Temporal trend of green space coverage in China and its relationship with urbanization over the last two decades.

PubMed

Zhao, Juanjuan; Chen, Shengbin; Jiang, Bo; Ren, Yin; Wang, Hua; Vause, Jonathan; Yu, Haidong

2013-01-01

Irrespective of which side is taken in the densification-sprawl debate, insights into the relationship between urban green space coverage and urbanization have been recognized as essential for guiding sustainable urban development. However, knowledge of the relationships between socio-economic variables of urbanization and long-term green space change is still limited. In this paper, using simple regression, hierarchical partitioning and multi-regression, the temporal trend in green space coverage and its relationship with urbanization were investigated using data from 286 cities between 1989 and 2009, covering all provinces in mainland China with the exception of Tibet. We found that: [1] average green space coverage of cities investigated increased steadily from 17.0% in 1989 to 37.3% in 2009; [2] cities with higher recent green space coverage also had relatively higher green space coverage historically; [3] cities in the same region exhibited similar long-term trends in green space coverage; [4] eight of the nine variables characterizing urbanization showed a significant positive linear relationship with green space coverage, with 'per capita GDP' having the highest independent contribution (24.2%); [5] among the climatic and geographic factors investigated, only mean elevation showed a significant effect; and [6] using the seven largest contributing individual factors, a linear model to predict variance in green space coverage was constructed. Here, we demonstrated that green space coverage in built-up areas tended to reflect the effects of urbanization rather than those of climatic or geographic factors. Quantification of the urbanization effects and the characteristics of green space development in China may provide a valuable reference for research into the processes of urban sprawl and its relationship with green space change. Copyright © 2012 Elsevier B.V. All rights reserved.

Novel methods to optimize genotypic imputation for low-coverage, next-generation sequence data in crop plants

USDA-ARS?s Scientific Manuscript database

Next-generation sequencing technology such as genotyping-by-sequencing (GBS) made low-cost, but often low-coverage, whole-genome sequencing widely available. Extensive inbreeding in crop plants provides an untapped, high quality source of phased haplotypes for imputing missing genotypes. We introduc...

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

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/.

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

An Enumerative Combinatorics Model for Fragmentation Patterns in RNA Sequencing Provides Insights into Nonuniformity of the Expected Fragment Starting-Point and Coverage Profile.

PubMed

Prakash, Celine; Haeseler, Arndt Von

2017-03-01

RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment.

An Enumerative Combinatorics Model for Fragmentation Patterns in RNA Sequencing Provides Insights into Nonuniformity of the Expected Fragment Starting-Point and Coverage Profile

PubMed Central

Haeseler, Arndt Von

2017-01-01

Abstract RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment. PMID:27661099

Three-Dimensional Analysis of Deep Space Network Antenna Coverage

NASA Technical Reports Server (NTRS)

Kegege, Obadiah; Fuentes, Michael; Meyer, Nicholas; Sil, Amy

2012-01-01

There is a need to understand NASA s Deep Space Network (DSN) coverage gaps and any limitations to provide redundant communication coverage for future deep space missions, especially for manned missions to Moon and Mars. The DSN antennas are required to provide continuous communication coverage for deep space flights, interplanetary missions, and deep space scientific observations. The DSN consists of ground antennas located at three sites: Goldstone in USA, Canberra in Australia, and Madrid in Spain. These locations are not separated by the exactly 120 degrees and some DSN antennas are located in the bowl-shaped mountainous terrain to shield against radiofrequency interference resulting in a coverage gap in the southern hemisphere for the current DSN architecture. To analyze the extent of this gap and other coverage limitations, simulations of the DSN architecture were performed. In addition to the physical properties of the DSN assets, the simulation incorporated communication forward link calculations and azimuth/elevation masks that constrain the effects of terrain for each DSN antenna. Analysis of the simulation data was performed to create coverage profiles with the receiver settings at a deep space altitudes ranging from 2 million to 10 million km and a spherical grid resolution of 0.25 degrees with respect to longitude and latitude. With the results of these simulations, two- and three-dimensional representations of the area without communication coverage and area with coverage were developed, showing the size and shape of the communication coverage gap projected in space. Also, the significance of this communication coverage gap is analyzed from the simulation data.

Utilization of sequence on relatives to improve analysis of individuals' low-coverage NGS data

USDA-ARS?s Scientific Manuscript database

Low-coverage sequence data is expected to have low call rates under the prevailing paradigm that genotypes are first “called” from sequence data of each individual independently and subsequent analyses (including determination of haplotypes) are dependent on those called genotypes. However, provide...

A PRESTO-SENSE sequence with alternating partial-Fourier encoding for rapid susceptibility-weighted 3D MRI time series.

PubMed

Klarhöfer, Markus; Dilharreguy, Bixente; van Gelderen, Peter; Moonen, Chrit T W

2003-10-01

A 3D sequence for dynamic susceptibility imaging is proposed which combines echo-shifting principles (such as PRESTO), sensitivity encoding (SENSE), and partial-Fourier acquisition. The method uses a moderate SENSE factor of 2 and takes advantage of an alternating partial k-space acquisition in the "slow" phase encode direction allowing an iterative reconstruction using high-resolution phase estimates. Offering an isotropic spatial resolution of 4 x 4 x 4 mm(3), the novel sequence covers the whole brain including parts of the cerebellum in 0.5 sec. Its temporal signal stability is comparable to that of a full-Fourier, full-FOV EPI sequence having the same dynamic scan time but much less brain coverage. Initial functional MRI experiments showed consistent activation in the motor cortex with an average signal change slightly less than that of EPI. Copyright 2003 Wiley-Liss, Inc.

Lessons learned from the initial sequencing of the pig genome: comparative analysis of an 8 Mb region of pig chromosome 17

PubMed Central

Hart, Elizabeth A; Caccamo, Mario; Harrow, Jennifer L; Humphray, Sean J; Gilbert, James GR; Trevanion, Steve; Hubbard, Tim; Rogers, Jane; Rothschild, Max F

2007-01-01

Background We describe here the sequencing, annotation and comparative analysis of an 8 Mb region of pig chromosome 17, which provides a useful test region to assess coverage and quality for the pig genome sequencing project. We report our findings comparing the annotation of draft sequence assembled at different depths of coverage. Results Within this region we annotated 71 loci, of which 53 are orthologous to human known coding genes. When compared to the syntenic regions in human (20q13.13-q13.33) and mouse (chromosome 2, 167.5 Mb-178.3 Mb), this region was found to be highly conserved with respect to gene order. The most notable difference between the three species is the presence of a large expansion of zinc finger coding genes and pseudogenes on mouse chromosome 2 between Edn3 and Phactr3 that is absent from pig and human. All of our annotation has been made publicly available in the Vertebrate Genome Annotation browser, VEGA. We assessed the impact of coverage on sequence assembly across this region and found, as expected, that increased sequence depth resulted in fewer, longer contigs. One-third of our annotated loci could not be fully re-aligned back to the low coverage version of the sequence, principally because the transcripts are fragmented over several contigs. Conclusion We have demonstrated the considerable advantages of sequencing at increased read depths and discuss the implications that lower coverage sequence may have on subsequent comparative and functional studies, particularly those involving complex loci such as GNAS. PMID:17705864

Carotid arterial wall MRI at 3T using 3D variable-flip-angle turbo spin-echo (TSE) with flow-sensitive dephasing (FSD).

