylogenetic analysis, Aconitum species positon among the Ranunculaceae was determined with other family cp genomes in the Ranunculales. We obtained a barcoding target sequence in a divergent region, ndhC-trnV, and successfully developed a SCAR (sequence characterized amplified region) marker for discrimination of A. coreanum. Our results provide useful genetic information and a specific barcode for discrimination of Aconitum species.

Whole-genome sequencing reveals that Shewanella haliotis Kim et al. 2007 can be considered a later heterotypic synonym of Shewanella algae Simidu et al. 1990.

PubMed

Szeinbaum, Nadia; Kellum, Cailin E; Glass, Jennifer B; Janda, J Michael; DiChristina, Thomas J

2018-04-01

Previously, experimental DNA-DNA hybridization (DDH) between Shewanellahaliotis JCM 14758 T and Shewanellaalgae JCM 21037 T had suggested that the two strains could be considered different species, despite minimal phenotypic differences. The recent isolation of Shewanella sp. MN-01, with 99 % 16S rRNA gene identity to S. algae and S. haliotis, revealed a potential taxonomic problem between these two species. In this study, we reassessed the nomenclature of S. haliotis and S. algae using available whole-genome sequences. The whole-genome sequence of S. haliotis JCM 14758 T and ten S. algae strains showed ≥97.7 % average nucleotide identity and >78.9 % digital DDH, clearly above the recommended species thresholds. According to the rules of priority and in view of the results obtained, S. haliotis is to be considered a later heterotypic synonym of S. algae. Because the whole-genome sequence of Shewanella sp. strain MN-01 shares >99 % ANI with S. algae JCM 14758 T , it can be confidently identified as S. algae.

Organization of nif gene cluster in Frankia sp. EuIK1 strain, a symbiont of Elaeagnus umbellata.

PubMed

Oh, Chang Jae; Kim, Ho Bang; Kim, Jitae; Kim, Won Jin; Lee, Hyoungseok; An, Chung Sun

2012-01-01

The nucleotide sequence of a 20.5-kb genomic region harboring nif genes was determined and analyzed. The fragment was obtained from Frankia sp. EuIK1 strain, an indigenous symbiont of Elaeagnus umbellata. A total of 20 ORFs including 12 nif genes were identified and subjected to comparative analysis with the genome sequences of 3 Frankia strains representing diverse host plant specificities. The nucleotide and deduced amino acid sequences showed highest levels of identity with orthologous genes from an Elaeagnus-infecting strain. The gene organization patterns around the nif gene clusters were well conserved among all 4 Frankia strains. However, characteristic features appeared in the location of the nifV gene for each Frankia strain, depending on the type of host plant. Sequence analysis was performed to determine the transcription units and suggested that there could be an independent operon starting from the nifW gene in the EuIK strain. Considering the organization patterns and their total extensions on the genome, we propose that the nif gene clusters remained stable despite genetic variations occurring in the Frankia genomes.

Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity.

PubMed

Edger, Patrick P; VanBuren, Robert; Colle, Marivi; Poorten, Thomas J; Wai, Ching Man; Niederhuth, Chad E; Alger, Elizabeth I; Ou, Shujun; Acharya, Charlotte B; Wang, Jie; Callow, Pete; McKain, Michael R; Shi, Jinghua; Collier, Chad; Xiong, Zhiyong; Mower, Jeffrey P; Slovin, Janet P; Hytönen, Timo; Jiang, Ning; Childs, Kevin L; Knapp, Steven J

2018-02-01

Although draft genomes are available for most agronomically important plant species, the majority are incomplete, highly fragmented, and often riddled with assembly and scaffolding errors. These assembly issues hinder advances in tool development for functional genomics and systems biology. Here we utilized a robust, cost-effective approach to produce high-quality reference genomes. We report a near-complete genome of diploid woodland strawberry (Fragaria vesca) using single-molecule real-time sequencing from Pacific Biosciences (PacBio). This assembly has a contig N50 length of ∼7.9 million base pairs (Mb), representing a ∼300-fold improvement of the previous version. The vast majority (>99.8%) of the assembly was anchored to 7 pseudomolecules using 2 sets of optical maps from Bionano Genomics. We obtained ∼24.96 Mb of sequence not present in the previous version of the F. vesca genome and produced an improved annotation that includes 1496 new genes. Comparative syntenic analyses uncovered numerous, large-scale scaffolding errors present in each chromosome in the previously published version of the F. vesca genome. Our results highlight the need to improve existing short-read based reference genomes. Furthermore, we demonstrate how genome quality impacts commonly used analyses for addressing both fundamental and applied biological questions. © The Authors 2017. Published by Oxford University Press.

Analysis of the entire genomes of fifteen torque teno midi virus variants classifiable into a third group of genus Anellovirus.

PubMed

Ninomiya, M; Takahashi, M; Shimosegawa, T; Okamoto, H

2007-01-01

Recently, we identified a novel human virus with a circular DNA genome of 3.2 kb, tentatively designated as torque teno midi virus (TTMDV), with a genomic organization resembling those of torque teno virus (TTV) of 3.8-3.9 kb and torque teno mini virus (TTMV) of 2.8-2.9 kb. To investigate the extent of genomic variability of TTMDV genomes, the full-length sequence was determined for 15 TTMDV isolates obtained from viremic individuals in Japan. The 15 TTMDV isolates comprised 3175-3230 bases and shared 67.0-90.3% identities with each other, and were only 68.4-73.0% identical to the 3 reported TTMDV isolates over the entire genome. TTMDV possessed a genomic organization with four open reading frames (ORF1-ORF4) with characteristic sequence motifs and stem and loop structures with high GC content, similar to TTV and TTMV. The total of 18 TTMDV genomes differed by up to 60.7% from each other in the amino acid sequence of ORF1 (658-677 amino acids), but segregated phylogenetically into the same cluster, which was distantly related to the TTVs and TTMVs. These results indicate that TTMDV with a circular DNA genome of 3.2 kb, has an extremely high degree of genomic variability, and is classifiable into a third group in the genus Anellovirus.

Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.

PubMed

Jia, Xianbo; Lin, Xinjian; Chen, Jichen

2017-11-02

Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

GenColors: annotation and comparative genomics of prokaryotes made easy.

PubMed

Romualdi, Alessandro; Felder, Marius; Rose, Dominic; Gausmann, Ulrike; Schilhabel, Markus; Glöckner, Gernot; Platzer, Matthias; Sühnel, Jürgen

2007-01-01

GenColors (gencolors.fli-leibniz.de) is a new web-based software/database system aimed at an improved and accelerated annotation of prokaryotic genomes considering information on related genomes and making extensive use of genome comparison. It offers a seamless integration of data from ongoing sequencing projects and annotated genomic sequences obtained from GenBank. A variety of export/import filters manages an effective data flow from sequence assembly and manipulation programs (e.g., GAP4) to GenColors and back as well as to standard GenBank file(s). The genome comparison tools include best bidirectional hits, gene conservation, syntenies, and gene core sets. Precomputed UniProt matches allow annotation and analysis in an effective manner. In addition to these analysis options, base-specific quality data (coverage and confidence) can also be handled if available. The GenColors system can be used both for annotation purposes in ongoing genome projects and as an analysis tool for finished genomes. GenColors comes in two types, as dedicated genome browsers and as the Jena Prokaryotic Genome Viewer (JPGV). Dedicated genome browsers contain genomic information on a set of related genomes and offer a large number of options for genome comparison. The system has been efficiently used in the genomic sequencing of Borrelia garinii and is currently applied to various ongoing genome projects on Borrelia, Legionella, Escherichia, and Pseudomonas genomes. One of these dedicated browsers, the Spirochetes Genome Browser (sgb.fli-leibniz.de) with Borrelia, Leptospira, and Treponema genomes, is freely accessible. The others will be released after finalization of the corresponding genome projects. JPGV (jpgv.fli-leibniz.de) offers information on almost all finished bacterial genomes, as compared to the dedicated browsers with reduced genome comparison functionality, however. As of January 2006, this viewer includes 632 genomic elements (e.g., chromosomes and plasmids) of 293 species. The system provides versatile quick and advanced search options for all currently known prokaryotic genomes and generates circular and linear genome plots. Gene information sheets contain basic gene information, database search options, and links to external databases. GenColors is also available on request for local installation.

One Size Doesn't Fit All - RefEditor: Building Personalized Diploid Reference Genome to Improve Read Mapping and Genotype Calling in Next Generation Sequencing Studies

PubMed Central

Yuan, Shuai; Johnston, H. Richard; Zhang, Guosheng; Li, Yun; Hu, Yi-Juan; Qin, Zhaohui S.

2015-01-01

With rapid decline of the sequencing cost, researchers today rush to embrace whole genome sequencing (WGS), or whole exome sequencing (WES) approach as the next powerful tool for relating genetic variants to human diseases and phenotypes. A fundamental step in analyzing WGS and WES data is mapping short sequencing reads back to the reference genome. This is an important issue because incorrectly mapped reads affect the downstream variant discovery, genotype calling and association analysis. Although many read mapping algorithms have been developed, the majority of them uses the universal reference genome and do not take sequence variants into consideration. Given that genetic variants are ubiquitous, it is highly desirable if they can be factored into the read mapping procedure. In this work, we developed a novel strategy that utilizes genotypes obtained a priori to customize the universal haploid reference genome into a personalized diploid reference genome. The new strategy is implemented in a program named RefEditor. When applying RefEditor to real data, we achieved encouraging improvements in read mapping, variant discovery and genotype calling. Compared to standard approaches, RefEditor can significantly increase genotype calling consistency (from 43% to 61% at 4X coverage; from 82% to 92% at 20X coverage) and reduce Mendelian inconsistency across various sequencing depths. Because many WGS and WES studies are conducted on cohorts that have been genotyped using array-based genotyping platforms previously or concurrently, we believe the proposed strategy will be of high value in practice, which can also be applied to the scenario where multiple NGS experiments are conducted on the same cohort. The RefEditor sources are available at https://github.com/superyuan/refeditor. PMID:26267278

Final Report for LDRD Project 02-ERD-069: Discovering the Unknown Mechanism(s) of Virulence in a BW, Class A Select Agent

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

Chain, P; Garcia, E

2003-02-06

The goal of this proposed effort was to assess the difficulty in identifying and characterizing virulence candidate genes in an organism for which very limited data exists. This was accomplished by first addressing the finishing phase of draft-sequenced F. tularensis genomes and conducting comparative analyses to determine the coding potential of each genome; to discover the differences in genome structure and content, and to identify potential genes whose products may be involved in the F. tularensis virulence process. The project was divided into three parts: (1) Genome finishing: This part involves determining the order and orientation of the consensus sequencesmore » of contigs obtained from Phrap assemblies of random draft genomic sequences. This tedious process consists of linking contig ends using information embedded in each sequence file that relates the sequence to the original cloned insert. Since inserts are sequenced from both ends, we can establish a link between these paired-ends in different contigs and thus order and orient contigs. Since these genomes carry numerous copies of insertion sequences, these repeated elements ''confuse'' the Phrap assembly program. It is thus necessary to break these contigs apart at the repeated sequences and individually join the proper flanking regions using paired-end information, or using results of comparisons against a similar genome. Larger repeated elements such as the small subunit ribosomal RNA operon require verification with PCR. Tandem repeats require manual intervention and typically rely on single nucleotide polymorphisms to be resolved. Remaining gaps require PCR reactions and sequencing. Once the genomes have been ''closed'', low quality regions are addressed by resequencing reactions. (2) Genome analysis: The final consensus sequences are processed by combining the results of three gene modelers: Glimmer, Critica and Generation. The final gene models are submitted to a battery of homology searches and domain prediction programs in order to annotate them (e.g. BLAST, Pfam, TIGRfam, COG, KEGG, InterPro, TMhmm, SignalP). The genome structure is also assessed in terms of G+C content, GC bias (GC skew), and locations of repeated regions (e.g. IS elements) and phage-like genes. (3) Comparative genomics: The results of the various genome analyses are compared between the finished (or almost finished) genomes. Here, we have compared the F. tularensis genomes from the extremely lethal strain Schu4 (subsp. tularensis), the vaccine strain LVS (subsp. holartica), and strain UT01-4992 of the less virulent, opportunistic subsp. novicida. Regions present in the highly virulent strain that are absent from the other less virulent strains may provide insight into what factors are required for the high level of virulence.« less

Improved hybrid de novo genome assembly of domesticated apple (Malus x domestica).

PubMed

Li, Xuewei; Kui, Ling; Zhang, Jing; Xie, Yinpeng; Wang, Liping; Yan, Yan; Wang, Na; Xu, Jidi; Li, Cuiying; Wang, Wen; van Nocker, Steve; Dong, Yang; Ma, Fengwang; Guan, Qingmei

2016-08-08

Domesticated apple (Malus × domestica Borkh) is a popular temperate fruit with high nutrient levels and diverse flavors. In 2012, global apple production accounted for at least one tenth of all harvested fruits. A high-quality apple genome assembly is crucial for the selection and breeding of new cultivars. Currently, a single reference genome is available for apple, assembled from 16.9 × genome coverage short reads via Sanger and 454 sequencing technologies. Although a useful resource, this assembly covers only ~89 % of the non-repetitive portion of the genome, and has a relatively short (16.7 kb) contig N50 length. These downsides make it difficult to apply this reference in transcriptive or whole-genome re-sequencing analyses. Here we present an improved hybrid de novo genomic assembly of apple (Golden Delicious), which was obtained from 76 Gb (~102 × genome coverage) Illumina HiSeq data and 21.7 Gb (~29 × genome coverage) PacBio data. The final draft genome is approximately 632.4 Mb, representing ~ 90 % of the estimated genome. The contig N50 size is 111,619 bp, representing a 7 fold improvement. Further annotation analyses predicted 53,922 protein-coding genes and 2,765 non-coding RNA genes. The new apple genome assembly will serve as a valuable resource for investigating complex apple traits at the genomic level. It is not only suitable for genome editing and gene cloning, but also for RNA-seq and whole-genome re-sequencing studies.

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes.

PubMed

Yasui, Yasuo; Hirakawa, Hideki; Ueno, Mariko; Matsui, Katsuhiro; Katsube-Tanaka, Tomoyuki; Yang, Soo Jung; Aii, Jotaro; Sato, Shingo; Mori, Masashi

2016-06-01

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Optimizing and evaluating the reconstruction of Metagenome-assembled microbial genomes.

PubMed

Papudeshi, Bhavya; Haggerty, J Matthew; Doane, Michael; Morris, Megan M; Walsh, Kevin; Beattie, Douglas T; Pande, Dnyanada; Zaeri, Parisa; Silva, Genivaldo G Z; Thompson, Fabiano; Edwards, Robert A; Dinsdale, Elizabeth A

2017-11-28

Microbiome/host interactions describe characteristics that affect the host's health. Shotgun metagenomics includes sequencing a random subset of the microbiome to analyze its taxonomic and metabolic potential. Reconstruction of DNA fragments into genomes from metagenomes (called metagenome-assembled genomes) assigns unknown fragments to taxa/function and facilitates discovery of novel organisms. Genome reconstruction incorporates sequence assembly and sorting of assembled sequences into bins, characteristic of a genome. However, the microbial community composition, including taxonomic and phylogenetic diversity may influence genome reconstruction. We determine the optimal reconstruction method for four microbiome projects that had variable sequencing platforms (IonTorrent and Illumina), diversity (high or low), and environment (coral reefs and kelp forests), using a set of parameters to select for optimal assembly and binning tools. We tested the effects of the assembly and binning processes on population genome reconstruction using 105 marine metagenomes from 4 projects. Reconstructed genomes were obtained from each project using 3 assemblers (IDBA, MetaVelvet, and SPAdes) and 2 binning tools (GroopM and MetaBat). We assessed the efficiency of assemblers using statistics that including contig continuity and contig chimerism and the effectiveness of binning tools using genome completeness and taxonomic identification. We concluded that SPAdes, assembled more contigs (143,718 ± 124 contigs) of longer length (N50 = 1632 ± 108 bp), and incorporated the most sequences (sequences-assembled = 19.65%). The microbial richness and evenness were maintained across the assembly, suggesting low contig chimeras. SPAdes assembly was responsive to the biological and technological variations within the project, compared with other assemblers. Among binning tools, we conclude that MetaBat produced bins with less variation in GC content (average standard deviation: 1.49), low species richness (4.91 ± 0.66), and higher genome completeness (40.92 ± 1.75) across all projects. MetaBat extracted 115 bins from the 4 projects of which 66 bins were identified as reconstructed metagenome-assembled genomes with sequences belonging to a specific genus. We identified 13 novel genomes, some of which were 100% complete, but show low similarity to genomes within databases. In conclusion, we present a set of biologically relevant parameters for evaluation to select for optimal assembly and binning tools. For the tools we tested, SPAdes assembler and MetaBat binning tools reconstructed quality metagenome-assembled genomes for the four projects. We also conclude that metagenomes from microbial communities that have high coverage of phylogenetically distinct, and low taxonomic diversity results in highest quality metagenome-assembled genomes.