PubMed

Fan, Zhaoyang; Zhang, Zhuoli; Chung, Yiu-Cho; Weale, Peter; Zuehlsdorff, Sven; Carr, James; Li, Debiao

2010-03-01

To evaluate the effectiveness of flow-sensitive dephasing (FSD) magnetization preparation in improving blood signal suppression of three-dimensional (3D) turbo spin-echo (TSE) sequence (SPACE) for isotropic high-spatial-resolution carotid arterial wall imaging at 3T. The FSD-prepared SPACE sequence (FSD-SPACE) was implemented by adding two identical FSD gradient pulses right before and after the first refocusing 180 degrees -pulse of the SPACE sequence in all three orthogonal directions. Nine healthy volunteers were imaged at 3T with SPACE, FSD-SPACE, and multislice T2-weighted 2D TSE coupled with saturation band (SB-TSE). Apparent carotid wall-lumen contrast-to-noise ratio (aCNR(w-l)) and apparent lumen area (aLA) at the locations with residual-blood (rb) signal shown on SPACE images were compared between SPACE and FSD-SPACE. Carotid aCNR(w-l) and lumen (LA) and wall area (WA) measured from FSD-SPACE were compared to those measured from SB-TSE. Plaque-mimicking flow artifacts identified in seven carotids on SPACE images were eliminated on FSD-SPACE images. The FSD preparation resulted in slightly reduced aCNR(w-l) (P = 0.025), but significantly improved aCNR between the wall and rb regions (P < 0.001) and larger aLA (P < 0.001). Compared to SB-TSE, FSD-SPACE offered comparable aCNR(w-l) with much higher spatial resolution, shorter imaging time, and larger artery coverage. The LA and WA measurements from the two techniques were in good agreement based on intraclasss correlation coefficient (0.988 and 0.949, respectively; P < 0.001) and Bland-Altman analyses. FSD-SPACE is a time-efficient 3D imaging technique for carotid arterial wall with superior spatial resolution and blood signal suppression.

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.

Multi-Objective Optimization of Spacecraft Trajectories for Small-Body Coverage Missions

NASA Technical Reports Server (NTRS)

Hinckley, David, Jr.; Englander, Jacob; Hitt, Darren

2017-01-01

Visual coverage of surface elements of a small-body object requires multiple images to be taken that meet many requirements on their viewing angles, illumination angles, times of day, and combinations thereof. Designing trajectories capable of maximizing total possible coverage may not be useful since the image target sequence and the feasibility of said sequence given the rotation-rate limitations of the spacecraft are not taken into account. This work presents a means of optimizing, in a multi-objective manner, surface target sequences that account for such limitations.

A fast screening protocol for carotid plaques imaging using 3D multi-contrast MRI without contrast agent.

PubMed

Zhang, Na; Zhang, Lei; Yang, Qi; Pei, Anqi; Tong, Xiaoxin; Chung, Yiu-Cho; Liu, Xin

2017-06-01

To implement a fast (~15min) MRI protocol for carotid plaque screening using 3D multi-contrast MRI sequences without contrast agent on a 3Tesla MRI scanner. 7 healthy volunteers and 25 patients with clinically confirmed transient ischemic attack or suspected cerebrovascular ischemia were included in this study. The proposed protocol, including 3D T1-weighted and T2-weighted SPACE (variable-flip-angle 3D turbo spin echo), and T1-weighted magnetization prepared rapid acquisition gradient echo (MPRAGE) was performed first and was followed by 2D T1-weighted and T2-weighted turbo spin echo, and post-contrast T1-weighted SPACE sequences. Image quality, number of plaques, and vessel wall thicknesses measured at the intersection of the plaques were evaluated and compared between sequences. Average examination time of the proposed protocol was 14.6min. The average image quality scores of 3D T1-weighted, T2-weighted SPACE, and T1-weighted magnetization prepared rapid acquisition gradient echo were 3.69, 3.75, and 3.48, respectively. There was no significant difference in detecting the number of plaques and vulnerable plaques using pre-contrast 3D images with or without post-contrast T1-weighted SPACE. The 3D SPACE and 2D turbo spin echo sequences had excellent agreement (R=0.96 for T1-weighted and 0.98 for T2-weighted, p<0.001) regarding vessel wall thickness measurements. The proposed protocol demonstrated the feasibility of attaining carotid plaque screening within a 15-minute scan, which provided sufficient anatomical coverage and critical diagnostic information. This protocol offers the potential for rapid and reliable screening for carotid plaques without contrast agent. Copyright © 2016. Published by Elsevier Inc.

snpAD: An ancient DNA genotype caller.

PubMed

Prüfer, Kay

2018-06-21

The study of ancient genomes can elucidate the evolutionary past. However, analyses are complicated by base-modifications in ancient DNA molecules that result in errors in DNA sequences. These errors are particularly common near the ends of sequences and pose a challenge for genotype calling. I describe an iterative method that estimates genotype frequencies and errors along sequences to allow for accurate genotype calling from ancient sequences. The implementation of this method, called snpAD, performs well on high-coverage ancient data, as shown by simulations and by subsampling the data of a high-coverage Neandertal genome. Although estimates for low-coverage genomes are less accurate, I am able to derive approximate estimates of heterozygosity from several low-coverage Neandertals. These estimates show that low heterozygosity, compared to modern humans, was common among Neandertals. The C ++ code of snpAD is freely available at http://bioinf.eva.mpg.de/snpAD/. Supplementary data are available at Bioinformatics online.

Indexcov: fast coverage quality control for whole-genome sequencing.

PubMed

Pedersen, Brent S; Collins, Ryan L; Talkowski, Michael E; Quinlan, Aaron R

2017-11-01

The BAM and CRAM formats provide a supplementary linear index that facilitates rapid access to sequence alignments in arbitrary genomic regions. Comparing consecutive entries in a BAM or CRAM index allows one to infer the number of alignment records per genomic region for use as an effective proxy of sequence depth in each genomic region. Based on these properties, we have developed indexcov, an efficient estimator of whole-genome sequencing coverage to rapidly identify samples with aberrant coverage profiles, reveal large-scale chromosomal anomalies, recognize potential batch effects, and infer the sex of a sample. Indexcov is available at https://github.com/brentp/goleft under the MIT license. © The Authors 2017. Published by Oxford University Press.

Assessing the Relationship of Ancient and Modern Populations

PubMed Central

Schraiber, Joshua G.

2018-01-01

Genetic material sequenced from ancient samples is revolutionizing our understanding of the recent evolutionary past. However, ancient DNA is often degraded, resulting in low coverage, error-prone sequencing. Several solutions exist to this problem, ranging from simple approach, such as selecting a read at random for each site, to more complicated approaches involving genotype likelihoods. In this work, we present a novel method for assessing the relationship of an ancient sample with a modern population, while accounting for sequencing error and postmortem damage by analyzing raw reads from multiple ancient individuals simultaneously. We show that, when analyzing SNP data, it is better to sequence more ancient samples to low coverage: two samples sequenced to 0.5× coverage provide better resolution than a single sample sequenced to 2× coverage. We also examined the power to detect whether an ancient sample is directly ancestral to a modern population, finding that, with even a few high coverage individuals, even ancient samples that are very slightly diverged from the modern population can be detected with ease. When we applied our approach to European samples, we found that no ancient samples represent direct ancestors of modern Europeans. We also found that, as shown previously, the most ancient Europeans appear to have had the smallest effective population sizes, indicating a role for agriculture in modern population growth. PMID:29167200

Launch mission summary and sequence of events Telesat-F(anik-D1)/Delta-164

NASA Technical Reports Server (NTRS)

1982-01-01

The launch vehicle, spacecraft, and mission are summarized. Launch window information, vehicle telemetry coverage, real time data flow, telemetry coverage by station, selected trajectory information, and a brief sequence of flight events are included.

De novo protein sequencing by combining top-down and bottom-up tandem mass spectra.

PubMed

Liu, Xiaowen; Dekker, Lennard J M; Wu, Si; Vanduijn, Martijn M; Luider, Theo M; Tolić, Nikola; Kou, Qiang; Dvorkin, Mikhail; Alexandrova, Sonya; Vyatkina, Kira; Paša-Tolić, Ljiljana; Pevzner, Pavel A

2014-07-03

There are two approaches for de novo protein sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Experiments on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.

Rapid genotyping by low-coverage resequencing to construct genetic linkage maps of fungi: a case study in Lentinula edodes

PubMed Central

2013-01-01

Background Genetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system. Findings Shiitake mushroom, Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB. Conclusions We used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly. PMID:23915543

Defining epitope coverage requirements for T cell-based HIV vaccines: Theoretical considerations and practical applications

PubMed Central

2011-01-01

Background HIV vaccine development must address the genetic diversity and plasticity of the virus that permits the presentation of diverse genetic forms to the immune system and subsequent escape from immune pressure. Assessment of potential HIV strain coverage by candidate T cell-based vaccines (whether natural sequence or computationally optimized products) is now a critical component in interpreting candidate vaccine suitability. Methods We have utilized an N-mer identity algorithm to represent T cell epitopes and explore potential coverage of the global HIV pandemic using natural sequences derived from candidate HIV vaccines. Breadth (the number of T cell epitopes generated) and depth (the variant coverage within a T cell epitope) analyses have been incorporated into the model to explore vaccine coverage requirements in terms of the number of discrete T cell epitopes generated. Results We show that when multiple epitope generation by a vaccine product is considered a far more nuanced appraisal of the potential HIV strain coverage of the vaccine product emerges. By considering epitope breadth and depth several important observations were made: (1) epitope breadth requirements to reach particular levels of vaccine coverage, even for natural sequence-based vaccine products is not necessarily an intractable problem for the immune system; (2) increasing the valency (number of T cell epitope variants present) of vaccine products dramatically decreases the epitope requirements to reach particular coverage levels for any epidemic; (3) considering multiple-hit models (more than one exact epitope match with an incoming HIV strain) places a significantly higher requirement upon epitope breadth in order to reach a given level of coverage, to the point where low valency natural sequence based products would not practically be able to generate sufficient epitopes. Conclusions When HIV vaccine sequences are compared against datasets of potential incoming viruses important metrics such as the minimum epitope count required to reach a desired level of coverage can be easily calculated. We propose that such analyses can be applied early in the planning stages and during the execution phase of a vaccine trial to explore theoretical and empirical suitability of a vaccine product to a particular epidemic setting. PMID:22152192

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

Camps 2.0: exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins.

PubMed

Neumann, Sindy; Hartmann, Holger; Martin-Galiano, Antonio J; Fuchs, Angelika; Frishman, Dmitrij

2012-03-01

Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/. Copyright © 2011 Wiley Periodicals, Inc.

Estimating genotype error rates from high-coverage next-generation sequence data.

PubMed

Wall, Jeffrey D; Tang, Ling Fung; Zerbe, Brandon; Kvale, Mark N; Kwok, Pui-Yan; Schaefer, Catherine; Risch, Neil

2014-11-01

Exome and whole-genome sequencing studies are becoming increasingly common, but little is known about the accuracy of the genotype calls made by the commonly used platforms. Here we use replicate high-coverage sequencing of blood and saliva DNA samples from four European-American individuals to estimate lower bounds on the error rates of Complete Genomics and Illumina HiSeq whole-genome and whole-exome sequencing. Error rates for nonreference genotype calls range from 0.1% to 0.6%, depending on the platform and the depth of coverage. Additionally, we found (1) no difference in the error profiles or rates between blood and saliva samples; (2) Complete Genomics sequences had substantially higher error rates than Illumina sequences had; (3) error rates were higher (up to 6%) for rare or unique variants; (4) error rates generally declined with genotype quality (GQ) score, but in a nonlinear fashion for the Illumina data, likely due to loss of specificity of GQ scores greater than 60; and (5) error rates increased with increasing depth of coverage for the Illumina data. These findings, especially (3)-(5), suggest that caution should be taken in interpreting the results of next-generation sequencing-based association studies, and even more so in clinical application of this technology in the absence of validation by other more robust sequencing or genotyping methods. © 2014 Wall et al.; Published by Cold Spring Harbor Laboratory Press.

CoMet: a workflow using contig coverage and composition for binning a metagenomic sample with high precision.