Elucidating and mining the Tulipa and Lilium transcriptomes.

PubMed

Moreno-Pachon, Natalia M; Leeggangers, Hendrika A C F; Nijveen, Harm; Severing, Edouard; Hilhorst, Henk; Immink, Richard G H

2016-10-01

Genome sequencing remains a challenge for species with large and complex genomes containing extensive repetitive sequences, of which the bulbous and monocotyledonous plants tulip and lily are examples. In such a case, sequencing of only the active part of the genome, represented by the transcriptome, is a good alternative to obtain information about gene content. In this study we aimed to generate a high quality transcriptome of tulip and lily and to make this data available as an open-access resource via a user-friendly web-based interface. The Illumina HiSeq 2000 platform was applied and the transcribed RNA was sequenced from a collection of different lily and tulip tissues, respectively. In order to obtain good transcriptome coverage and to facilitate effective data mining, assembly was done using different filtering parameters for clearing out contamination and noise of the RNAseq datasets. This analysis revealed limitations of commonly applied methods and parameter settings used in de novo transcriptome assembly. The final created transcriptomes are publicly available via a user friendly Transcriptome browser ( http://www.bioinformatics.nl/bulbs/db/species/index ). The usefulness of this resource has been exemplified by a search for all potential transcription factors in lily and tulip, with special focus on the TCP transcription factor family. This analysis and other quality parameters point out the quality of the transcriptomes, which can serve as a basis for further genomics studies in lily, tulip, and bulbous plants in general.

Draft genome sequence of field isolate Brucella melitensis strain 2007BM/1 from India.

PubMed

Singh, D K; Kumar, Bablu; Shrinet, Garima; Singh, R P; Das, Aparajita; Mantur, B G; Abhishek; Pandey, Aruna; Mondal, Piyali; Sajjanar, B K; Doimari, Soni; Singh, Vijayata; Kumari, Reena; Tiwari, A K; Gandham, Ravi Kumar

2018-04-21

Brucellosis is among one of the most widespread important global zoonotic diseases that is endemic in many parts of India. Brucella melitensis is supposed to be the most pathogenic species for humans. Here we report the draft genome sequence of B. melitensis strain 2007BM/1 isolated from a human in India. Genomic DNA was extracted from Brucella culture and was sequenced using an Illumina MiSeq platform. The generated reads were assembled using three de novo assemblers and the draft genome was annotated. This monoisolate, with a genome length of 3268756bp, was found to be resistant to azithromycin and trimethoprim/sulfamethoxazole but susceptible to tetracycline, ofloxacin, rifampicin, ciprofloxacin and doxycycline. The presence of virulence genes in the strain was identified. The results obtained will help in understanding drug resistance mechanisms and virulence factors in highly zoonotic B. melitensis and suggest the need for judicious use of antibiotics in livestock health and management practices. Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Opportunities and challenges associated with clinical diagnostic genome sequencing: a report of the Association for Molecular Pathology.

PubMed

Schrijver, Iris; Aziz, Nazneen; Farkas, Daniel H; Furtado, Manohar; Gonzalez, Andrea Ferreira; Greiner, Timothy C; Grody, Wayne W; Hambuch, Tina; Kalman, Lisa; Kant, Jeffrey A; Klein, Roger D; Leonard, Debra G B; Lubin, Ira M; Mao, Rong; Nagan, Narasimhan; Pratt, Victoria M; Sobel, Mark E; Voelkerding, Karl V; Gibson, Jane S

2012-11-01

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Bradyrhizobium paxllaeri sp. nov. and Bradyrhizobium icense sp. nov., nitrogen-fixing rhizobial symbionts of Lima bean (Phaseolus lunatus L.) in Peru.

PubMed

Durán, David; Rey, Luis; Mayo, Juan; Zúñiga-Dávila, Doris; Imperial, Juan; Ruiz-Argüeso, Tomás; Martínez-Romero, Esperanza; Ormeño-Orrillo, Ernesto

2014-06-01

A group of strains isolated from root nodules of Phaseolus lunatus (Lima bean) in Peru were characterized by genotypic, genomic and phenotypic methods. All strains possessed identical 16S rRNA gene sequences that were 99.9% identical to that of Bradyrhizobium lablabi CCBAU 23086(T). Despite having identical 16S rRNA gene sequences, the Phaseolus lunatus strains could be divided into two clades by sequence analysis of recA, atpD, glnII, dnaK and gyrB genes. The genome sequence of a representative of each clade was obtained and compared to the genomes of closely related species of the genus Bradyrhizobium. Average nucleotide identity values below the species circumscription threshold were obtained when comparing the two clades to each other (88.6%) and with all type strains of the genus Bradyrhizobium (≤92.9%). Phenotypes distinguishing both clades from all described and closely related species of the genus Bradyrhizobium were found. On the basis of the results obtained, two novel species, Bradyrhizobium paxllaeri sp. nov. (type strain LMTR 21(T) = DSM 18454(T) = HAMBI 2911(T)) and Bradyrhizobium icense sp. nov. (type strain LMTR 13(T) = HAMBI 3584(T) = CECT 8509(T) = CNPSo 2583(T)), are proposed to accommodate the uncovered clades of Phaseolus lunatus bradyrhizobia. These species share highly related but distinct nifH and nodC symbiosis genes. © 2014 IUMS.

A novel method of genomic DNA extraction for Cactaceae1

PubMed Central

Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

2013-01-01

• Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India.

PubMed

Bondre, Vijay P; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N

2016-11-01

Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections.

Genetic characterization of human herpesvirus type 1: Full-length genome sequence of strain obtained from an encephalitis case from India

PubMed Central

Bondre, Vijay P.; Sankararaman, Vasudha; Andhare, Vijaysinh; Tupekar, Manisha; Sapkal, Gajanan N.

2016-01-01

Background & objectives: Human herpes simplex virus 1 (HSV-1) is the most common cause of sporadic encephalitis in humans that contributes to >10 per cent of the encephalitis cases occurring worldwide. Availability of limited full genome sequences from a small number of isolates resulted in poor understanding of host and viral factors responsible for variable clinical outcome. In this study genetic relationship, extent and source of recombination using full-length genome sequence derived from a newly isolated HSV-1 isolate was studied in comparison with those sampled from patients with varied clinical outcome. Methods: Full genome sequence of HSV-1 isolated from cerebrospinal fluid (CSF) of a patient with acute encephalitis syndrome (AES) by inoculation in baby hamster kidney-21 (BHK-21) cells was determined using next-generation sequencing (NGS) technology. Phylogenetic analysis of the newly generated sequence in comparison with 33 additional full-length genomes defined genetic relationship with worldwide distributed strains. The bootscan and similarity plot analysis defined recombination crossovers and similarities between newly isolated Indian HSV-1 with six Asian and a total of 34 worldwide isolated strains. Results: Mapping of 376,332 reads amplified from HSV-1 DNA by NGS generated full-length genome of 151,024 bp from newly isolated Indian HSV-1. Phylogenetic analysis classified worldwide distributed strains into three major evolutionary lineages correlating to their geographic distribution. Lineage 1 containing strains were isolated from America and Europe; lineage 2 contained all the strains from Asian countries along with the North American KOS and RE strains whereas the South African isolates were distributed into two groups under lineage 3. Recombination analysis confirmed events of recombination in Indian HSV-1 genome resulting from mixing of different strains evolved in Asian countries. Interpretation & conclusions: Our results showed that the full-length genome sequence generated from an Indian HSV-1 isolate shared close genetic relationship with the American KOS and Chinese CR38 strains which belonged to the Asian genetic lineage. Recombination analysis of Indian isolate demonstrated multiple recombination crossover points throughout the genome. This full-length genome sequence amplified from the Indian isolate would be helpful to study HSV evolution, genetic basis of differential pathogenesis, host-virus interactions and viral factors contributing towards differential clinical outcome in human infections. PMID:28361829

MALINA: a web service for visual analytics of human gut microbiota whole-genome metagenomic reads.

PubMed

Tyakht, Alexander V; Popenko, Anna S; Belenikin, Maxim S; Altukhov, Ilya A; Pavlenko, Alexander V; Kostryukova, Elena S; Selezneva, Oksana V; Larin, Andrei K; Karpova, Irina Y; Alexeev, Dmitry G

2012-12-07

MALINA is a web service for bioinformatic analysis of whole-genome metagenomic data obtained from human gut microbiota sequencing. As input data, it accepts metagenomic reads of various sequencing technologies, including long reads (such as Sanger and 454 sequencing) and next-generation (including SOLiD and Illumina). It is the first metagenomic web service that is capable of processing SOLiD color-space reads, to authors' knowledge. The web service allows phylogenetic and functional profiling of metagenomic samples using coverage depth resulting from the alignment of the reads to the catalogue of reference sequences which are built into the pipeline and contain prevalent microbial genomes and genes of human gut microbiota. The obtained metagenomic composition vectors are processed by the statistical analysis and visualization module containing methods for clustering, dimension reduction and group comparison. Additionally, the MALINA database includes vectors of bacterial and functional composition for human gut microbiota samples from a large number of existing studies allowing their comparative analysis together with user samples, namely datasets from Russian Metagenome project, MetaHIT and Human Microbiome Project (downloaded from http://hmpdacc.org). MALINA is made freely available on the web at http://malina.metagenome.ru. The website is implemented in JavaScript (using Ext JS), Microsoft .NET Framework, MS SQL, Python, with all major browsers supported.

Whole-genome characterization of Uruguayan strains of avian infectious bronchitis virus reveals extensive recombination between the two major South American lineages.

PubMed

Marandino, Ana; Tomás, Gonzalo; Panzera, Yanina; Greif, Gonzalo; Parodi-Talice, Adriana; Hernández, Martín; Techera, Claudia; Hernández, Diego; Pérez, Ruben

2017-10-01

Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus that causes one of the most persistent respiratory diseases in poultry. The virus is classified in genotypes and lineages with different epidemiological relevance. Two lineages of the GI genotype (11 and 16) have been widely circulating for decades in South America. GI-11 is an exclusive South American lineage while the GI-16 lineage is distributed in Asia, Europe and South America. Here, we obtained the whole genome of two Uruguayan strains of the GI-11 and GI-16 lineages using Illumina high-throughput sequencing. The strains here sequenced are the first obtained in South America for the infectious bronchitis virus and provide new insights into the origin, spreading and evolution of viral variants. The complete genome of the GI-11 and GI-16 strains have 27,621 and 27,638 nucleotides, respectively, and possess the same genomic organization. Phylogenetic incongruence analysis reveals that both strains have a mosaic genome that arose by recombination between Euro Asiatic strains of the GI-16 lineage and ancestral South American GI-11 viruses. The recombination occurred in South America and produced two viral variants that have retained the full-length S1 sequences of the parental lineages but are extremely similar in the rest of their genomes. These recombinant virus have been extraordinary successful, persisting in the continent for several years with a notorious wide geographic distribution. Our findings reveal a singular viral dynamics and emphasize the importance of complete genomic characterization to understand the emergence and evolutionary history of viral variants. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficient analysis of mouse genome sequences reveal many nonsense variants

PubMed Central

Steeland, Sophie; Timmermans, Steven; Van Ryckeghem, Sara; Hulpiau, Paco; Saeys, Yvan; Van Montagu, Marc; Vandenbroucke, Roosmarijn E.; Libert, Claude

2016-01-01

Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1. PMID:27147605

Mobile Genome Express (MGE): A comprehensive automatic genetic analyses pipeline with a mobile device.

PubMed

Yoon, Jun-Hee; Kim, Thomas W; Mendez, Pedro; Jablons, David M; Kim, Il-Jin

2017-01-01

The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or genome. However, data analysis is still the biggest bottleneck for its wide implementation. Most laboratories still depend on manual procedures for data handling and analyses, which translates into a delay and decreased efficiency in the delivery of NGS results to doctors and patients. Thus, there is high demand for developing an automatic and an easy-to-use NGS data analyses system. We developed comprehensive, automatic genetic analyses controller named Mobile Genome Express (MGE) that works in smartphones or other mobile devices. MGE can handle all the steps for genetic analyses, such as: sample information submission, sequencing run quality check from the sequencer, secured data transfer and results review. We sequenced an Actrometrix control DNA containing multiple proven human mutations using a targeted sequencing panel, and the whole analysis was managed by MGE, and its data reviewing program called ELECTRO. All steps were processed automatically except for the final sequencing review procedure with ELECTRO to confirm mutations. The data analysis process was completed within several hours. We confirmed the mutations that we have identified were consistent with our previous results obtained by using multi-step, manual pipelines.

Draft Genome Sequence of a Dictyoglomus sp. from an Enrichment Culture of a New Zealand Geothermal Spring

PubMed Central

Donaho, John A.; Kelley, John F.; St. John, Emily; Turner, Christina; Podar, Mircea; Stott, Matthew B.

2018-01-01

ABSTRACT A draft genome of a novel Dictyoglomus sp., NZ13-RE01, was obtained from a New Zealand hot spring enrichment culture. The 1,927,012-bp genome is similar in both size and G+C content to other Dictyoglomus spp. Like its relatives, Dictyoglomus sp. NZ13-RE01 encodes many genes involved in complex carbohydrate metabolism. PMID:29545298

Draft Genome Sequence of Geobacillus sp. LEMMY01, a Thermophilic Bacterium Isolated from the Site of a Burning Grass Pile

PubMed Central

de Souza, Yuri Pinheiro Alves; da Mota, Fábio Faria

2017-01-01

ABSTRACT We report here the 3,586,065-bp draft genome of Geobacillus sp. LEMMY01, which was isolated (axenic culture) from a thermophilic chemolitoautotrophic consortium obtained from the site of a burning grass pile. The genome contains biosynthetic gene clusters coding for secondary metabolites, such as terpene and lantipeptide, confirming the biotechnological potential of this strain. PMID:28495764

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge.

PubMed

Brownstein, Catherine A; Beggs, Alan H; Homer, Nils; Merriman, Barry; Yu, Timothy W; Flannery, Katherine C; DeChene, Elizabeth T; Towne, Meghan C; Savage, Sarah K; Price, Emily N; Holm, Ingrid A; Luquette, Lovelace J; Lyon, Elaine; Majzoub, Joseph; Neupert, Peter; McCallie, David; Szolovits, Peter; Willard, Huntington F; Mendelsohn, Nancy J; Temme, Renee; Finkel, Richard S; Yum, Sabrina W; Medne, Livija; Sunyaev, Shamil R; Adzhubey, Ivan; Cassa, Christopher A; de Bakker, Paul I W; Duzkale, Hatice; Dworzyński, Piotr; Fairbrother, William; Francioli, Laurent; Funke, Birgit H; Giovanni, Monica A; Handsaker, Robert E; Lage, Kasper; Lebo, Matthew S; Lek, Monkol; Leshchiner, Ignaty; MacArthur, Daniel G; McLaughlin, Heather M; Murray, Michael F; Pers, Tune H; Polak, Paz P; Raychaudhuri, Soumya; Rehm, Heidi L; Soemedi, Rachel; Stitziel, Nathan O; Vestecka, Sara; Supper, Jochen; Gugenmus, Claudia; Klocke, Bernward; Hahn, Alexander; Schubach, Max; Menzel, Mortiz; Biskup, Saskia; Freisinger, Peter; Deng, Mario; Braun, Martin; Perner, Sven; Smith, Richard J H; Andorf, Janeen L; Huang, Jian; Ryckman, Kelli; Sheffield, Val C; Stone, Edwin M; Bair, Thomas; Black-Ziegelbein, E Ann; Braun, Terry A; Darbro, Benjamin; DeLuca, Adam P; Kolbe, Diana L; Scheetz, Todd E; Shearer, Aiden E; Sompallae, Rama; Wang, Kai; Bassuk, Alexander G; Edens, Erik; Mathews, Katherine; Moore, Steven A; Shchelochkov, Oleg A; Trapane, Pamela; Bossler, Aaron; Campbell, Colleen A; Heusel, Jonathan W; Kwitek, Anne; Maga, Tara; Panzer, Karin; Wassink, Thomas; Van Daele, Douglas; Azaiez, Hela; Booth, Kevin; Meyer, Nic; Segal, Michael M; Williams, Marc S; Tromp, Gerard; White, Peter; Corsmeier, Donald; Fitzgerald-Butt, Sara; Herman, Gail; Lamb-Thrush, Devon; McBride, Kim L; Newsom, David; Pierson, Christopher R; Rakowsky, Alexander T; Maver, Aleš; Lovrečić, Luca; Palandačić, Anja; Peterlin, Borut; Torkamani, Ali; Wedell, Anna; Huss, Mikael; Alexeyenko, Andrey; Lindvall, Jessica M; Magnusson, Måns; Nilsson, Daniel; Stranneheim, Henrik; Taylan, Fulya; Gilissen, Christian; Hoischen, Alexander; van Bon, Bregje; Yntema, Helger; Nelen, Marcel; Zhang, Weidong; Sager, Jason; Zhang, Lu; Blair, Kathryn; Kural, Deniz; Cariaso, Michael; Lennon, Greg G; Javed, Asif; Agrawal, Saloni; Ng, Pauline C; Sandhu, Komal S; Krishna, Shuba; Veeramachaneni, Vamsi; Isakov, Ofer; Halperin, Eran; Friedman, Eitan; Shomron, Noam; Glusman, Gustavo; Roach, Jared C; Caballero, Juan; Cox, Hannah C; Mauldin, Denise; Ament, Seth A; Rowen, Lee; Richards, Daniel R; San Lucas, F Anthony; Gonzalez-Garay, Manuel L; Caskey, C Thomas; Bai, Yu; Huang, Ying; Fang, Fang; Zhang, Yan; Wang, Zhengyuan; Barrera, Jorge; Garcia-Lobo, Juan M; González-Lamuño, Domingo; Llorca, Javier; Rodriguez, Maria C; Varela, Ignacio; Reese, Martin G; De La Vega, Francisco M; Kiruluta, Edward; Cargill, Michele; Hart, Reece K; Sorenson, Jon M; Lyon, Gholson J; Stevenson, David A; Bray, Bruce E; Moore, Barry M; Eilbeck, Karen; Yandell, Mark; Zhao, Hongyu; Hou, Lin; Chen, Xiaowei; Yan, Xiting; Chen, Mengjie; Li, Cong; Yang, Can; Gunel, Murat; Li, Peining; Kong, Yong; Alexander, Austin C; Albertyn, Zayed I; Boycott, Kym M; Bulman, Dennis E; Gordon, Paul M K; Innes, A Micheil; Knoppers, Bartha M; Majewski, Jacek; Marshall, Christian R; Parboosingh, Jillian S; Sawyer, Sarah L; Samuels, Mark E; Schwartzentruber, Jeremy; Kohane, Isaac S; Margulies, David M