PubMed

Herath, Damayanthi; Tang, Sen-Lin; Tandon, Kshitij; Ackland, David; Halgamuge, Saman Kumara

2017-12-28

In metagenomics, the separation of nucleotide sequences belonging to an individual or closely matched populations is termed binning. Binning helps the evaluation of underlying microbial population structure as well as the recovery of individual genomes from a sample of uncultivable microbial organisms. Both supervised and unsupervised learning methods have been employed in binning; however, characterizing a metagenomic sample containing multiple strains remains a significant challenge. In this study, we designed and implemented a new workflow, Coverage and composition based binning of Metagenomes (CoMet), for binning contigs in a single metagenomic sample. CoMet utilizes coverage values and the compositional features of metagenomic contigs. The binning strategy in CoMet includes the initial grouping of contigs in guanine-cytosine (GC) content-coverage space and refinement of bins in tetranucleotide frequencies space in a purely unsupervised manner. With CoMet, the clustering algorithm DBSCAN is employed for binning contigs. The performances of CoMet were compared against four existing approaches for binning a single metagenomic sample, including MaxBin, Metawatt, MyCC (default) and MyCC (coverage) using multiple datasets including a sample comprised of multiple strains. Binning methods based on both compositional features and coverages of contigs had higher performances than the method which is based only on compositional features of contigs. CoMet yielded higher or comparable precision in comparison to the existing binning methods on benchmark datasets of varying complexities. MyCC (coverage) had the highest ranking score in F1-score. However, the performances of CoMet were higher than MyCC (coverage) on the dataset containing multiple strains. Furthermore, CoMet recovered contigs of more species and was 18 - 39% higher in precision than the compared existing methods in discriminating species from the sample of multiple strains. CoMet resulted in higher precision than MyCC (default) and MyCC (coverage) on a real metagenome. The approach proposed with CoMet for binning contigs, improves the precision of binning while characterizing more species in a single metagenomic sample and in a sample containing multiple strains. The F1-scores obtained from different binning strategies vary with different datasets; however, CoMet yields the highest F1-score with a sample comprised of multiple strains.

Electron-Transfer Ion/Ion Reactions of Doubly Protonated Peptides: Effect of Elevated Bath Gas Temperature

PubMed Central

Pitteri, Sharon J.; Chrisman, Paul A.; McLuckey, Scott A.

2005-01-01

In this study, the electron-transfer dissociation (ETD) behavior of cations derived from 27 different peptides (22 of which are tryptic peptides) has been studied in a 3D quadrupole ion trap mass spectrometer. Ion/ion reactions between peptide cations and nitrobenzene anions have been examined at both room temperature and in an elevated temperature bath gas environment to form ETD product ions. From the peptides studied, the ETD sequence coverage tends to be inversely related to peptide size. At room temperature, very high sequence coverage (~100%) was observed for small peptides (≤7 amino acids). For medium-sized peptides composed of 8–11 amino acids, the average sequence coverage was 46%. Larger peptides with 14 or more amino acids yielded an average sequence coverage of 23%. Elevated-temperature ETD provided increased sequence coverage over room-temperature experiments for the peptides of greater than 7 residues, giving an average of 67% for medium-sized peptides and 63% for larger peptides. Percent ETD, a measure of the extent of electron transfer, has also been calculated for the peptides and also shows an inverse relation with peptide size. Bath gas temperature does not have a consistent effect on percent ETD, however. For the tryptic peptides, fragmentation is localized at the ends of the peptides suggesting that the distribution of charge within the peptide may play an important role in determining fragmentation sites. A triply protonated peptide has also been studied and shows behavior similar to the doubly charged peptides. These preliminary results suggest that for a given charge state there is a maximum size for which high sequence coverage is obtained and that increasing the bath gas temperature can increase this maximum. PMID:16131079

Directions for Space-Based Low-Frequency Radio Astronomy 2. Telescopes

NASA Astrophysics Data System (ADS)

Basart, J. P.; Burns, J. O.; Dennison, B. K.; Weiler, K. W.; Kassim, N. E.; Castillo, S. P.; McCune, B. M.

Astronomical studies of celestial sources at low radio frequencies (0.3 to 30 MHz) lag far behind the investigations of celestial sources at high radio frequencies. In a companion paper [Basart et al., this issue] we discussed the need for low-frequency investigations, and in this paper we discuss the telescopes required to make the observations. Radio telescopes for use in the low-frequency range can be built principally from ``off-the-shelf'' components. For relatively little cost for a space mission, great strides can be made in deploying arrays of antennas and receivers in space that would produce data contributing significantly to our understanding of galaxies and galactic nebulae. In this paper we discuss an evolutionary sequence of telescopes, antenna systems, receivers, and (u,v) plane coverage. The telescopes are space-based because of the disruptive aspects of the Earth's ionosphere on low-frequency celestial signals traveling to the Earth's surface. Orbiting antennas consisting of array elements deposited on a Kevlar balloon have strong advantages of nearly identical multiple beams over 4π steradians and few mechanical aspects in deployment and operation. The relatively narrow beam width of these antennas can significantly help reduce the ``confusion'' problem. The evolutionary sequence of telescopes starts with an Earth-orbiting spectrometer to measure the low-frequency radio environment in space, proceeds to a two-element interferometer, then to an orbiting array, and ends with a telescope on the lunar farside. The sequence is in the order of increasing capability which is also the order of increasing complexity and cost. All the missions can be accomplished with current technology.

Nonpareil 3: Fast Estimation of Metagenomic Coverage and Sequence Diversity.

PubMed

Rodriguez-R, Luis M; Gunturu, Santosh; Tiedje, James M; Cole, James R; Konstantinidis, Konstantinos T

2018-01-01

Estimations of microbial community diversity based on metagenomic data sets are affected, often to an unknown degree, by biases derived from insufficient coverage and reference database-dependent estimations of diversity. For instance, the completeness of reference databases cannot be generally estimated since it depends on the extant diversity sampled to date, which, with the exception of a few habitats such as the human gut, remains severely undersampled. Further, estimation of the degree of coverage of a microbial community by a metagenomic data set is prohibitively time-consuming for large data sets, and coverage values may not be directly comparable between data sets obtained with different sequencing technologies. Here, we extend Nonpareil, a database-independent tool for the estimation of coverage in metagenomic data sets, to a high-performance computing implementation that scales up to hundreds of cores and includes, in addition, a k -mer-based estimation as sensitive as the original alignment-based version but about three hundred times as fast. Further, we propose a metric of sequence diversity ( N d ) derived directly from Nonpareil curves that correlates well with alpha diversity assessed by traditional metrics. We use this metric in different experiments demonstrating the correlation with the Shannon index estimated on 16S rRNA gene profiles and show that N d additionally reveals seasonal patterns in marine samples that are not captured by the Shannon index and more precise rankings of the magnitude of diversity of microbial communities in different habitats. Therefore, the new version of Nonpareil, called Nonpareil 3, advances the toolbox for metagenomic analyses of microbiomes. IMPORTANCE Estimation of the coverage provided by a metagenomic data set, i.e., what fraction of the microbial community was sampled by DNA sequencing, represents an essential first step of every culture-independent genomic study that aims to robustly assess the sequence diversity present in a sample. However, estimation of coverage remains elusive because of several technical limitations associated with high computational requirements and limiting statistical approaches to quantify diversity. Here we described Nonpareil 3, a new bioinformatics algorithm that circumvents several of these limitations and thus can facilitate culture-independent studies in clinical or environmental settings, independent of the sequencing platform employed. In addition, we present a new metric of sequence diversity based on rarefied coverage and demonstrate its use in communities from diverse ecosystems.

Haplotype estimation using sequencing reads.