2014-03-25

There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge

PubMed Central

2014-01-01

Background There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. Results A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. Conclusions The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups. PMID:24667040

Associations of Perceived Norms With Intentions to Learn Genomic Sequencing Results: Roles for Attitudes and Ambivalence

PubMed Central

Reid, Allecia E.; Taber, Jennifer M.; Ferrer, Rebecca A.; Biesecker, Barbara B.; Lewis, Katie L.; Biesecker, Leslie G.; Klein, William M. P.

2018-01-01

Objective Genomic sequencing is becoming increasingly accessible, highlighting the need to understand the social and psychological factors that drive interest in receiving testing results. These decisions may depend on perceived descriptive norms (how most others behave) and injunctive norms (what is approved of by others). We predicted that descriptive norms would be directly associated with intentions to learn genomic sequencing results, whereas injunctive norms would be associated indirectly, via attitudes. These differential associations with intentions versus attitudes were hypothesized to be strongest when individuals held ambivalent attitudes toward obtaining results. Methods Participants enrolled in a genomic sequencing trial (n=372) reported intentions to learn medically actionable, non-medically actionable, and carrier sequencing results. Descriptive norms items referenced other study participants. Injunctive norms were analyzed separately for close friends and family members. Attitudes, attitudinal ambivalence, and sociodemographic covariates were also assessed. Results In structural equation models, both descriptive norms and friend injunctive norms were associated with intentions to receive all sequencing results (ps<.004). Attitudes consistently mediated all friend injunctive norms-intentions associations, but not the descriptive norms-intentions associations. Attitudinal ambivalence moderated the association between friend injunctive norms (p≤.001), but not descriptive norms (p=.16), and attitudes. Injunctive norms were significantly associated with attitudes when ambivalence was high, but were unrelated when ambivalence was low. Results replicated for family injunctive norms. Conclusions Descriptive and injunctive norms play roles in genomic sequencing decisions. Considering mediators and moderators of these processes enhances ability to optimize use of normative information to support informed decision making. PMID:29745680

Diagnostics for Yaws Eradication: Insights From Direct Next-Generation Sequencing of Cutaneous Strains of Treponema pallidum

PubMed Central

Marks, Michael; Fookes, Maria; Wagner, Josef; Butcher, Robert; Ghinai, Rosanna; Sokana, Oliver; Sarkodie, Yaw-Adu; Lukehart, Sheila A; Solomon, Anthony W; Mabey, David C W; Thomson, Nicholas

2018-01-01

Abstract Background Yaws-like chronic ulcers can be caused by Treponema pallidum subspecies pertenue, Haemophilus ducreyi, or other, still-undefined bacteria. To permit accurate evaluation of yaws elimination efforts, programmatic use of molecular diagnostics is required. The accuracy and sensitivity of current tools remain unclear because our understanding of T. pallidum diversity is limited by the low number of sequenced genomes. Methods We tested samples from patients with suspected yaws collected in the Solomon Islands and Ghana. All samples were from patients whose lesions had previously tested negative using the Centers for Disease Control and Prevention (CDC) diagnostic assay in widespread use. However, some of these patients had positive serological assays for yaws on blood. We used direct whole-genome sequencing to identify T. pallidum subsp pertenue strains missed by the current assay. Results From 45 Solomon Islands and 27 Ghanaian samples, 11 were positive for T. pallidum DNA using the species-wide quantitative polymerase chain reaction (PCR) assay, from which we obtained 6 previously undetected T. pallidum subsp pertenue whole-genome sequences. These show that Solomon Islands sequences represent distinct T. pallidum subsp pertenue clades. These isolates were invisible to the CDC diagnostic PCR assay, due to sequence variation in the primer binding site. Conclusions Our data double the number of published T. pallidum subsp pertenue genomes. We show that Solomon Islands strains are undetectable by the PCR used in many studies and by health ministries. This assay is therefore not adequate for the eradication program. Next-generation genome sequence data are essential for these efforts. PMID:29045605

Dichlorvos Exposure Results in Large Scale Disruption of Energy Metabolism in the Liver of the Zebra Fish, Danio Rerio

DTIC Science & Technology

2015-10-24

zebrafish reference genome sequence and its relationship to the human genome . Nature. 2013;496(7446):498–503. 21. Linney E, Upchurch L, Donerly S. Zebrafish...To obtain a broader understanding of the effects of dichlorvos on liver metabolism, we per- formed a genome -wide analysis of gene expression in the ...condition) for whole genome transcript ana- lysis, and fixed another set of fish for histological evaluation (n = 5/condition). We determined the target

Genome Sequence, Assembly and Characterization of Two Metschnikowia fructicola Strains Used as Biocontrol Agents of Postharvest Diseases

PubMed Central

Piombo, Edoardo; Sela, Noa; Wisniewski, Michael; Hoffmann, Maria; Gullino, Maria L.; Allard, Marc W.; Levin, Elena; Spadaro, Davide; Droby, Samir

2018-01-01

The yeast Metschnikowia fructicola was reported as an efficient biological control agent of postharvest diseases of fruits and vegetables, and it is the bases of the commercial formulated product “Shemer.” Several mechanisms of action by which M. fructicola inhibits postharvest pathogens were suggested including iron-binding compounds, induction of defense signaling genes, production of fungal cell wall degrading enzymes and relatively high amounts of superoxide anions. We assembled the whole genome sequence of two strains of M. fructicola using PacBio and Illumina shotgun sequencing technologies. Using the PacBio, a high-quality draft genome consisting of 93 contigs, with an estimated genome size of approximately 26 Mb, was obtained. Comparative analysis of M. fructicola proteins with the other three available closely related genomes revealed a shared core of homologous proteins coded by 5,776 genes. Comparing the genomes of the two M. fructicola strains using a SNP calling approach resulted in the identification of 564,302 homologous SNPs with 2,004 predicted high impact mutations. The size of the genome is exceptionally high when compared with those of available closely related organisms, and the high rate of homology among M. fructicola genes points toward a recent whole-genome duplication event as the cause of this large genome. Based on the assembled genome, sequences were annotated with a gene description and gene ontology (GO term) and clustered in functional groups. Analysis of CAZymes family genes revealed 1,145 putative genes, and transcriptomic analysis of CAZyme expression levels in M. fructicola during its interaction with either grapefruit peel tissue or Penicillium digitatum revealed a high level of CAZyme gene expression when the yeast was placed in wounded fruit tissue. PMID:29666611

Optofluidic Single-Cell Genome Amplification of Sub-micron Bacteria in the Ocean Subsurface

PubMed Central

Landry, Zachary C.; Vergin, Kevin; Mannenbach, Christopher; Block, Stephen; Yang, Qiao; Blainey, Paul; Carlson, Craig; Giovannoni, Stephen

2018-01-01

Optofluidic single-cell genome amplification was used to obtain genome sequences from sub-micron cells collected from the euphotic and mesopelagic zones of the northwestern Sargasso Sea. Plankton cells were visually selected and manually sorted with an optical trap, yielding 20 partial genome sequences representing seven bacterial phyla. Two organisms, E01-9C-26 (Gammaproteobacteria), represented by four single cell genomes, and Opi.OSU.00C, an uncharacterized Verrucomicrobia, were the first of their types retrieved by single cell genome sequencing and were studied in detail. Metagenomic data showed that E01-9C-26 is found throughout the dark ocean, while Opi.OSU.00C was observed to bloom transiently in the nutrient-depleted euphotic zone of the late spring and early summer. The E01-9C-26 genomes had an estimated size of 4.76–5.05 Mbps, and contained “O” and “W”-type monooxygenase genes related to methane and ammonium monooxygenases that were previously reported from ocean metagenomes. Metabolic reconstruction indicated E01-9C-26 are likely versatile methylotrophs capable of scavenging C1 compounds, methylated compounds, reduced sulfur compounds, and a wide range of amines, including D-amino acids. The genome sequences identified E01-9C-26 as a source of “O” and “W”-type monooxygenase genes related to methane and ammonium monooxygenases that were previously reported from ocean metagenomes, but are of unknown function. In contrast, Opi.OSU.00C genomes encode genes for catabolizing carbohydrate compounds normally associated with eukaryotic phytoplankton. This exploration of optofluidics showed that it was effective for retrieving diverse single-cell bacterioplankton genomes and has potential advantages in microbiology applications that require working with small sample volumes or targeting cells by their morphology.

The Rosa genome provides new insights into the domestication of modern roses.

PubMed

Raymond, Olivier; Gouzy, Jérôme; Just, Jérémy; Badouin, Hélène; Verdenaud, Marion; Lemainque, Arnaud; Vergne, Philippe; Moja, Sandrine; Choisne, Nathalie; Pont, Caroline; Carrère, Sébastien; Caissard, Jean-Claude; Couloux, Arnaud; Cottret, Ludovic; Aury, Jean-Marc; Szécsi, Judit; Latrasse, David; Madoui, Mohammed-Amin; François, Léa; Fu, Xiaopeng; Yang, Shu-Hua; Dubois, Annick; Piola, Florence; Larrieu, Antoine; Perez, Magali; Labadie, Karine; Perrier, Lauriane; Govetto, Benjamin; Labrousse, Yoan; Villand, Priscilla; Bardoux, Claudia; Boltz, Véronique; Lopez-Roques, Céline; Heitzler, Pascal; Vernoux, Teva; Vandenbussche, Michiel; Quesneville, Hadi; Boualem, Adnane; Bendahmane, Abdelhafid; Liu, Chang; Le Bris, Manuel; Salse, Jérôme; Baudino, Sylvie; Benhamed, Moussa; Wincker, Patrick; Bendahmane, Mohammed

2018-06-01

Roses have high cultural and economic importance as ornamental plants and in the perfume industry. We report the rose whole-genome sequencing and assembly and resequencing of major genotypes that contributed to rose domestication. We generated a homozygous genotype from a heterozygous diploid modern rose progenitor, Rosa chinensis 'Old Blush'. Using single-molecule real-time sequencing and a meta-assembly approach, we obtained one of the most comprehensive plant genomes to date. Diversity analyses highlighted the mosaic origin of 'La France', one of the first hybrids combining the growth vigor of European species and the recurrent blooming of Chinese species. Genomic segments of Chinese ancestry identified new candidate genes for recurrent blooming. Reconstructing regulatory and secondary metabolism pathways allowed us to propose a model of interconnected regulation of scent and flower color. This genome provides a foundation for understanding the mechanisms governing rose traits and should accelerate improvement in roses, Rosaceae and ornamentals.

Recovery of a Medieval Brucella melitensis Genome Using Shotgun Metagenomics

PubMed Central

Kay, Gemma L.; Sergeant, Martin J.; Giuffra, Valentina; Bandiera, Pasquale; Milanese, Marco; Bramanti, Barbara

2014-01-01

ABSTRACT Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. PMID:25028426

Whole genome analysis of halotolerant and alkalotolerant plant growth-promoting rhizobacterium Klebsiella sp. D5A

NASA Astrophysics Data System (ADS)

Liu, Wuxing; Wang, Qingling; Hou, Jinyu; Tu, Chen; Luo, Yongming; Christie, Peter

2016-05-01

This research undertook the systematic analysis of the Klebsiella sp. D5A genome and identification of genes that contribute to plant growth-promoting (PGP) traits, especially genes related to salt tolerance and wide pH adaptability. The genome sequence of isolate D5A was obtained using an Illumina HiSeq 2000 sequencing system with average coverages of 174.7× and 200.1× using the paired-end and mate-pair sequencing, respectively. Predicted and annotated gene sequences were analyzed for similarity with the Kyoto Encyclopedia of Genes and Genomes (KEGG) enzyme database followed by assignment of each gene into the KEGG pathway charts. The results show that the Klebsiella sp. D5A genome has a total of 5,540,009 bp with 57.15% G + C content. PGP conferring genes such as indole-3-acetic acid (IAA) biosynthesis, phosphate solubilization, siderophore production, acetoin and 2,3-butanediol synthesis, and N2 fixation were determined. Moreover, genes putatively responsible for resistance to high salinity including glycine-betaine synthesis, trehalose synthesis and a number of osmoregulation receptors and transport systems were also observed in the D5A genome together with numerous genes that contribute to pH homeostasis. These genes reveal the genetic adaptation of D5A to versatile environmental conditions and the effectiveness of the isolate to serve as a plant growth stimulator.

High-Quality Draft Genome Sequence of Candida apicola NRRL Y-50540

PubMed Central

Vega-Alvarado, Leticia; Gómez-Angulo, Jorge; Escalante-García, Zazil; Grande, Ricardo; Gschaedler-Mathis, Anne; Amaya-Delgado, Lorena

2015-01-01

Candida apicola, a highly osmotolerant ascomycetes yeast, produces sophorolipids (biosurfactants), membrane fatty acids, and enzymes of biotechnological interest. The genome obtained has a high-quality draft for this species and can be used as a reference to perform further analyses, such as differential gene expression in yeast from Candida genera. PMID:26067948

A nine-scaffold genome assembly of the nine chromosome sugar beet

USDA-ARS?s Scientific Manuscript database

Over the course of 20 months, we assembled a sugar beet genome (700 - 800 Mb) into a close representation of the nine haploid chromosomes of beet. This result was obtained by sequentially assembling sequences >40 kb in length, orienting these assemblies via optical mapping, and scaffolding with in v...

From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

PubMed Central

2014-01-01

Background Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users. Results Here we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling). Conclusions All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources. PMID:24460871

Health Care Infrastructure for Financially Sustainable Clinical Genomics.