PubMed

Delaneau, Olivier; Howie, Bryan; Cox, Anthony J; Zagury, Jean-François; Marchini, Jonathan

2013-10-03

High-throughput sequencing technologies produce short sequence reads that can contain phase information if they span two or more heterozygote genotypes. This information is not routinely used by current methods that infer haplotypes from genotype data. We have extended the SHAPEIT2 method to use phase-informative sequencing reads to improve phasing accuracy. Our model incorporates the read information in a probabilistic model through base quality scores within each read. The method is primarily designed for high-coverage sequence data or data sets that already have genotypes called. One important application is phasing of single samples sequenced at high coverage for use in medical sequencing and studies of rare diseases. Our method can also use existing panels of reference haplotypes. We tested the method by using a mother-father-child trio sequenced at high-coverage by Illumina together with the low-coverage sequence data from the 1000 Genomes Project (1000GP). We found that use of phase-informative reads increases the mean distance between switch errors by 22% from 274.4 kb to 328.6 kb. We also used male chromosome X haplotypes from the 1000GP samples to simulate sequencing reads with varying insert size, read length, and base error rate. When using short 100 bp paired-end reads, we found that using mixtures of insert sizes produced the best results. When using longer reads with high error rates (5-20 kb read with 4%-15% error per base), phasing performance was substantially improved. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

CORNAS: coverage-dependent RNA-Seq analysis of gene expression data without biological replicates.

PubMed

Low, Joel Z B; Khang, Tsung Fei; Tammi, Martti T

2017-12-28

In current statistical methods for calling differentially expressed genes in RNA-Seq experiments, the assumption is that an adjusted observed gene count represents an unknown true gene count. This adjustment usually consists of a normalization step to account for heterogeneous sample library sizes, and then the resulting normalized gene counts are used as input for parametric or non-parametric differential gene expression tests. A distribution of true gene counts, each with a different probability, can result in the same observed gene count. Importantly, sequencing coverage information is currently not explicitly incorporated into any of the statistical models used for RNA-Seq analysis. We developed a fast Bayesian method which uses the sequencing coverage information determined from the concentration of an RNA sample to estimate the posterior distribution of a true gene count. Our method has better or comparable performance compared to NOISeq and GFOLD, according to the results from simulations and experiments with real unreplicated data. We incorporated a previously unused sequencing coverage parameter into a procedure for differential gene expression analysis with RNA-Seq data. Our results suggest that our method can be used to overcome analytical bottlenecks in experiments with limited number of replicates and low sequencing coverage. The method is implemented in CORNAS (Coverage-dependent RNA-Seq), and is available at https://github.com/joel-lzb/CORNAS .

NASA STI Database, Aerospace Database and ARIN coverage of 'space law'

NASA Technical Reports Server (NTRS)

Buchan, Ronald L.

1992-01-01

The space-law coverage provided by the NASA STI Database, the Aerospace Database, and ARIN is briefly described. Particular attention is given to the space law content of the two Databases and of ARIN, the NASA Thesauras space law terminology, space law publication forms, and the availability of the space law literature.

Space Shuttle Communications Coverage Analysis for Thermal Tile Inspection

NASA Technical Reports Server (NTRS)

Kroll, Quin D.; Hwu, Shian U.; Upanavage, Matthew; Boster, John P.; Chavez, Mark A.

2009-01-01

The space shuttle ultra-high frequency Space-to-Space Communication System has to provide adequate communication coverage for astronauts who are performing thermal tile inspection and repair on the underside of the space shuttle orbiter (SSO). Careful planning and quantitative assessment are necessary to ensure successful system operations and mission safety in this work environment. This study assesses communication systems performance for astronauts who are working in the underside, non-line-of-sight shadow region on the space shuttle. All of the space shuttle and International Space Station (ISS) transmitting antennas are blocked by the SSO structure. To ensure communication coverage at planned inspection worksites, the signal strength and link margin between the SSO/ISS antennas and the extravehicular activity astronauts, whose line-of-sight is blocked by vehicle structure, was analyzed. Investigations were performed using rigorous computational electromagnetic modeling techniques. Signal strength was obtained by computing the reflected and diffracted fields along the signal propagation paths between transmitting and receiving antennas. Radio frequency (RF) coverage was determined for thermal tile inspection and repair missions using the results of this computation. Analysis results from this paper are important in formulating the limits on reliable communication range and RF coverage at planned underside inspection and repair worksites.

High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic.

PubMed

Sealfon, Rachel; Gire, Stephen; Ellis, Crystal; Calderwood, Stephen; Qadri, Firdausi; Hensley, Lisa; Kellis, Manolis; Ryan, Edward T; LaRocque, Regina C; Harris, Jason B; Sabeti, Pardis C

2012-09-11

Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

FY11 Report on Metagenome Analysis using Pathogen Marker Libraries

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

Gardner, Shea N.; Allen, Jonathan E.; McLoughlin, Kevin S.

2011-06-02

A method, sequence library, and software suite was invented to rapidly assess whether any member of a pre-specified list of threat organisms or their near neighbors is present in a metagenome. The system was designed to handle mega- to giga-bases of FASTA-formatted raw sequence reads from short or long read next generation sequencing platforms. The approach is to pre-calculate a viral and a bacterial "Pathogen Marker Library" (PML) containing sub-sequences specific to pathogens or their near neighbors. A list of expected matches comparing every bacterial or viral genome against the PML sequences is also pre-calculated. To analyze a metagenome, readsmore » are compared to the PML, and observed PML-metagenome matches are compared to the expected PML-genome matches, and the ratio of observed relative to expected matches is reported. In other words, a 3-way comparison among the PML, metagenome, and existing genome sequences is used to quickly assess which (if any) species included in the PML is likely to be present in the metagenome, based on available sequence data. Our tests showed that the species with the most PML matches correctly indicated the organism sequenced for empirical metagenomes consisting of a cultured, relatively pure isolate. These runs completed in 1 minute to 3 hours on 12 CPU (1 thread/CPU), depending on the metagenome and PML. Using more threads on the same number of CPU resulted in speed improvements roughly proportional to the number of threads. Simulations indicated that detection sensitivity depends on both sequencing coverage levels for a species and the size of the PML: species were correctly detected even at ~0.003x coverage by the large PMLs, and at ~0.03x coverage by the smaller PMLs. Matches to true positive species were 3-4 orders of magnitude higher than to false positives. Simulations with short reads (36 nt and ~260 nt) showed that species were usually detected for metagenome coverage above 0.005x and coverage in the PML above 0.05x, and detection probability appears to be a function of both coverages. Multiple species could be detected simultaneously in a simulated low-coverage, complex metagenome, and the largest PML gave no false negative species and no false positive genera. The presence of multiple species was predicted in a complex metagenome from a human gut microbiome with 1.9 GB of short reads (75 nt); the species predicted were reasonable gut flora and no biothreat agents were detected, showing the feasibility of PML analysis of empirical complex metagenomes.« less

Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics.

PubMed

Tin, Mandy Man-Ying; Economo, Evan Philip; Mikheyev, Alexander Sergeyevich

2014-01-01

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37 ± 0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

Extending CATH: increasing coverage of the protein structure universe and linking structure with function

PubMed Central

Cuff, Alison L.; Sillitoe, Ian; Lewis, Tony; Clegg, Andrew B.; Rentzsch, Robert; Furnham, Nicholas; Pellegrini-Calace, Marialuisa; Jones, David; Thornton, Janet; Orengo, Christine A.

2011-01-01

CATH version 3.3 (class, architecture, topology, homology) contains 128 688 domains, 2386 homologous superfamilies and 1233 fold groups, and reflects a major focus on classifying structural genomics (SG) structures and transmembrane proteins, both of which are likely to add structural novelty to the database and therefore increase the coverage of protein fold space within CATH. For CATH version 3.4 we have significantly improved the presentation of sequence information and associated functional information for CATH superfamilies. The CATH superfamily pages now reflect both the functional and structural diversity within the superfamily and include structural alignments of close and distant relatives within the superfamily, annotated with functional information and details of conserved residues. A significantly more efficient search function for CATH has been established by implementing the search server Solr (http://lucene.apache.org/solr/). The CATH v3.4 webpages have been built using the Catalyst web framework. PMID:21097779

Use of low-coverage, large-insert, short-read data for rapid and accurate generation of enhanced-quality draft Pseudomonas genome sequences.

PubMed

O'Brien, Heath E; Gong, Yunchen; Fung, Pauline; Wang, Pauline W; Guttman, David S

2011-01-01

Next-generation genomic technology has both greatly accelerated the pace of genome research as well as increased our reliance on draft genome sequences. While groups such as the Genomics Standards Consortium have made strong efforts to promote genome standards there is a still a general lack of uniformity among published draft genomes, leading to challenges for downstream comparative analyses. This lack of uniformity is a particular problem when using standard draft genomes that frequently have large numbers of low-quality sequencing tracts. Here we present a proposal for an "enhanced-quality draft" genome that identifies at least 95% of the coding sequences, thereby effectively providing a full accounting of the genic component of the genome. Enhanced-quality draft genomes are easily attainable through a combination of small- and large-insert next-generation, paired-end sequencing. We illustrate the generation of an enhanced-quality draft genome by re-sequencing the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola 1448A (Pph 1448A), which has a published, closed genome sequence of 5.93 Mbp. We use a combination of Illumina paired-end and mate-pair sequencing, and surprisingly find that de novo assemblies with 100x paired-end coverage and mate-pair sequencing with as low as low as 2-5x coverage are substantially better than assemblies based on higher coverage. The rapid and low-cost generation of large numbers of enhanced-quality draft genome sequences will be of particular value for microbial diagnostics and biosecurity, which rely on precise discrimination of potentially dangerous clones from closely related benign strains.

14 CFR 198.5 - Types of insurance coverage available.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 3 2013-01-01 2013-01-01 false Types of insurance coverage available. 198.5 Section 198.5 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) WAR RISK INSURANCE AVIATION INSURANCE § 198.5 Types of insurance coverage available. Application...

14 CFR 198.5 - Types of insurance coverage available.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 3 2012-01-01 2012-01-01 false Types of insurance coverage available. 198.5 Section 198.5 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) WAR RISK INSURANCE AVIATION INSURANCE § 198.5 Types of insurance coverage available. Application...

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

Treesearch

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

2011-01-01

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...

Breaking Lander-Waterman’s Coverage Bound

PubMed Central

Nashta-ali, Damoun; Motahari, Seyed Abolfazl; Hosseinkhalaj, Babak

2016-01-01

Lander-Waterman’s coverage bound establishes the total number of reads required to cover the whole genome of size G bases. In fact, their bound is a direct consequence of the well-known solution to the coupon collector’s problem which proves that for such genome, the total number of bases to be sequenced should be O(G ln G). Although the result leads to a tight bound, it is based on a tacit assumption that the set of reads are first collected through a sequencing process and then are processed through a computation process, i.e., there are two different machines: one for sequencing and one for processing. In this paper, we present a significant improvement compared to Lander-Waterman’s result and prove that by combining the sequencing and computing processes, one can re-sequence the whole genome with as low as O(G) sequenced bases in total. Our approach also dramatically reduces the required computational power for the combined process. Simulation results are performed on real genomes with different sequencing error rates. The results support our theory predicting the log G improvement on coverage bound and corresponding reduction in the total number of bases required to be sequenced. PMID:27806058

A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data.

PubMed

Sepúlveda, Nuno; Campino, Susana G; Assefa, Samuel A; Sutherland, Colin J; Pain, Arnab; Clark, Taane G

2013-02-26

The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data.

Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes.

PubMed

Fu, Yong-Bi; Peterson, Gregory W; Dong, Yibo

2016-04-07

Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or pair of restriction enzymes on one sequenced genome, and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal, and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to eight times more genome coverage for the new combinations consisted of pairing four- or five-cutter restriction enzymes than the commonly used enzyme combination PstI + MspI. The empirical evaluation of the new enzyme combination (HinfI + HpyCH4IV) in 12 plant species showed 1.7-6 times more genome coverage than PstI + MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI + HpyCH4IV generated 7 and 1.3 times more SNPs (with 0-16.7% missing observations) than PstI + MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications. Copyright © 2016 Fu et al.

Expanding proteome coverage with orthogonal-specificity α-Lytic proteases

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

Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.

2014-03-01

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage by cleavage at sequences complimentary to trypsin may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type alpha-lytic protease (WaLP), and an active site mutant of WaLP, M190A alpha-lytic protease (MaLP). We assess several relevant factors including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. Bymore » combining data from separate digestions with trypsin, LysC, WaLP, and MaLP, proteome coverage was increased 101% compared to trypsin digestion alone. To demonstrate how the gained sequence coverage can access additional PTM information, we show identification of a number of novel phosphorylation sites in the S. pombe proteome and include an illustrative example from the protein MPD2, wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.« less

14 CFR 198.7 - Amount of insurance coverage available.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 3 2012-01-01 2012-01-01 false Amount of insurance coverage available. 198.7 Section 198.7 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) WAR RISK INSURANCE AVIATION INSURANCE § 198.7 Amount of insurance coverage available. (a) For...

14 CFR 198.7 - Amount of insurance coverage available.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 3 2013-01-01 2013-01-01 false Amount of insurance coverage available. 198.7 Section 198.7 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) WAR RISK INSURANCE AVIATION INSURANCE § 198.7 Amount of insurance coverage available. (a) For...

14 CFR 198.17 - Ground support and other coverage.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 3 2011-01-01 2011-01-01 false Ground support and other coverage. 198.17 Section 198.17 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) WAR RISK INSURANCE AVIATION INSURANCE § 198.17 Ground support and other coverage. An aircraft...