PubMed

Lennerz, Jochen K; McLaughlin, Heather M; Baron, Jason M; Rasmussen, David; Sumbada Shin, Meini; Berners-Lee, Nancy; Miller Batten, Julie; Swoboda, Kathryn J; Gala, Manish K; Winter, Harland S; Schmahmann, Jeremy D; Sweetser, David A; Boswell, Marianne; Pacula, Maciej; Stenzinger, Albrecht; Le, Long P; Hynes, William; Rehm, Heidi L; Klibanski, Anne; Black-Schaffer, Stephen W; Golden, Jeffrey A; Louis, David N; Weiss, Scott T; Iafrate, A John

2016-09-01

Next-generation sequencing has evolved technically and economically into the method of choice for interrogating the genome in cancer and inherited disorders. The introduction of procedural code sets for whole-exome and genome sequencing is a milestone toward financially sustainable clinical implementation; however, achieving reimbursement is currently a major challenge. As part of a prospective quality-improvement initiative to implement the new code sets, we adopted Agile, a development methodology originally devised in software development. We implemented eight functionally distinct modules (request review, cost estimation, preauthorization, accessioning, prebilling, testing, reporting, and reimbursement consultation) and obtained feedback via an anonymous survey. We managed 50 clinical requests (January to June 2015). The fraction of pursued-to-requested cases (n = 15/50; utilization management fraction, 0.3) aimed for a high rate of preauthorizations. In 13 of 15 patients the insurance plan required preauthorization, which we obtained in 70% and ultimately achieved reimbursement in 50%. Interoperability enabled assessment of 12 different combinations of modules that underline the importance of an adaptive workflow and policy tailoring to achieve higher yields of reimbursement. The survey confirmed a positive attitude toward self-organizing teams. We acknowledge the individuals and their interactions and termed the infrastructure: human pipeline. Nontechnical barriers currently are limiting the scope and availability of clinical genomic sequencing. The presented human pipeline is one approach toward long-term financial sustainability of clinical genomics. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A survey of single nucleotide polymorphisms identified from whole-genome sequencing and their functional effect in the porcine genome.

PubMed

Keel, B N; Nonneman, D J; Rohrer, G A

2017-08-01

Genetic variants detected from sequence have been used to successfully identify causal variants and map complex traits in several organisms. High and moderate impact variants, those expected to alter or disrupt the protein coded by a gene and those that regulate protein production, likely have a more significant effect on phenotypic variation than do other types of genetic variants. Hence, a comprehensive list of these functional variants would be of considerable interest in swine genomic studies, particularly those targeting fertility and production traits. Whole-genome sequence was obtained from 72 of the founders of an intensely phenotyped experimental swine herd at the U.S. Meat Animal Research Center (USMARC). These animals included all 24 of the founding boars (12 Duroc and 12 Landrace) and 48 Yorkshire-Landrace composite sows. Sequence reads were mapped to the Sscrofa10.2 genome build, resulting in a mean of 6.1 fold (×) coverage per genome. A total of 22 342 915 high confidence SNPs were identified from the sequenced genomes. These included 21 million previously reported SNPs and 79% of the 62 163 SNPs on the PorcineSNP60 BeadChip assay. Variation was detected in the coding sequence or untranslated regions (UTRs) of 87.8% of the genes in the porcine genome: loss-of-function variants were predicted in 504 genes, 10 202 genes contained nonsynonymous variants, 10 773 had variation in UTRs and 13 010 genes contained synonymous variants. Approximately 139 000 SNPs were classified as loss-of-function, nonsynonymous or regulatory, which suggests that over 99% of the variation detected in our pigs could potentially be ignored, allowing us to focus on a much smaller number of functional SNPs during future analyses. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions.

PubMed

Schmidt, Martin; Van Bel, Michiel; Woloszynska, Magdalena; Slabbinck, Bram; Martens, Cindy; De Block, Marc; Coppens, Frederik; Van Lijsebettens, Mieke

2017-07-06

Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Plant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

proGenomes: a resource for consistent functional and taxonomic annotations of prokaryotic genomes.

PubMed

Mende, Daniel R; Letunic, Ivica; Huerta-Cepas, Jaime; Li, Simone S; Forslund, Kristoffer; Sunagawa, Shinichi; Bork, Peer

2017-01-04

The availability of microbial genomes has opened many new avenues of research within microbiology. This has been driven primarily by comparative genomics approaches, which rely on accurate and consistent characterization of genomic sequences. It is nevertheless difficult to obtain consistent taxonomic and integrated functional annotations for defined prokaryotic clades. Thus, we developed proGenomes, a resource that provides user-friendly access to currently 25 038 high-quality genomes whose sequences and consistent annotations can be retrieved individually or by taxonomic clade. These genomes are assigned to 5306 consistent and accurate taxonomic species clusters based on previously established methodology. proGenomes also contains functional information for almost 80 million protein-coding genes, including a comprehensive set of general annotations and more focused annotations for carbohydrate-active enzymes and antibiotic resistance genes. Additionally, broad habitat information is provided for many genomes. All genomes and associated information can be downloaded by user-selected clade or multiple habitat-specific sets of representative genomes. We expect that the availability of high-quality genomes with comprehensive functional annotations will promote advances in clinical microbial genomics, functional evolution and other subfields of microbiology. proGenomes is available at http://progenomes.embl.de. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Relative stability of DNA as a generic criterion for promoter prediction: whole genome annotation of microbial genomes with varying nucleotide base composition.

PubMed

Rangannan, Vetriselvi; Bansal, Manju

2009-12-01

The rapid increase in genome sequence information has necessitated the annotation of their functional elements, particularly those occurring in the non-coding regions, in the genomic context. Promoter region is the key regulatory region, which enables the gene to be transcribed or repressed, but it is difficult to determine experimentally. Hence an in silico identification of promoters is crucial in order to guide experimental work and to pin point the key region that controls the transcription initiation of a gene. In this analysis, we demonstrate that while the promoter regions are in general less stable than the flanking regions, their average free energy varies depending on the GC composition of the flanking genomic sequence. We have therefore obtained a set of free energy threshold values, for genomic DNA with varying GC content and used them as generic criteria for predicting promoter regions in several microbial genomes, using an in-house developed tool PromPredict. On applying it to predict promoter regions corresponding to the 1144 and 612 experimentally validated TSSs in E. coli (50.8% GC) and B. subtilis (43.5% GC) sensitivity of 99% and 95% and precision values of 58% and 60%, respectively, were achieved. For the limited data set of 81 TSSs available for M. tuberculosis (65.6% GC) a sensitivity of 100% and precision of 49% was obtained.

Congruent Deep Relationships in the Grape Family (Vitaceae) Based on Sequences of Chloroplast Genomes and Mitochondrial Genes via Genome Skimming

PubMed Central

Zhang, Ning; Wen, Jun; Zimmer, Elizabeth A.

2015-01-01

Vitaceae is well-known for having one of the most economically important fruits, i.e., the grape (Vitis vinifera). The deep phylogeny of the grape family was not resolved until a recent phylogenomic analysis of 417 nuclear genes from transcriptome data. However, it has been reported extensively that topologies based on nuclear and organellar genes may be incongruent due to differences in their evolutionary histories. Therefore, it is important to reconstruct a backbone phylogeny of the grape family using plastomes and mitochondrial genes. In this study, next-generation sequencing data sets of 27 species were obtained using genome skimming with total DNAs from silica-gel preserved tissue samples on an Illumina HiSeq 2500 instrument. Plastomes were assembled using the combination of de novo and reference genome (of V. vinifera) methods. Sixteen mitochondrial genes were also obtained via genome skimming using the reference genome of V. vinifera. Extensive phylogenetic analyses were performed using maximum likelihood and Bayesian methods. The topology based on either plastome data or mitochondrial genes is congruent with the one using hundreds of nuclear genes, indicating that the grape family did not exhibit significant reticulation at the deep level. The results showcase the power of genome skimming in capturing extensive phylogenetic data: especially from chloroplast and mitochondrial DNAs. PMID:26656830

Congruent Deep Relationships in the Grape Family (Vitaceae) Based on Sequences of Chloroplast Genomes and Mitochondrial Genes via Genome Skimming.

PubMed

Zhang, Ning; Wen, Jun; Zimmer, Elizabeth A

2015-01-01

Vitaceae is well-known for having one of the most economically important fruits, i.e., the grape (Vitis vinifera). The deep phylogeny of the grape family was not resolved until a recent phylogenomic analysis of 417 nuclear genes from transcriptome data. However, it has been reported extensively that topologies based on nuclear and organellar genes may be incongruent due to differences in their evolutionary histories. Therefore, it is important to reconstruct a backbone phylogeny of the grape family using plastomes and mitochondrial genes. In this study,next-generation sequencing data sets of 27 species were obtained using genome skimming with total DNAs from silica-gel preserved tissue samples on an Illumina NextSeq 500 instrument [corrected]. Plastomes were assembled using the combination of de novo and reference genome (of V. vinifera) methods. Sixteen mitochondrial genes were also obtained via genome skimming using the reference genome of V. vinifera. Extensive phylogenetic analyses were performed using maximum likelihood and Bayesian methods. The topology based on either plastome data or mitochondrial genes is congruent with the one using hundreds of nuclear genes, indicating that the grape family did not exhibit significant reticulation at the deep level. The results showcase the power of genome skimming in capturing extensive phylogenetic data: especially from chloroplast and mitochondrial DNAs.

[Isolation and identification of specific sequences correlated to cytoplasmic male sterility and fertile maintenance in cauliflower (Brassica oleracea var. botrytis)].

PubMed

Wang, Chun Guo; Chen, Xiao Qiang; Li, Hui; Zhao, Qian Cheng; Sun, De Ling; Song, Wen Qin

2008-02-01

Analysis of ISSR (Inter-Simple Sequence Repeat) and DDRT-PCR (Differential Display Reverse Transcriptase Polymerase Chain Reaction) was performed between cytoplasmic male sterility cauliflower ogura-A and its corresponding maintainer line ogura-B. Totally, 306 detectable bands were obtained by ISSR using thirty oligonucleotide primers. Commonly, six to twelve bands were produced per primer. Among all these primers only the amplification of primer ISSR3 was polymorphic, an 1100 bp specific band was only detected in maintainer line, named ISSR3(1100). Analysis of this sequence indicated that ISSR3(1100) was high homologous with the corresponding sequences of mitochondrial genome in Brassica napus and Arabidopsis thaliana,which suggested that ISSR3(1100) may derive from mitochondrial genome in cauliflower. To carry out DDRT-PCR analysis, three anchor primers and fifteen random primers were selected to combine. Totally, 1122 bands from 1 000 bp to 50 bp were detected. However, only four bands, named ogura-A 205, ogura-A383, ogura-B307 and ogura-B352, were confirmed to be different display in both lines. This result was further identified by reverse Northern dot blotting analysis. Among these four bands, ogura-A205 and ogura-A383 only express in cytoplasmic male sterility line, while ogura-B307 and ogura-B352 were only detected in maintainer line. Analysis of these sequences indicated that it was the first time that these four sequences were reported in cauliflower. Interestingly, ogura-A205 and ogura-B307 did not exhibit any similarities to other reported sequences in other species, more investigations were required to obtain further information. ogura-A383 and ogura-B352 were also two new sequences, they showed high similarities to corresponding chloroplast sequences of Arabidopsis thaliana and Brassica rapa subsp. pekinensis. So we speculated that these two sequences may derive from chloroplast genome. All these results obtained in this study offer new and significant information to investigate the molecular mechanism of cytoplasmic male sterility and fertile maintenance in cauliflower.

Genomic resources and draft assemblies of the human and porcine varieties of scabies mites, Sarcoptes scabiei var. hominis and var. suis.

PubMed

Mofiz, Ehtesham; Holt, Deborah C; Seemann, Torsten; Currie, Bart J; Fischer, Katja; Papenfuss, Anthony T

2016-06-02

The scabies mite, Sarcoptes scabiei, is a parasitic arachnid and cause of the infectious skin disease scabies in humans and mange in other animal species. Scabies infections are a major health problem, particularly in remote Indigenous communities in Australia, where secondary group A streptococcal and Staphylococcus aureus infections of scabies sores are thought to drive the high rate of rheumatic heart disease and chronic kidney disease. We sequenced the genome of two samples of Sarcoptes scabiei var. hominis obtained from unrelated patients with crusted scabies located in different parts of northern Australia using the Illumina HiSeq. We also sequenced samples of Sarcoptes scabiei var. suis from a pig model. Because of the small size of the scabies mite, these data are derived from pools of thousands of mites and are metagenomic, including host and microbiome DNA. We performed cleaning and de novo assembly and present Sarcoptes scabiei var. hominis and var. suis draft reference genomes. We have constructed a preliminary annotation of this reference comprising 13,226 putative coding sequences based on sequence similarity to known proteins. We have developed extensive genomic resources for the scabies mite, including reference genomes and a preliminary annotation.

GAMES identifies and annotates mutations in next-generation sequencing projects.

PubMed

Sana, Maria Elena; Iascone, Maria; Marchetti, Daniela; Palatini, Jeff; Galasso, Marco; Volinia, Stefano

2011-01-01

Next-generation sequencing (NGS) methods have the potential for changing the landscape of biomedical science, but at the same time pose several problems in analysis and interpretation. Currently, there are many commercial and public software packages that analyze NGS data. However, the limitations of these applications include output which is insufficiently annotated and of difficult functional comprehension to end users. We developed GAMES (Genomic Analysis of Mutations Extracted by Sequencing), a pipeline aiming to serve as an efficient middleman between data deluge and investigators. GAMES attains multiple levels of filtering and annotation, such as aligning the reads to a reference genome, performing quality control and mutational analysis, integrating results with genome annotations and sorting each mismatch/deletion according to a range of parameters. Variations are matched to known polymorphisms. The prediction of functional mutations is achieved by using different approaches. Overall GAMES enables an effective complexity reduction in large-scale DNA-sequencing projects. GAMES is available free of charge to academic users and may be obtained from http://aqua.unife.it/GAMES.

BAC sequencing using pooled methods.

PubMed

Saski, Christopher A; Feltus, F Alex; Parida, Laxmi; Haiminen, Niina

2015-01-01

Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly.

Draft Genome Sequence of Enterococcus casseliflavus PAVET15 Obtained from the Oviduct Infection of the Cattle Tick (Rhipicephalus microplus) in Jiutepec, Morelos, Mexico.

PubMed

Cossío-Bayúgar, R; Miranda-Miranda, E; Arreguín-Pérez, C A; Lozano, L; Peréz de la Rosa, D; Rocha-Martínez, M K; Bravo-Díaz, M A; Sachman-Ruiz, B

2017-04-20

Enterococcus spp. are Gram-positive lactic acid-producing bacteria found in the intestinal tracts of animals, like mammals, birds, and arthropods. Enterococcus spp. may cause oportunistic infections in vertebrate and invertebrate hosts. We report here the draft genome sequence of Enterococcus casseliflavus PAVET15 containing 3,722,480 bp, with 80 contigs, an N 50 of 179,476 bp, and 41.93% G+C content. Copyright © 2017 Cossío-Bayúgar et al.

Reduced representation bisulphite sequencing of the cattle genome reveals DNA methylation patterns

USDA-ARS?s Scientific Manuscript database

Using reduced representation bisulphite sequencing (RRBS), we obtained the first single-base-resolution maps of bovine DNA methylation in ten somatic tissues. In total, we observed 1,868,049 cytosines in the CG-enriched regions. Similar to the methylation patterns in other species, the CG context wa...

Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus Cuscuta.

PubMed

McNeal, Joel R; Kuehl, Jennifer V; Boore, Jeffrey L; de Pamphilis, Claude W

2007-10-24

Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle. Aside from loss of all ndh genes, Cuscuta exaltata retains photosynthetic and photorespiratory genes that evolve under strong selective constraint. Cuscuta obtusiflora has incurred substantially more change to its plastid genome, including loss of all genes for the plastid-encoded RNA polymerase. Despite extensive change in gene content and greatly increased rate of overall nucleotide substitution, C. obtusiflora also retains all photosynthetic and photorespiratory genes with only one minor exception. Although Epifagus virginiana, the only other parasitic plant with its plastid genome sequenced to date, has lost a largely overlapping set of transfer-RNA and ribosomal genes as Cuscuta, it has lost all genes related to photosynthesis and maintains a set of genes which are among the most divergent in Cuscuta. Analyses demonstrate photosynthetic genes are under the highest constraint of any genes within the plastid genomes of Cuscuta, indicating a function involving RuBisCo and electron transport through photosystems is still the primary reason for retention of the plastid genome in these species.

Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus Cuscuta

PubMed Central

McNeal, Joel R; Kuehl, Jennifer V; Boore, Jeffrey L; de Pamphilis, Claude W

2007-01-01

Background Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle. Results Aside from loss of all ndh genes, Cuscuta exaltata retains photosynthetic and photorespiratory genes that evolve under strong selective constraint. Cuscuta obtusiflora has incurred substantially more change to its plastid genome, including loss of all genes for the plastid-encoded RNA polymerase. Despite extensive change in gene content and greatly increased rate of overall nucleotide substitution, C. obtusiflora also retains all photosynthetic and photorespiratory genes with only one minor exception. Conclusion Although Epifagus virginiana, the only other parasitic plant with its plastid genome sequenced to date, has lost a largely overlapping set of transfer-RNA and ribosomal genes as Cuscuta, it has lost all genes related to photosynthesis and maintains a set of genes which are among the most divergent in Cuscuta. Analyses demonstrate photosynthetic genes are under the highest constraint of any genes within the plastid genomes of Cuscuta, indicating a function involving RuBisCo and electron transport through photosystems is still the primary reason for retention of the plastid genome in these species. PMID:17956636

Quantifying the Number of Independent Organelle DNA Insertions in Genome Evolution and Human Health.