14 CFR 198.17 - Ground support and other coverage.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Ground support and other coverage. 198.17 Section 198.17 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) WAR RISK INSURANCE AVIATION INSURANCE § 198.17 Ground support and other coverage. An aircraft...

Fully immunized child: coverage, timing and sequencing of routine immunization in an urban poor settlement in Nairobi, Kenya.

PubMed

Mutua, Martin Kavao; Kimani-Murage, Elizabeth; Ngomi, Nicholas; Ravn, Henrik; Mwaniki, Peter; Echoka, Elizabeth

2016-01-01

More efforts have been put in place to increase full immunization coverage rates in the last decade. Little is known about the levels and consequences of delaying or vaccinating children in different schedules. Vaccine effectiveness depends on the timing of its administration, and it is not optimal if given early, delayed or not given as recommended. Evidence of non-specific effects of vaccines is well documented and could be linked to timing and sequencing of immunization. This paper documents the levels of coverage, timing and sequencing of routine childhood vaccines. The study was conducted between 2007 and 2014 in two informal urban settlements in Nairobi. A total of 3856 children, aged 12-23 months and having a vaccination card seen were included in analysis. Vaccination dates recorded from the cards seen were used to define full immunization coverage, timeliness and sequencing. Proportions, medians and Kaplan-Meier curves were used to assess and describe the levels of full immunization coverage, vaccination delays and sequencing. The findings indicate that 67 % of the children were fully immunized by 12 months of age. Missing measles and third doses of polio and pentavalent vaccine were the main reason for not being fully immunized. Delays were highest for third doses of polio and pentavalent and measles. About 22 % of fully immunized children had vaccines in an out-of-sequence manner with 18 % not receiving pentavalent together with polio vaccine as recommended. Results show higher levels of missed opportunities and low coverage of routine childhood vaccinations given at later ages. New strategies are needed to enable health care providers and parents/guardians to work together to increase the levels of completion of all required vaccinations. In particular, more focus is needed on vaccines given in multiple doses (polio, pentavalent and pneumococcal conjugate vaccines).

Low-coverage MiSeq next generation sequencing reveals the mitochondrial genome of the Eastern Rock Lobster, Sagmariasus verreauxi.

PubMed

Doyle, Stephen R; Griffith, Ian S; Murphy, Nick P; Strugnell, Jan M

2015-01-01

The complete mitochondrial genome of the Eastern Rock lobster, Sagmariasus verreauxi, is reported for the first time. Using low-coverage, long read MiSeq next generation sequencing, we constructed and determined the mtDNA genome organization of the 15,470 bp sequence from two isolates from Eastern Tasmania, Australia and Northern New Zealand, and identified 46 polymorphic nucleotides between the two sequences. This genome sequence and its genetic polymorphisms will likely be useful in understanding the distribution and population connectivity of the Eastern Rock Lobster, and in the fisheries management of this commercially important species.

Comparing de novo genome assembly: the long and short of it.

PubMed

Narzisi, Giuseppe; Mishra, Bud

2011-04-29

Recent advances in DNA sequencing technology and their focal role in Genome Wide Association Studies (GWAS) have rekindled a growing interest in the whole-genome sequence assembly (WGSA) problem, thereby, inundating the field with a plethora of new formalizations, algorithms, heuristics and implementations. And yet, scant attention has been paid to comparative assessments of these assemblers' quality and accuracy. No commonly accepted and standardized method for comparison exists yet. Even worse, widely used metrics to compare the assembled sequences emphasize only size, poorly capturing the contig quality and accuracy. This paper addresses these concerns: it highlights common anomalies in assembly accuracy through a rigorous study of several assemblers, compared under both standard metrics (N50, coverage, contig sizes, etc.) as well as a more comprehensive metric (Feature-Response Curves, FRC) that is introduced here; FRC transparently captures the trade-offs between contigs' quality against their sizes. For this purpose, most of the publicly available major sequence assemblers--both for low-coverage long (Sanger) and high-coverage short (Illumina) reads technologies--are compared. These assemblers are applied to microbial (Escherichia coli, Brucella, Wolbachia, Staphylococcus, Helicobacter) and partial human genome sequences (Chr. Y), using sequence reads of various read-lengths, coverages, accuracies, and with and without mate-pairs. It is hoped that, based on these evaluations, computational biologists will identify innovative sequence assembly paradigms, bioinformaticists will determine promising approaches for developing "next-generation" assemblers, and biotechnologists will formulate more meaningful design desiderata for sequencing technology platforms. A new software tool for computing the FRC metric has been developed and is available through the AMOS open-source consortium.

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.

PubMed

VanBuren, Robert; Bryant, Doug; Edger, Patrick P; Tang, Haibao; Burgess, Diane; Challabathula, Dinakar; Spittle, Kristi; Hall, Richard; Gu, Jenny; Lyons, Eric; Freeling, Michael; Bartels, Dorothea; Ten Hallers, Boudewijn; Hastie, Alex; Michael, Todd P; Mockler, Todd C

2015-11-26

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

Vaccination coverage and out-of-sequence vaccinations in rural Guinea-Bissau: an observational cohort study

PubMed Central

Hornshøj, Linda; Benn, Christine Stabell; Fernandes, Manuel; Rodrigues, Amabelia; Aaby, Peter; Fisker, Ane Bærent

2012-01-01

Objective The WHO aims for 90% coverage of the Expanded Program on Immunization (EPI), which in Guinea-Bissau included BCG vaccine at birth, three doses of diphtheria−tetanus−pertussis vaccine (DTP) and oral polio vaccine (OPV) at 6, 10 and 14 weeks and measles vaccine (MV) at 9 months when this study was conducted. The WHO assesses coverage by 12 months of age. The sequence of vaccines may have an effect on child mortality, but is not considered in official statistics or assessments of programme performance. We assessed vaccination coverage and frequency of out-of-sequence vaccinations by 12 and 24 months of age. Design Observational cohort study. Setting and participants The Bandim Health Project's (BHP) rural Health and Demographic Surveillance site covers 258 randomly selected villages in all regions of Guinea-Bissau. Villages are visited biannually and vaccination cards inspected to ascertain vaccination status. Between 2003 and 2009 vaccination status by 12 months of age was assessed for 5806 children aged 12–23 months; vaccination status by 24 months of age was assessed for 3792 children aged 24–35 months. Outcome measures Coverage of EPI vaccinations and frequency of out-of-sequence vaccinations. Results Half of 12-month-old children and 65% of 24-month-old children had completed all EPI vaccinations. Many children received vaccines out of sequence: by 12 months of age 54% of BCG-vaccinated children had received DTP with or before BCG and 28% of measles-vaccinated children had received DTP with or after MV. By 24 months of age the proportion of out-of-sequence vaccinations was 58% and 35%, respectively, for BCG and MV. Conclusions In rural Guinea-Bissau vaccination coverage by 12 months of age was low, but continued to increase beyond 12 months of age. More than half of all children received vaccinations out of sequence. This highlights the need to improve vaccination services. PMID:23166127

Lessons for livestock genomics from genome and transcriptome sequencing in cattle and other mammals.