PubMed

Hazkani-Covo, Einat; Martin, William F

2017-05-01

Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy. Because numts and nupts are typically identified through sequence comparison tools such as BLAST, disruption of insertions into smaller fragments can lead to systematic overestimation of numt and nupt frequencies. Accurate identification of numts and nupts is important, however, both for better understanding of their role during evolution, and for monitoring their increasingly evident role in human disease. Human populations are polymorphic for 141 numt loci, five numts are causal to genetic disease, and cancer genomic studies are revealing an abundance of numts associated with tumor progression. Here, we report investigation of salient parameters involved in obtaining accurate estimates of numt and nupt numbers in genome sequence data. Numts and nupts from 44 sequenced eukaryotic genomes reveal lineage-specific differences in the number, relative age and frequency of insertional events as well as lineage-specific dynamics of their postinsertional fragmentation. Our findings outline the main technical parameters influencing accurate identification and frequency estimation of numts in genomic studies pertinent to both evolution and human health. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Secure distributed genome analysis for GWAS and sequence comparison computation.

PubMed

Zhang, Yihua; Blanton, Marina; Almashaqbeh, Ghada

2015-01-01

The rapid increase in the availability and volume of genomic data makes significant advances in biomedical research possible, but sharing of genomic data poses challenges due to the highly sensitive nature of such data. To address the challenges, a competition for secure distributed processing of genomic data was organized by the iDASH research center. In this work we propose techniques for securing computation with real-life genomic data for minor allele frequency and chi-squared statistics computation, as well as distance computation between two genomic sequences, as specified by the iDASH competition tasks. We put forward novel optimizations, including a generalization of a version of mergesort, which might be of independent interest. We provide implementation results of our techniques based on secret sharing that demonstrate practicality of the suggested protocols and also report on performance improvements due to our optimization techniques. This work describes our techniques, findings, and experimental results developed and obtained as part of iDASH 2015 research competition to secure real-life genomic computations and shows feasibility of securely computing with genomic data in practice.

Secure distributed genome analysis for GWAS and sequence comparison computation

PubMed Central

2015-01-01

Background The rapid increase in the availability and volume of genomic data makes significant advances in biomedical research possible, but sharing of genomic data poses challenges due to the highly sensitive nature of such data. To address the challenges, a competition for secure distributed processing of genomic data was organized by the iDASH research center. Methods In this work we propose techniques for securing computation with real-life genomic data for minor allele frequency and chi-squared statistics computation, as well as distance computation between two genomic sequences, as specified by the iDASH competition tasks. We put forward novel optimizations, including a generalization of a version of mergesort, which might be of independent interest. Results We provide implementation results of our techniques based on secret sharing that demonstrate practicality of the suggested protocols and also report on performance improvements due to our optimization techniques. Conclusions This work describes our techniques, findings, and experimental results developed and obtained as part of iDASH 2015 research competition to secure real-life genomic computations and shows feasibility of securely computing with genomic data in practice. PMID:26733307

Differential repetitive DNA composition in the centromeric region of chromosomes of Amazonian lizard species in the family Teiidae

PubMed Central

Carvalho, Natalia D. M.; Carmo, Edson; Neves, Rogerio O.; Schneider, Carlos Henrique; Gross, Maria Claudia

2016-01-01

Abstract Differences in heterochromatin distribution patterns and its composition were observed in Amazonian teiid species. Studies have shown repetitive DNA harbors heterochromatic blocks which are located in centromeric and telomeric regions in Ameiva ameiva (Linnaeus, 1758), Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868), and Tupinambis teguixin (Linnaeus, 1758). In Cnemidophorus sp.1, repetitive DNA has multiple signals along all chromosomes. The aim of this study was to characterize moderately and highly repetitive DNA sequences by Cot1-DNA from Ameiva ameiva and Cnemidophorus sp.1 genomes through cloning and DNA sequencing, as well as mapping them chromosomally to better understand its organization and genome dynamics. The results of sequencing of DNA libraries obtained by Cot1-DNA showed that different microsatellites, transposons, retrotransposons, and some gene families also comprise the fraction of repetitive DNA in the teiid species. FISH using Cot1-DNA probes isolated from both Ameiva ameiva and Cnemidophorus sp.1 showed these sequences mainly located in heterochromatic centromeric, and telomeric regions in Ameiva ameiva, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin chromosomes, indicating they play structural and functional roles in the genome of these species. In Cnemidophorus sp.1, Cot1-DNA probe isolated from Ameiva ameiva had multiple interstitial signals on chromosomes, whereas mapping of Cot1-DNA isolated from the Ameiva ameiva and Cnemidophorus sp.1 highlighted centromeric regions of some chromosomes. Thus, the data obtained showed that many repetitive DNA classes are part of the genome of Ameiva ameiva, Cnemidophorus sp.1, Kentroyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin, and these sequences are shared among the analyzed teiid species, but they were not always allocated at the same chromosome position. PMID:27551343

Differential repetitive DNA composition in the centromeric region of chromosomes of Amazonian lizard species in the family Teiidae.

PubMed

Carvalho, Natalia D M; Carmo, Edson; Neves, Rogerio O; Schneider, Carlos Henrique; Gross, Maria Claudia

2016-01-01

Differences in heterochromatin distribution patterns and its composition were observed in Amazonian teiid species. Studies have shown repetitive DNA harbors heterochromatic blocks which are located in centromeric and telomeric regions in Ameiva ameiva (Linnaeus, 1758), Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868), and Tupinambis teguixin (Linnaeus, 1758). In Cnemidophorus sp.1, repetitive DNA has multiple signals along all chromosomes. The aim of this study was to characterize moderately and highly repetitive DNA sequences by C ot1-DNA from Ameiva ameiva and Cnemidophorus sp.1 genomes through cloning and DNA sequencing, as well as mapping them chromosomally to better understand its organization and genome dynamics. The results of sequencing of DNA libraries obtained by C ot1-DNA showed that different microsatellites, transposons, retrotransposons, and some gene families also comprise the fraction of repetitive DNA in the teiid species. FISH using C ot1-DNA probes isolated from both Ameiva ameiva and Cnemidophorus sp.1 showed these sequences mainly located in heterochromatic centromeric, and telomeric regions in Ameiva ameiva, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin chromosomes, indicating they play structural and functional roles in the genome of these species. In Cnemidophorus sp.1, C ot1-DNA probe isolated from Ameiva ameiva had multiple interstitial signals on chromosomes, whereas mapping of C ot1-DNA isolated from the Ameiva ameiva and Cnemidophorus sp.1 highlighted centromeric regions of some chromosomes. Thus, the data obtained showed that many repetitive DNA classes are part of the genome of Ameiva ameiva, Cnemidophorus sp.1, Kentroyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin, and these sequences are shared among the analyzed teiid species, but they were not always allocated at the same chromosome position.

Genome-wide analysis of wild-type Epstein-Barr virus genomes derived from healthy individuals of the 1,000 Genomes Project.

PubMed

Santpere, Gabriel; Darre, Fleur; Blanco, Soledad; Alcami, Antonio; Villoslada, Pablo; Mar Albà, M; Navarro, Arcadi

2014-04-01

Most people in the world (∼90%) are infected by the Epstein-Barr virus (EBV), which establishes itself permanently in B cells. Infection by EBV is related to a number of diseases including infectious mononucleosis, multiple sclerosis, and different types of cancer. So far, only seven complete EBV strains have been described, all of them coming from donors presenting EBV-related diseases. To perform a detailed comparative genomic analysis of EBV including, for the first time, EBV strains derived from healthy individuals, we reconstructed EBV sequences infecting lymphoblastoid cell lines (LCLs) from the 1000 Genomes Project. As strain B95-8 was used to transform B cells to obtain LCLs, it is always present, but a specific deletion in its genome sets it apart from natural EBV strains. After studying hundreds of individuals, we determined the presence of natural EBV in at least 10 of them and obtained a set of variants specific to wild-type EBV. By mapping the natural EBV reads into the EBV reference genome (NC007605), we constructed nearly complete wild-type viral genomes from three individuals. Adding them to the five disease-derived EBV genomic sequences available in the literature, we performed an in-depth comparative genomic analysis. We found that latency genes harbor more nucleotide diversity than lytic genes and that six out of nine latency-related genes, as well as other genes involved in viral attachment and entry into host cells, packaging, and the capsid, present the molecular signature of accelerated protein evolution rates, suggesting rapid host-parasite coevolution.

The complete mitochondrial genome of the Anabas testudineus (Perciformes, Anabantidae) and its comparison with other related fish species.

PubMed

Behera, Bijay Kumar; Baisvar, Vishwamitra Singh; Kumari, Kavita; Rout, Ajaya Kumar; Pakrashi, Sudip; Paria, Prasenjet; Rao, A R; Rai, Anil

2017-03-01

In the present study, the complete mitochondrial genome sequence of Anabas testudineusis reported using PGM sequencer (Ion Torrent, Life Technologies, La Jolla, CA). The complete mitogenome of climbing perch, A. testudineusis obtained by the de novo sequences assembly of genomic reads using the Torrent Mapping Alignment Program (TMAP), which is 16 603 bp in length. The mitogenome of A. testudineus composed of 13 protein- coding genes, two rRNA, and 22 tRNAs. Here, 20 tRNAs genes showed typical clover leaf model, and D-Loop as the control region along with gene order and organization, being closely similar to Osphronemidae and most of other Perciformes fish mitogenomes of NCBI databases. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of earlier reported A. testudineus. The phylogenetic analysis of Anabantidae depicted that their mitogenomes are closely related to each other. The complete mitogenome sequence of A. testudineus would be helpful in understanding the population genetics, phylogenetics, and evolution of Anabantidae.

Integrative Clinical Genomics of Metastatic Cancer

PubMed Central

Robinson, Dan R.; Wu, Yi-Mi; Lonigro, Robert J.; Vats, Pankaj; Cobain, Erin; Everett, Jessica; Cao, Xuhong; Rabban, Erica; Kumar-Sinha, Chandan; Raymond, Victoria; Schuetze, Scott; Alva, Ajjai; Siddiqui, Javed; Chugh, Rashmi; Worden, Francis; Zalupski, Mark M.; Innis, Jeffrey; Mody, Rajen J.; Tomlins, Scott A.; Lucas, David; Baker, Laurence H.; Ramnath, Nithya; Schott, Ann F.; Hayes, Daniel F.; Vijai, Joseph; Offit, Kenneth; Stoffel, Elena M.; Roberts, J. Scott; Smith, David C.; Kunju, Lakshmi P.; Talpaz, Moshe; Cieslik, Marcin; Chinnaiyan, Arul M.

2017-01-01

SUMMARY Metastasis is the primary cause of cancer-related deaths. While The Cancer Genome Atlas (TCGA) has sequenced primary tumor types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here, we perform whole exome and transcriptome sequencing of 500 adult patients with metastatic solid tumors of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53, CDKN2A, PTEN, PIK3CA, and RB1. Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing for the identification of gene fusions, pathway activation, and immune profiling. Integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers. PMID:28783718

Evaluation and integration of functional annotation pipelines for newly sequenced organisms: the potato genome as a test case.

PubMed

Amar, David; Frades, Itziar; Danek, Agnieszka; Goldberg, Tatyana; Sharma, Sanjeev K; Hedley, Pete E; Proux-Wera, Estelle; Andreasson, Erik; Shamir, Ron; Tzfadia, Oren; Alexandersson, Erik

2014-12-05

For most organisms, even if their genome sequence is available, little functional information about individual genes or proteins exists. Several annotation pipelines have been developed for functional analysis based on sequence, 'omics', and literature data. However, researchers encounter little guidance on how well they perform. Here, we used the recently sequenced potato genome as a case study. The potato genome was selected since its genome is newly sequenced and it is a non-model plant even if there is relatively ample information on individual potato genes, and multiple gene expression profiles are available. We show that the automatic gene annotations of potato have low accuracy when compared to a "gold standard" based on experimentally validated potato genes. Furthermore, we evaluate six state-of-the-art annotation pipelines and show that their predictions are markedly dissimilar (Jaccard similarity coefficient of 0.27 between pipelines on average). To overcome this discrepancy, we introduce a simple GO structure-based algorithm that reconciles the predictions of the different pipelines. We show that the integrated annotation covers more genes, increases by over 50% the number of highly co-expressed GO processes, and obtains much higher agreement with the gold standard. We find that different annotation pipelines produce different results, and show how to integrate them into a unified annotation that is of higher quality than each single pipeline. We offer an improved functional annotation of both PGSC and ITAG potato gene models, as well as tools that can be applied to additional pipelines and improve annotation in other organisms. This will greatly aid future functional analysis of '-omics' datasets from potato and other organisms with newly sequenced genomes. The new potato annotations are available with this paper.

The human homolog of S. cerevisiae CDC27, CDC27 Hs, is encoded by a highly conserved intronless gene present in multiple copies in the human genome

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

Devor, E.J.; Dill-Devor, R.M.

1994-09-01

We have obtained a number of unique sequences via PCR amplification of human genomic DNA using degenerate primers under low stringency (42{degrees}C). One of these, an 853 bp product, has been identified as a partial genomic sequence of the human homolog of the S. cerevisiae CDC27 gene, CDC27Hs (GenBank No. U00001). This gene, reported by Turgendreich et al. is also designated EST00556 from Adams et al. We have undertaken a more detailed examination of our sequence, MCP34N, and have found that: 1. the genomic sequence is nearly identical to CDC27Hs over its entire 853 bp length; 2. an MCP34N-specific PCRmore » assay of several non-human primate species reveals amplification products in chimpanzee and gorilla genomes having greater than 90% sequence identity with CDC27Hs; and 3. an MCP34N-specific PCR assay of the BIOS hybrid cell line panel gives a discordancy pattern suggesting multiple loci. Based upon these data, we present the following initial characterization: 1. the complete MCP34N sequence identity with CDC27Hs indicates that the latter is encoded by an intronless gene; 2. CDC27Hs is highly conserved among higher primates; and 3. CDC27Hs is present in multiple copies in the human genome. These characteristics, taken together with those initially reported for CDC27Hs, suggest that this is an old gene that carries out an important but, as yet, unknown function in the human brain.« less

The first complete mitochondrial genome of Bactrocera tsuneonis (Miyake) (Diptera: Tephritidae) by next-generation sequencing and its phylogenetic implications.

PubMed

Zhang, Yue; Feng, Shiqian; Zeng, Yiying; Ning, Hong; Liu, Lijun; Zhao, Zihua; Jiang, Fan; Li, Zhihong

2018-06-23

Bactrocera tsuneonis (Miyake), generally known as the Japanese orange fly, is considered to be a major pest of commercial citrus crops. It has a limited distribution in China, Japan and Vietnam, but it has the potential to invade areas outside of Asia. More genetic information of B. tsuneonis should be obtained in order to develop effective methodologies for rapid and accurate molecular identification due to the difficulty of distinguishing it from Bactrocera minax based on morphological features. We report here the whole mitochondrial genome of B. tsuneonis sequenced by next-generation sequencing. This mitogenome sequence had a total length of 15,865 bp, a typical circular molecule comprising 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region (A + T-rich control region). The structure and organization of the molecule were typical and similar compared with the published homologous sequences of other fruit flies in Tephritidae. The phylogenetic analyses based on the mitochondrial genome data presented a close genetic relationship between B. tsuneonis and B. minax. This is the first report of the complete mitochondrial genome of B. tsuneonis, and it can be used in further studies of species diagnosis, evolutionary biology, prevention and control. Copyright © 2018. Published by Elsevier B.V.

Coval: Improving Alignment Quality and Variant Calling Accuracy for Next-Generation Sequencing Data

PubMed Central

Kosugi, Shunichi; Natsume, Satoshi; Yoshida, Kentaro; MacLean, Daniel; Cano, Liliana; Kamoun, Sophien; Terauchi, Ryohei

2013-01-01

Accurate identification of DNA polymorphisms using next-generation sequencing technology is challenging because of a high rate of sequencing error and incorrect mapping of reads to reference genomes. Currently available short read aligners and DNA variant callers suffer from these problems. We developed the Coval software to improve the quality of short read alignments. Coval is designed to minimize the incidence of spurious alignment of short reads, by filtering mismatched reads that remained in alignments after local realignment and error correction of mismatched reads. The error correction is executed based on the base quality and allele frequency at the non-reference positions for an individual or pooled sample. We demonstrated the utility of Coval by applying it to simulated genomes and experimentally obtained short-read data of rice, nematode, and mouse. Moreover, we found an unexpectedly large number of incorrectly mapped reads in ‘targeted’ alignments, where the whole genome sequencing reads had been aligned to a local genomic segment, and showed that Coval effectively eliminated such spurious alignments. We conclude that Coval significantly improves the quality of short-read sequence alignments, thereby increasing the calling accuracy of currently available tools for SNP and indel identification. Coval is available at http://sourceforge.net/projects/coval105/. PMID:24116042

IVAG: An Integrative Visualization Application for Various Types of Genomic Data Based on R-Shiny and the Docker Platform.

PubMed

Lee, Tae-Rim; Ahn, Jin Mo; Kim, Gyuhee; Kim, Sangsoo

2017-12-01

Next-generation sequencing (NGS) technology has become a trend in the genomics research area. There are many software programs and automated pipelines to analyze NGS data, which can ease the pain for traditional scientists who are not familiar with computer programming. However, downstream analyses, such as finding differentially expressed genes or visualizing linkage disequilibrium maps and genome-wide association study (GWAS) data, still remain a challenge. Here, we introduce a dockerized web application written in R using the Shiny platform to visualize pre-analyzed RNA sequencing and GWAS data. In addition, we have integrated a genome browser based on the JBrowse platform and an automated intermediate parsing process required for custom track construction, so that users can easily build and navigate their personal genome tracks with in-house datasets. This application will help scientists perform series of downstream analyses and obtain a more integrative understanding about various types of genomic data by interactively visualizing them with customizable options.

Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast

PubMed Central

Oud, Bart; Maris, Antonius J A; Daran, Jean-Marc; Pronk, Jack T

2012-01-01

Successful reverse engineering of mutants that have been obtained by nontargeted strain improvement has long presented a major challenge in yeast biotechnology. This paper reviews the use of genome-wide approaches for analysis of Saccharomyces cerevisiae strains originating from evolutionary engineering or random mutagenesis. On the basis of an evaluation of the strengths and weaknesses of different methods, we conclude that for the initial identification of relevant genetic changes, whole genome sequencing is superior to other analytical techniques, such as transcriptome, metabolome, proteome, or array-based genome analysis. Key advantages of this technique over gene expression analysis include the independency of genome sequences on experimental context and the possibility to directly and precisely reproduce the identified changes in naive strains. The predictive value of genome-wide analysis of strains with industrially relevant characteristics can be further improved by classical genetics or simultaneous analysis of strains derived from parallel, independent strain improvement lineages. PMID:22152095

Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast.

PubMed

Oud, Bart; van Maris, Antonius J A; Daran, Jean-Marc; Pronk, Jack T

2012-03-01

Successful reverse engineering of mutants that have been obtained by nontargeted strain improvement has long presented a major challenge in yeast biotechnology. This paper reviews the use of genome-wide approaches for analysis of Saccharomyces cerevisiae strains originating from evolutionary engineering or random mutagenesis. On the basis of an evaluation of the strengths and weaknesses of different methods, we conclude that for the initial identification of relevant genetic changes, whole genome sequencing is superior to other analytical techniques, such as transcriptome, metabolome, proteome, or array-based genome analysis. Key advantages of this technique over gene expression analysis include the independency of genome sequences on experimental context and the possibility to directly and precisely reproduce the identified changes in naive strains. The predictive value of genome-wide analysis of strains with industrially relevant characteristics can be further improved by classical genetics or simultaneous analysis of strains derived from parallel, independent strain improvement lineages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data.

PubMed

Park, Doori; Park, Su-Hyun; Ban, Yong Wook; Kim, Youn Shic; Park, Kyoung-Cheul; Kim, Nam-Soo; Kim, Ju-Kon; Choi, Ik-Young

2017-08-15

Genetically modified crops (GM crops) have been developed to improve the agricultural traits of modern crop cultivars. Safety assessments of GM crops are of paramount importance in research at developmental stages and before releasing transgenic plants into the marketplace. Sequencing technology is developing rapidly, with higher output and labor efficiencies, and will eventually replace existing methods for the molecular characterization of genetically modified organisms. To detect the transgenic insertion locations in the three GM rice gnomes, Illumina sequencing reads are mapped and classified to the rice genome and plasmid sequence. The both mapped reads are classified to characterize the junction site between plant and transgene sequence by sequence alignment. Herein, we present a next generation sequencing (NGS)-based molecular characterization method, using transgenic rice plants SNU-Bt9-5, SNU-Bt9-30, and SNU-Bt9-109. Specifically, using bioinformatics tools, we detected the precise insertion locations and copy numbers of transfer DNA, genetic rearrangements, and the absence of backbone sequences, which were equivalent to results obtained from Southern blot analyses. NGS methods have been suggested as an effective means of characterizing and detecting transgenic insertion locations in genomes. Our results demonstrate the use of a combination of NGS technology and bioinformatics approaches that offers cost- and time-effective methods for assessing the safety of transgenic plants.

Personalized medicine in thrombosis: back to the future

PubMed Central

Nagalla, Srikanth

2016-01-01

Most physicians believe they practiced personalized medicine prior to the genomics era that followed the sequencing of the human genome. The focus of personalized medicine has been primarily genomic medicine, wherein it is hoped that the nucleotide dissimilarities among different individuals would provide clinicians with more precise understanding of physiology, more refined diagnoses, better disease risk assessment, earlier detection and monitoring, and tailored treatments to the individual patient. However, to date, the “genomic bench” has not worked itself to the clinical thrombosis bedside. In fact, traditional plasma-based hemostasis-thrombosis laboratory testing, by assessing functional pathways of coagulation, may better help manage venous thrombotic disease than a single DNA variant with a small effect size. There are some new and exciting discoveries in the genetics of platelet reactivity pertaining to atherothrombotic disease. Despite a plethora of genetic/genomic data on platelet reactivity, there are relatively little actionable pharmacogenetic data with antiplatelet agents. Nevertheless, it is crucial for genome-wide DNA/RNA sequencing to continue in research settings for causal gene discovery, pharmacogenetic purposes, and gene-gene and gene-environment interactions. The potential of genomics to advance medicine will require integration of personal data that are obtained in the patient history: environmental exposures, diet, social data, etc. Furthermore, without the ritual of obtaining this information, we will have depersonalized medicine, which lacks the precision needed for the research required to eventually incorporate genomics into routine, optimal, and value-added clinical care. PMID:26847245

Cloning, analysis and functional annotation of expressed sequence tags from the Earthworm Eisenia fetida

PubMed Central

Pirooznia, Mehdi; Gong, Ping; Guan, Xin; Inouye, Laura S; Yang, Kuan; Perkins, Edward J; Deng, Youping

2007-01-01

Background Eisenia fetida, commonly known as red wiggler or compost worm, belongs to the Lumbricidae family of the Annelida phylum. Little is known about its genome sequence although it has been extensively used as a test organism in terrestrial ecotoxicology. In order to understand its gene expression response to environmental contaminants, we cloned 4032 cDNAs or expressed sequence tags (ESTs) from two E. fetida libraries enriched with genes responsive to ten ordnance related compounds using suppressive subtractive hybridization-PCR. Results A total of 3144 good quality ESTs (GenBank dbEST accession number EH669363–EH672369 and EL515444–EL515580) were obtained from the raw clone sequences after cleaning. Clustering analysis yielded 2231 unique sequences including 448 contigs (from 1361 ESTs) and 1783 singletons. Comparative genomic analysis showed that 743 or 33% of the unique sequences shared high similarity with existing genes in the GenBank nr database. Provisional function annotation assigned 830 Gene Ontology terms to 517 unique sequences based on their homology with the annotated genomes of four model organisms Drosophila melanogaster, Mus musculus, Saccharomyces cerevisiae, and Caenorhabditis elegans. Seven percent of the unique sequences were further mapped to 99 Kyoto Encyclopedia of Genes and Genomes pathways based on their matching Enzyme Commission numbers. All the information is stored and retrievable at a highly performed, web-based and user-friendly relational database called EST model database or ESTMD version 2. Conclusion The ESTMD containing the sequence and annotation information of 4032 E. fetida ESTs is publicly accessible at . PMID:18047730

Mitochondrial genome of the endangered marine gastropod Strombus gigas Linnaeus, 1758 (Mollusca: Gastropoda).

PubMed

Márquez, Edna J; Castro, Erick R; Alzate, Juan F

2016-01-01

The queen conch Strombus gigas is an endangered marine gastropod of significant economic importance across the Greater Caribbean region. This work reports for the first time the complete mitochondrial genome of S. gigas, obtained by FLX 454 pyrosequencing. The mtDNA genome encodes for 13 proteins, 22 tRNAs and 2 ribosomal RNAs. In addition, the coding sequences and gene synteny were similar to other previously reported mitogenomes of gastropods.

Draft Genome Sequence of Geobacillus sp. LEMMY01, a Thermophilic Bacterium Isolated from the Site of a Burning Grass Pile.

PubMed

de Souza, Yuri Pinheiro Alves; da Mota, Fábio Faria; Rosado, Alexandre Soares

2017-05-11

We report here the 3,586,065-bp draft genome of Geobacillus sp. LEMMY01, which was isolated (axenic culture) from a thermophilic chemolitoautotrophic consortium obtained from the site of a burning grass pile. The genome contains biosynthetic gene clusters coding for secondary metabolites, such as terpene and lantipeptide, confirming the biotechnological potential of this strain. Copyright © 2017 de Souza et al.

Draft Genome Sequence of a Dictyoglomus sp. from an Enrichment Culture of a New Zealand Geothermal Spring.

PubMed

Reysenbach, Anna-Louise; Donaho, John A; Kelley, John F; St John, Emily; Turner, Christina; Podar, Mircea; Stott, Matthew B

2018-03-15

A draft genome of a novel Dictyoglomus sp., NZ13-RE01, was obtained from a New Zealand hot spring enrichment culture. The 1,927,012-bp genome is similar in both size and G+C content to other Dictyoglomus spp. Like its relatives, Dictyoglomus sp. NZ13-RE01 encodes many genes involved in complex carbohydrate metabolism. Copyright © 2018 Reysenbach et al.

Complete chloroplast genome sequences of Solanum commersonii and its application to chloroplast genotype in somatic hybrids with Solanum tuberosum.

PubMed

Cho, Kwang-Soo; Cheon, Kyeong-Sik; Hong, Su-Young; Cho, Ji-Hong; Im, Ju-Seong; Mekapogu, Manjulatha; Yu, Yei-Soo; Park, Tae-Ho

2016-10-01

Chloroplast genome of Solanum commersonii and S olanum tuberosum were completely sequenced, and Indel markers were successfully applied to distinguish chlorotypes demonstrating the chloroplast genome was randomly distributed during protoplast fusion. Somatic hybridization has been widely employed for the introgression of resistance to several diseases from wild Solanum species to overcome sexual barriers in potato breeding. Solanum commersonii is a major resource used as a parent line in somatic hybridization to improve bacterial wilt resistance in interspecies transfer to cultivated potato (S. tuberosum). Here, we sequenced the complete chloroplast genomes of Lz3.2 (S. commersonii) and S. tuberosum (PT56), which were used to develop fusion products, then compared them with those of five members of the Solanaceae family, S. tuberosum, Capsicum annum, S. lycopersicum, S. bulbocastanum and S. nigrum and Coffea arabica as an out-group. We then developed Indel markers for application in chloroplast genotyping. The complete chloroplast genome of Lz3.2 is composed of 155,525 bp, which is larger than the PT56 genome with 155,296 bp. Gene content, order and orientation of the S. commersonii chloroplast genome were highly conserved with those of other Solanaceae species, and the phylogenetic tree revealed that S. commersonii is located within the same node of S. tuberosum. However, sequence alignment revealed nine Indels between S. commersonii and S. tuberosum in their chloroplast genomes, allowing two Indel markers to be developed. The markers could distinguish the two species and were successfully applied to chloroplast genotyping (chlorotype) in somatic hybrids and their progenies. The results obtained in this study confirmed the random distribution of the chloroplast genome during protoplast fusion and its maternal inheritance and can be applied to select proper plastid genotypes in potato breeding program.

Genomic characterization of a Helicobacter pylori isolate from a patient with gastric cancer in China

PubMed Central

2014-01-01

Background Helicobacter pylori is well known for its relationship with the occurrence of several severe gastric diseases. The mechanisms of pathogenesis triggered by H. pylori are less well known. In this study, we report the genome sequence and genomic characterizations of H. pylori strain HLJ039 that was isolated from a patient with gastric cancer in the Chinese province of Heilongjiang, where there is a high incidence of gastric cancer. To investigate potential genomic features that may be involved in pathogenesis of carcinoma, the genome was compared to three previously sequenced genomes in this area. Result We obtained 42 contigs with a total length of 1,611,192 bp and predicted 1,687 coding sequences. Compared to strains isolated from gastritis and ulcers in this area, 10 different regions were identified as being unique for HLJ039; they mainly encoded type II restriction-modification enzyme, type II m6A methylase, DNA-cytosine methyltransferase, DNA methylase, and hypothetical proteins. A unique 547-bp fragment sharing 93% identity with a hypothetical protein of Helicobacter cinaedi ATCC BAA-847 was not present in any other previous H. pylori strains. Phylogenetic analysis based on core genome single nucleotide polymorphisms shows that HLJ039 is defined as hspEAsia subgroup, which belongs to the hpEastAsia group. Conclusion DNA methylations, variations of the genomic regions involved in restriction and modification systems, are the “hot” regions that may be related to the mechanism of H. pylori-induced gastric cancer. The genome sequence will provide useful information for the deep mining of potential mechanisms related to East Asian gastric cancer. PMID:24565107

Improving draft genome contiguity with reference-derived in silico mate-pair libraries.

PubMed

Grau, José Horacio; Hackl, Thomas; Koepfli, Klaus-Peter; Hofreiter, Michael

2018-05-01

Contiguous genome assemblies are a highly valued biological resource because of the higher number of completely annotated genes and genomic elements that are usable compared to fragmented draft genomes. Nonetheless, contiguity is difficult to obtain if only low coverage data and/or only distantly related reference genome assemblies are available. In order to improve genome contiguity, we have developed Cross-Species Scaffolding-a new pipeline that imports long-range distance information directly into the de novo assembly process by constructing mate-pair libraries in silico. We show how genome assembly metrics and gene prediction dramatically improve with our pipeline by assembling two primate genomes solely based on ∼30x coverage of shotgun sequencing data.

Genome evolution in Reptilia: in silico chicken mapping of 12,000 BAC-end sequences from two reptiles and a basal bird

PubMed Central

2009-01-01

Background With the publication of the draft chicken genome and the recent production of several BAC clone libraries from non-avian reptiles and birds, it is now possible to undertake more detailed comparative genomic studies in Reptilia. Of interest in particular are the genomic events that transformed the large, repeat-rich genomes of mammals and non-avian reptiles into the minimalist chicken genome. We have used paired BAC end sequences (BESs) from the American alligator (Alligator mississippiensis), painted turtle (Chrysemys picta) and emu (Dromaius novaehollandiae) to investigate patterns of sequence divergence, gene and retroelement content, and microsynteny between these species and chicken. Results From a total of 11,967 curated BESs, we successfully mapped 725, 773 and 2597 sequences in alligator, turtle, and emu, respectively, to sites in the draft chicken genome using a stringent BLAST protocol. Most commonly, sequences mapped to a single site in the chicken genome. Of 1675, 1828 and 2936 paired BESs obtained for alligator, turtle, and emu, respectively, a total of 34 (alligator, 2%), 24 (turtle, 1.3%) and 479 (emu, 16.3%) pairs were found to map with high confidence and in the correct orientation and with BAC-sized intermarker distances to single chicken chromosomes, including 25 such paired hits in emu mapping to the chicken Z chromosome. By determining the insert sizes of a subset of BAC clones from these three species, we also found a significant correlation between the intermarker distance in alligator and turtle and in chicken, with slopes as expected on the basis of the ratio of the genome sizes. Conclusion Our results suggest that a large number of small-scale chromosomal rearrangements and deletions in the lineage leading to chicken have drastically reduced the number of detected syntenies observed between the chicken and alligator, turtle, and emu genomes and imply that small deletions occurring widely throughout the genomes of reptilian and avian ancestors led to the ~50% reduction in genome size observed in birds compared to reptiles. We have also mapped and identified likely gene regions in hundreds of new BAC clones from these species. PMID:19607659

Genome evolution in Reptilia: in silico chicken mapping of 12,000 BAC-end sequences from two reptiles and a basal bird.

PubMed

Chapus, Charles; Edwards, Scott V

2009-07-14

With the publication of the draft chicken genome and the recent production of several BAC clone libraries from non-avian reptiles and birds, it is now possible to undertake more detailed comparative genomic studies in Reptilia. Of interest in particular are the genomic events that transformed the large, repeat-rich genomes of mammals and non-avian reptiles into the minimalist chicken genome. We have used paired BAC end sequences (BESs) from the American alligator (Alligator mississippiensis), painted turtle (Chrysemys picta) and emu (Dromaius novaehollandiae) to investigate patterns of sequence divergence, gene and retroelement content, and microsynteny between these species and chicken. From a total of 11,967 curated BESs, we successfully mapped 725, 773 and 2597 sequences in alligator, turtle, and emu, respectively, to sites in the draft chicken genome using a stringent BLAST protocol. Most commonly, sequences mapped to a single site in the chicken genome. Of 1675, 1828 and 2936 paired BESs obtained for alligator, turtle, and emu, respectively, a total of 34 (alligator, 2%), 24 (turtle, 1.3%) and 479 (emu, 16.3%) pairs were found to map with high confidence and in the correct orientation and with BAC-sized intermarker distances to single chicken chromosomes, including 25 such paired hits in emu mapping to the chicken Z chromosome. By determining the insert sizes of a subset of BAC clones from these three species, we also found a significant correlation between the intermarker distance in alligator and turtle and in chicken, with slopes as expected on the basis of the ratio of the genome sizes. Our results suggest that a large number of small-scale chromosomal rearrangements and deletions in the lineage leading to chicken have drastically reduced the number of detected syntenies observed between the chicken and alligator, turtle, and emu genomes and imply that small deletions occurring widely throughout the genomes of reptilian and avian ancestors led to the ~50% reduction in genome size observed in birds compared to reptiles. We have also mapped and identified likely gene regions in hundreds of new BAC clones from these species.

Draft Genome of Scalindua rubra, Obtained from the Interface Above the Discovery Deep Brine in the Red Sea, Sheds Light on Potential Salt Adaptation Strategies in Anammox Bacteria.

PubMed

Speth, Daan R; Lagkouvardos, Ilias; Wang, Yong; Qian, Pei-Yuan; Dutilh, Bas E; Jetten, Mike S M

2017-07-01

Several recent studies have indicated that members of the phylum Planctomycetes are abundantly present at the brine-seawater interface (BSI) above multiple brine pools in the Red Sea. Planctomycetes include bacteria capable of anaerobic ammonium oxidation (anammox). Here, we investigated the possibility of anammox at BSI sites using metagenomic shotgun sequencing of DNA obtained from the BSI above the Discovery Deep brine pool. Analysis of sequencing reads matching the 16S rRNA and hzsA genes confirmed presence of anammox bacteria of the genus Scalindua. Phylogenetic analysis of the 16S rRNA gene indicated that this Scalindua sp. belongs to a distinct group, separate from the anammox bacteria in the seawater column, that contains mostly sequences retrieved from high-salt environments. Using coverage- and composition-based binning, we extracted and assembled the draft genome of the dominant anammox bacterium. Comparative genomic analysis indicated that this Scalindua species uses compatible solutes for osmoadaptation, in contrast to other marine anammox bacteria that likely use a salt-in strategy. We propose the name Candidatus Scalindua rubra for this novel species, alluding to its discovery in the Red Sea.

Sequencing thousands of single-cell genomes with combinatorial indexing.

PubMed

Vitak, Sarah A; Torkenczy, Kristof A; Rosenkrantz, Jimi L; Fields, Andrew J; Christiansen, Lena; Wong, Melissa H; Carbone, Lucia; Steemers, Frank J; Adey, Andrew

2017-03-01

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

Building a genome database using an object-oriented approach.

PubMed

Barbasiewicz, Anna; Liu, Lin; Lang, B Franz; Burger, Gertraud

2002-01-01

GOBASE is a relational database that integrates data associated with mitochondria and chloroplasts. The most important data in GOBASE, i. e., molecular sequences and taxonomic information, are obtained from the public sequence data repository at the National Center for Biotechnology Information (NCBI), and are validated by our experts. Maintaining a curated genomic database comes with a towering labor cost, due to the shear volume of available genomic sequences and the plethora of annotation errors and omissions in records retrieved from public repositories. Here we describe our approach to increase automation of the database population process, thereby reducing manual intervention. As a first step, we used Unified Modeling Language (UML) to construct a list of potential errors. Each case was evaluated independently, and an expert solution was devised, and represented as a diagram. Subsequently, the UML diagrams were used as templates for writing object-oriented automation programs in the Java programming language.

Diagnosis of mitochondrial disorders by concomitant next-generation sequencing of the exome and mitochondrial genome

PubMed Central

Dinwiddie, Darrell L.; Smith, Laurie D.; Miller, Neil A.; Atherton, Andrea M.; Farrow, Emily G.; Strenk, Meghan E.; Soden, Sarah E.; Saunders, Carol J.; Kingsmore, Stephen F.

2015-01-01

Mitochondrial diseases are notoriously difficult to diagnose due to extreme locus and allelic heterogeneity, with both nuclear and mitochondrial genomes potentially liable. Using exome sequencing we demonstrate the ability to rapidly and cost effectively evaluate both the nuclear and mitochondrial genomes to obtain a molecular diagnosis for four patients with three distinct mitochondrial disorders. One patient was found to have Leigh syndrome due to a mutation in MT-ATP6, two affected siblings were discovered to be compound heterozygous for mutations in the NDUFV1 gene, which causes mitochondrial complex I deficiency, and one patient was found to have coenzyme Q10 deficiency due to compound heterozygous mutations in COQ2. In all cases conventional diagnostic testing failed to identify a molecular diagnosis. We suggest that additional studies should be conducted to evaluate exome sequencing as a primary diagnostic test for mitochondrial diseases, including those due to mtDNA mutations. PMID:23631824

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.

Molecular characterization of the Great Lakes viral hemorrhagic septicemia virus (VHSV) isolate from USA

PubMed Central

Ammayappan, Arun; Vakharia, Vikram N

2009-01-01

Background Viral hemorrhagic septicemia virus (VHSV) is a highly contagious viral disease of fresh and saltwater fish worldwide. VHSV caused several large scale fish kills in the Great Lakes area and has been found in 28 different host species. The emergence of VHS in the Great Lakes began with the isolation of VHSV from a diseased muskellunge (Esox masquinongy) caught from Lake St. Clair in 2003. VHSV is a member of the genus Novirhabdovirus, within the family Rhabdoviridae. It has a linear single-stranded, negative-sense RNA genome of approximately 11 kbp, with six genes. VHSV replicates in the cytoplasm and produces six monocistronic mRNAs. The gene order of VHSV is 3'-N-P-M-G-NV-L-5'. This study describes molecular characterization of the Great Lakes VHSV strain (MI03GL), and its phylogenetic relationships with selected European and North American isolates. Results The complete genomic sequences of VHSV-MI03GL strain was determined from cloned cDNA of six overlapping fragments, obtained by RT-PCR amplification of genomic RNA. The complete genome sequence of MI03GL comprises 11,184 nucleotides (GenBank GQ385941) with the gene order of 3'-N-P-M-G-NV-L-5'. These genes are separated by conserved gene junctions, with di-nucleotide gene spacers. The first 4 nucleotides at the termini of the VHSV genome are complementary and identical to other novirhadoviruses genomic termini. Sequence homology and phylogenetic analysis show that the Great Lakes virus is closely related to the Japanese strains JF00Ehi1 (96%) and KRRV9822 (95%). Among other novirhabdoviruses, VHSV shares highest sequence homology (62%) with snakehead rhabdovirus. Conclusion Phylogenetic tree obtained by comparing 48 glycoprotein gene sequences of different VHSV strains demonstrate that the Great Lakes VHSV is closely related to the North American and Japanese genotype IVa, but forms a distinct genotype IVb, which is clearly different from the three European genotypes. Molecular characterization of the Great Lakes isolate will be helpful in studying the pathogenesis of VHSV using a reverse genetics approach and developing efficient control strategies. PMID:19852863

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution.

PubMed

Verde, Ignazio; Abbott, Albert G; Scalabrin, Simone; Jung, Sook; Shu, Shengqiang; Marroni, Fabio; Zhebentyayeva, Tatyana; Dettori, Maria Teresa; Grimwood, Jane; Cattonaro, Federica; Zuccolo, Andrea; Rossini, Laura; Jenkins, Jerry; Vendramin, Elisa; Meisel, Lee A; Decroocq, Veronique; Sosinski, Bryon; Prochnik, Simon; Mitros, Therese; Policriti, Alberto; Cipriani, Guido; Dondini, Luca; Ficklin, Stephen; Goodstein, David M; Xuan, Pengfei; Del Fabbro, Cristian; Aramini, Valeria; Copetti, Dario; Gonzalez, Susana; Horner, David S; Falchi, Rachele; Lucas, Susan; Mica, Erica; Maldonado, Jonathan; Lazzari, Barbara; Bielenberg, Douglas; Pirona, Raul; Miculan, Mara; Barakat, Abdelali; Testolin, Raffaele; Stella, Alessandra; Tartarini, Stefano; Tonutti, Pietro; Arús, Pere; Orellana, Ariel; Wells, Christina; Main, Dorrie; Vizzotto, Giannina; Silva, Herman; Salamini, Francesco; Schmutz, Jeremy; Morgante, Michele; Rokhsar, Daniel S

2013-05-01

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree

PubMed Central

2013-01-01

Background With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. Results The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. Conclusions Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology. PMID:23497206

Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (Vaccinium macrocarpon Ait.).

PubMed

Zhu, H; Senalik, D; McCown, B H; Zeldin, E L; Speers, J; Hyman, J; Bassil, N; Hummer, K; Simon, P W; Zalapa, J E

2012-01-01

The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar 'HyRed'. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540-590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2-15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.

Characterization of a Novel Simian Immunodeficiency Virus (SIVmonNG1) Genome Sequence from a Mona Monkey (Cercopithecus mona)

PubMed Central

Barlow, Katrina L.; Ajao, Adebowale Oluwafemi; Clewley, Jonathan P.

2003-01-01

A novel simian immunodeficiency virus (SIV) sequence has been recovered from RNA extracted from the serum of a mona monkey (Cercopithecus mona) wild born in Nigeria. The sequence was obtained by using novel generic (degenerate) PCR primers and spans from two-thirds into the gag gene to the 3′ poly(A) tail of the SIVmonNG1 RNA genome. Analysis of the open reading frames revealed that the SIVmonNG1 genome codes for a Vpu protein, in addition to Gag, Pol, Vif, Vpr, Tat, Rev, Env, and Nef proteins. Previously, only lentiviruses infecting humans (human immunodeficiency virus type 1 [HIV-1]) and chimpanzees (SIVcpz) were known to have a vpu gene; more recently, this has also been found in SIVgsn from Cercopithecus nictitans. Overall, SIVmonNG1 most closely resembles SIVgsn: the env gene sequence groups with HIV-1/SIVcpz env sequences, whereas the pol gene sequence clusters closely with the pol sequence of SIVsyk from Cercopithecus albogaris. By bootscanning and similarity plotting, the first half of pol resembles SIVsyk, whereas the latter part is closer to SIVcol from Colobus guereza. The similarities between the complex mosaic genomes of SIVmonNG1 and SIVgsn are consistent with a shared or common lineage. These data further highlight the intricate nature of the relationships between the SIVs from different primate species and will be helpful for unraveling these associations. PMID:12768007

Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?

PubMed

Seneca, Sara; Vancampenhout, Kim; Van Coster, Rudy; Smet, Joél; Lissens, Willy; Vanlander, Arnaud; De Paepe, Boel; Jonckheere, An; Stouffs, Katrien; De Meirleir, Linda

2015-01-01

Next-generation sequencing (NGS), an innovative sequencing technology that enables the successful analysis of numerous gene sequences in a massive parallel sequencing approach, has revolutionized the field of molecular biology. Although NGS was introduced in a rather recent past, the technology has already demonstrated its potential and effectiveness in many research projects, and is now on the verge of being introduced into the diagnostic setting of routine laboratories to delineate the molecular basis of genetic disease in undiagnosed patient samples. We tested a benchtop device on retrospective genomic DNA (gDNA) samples of controls and patients with a clinical suspicion of a mitochondrial DNA disorder. This Ion Torrent Personal Genome Machine platform is a high-throughput sequencer with a fast turnaround time and reasonable running costs. We challenged the chemistry and technology with the analysis and processing of a mutational spectrum composed of samples with single-nucleotide substitutions, indels (insertions and deletions) and large single or multiple deletions, occasionally in heteroplasmy. The output data were compared with previously obtained conventional dideoxy sequencing results and the mitochondrial revised Cambridge Reference Sequence (rCRS). We were able to identify the majority of all nucleotide alterations, but three false-negative results were also encountered in the data set. At the same time, the poor performance of the PGM instrument in regions associated with homopolymeric stretches generated many false-positive miscalls demanding additional manual curation of the data.

Sequence-based novel genomic microsatellite markers for robust genotyping purposes in foxtail millet [Setaria italica (L.) P. Beauv].

PubMed

Gupta, Sarika; Kumari, Kajal; Sahu, Pranav Pankaj; Vidapu, Sudhakar; Prasad, Manoj

2012-02-01

The unavailability of microsatellite markers and saturated genetic linkage map has restricted the genetic improvement of foxtail millet [Setaria italica (L.) P. Beauv.], despite the fact that in recent times it has been documented as a new model species for biofuel grasses. With the objective to generate a good number of microsatellite markers in foxtail millet cultivar 'Prasad', 690 clones were sequenced which generated 112.95 kb high quality sequences obtained from three genomic libraries each enriched with different microsatellite repeat motifs. Microsatellites were identified in 512 (74.2%) of the 690 positive clones and 172 primer pairs (pp) were successfully designed from 249 (48.6%) unique SSR-containing clones. The efficacies of the microsatellite containing genomic sequences were established by superior primer designing ability (69%), PCR amplification efficiency (85.5%) and polymorphic potential (52%) in the parents of F(2) mapping population. Out of 172 pp, functional 147 markers showed high level of cross-species amplification (~74%) in six grass species. Higher polymorphism rate and broad range of genetic diversity (0.30-0.69 averaging 0.58) obtained in constructed phylogenetic tree using 52 microsatellite markers, demonstrated the utility of markers in germplasm characterizations. In silico comparative mapping of 147 foxtail millet microsatellite containing sequences against the mapping data of sorghum (~18%), maize (~16%) and rice (~5%) indicated the presence of orthologous sequences of the foxtail millet in the respective species. The result thus demonstrates the applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species.

Draft Genome Sequence of a Novel Thermofilum sp. Strain from a New Zealand Hot Spring Enrichment Culture

DOE PAGES

Reysenbach, Anna-Louise; Donaho, John; Hinsch, Todd; ...

2018-02-22

A draft genome of a newThermofilumsp. strain was obtained from an enrichment culture metagenome. Like its relatives,Thermofilumsp. strain NZ13 is adapted to organic-rich thermal environments and has to depend on other organisms and the environment for some key amino acids, purines, and cofactors.

Draft Genome Sequence of a Novel Thermofilum sp. Strain from a New Zealand Hot Spring Enrichment Culture

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

Reysenbach, Anna-Louise; Donaho, John; Hinsch, Todd

A draft genome of a newThermofilumsp. strain was obtained from an enrichment culture metagenome. Like its relatives,Thermofilumsp. strain NZ13 is adapted to organic-rich thermal environments and has to depend on other organisms and the environment for some key amino acids, purines, and cofactors.

High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus

USDA-ARS?s Scientific Manuscript database

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a ‘fosmid-to-fosmid’ strategy, to better understand its race evolution and pathogenesis. The Pst genome is hi...

Genome Sequence of Klebsiella pneumoniae KpQ3, a DHA-1 β-Lactamase-Producing Nosocomial Isolate

PubMed Central

Tobes, Raquel; Codoñer, Francisco M.; López-Camacho, Elena; Salanueva, Iñigo J.; Manrique, Marina; Brozynska, Marta; Gómez-Gil, Rosa; Martínez-Blanch, Juan F.; Álvarez-Tejado, Miguel; Pareja, Eduardo

2013-01-01

Klebsiella pneumoniae KpQ3 is a multidrug-resistant isolate obtained from a blood culture of a patient in a burn unit in the Hospital Universitario La Paz (Madrid, Spain) in 2008. The genome contains multiple antibiotic resistance genes, including a plasmid-mediated DHA-1 cephalosporinase gene. PMID:23469341

Whole-genome sequencing for comparative genomics and de novo genome assembly.

PubMed

Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

2015-01-01

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

Functions of the 3′ and 5′ genome RNA regions of members of the genus Flavivirus

PubMed Central

Brinton, Margo A.; Basu, Mausumi

2015-01-01

The positive sense genomes of members of the genus Flavivirus in the family Flaviviridae are ~11 kb nts in length and have a 5′ type I cap but no 3′ poly A. The 5′ and 3′ terminal regions contain short conserved sequences that are proposed to be repeated remnants of an ancient sequence. However, the functions of most of these conserved sequences have not yet been determined. The terminal regions of the genome also contain multiple conserved RNA structures. Functional data for many of these structures has been obtained. Three sets of complementary 3′ and 5′ terminal region sequences, some of which are located in conserved RNA structures, interact to form a panhandle structure that is required for initiation of minus strand RNA synthesis with the 5′ terminal structure functioning as the promoter. How the switch from the terminal RNA structure base pairing to the long distance RNA-RNA interaction is triggered and regulated is not well understood but evidence suggests involvement of a cell protein binding to three sites on the 3′ terminal RNA structures and a cis-acting metastable 3′ RNA element in the 3′ terminal structure. Cell proteins may also be involved in facilitating exponential replication of nascent genomic RNA within replication vesicles at later times of infection cycle. Other conserved RNA structures and/or sequences in the 5′ and 3′ terminal regions have been proposed to regulate genome translation. Additional functions of the 5′ and 3′ terminal sequences have also been reported. PMID:25683510

Complete mitochondrial genome of freshwater shark Wallago attu (Bloch & Schneider) from Indus River Sindh, Pakistan.

PubMed

Laghari, Muhammad Younis; Lashari, Punhal; Xu, Peng; Zhao, Zixia; Jiang, Li; Narejo, Naeem Tariq; Xin, Baoping; Sun, Xiaowen; Zhang, Yan

2016-01-01

Complete mitochondrial genome of fresh water giant catfish, Wallago attu, was isolated by LA PCR (TakaRa LAtaq, Dalian, China); and sequenced by Sanger's method to obtain the complete mitochondrial genome. The complete mitogenome was 15,639 bp in length and contains 13 typical vertebrate protein-coding genes, 2 rRNA and 22 tRNA genes. The whole genome base composition was estimated to be 31.17% A, 28.15% C, 15.55% G and 25.12% T. The complete mitochondrial genome of catfish, W. attu, provides the fundamental tools for genetic breeding.

From algae to angiosperms–inferring the phylogeny of green plants (Viridiplantae) from 360 plastid genomes

PubMed Central

2014-01-01

Background Next-generation sequencing has provided a wealth of plastid genome sequence data from an increasingly diverse set of green plants (Viridiplantae). Although these data have helped resolve the phylogeny of numerous clades (e.g., green algae, angiosperms, and gymnosperms), their utility for inferring relationships across all green plants is uncertain. Viridiplantae originated 700-1500 million years ago and may comprise as many as 500,000 species. This clade represents a major source of photosynthetic carbon and contains an immense diversity of life forms, including some of the smallest and largest eukaryotes. Here we explore the limits and challenges of inferring a comprehensive green plant phylogeny from available complete or nearly complete plastid genome sequence data. Results We assembled protein-coding sequence data for 78 genes from 360 diverse green plant taxa with complete or nearly complete plastid genome sequences available from GenBank. Phylogenetic analyses of the plastid data recovered well-supported backbone relationships and strong support for relationships that were not observed in previous analyses of major subclades within Viridiplantae. However, there also is evidence of systematic error in some analyses. In several instances we obtained strongly supported but conflicting topologies from analyses of nucleotides versus amino acid characters, and the considerable variation in GC content among lineages and within single genomes affected the phylogenetic placement of several taxa. Conclusions Analyses of the plastid sequence data recovered a strongly supported framework of relationships for green plants. This framework includes: i) the placement of Zygnematophyceace as sister to land plants (Embryophyta), ii) a clade of extant gymnosperms (Acrogymnospermae) with cycads + Ginkgo sister to remaining extant gymnosperms and with gnetophytes (Gnetophyta) sister to non-Pinaceae conifers (Gnecup trees), and iii) within the monilophyte clade (Monilophyta), Equisetales + Psilotales are sister to Marattiales + leptosporangiate ferns. Our analyses also highlight the challenges of using plastid genome sequences in deep-level phylogenomic analyses, and we provide suggestions for future analyses that will likely incorporate plastid genome sequence data for thousands of species. We particularly emphasize the importance of exploring the effects of different partitioning and character coding strategies. PMID:24533922

Diagnostics for Yaws Eradication: Insights From Direct Next-Generation Sequencing of Cutaneous Strains of Treponema pallidum.

PubMed

Marks, Michael; Fookes, Maria; Wagner, Josef; Butcher, Robert; Ghinai, Rosanna; Sokana, Oliver; Sarkodie, Yaw-Adu; Lukehart, Sheila A; Solomon, Anthony W; Mabey, David C W; Thomson, Nicholas

2018-03-05

Yaws-like chronic ulcers can be caused by Treponema pallidum subspecies pertenue, Haemophilus ducreyi, or other, still-undefined bacteria. To permit accurate evaluation of yaws elimination efforts, programmatic use of molecular diagnostics is required. The accuracy and sensitivity of current tools remain unclear because our understanding of T. pallidum diversity is limited by the low number of sequenced genomes. We tested samples from patients with suspected yaws collected in the Solomon Islands and Ghana. All samples were from patients whose lesions had previously tested negative using the Centers for Disease Control and Prevention (CDC) diagnostic assay in widespread use. However, some of these patients had positive serological assays for yaws on blood. We used direct whole-genome sequencing to identify T. pallidum subsp pertenue strains missed by the current assay. From 45 Solomon Islands and 27 Ghanaian samples, 11 were positive for T. pallidum DNA using the species-wide quantitative polymerase chain reaction (PCR) assay, from which we obtained 6 previously undetected T. pallidum subsp pertenue whole-genome sequences. These show that Solomon Islands sequences represent distinct T. pallidum subsp pertenue clades. These isolates were invisible to the CDC diagnostic PCR assay, due to sequence variation in the primer binding site. Our data double the number of published T. pallidum subsp pertenue genomes. We show that Solomon Islands strains are undetectable by the PCR used in many studies and by health ministries. This assay is therefore not adequate for the eradication program. Next-generation genome sequence data are essential for these efforts. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Azospirillum Genomes Reveal Transition of Bacteria from Aquatic to Terrestrial Environments

PubMed Central

Khalsa-Moyers, Gurusahai; Alexandre, Gladys; Sukharnikov, Leonid O.; Wuichet, Kristin; Hurst, Gregory B.; McDonald, W. Hayes; Robertson, Jon S.; Barbe, Valérie; Calteau, Alexandra; Rouy, Zoé; Mangenot, Sophie; Prigent-Combaret, Claire; Normand, Philippe; Boyer, Mickaël; Siguier, Patricia; Dessaux, Yves; Elmerich, Claudine; Condemine, Guy; Krishnen, Ganisan; Kennedy, Ivan; Paterson, Andrew H.; González, Victor; Mavingui, Patrick; Zhulin, Igor B.

2011-01-01

Fossil records indicate that life appeared in marine environments ∼3.5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2.5 Gyr. Sequence analysis suggests that “hydrobacteria” and “terrabacteria” might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land. PMID:22216014

Large-scale chromosome folding versus genomic DNA sequences: A discrete double Fourier transform technique.

PubMed

Chechetkin, V R; Lobzin, V V

2017-08-07

Using state-of-the-art techniques combining imaging methods and high-throughput genomic mapping tools leaded to the significant progress in detailing chromosome architecture of various organisms. However, a gap still remains between the rapidly growing structural data on the chromosome folding and the large-scale genome organization. Could a part of information on the chromosome folding be obtained directly from underlying genomic DNA sequences abundantly stored in the databanks? To answer this question, we developed an original discrete double Fourier transform (DDFT). DDFT serves for the detection of large-scale genome regularities associated with domains/units at the different levels of hierarchical chromosome folding. The method is versatile and can be applied to both genomic DNA sequences and corresponding physico-chemical parameters such as base-pairing free energy. The latter characteristic is closely related to the replication and transcription and can also be used for the assessment of temperature or supercoiling effects on the chromosome folding. We tested the method on the genome of E. coli K-12 and found good correspondence with the annotated domains/units established experimentally. As a brief illustration of further abilities of DDFT, the study of large-scale genome organization for bacteriophage PHIX174 and bacterium Caulobacter crescentus was also added. The combined experimental, modeling, and bioinformatic DDFT analysis should yield more complete knowledge on the chromosome architecture and genome organization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

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

Bejerman, Nicolás, E-mail: n.bejerman@uq.edu.au; Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072; Giolitti, Fabián

Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cellmore » periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses.« less

Metavir 2: new tools for viral metagenome comparison and assembled virome analysis

PubMed Central

2014-01-01

Background Metagenomics, based on culture-independent sequencing, is a well-fitted approach to provide insights into the composition, structure and dynamics of environmental viral communities. Following recent advances in sequencing technologies, new challenges arise for existing bioinformatic tools dedicated to viral metagenome (i.e. virome) analysis as (i) the number of viromes is rapidly growing and (ii) large genomic fragments can now be obtained by assembling the huge amount of sequence data generated for each metagenome. Results To face these challenges, a new version of Metavir was developed. First, all Metavir tools have been adapted to support comparative analysis of viromes in order to improve the analysis of multiple datasets. In addition to the sequence comparison previously provided, viromes can now be compared through their k-mer frequencies, their taxonomic compositions, recruitment plots and phylogenetic trees containing sequences from different datasets. Second, a new section has been specifically designed to handle assembled viromes made of thousands of large genomic fragments (i.e. contigs). This section includes an annotation pipeline for uploaded viral contigs (gene prediction, similarity search against reference viral genomes and protein domains) and an extensive comparison between contigs and reference genomes. Contigs and their annotations can be explored on the website through specifically developed dynamic genomic maps and interactive networks. Conclusions The new features of Metavir 2 allow users to explore and analyze viromes composed of raw reads or assembled fragments through a set of adapted tools and a user-friendly interface. PMID:24646187

Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline.

PubMed

Ye, Weixing; Zhu, Lei; Liu, Yingying; Crickmore, Neil; Peng, Donghai; Ruan, Lifang; Sun, Ming

2012-07-01

We have designed a high-throughput system for the identification of novel crystal protein genes (cry) from Bacillus thuringiensis strains. The system was developed with two goals: (i) to acquire the mixed plasmid-enriched genomic sequence of B. thuringiensis using next-generation sequencing biotechnology, and (ii) to identify cry genes with a computational pipeline (using BtToxin_scanner). In our pipeline method, we employed three different kinds of well-developed prediction methods, BLAST, hidden Markov model (HMM), and support vector machine (SVM), to predict the presence of Cry toxin genes. The pipeline proved to be fast (average speed, 1.02 Mb/min for proteins and open reading frames [ORFs] and 1.80 Mb/min for nucleotide sequences), sensitive (it detected 40% more protein toxin genes than a keyword extraction method using genomic sequences downloaded from GenBank), and highly specific. Twenty-one strains from our laboratory's collection were selected based on their plasmid pattern and/or crystal morphology. The plasmid-enriched genomic DNA was extracted from these strains and mixed for Illumina sequencing. The sequencing data were de novo assembled, and a total of 113 candidate cry sequences were identified using the computational pipeline. Twenty-seven candidate sequences were selected on the basis of their low level of sequence identity to known cry genes, and eight full-length genes were obtained with PCR. Finally, three new cry-type genes (primary ranks) and five cry holotypes, which were designated cry8Ac1, cry7Ha1, cry21Ca1, cry32Fa1, and cry21Da1 by the B. thuringiensis Toxin Nomenclature Committee, were identified. The system described here is both efficient and cost-effective and can greatly accelerate the discovery of novel cry genes.

The FLEXGene repository: exploiting the fruits of the genome projects by creating a needed resource to face the challenges of the post-genomic era.

PubMed

Brizuela, Leonardo; Richardson, Aaron; Marsischky, Gerald; Labaer, Joshua

2002-01-01

Thanks to the results of the multiple completed and ongoing genome sequencing projects and to the newly available recombination-based cloning techniques, it is now possible to build gene repositories with no precedent in their composition, formatting, and potential. This new type of gene repository is necessary to address the challenges imposed by the post-genomic era, i.e., experimentation on a genome-wide scale. We are building the FLEXGene (Full Length EXpression-ready) repository. This unique resource will contain clones representing the complete ORFeome of different organisms, including Homo sapiens as well as several pathogens and model organisms. It will consist of a comprehensive, characterized (sequence-verified), and arrayed gene repository. This resource will allow full exploitation of the genomic information by enabling genome-wide scale experimentation at the level of functional/phenotypic assays as well as at the level of protein expression, purification, and analysis. Here we describe the rationale and construction of this resource and focus on the data obtained from the Saccharomyces cerevisiae project.

National Science Foundation-sponsored workshop report. Draft plan for soybean genomics.

PubMed

Stacey, Gary; Vodkin, Lila; Parrott, Wayne A; Shoemaker, Randy C

2004-05-01

Recent efforts to coordinate and define a research strategy for soybean (Glycine max) genomics began with the establishment of a Soybean Genetics Executive Committee, which will serve as a communication focal point between the soybean research community and granting agencies. Secondly, a workshop was held to define a strategy to incorporate existing tools into a framework for advancing soybean genomics research. This workshop identified and ranked research priorities essential to making more informed decisions as to how to proceed with large scale sequencing and other genomics efforts. Most critical among these was the need to finalize a physical map and to obtain a better understanding of genome microstructure. Addressing these research needs will require pilot work on new technologies to demonstrate an ability to discriminate between recently duplicated regions in the soybean genome and pilot projects to analyze an adequate amount of random genome sequence to identify and catalog common repeats. The development of additional markers, reverse genetics tools, and bioinformatics is also necessary. Successful implementation of these goals will require close coordination among various working groups.

Improved maize reference genome with single-molecule technologies.

PubMed

Jiao, Yinping; Peluso, Paul; Shi, Jinghua; Liang, Tiffany; Stitzer, Michelle C; Wang, Bo; Campbell, Michael S; Stein, Joshua C; Wei, Xuehong; Chin, Chen-Shan; Guill, Katherine; Regulski, Michael; Kumari, Sunita; Olson, Andrew; Gent, Jonathan; Schneider, Kevin L; Wolfgruber, Thomas K; May, Michael R; Springer, Nathan M; Antoniou, Eric; McCombie, W Richard; Presting, Gernot G; McMullen, Michael; Ross-Ibarra, Jeffrey; Dawe, R Kelly; Hastie, Alex; Rank, David R; Ware, Doreen

2017-06-22

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

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

Shi, CY; Yang, H; Wei, CL

Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Using high-throughput Illumina RNA-seq, the transcriptome from poly (A){sup +} RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled intomore » 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR). An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis.« less

Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds

PubMed Central

2011-01-01

Background Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Results Using high-throughput Illumina RNA-seq, the transcriptome from poly (A)+ RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR). Conclusions An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis. PMID:21356090

A transcriptome atlas of rabbit revealed by PacBio single-molecule long-read sequencing.

PubMed

Chen, Shi-Yi; Deng, Feilong; Jia, Xianbo; Li, Cao; Lai, Song-Jia

2017-08-09

It is widely acknowledged that transcriptional diversity largely contributes to biological regulation in eukaryotes. Since the advent of second-generation sequencing technologies, a large number of RNA sequencing studies have considerably improved our understanding of transcriptome complexity. However, it still remains a huge challenge for obtaining full-length transcripts because of difficulties in the short read-based assembly. In the present study we employ PacBio single-molecule long-read sequencing technology for whole-transcriptome profiling in rabbit (Oryctolagus cuniculus). We totally obtain 36,186 high-confidence transcripts from 14,474 genic loci, among which more than 23% of genic loci and 66% of isoforms have not been annotated yet within the current reference genome. Furthermore, about 17% of transcripts are computationally revealed to be non-coding RNAs. Up to 24,797 alternative splicing (AS) and 11,184 alternative polyadenylation (APA) events are detected within this de novo constructed transcriptome, respectively. The results provide a comprehensive set of reference transcripts and hence contribute to the improved annotation of rabbit genome.

The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile.

PubMed

Økland, Arnfinn Lodden; Skoge, Renate Hvidsten; Nylund, Are

2018-06-01

We have determined the complete genome sequence of a new rhabdovirus, tentatively named Caligus rogercresseyi rhabdovirus Ch01 (CrRV-Ch01), which was found in the parasite Caligus rogercresseyi, present on farmed Atlantic salmon (Salmo salar) in Chile. The genome encodes the five canonical rhabdovirus proteins in addition to an unknown protein, in the order N-P-M-U (unknown)-G-L. Phylogenetic analysis showed that the virus clusters with two rhabdoviruses (Lepeophtheirus salmonis rhabdovirus No9 and Lepeophtheirus salmonis rhabdovirus No127) obtained from another parasitic caligid, Lepeophtheirus salmonis, present on farmed Atlantic salmon on the west coast of Norway.

Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

PubMed Central

Su, Chang; Wang, Chao; He, Lin; Yang, Chuanping; Wang, Yucheng

2014-01-01

DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch) by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees. PMID:25514241