PubMed

Taylor, Jeremy F; Whitacre, Lynsey K; Hoff, Jesse L; Tizioto, Polyana C; Kim, JaeWoo; Decker, Jared E; Schnabel, Robert D

2016-08-17

Decreasing sequencing costs and development of new protocols for characterizing global methylation, gene expression patterns and regulatory regions have stimulated the generation of large livestock datasets. Here, we discuss experiences in the analysis of whole-genome and transcriptome sequence data. We analyzed whole-genome sequence (WGS) data from 132 individuals from five canid species (Canis familiaris, C. latrans, C. dingo, C. aureus and C. lupus) and 61 breeds, three bison (Bison bison), 64 water buffalo (Bubalus bubalis) and 297 bovines from 17 breeds. By individual, data vary in extent of reference genome depth of coverage from 4.9X to 64.0X. We have also analyzed RNA-seq data for 580 samples representing 159 Bos taurus and Rattus norvegicus animals and 98 tissues. By aligning reads to a reference assembly and calling variants, we assessed effects of average depth of coverage on the actual coverage and on the number of called variants. We examined the identity of unmapped reads by assembling them and querying produced contigs against the non-redundant nucleic acids database. By imputing high-density single nucleotide polymorphism data on 4010 US registered Angus animals to WGS using Run4 of the 1000 Bull Genomes Project and assessing the accuracy of imputation, we identified misassembled reference sequence regions. We estimate that a 24X depth of coverage is required to achieve 99.5 % coverage of the reference assembly and identify 95 % of the variants within an individual's genome. Genomes sequenced to low average coverage (e.g., <10X) may fail to cover 10 % of the reference genome and identify <75 % of variants. About 10 % of genomic DNA or transcriptome sequence reads fail to align to the reference assembly. These reads include loci missing from the reference assembly and misassembled genes and interesting symbionts, commensal and pathogenic organisms. Assembly errors and a lack of annotation of functional elements significantly limit the utility of the current draft livestock reference assemblies. The Functional Annotation of Animal Genomes initiative seeks to annotate functional elements, while a 70X Pac-Bio assembly for cow is underway and may result in a significantly improved reference assembly.

Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) are large insertions, deletions or duplications in the genome that vary between members of a species and are known to affect a wide variety of phenotypic traits. In this study, we identified CNVs in a population of bulls using low coverage next-generation sequence data....

Initial sequence and comparative analysis of the cat genome

PubMed Central

Pontius, Joan U.; Mullikin, James C.; Smith, Douglas R.; Lindblad-Toh, Kerstin; Gnerre, Sante; Clamp, Michele; Chang, Jean; Stephens, Robert; Neelam, Beena; Volfovsky, Natalia; Schäffer, Alejandro A.; Agarwala, Richa; Narfström, Kristina; Murphy, William J.; Giger, Urs; Roca, Alfred L.; Antunes, Agostinho; Menotti-Raymond, Marilyn; Yuhki, Naoya; Pecon-Slattery, Jill; Johnson, Warren E.; Bourque, Guillaume; Tesler, Glenn; O’Brien, Stephen J.

2007-01-01

The genome sequence (1.9-fold coverage) of an inbred Abyssinian domestic cat was assembled, mapped, and annotated with a comparative approach that involved cross-reference to annotated genome assemblies of six mammals (human, chimpanzee, mouse, rat, dog, and cow). The results resolved chromosomal positions for 663,480 contigs, 20,285 putative feline gene orthologs, and 133,499 conserved sequence blocks (CSBs). Additional annotated features include repetitive elements, endogenous retroviral sequences, nuclear mitochondrial (numt) sequences, micro-RNAs, and evolutionary breakpoints that suggest historic balancing of translocation and inversion incidences in distinct mammalian lineages. Large numbers of single nucleotide polymorphisms (SNPs), deletion insertion polymorphisms (DIPs), and short tandem repeats (STRs), suitable for linkage or association studies were characterized in the context of long stretches of chromosome homozygosity. In spite of the light coverage capturing ∼65% of euchromatin sequence from the cat genome, these comparative insights shed new light on the tempo and mode of gene/genome evolution in mammals, promise several research applications for the cat, and also illustrate that a comparative approach using more deeply covered mammals provides an informative, preliminary annotation of a light (1.9-fold) coverage mammal genome sequence. PMID:17975172

Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.

PubMed

Nair, Shalima S; Luu, Phuc-Loi; Qu, Wenjia; Maddugoda, Madhavi; Huschtscha, Lily; Reddel, Roger; Chenevix-Trench, Georgia; Toso, Martina; Kench, James G; Horvath, Lisa G; Hayes, Vanessa M; Stricker, Phillip D; Hughes, Timothy P; White, Deborah L; Rasko, John E J; Wong, Justin J-L; Clark, Susan J

2018-05-28

Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing. In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×). Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.

RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing.

PubMed

Chang, Lun-Ching; Das, Biswajit; Lih, Chih-Jian; Si, Han; Camalier, Corinne E; McGregor, Paul M; Polley, Eric

2016-01-01

With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96-0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman's coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.

Flexible RF filter using a nonuniform SCISSOR.

PubMed

Zhuang, Leimeng

2016-03-15

This work presents a flexible radiofrequency (RF) filter using an integrated microwave photonic circuit that comprises a nonuniform side-coupled integrated spaced sequence of resonators (N-SCISSOR). The filter passband can be reconfigured by varying the N-SCISSOR parameters. When employing a dual-parallel Mach-Zechnder modulator, the filter is also able to perform frequency down-conversion. In the experiment, various filter response shapes are shown, ranging from a flat-top band-pass filter to a total opposite high-rejection (>40  dB) notch filter, with a frequency coverage of greater than two octaves. The frequency down-conversion function is also demonstrated.

Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics*

PubMed Central

Guo, Xiaofeng; Trudgian, David C.; Lemoff, Andrew; Yadavalli, Sivaramakrishna; Mirzaei, Hamid

2014-01-01

Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases. PMID:24696503

Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies.

PubMed

Card, Daren C; Schield, Drew R; Reyes-Velasco, Jacobo; Fujita, Matthew K; Andrew, Audra L; Oyler-McCance, Sara J; Fike, Jennifer A; Tomback, Diana F; Ruggiero, Robert P; Castoe, Todd A

2014-01-01

As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5-5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.

Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies

USGS Publications Warehouse

Card, Daren C.; Schield, Drew R.; Reyes-Velasco, Jacobo; Fujita, Matthre K.; Andrew, Audra L.; Oyler-McCance, Sara J.; Fike, Jennifer A.; Tomback, Diana F.; Ruggiero, Robert P.; Castoe, Todd A.

2014-01-01

As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (~3.5–5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.

Calibrating genomic and allelic coverage bias in single-cell sequencing.

PubMed

Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

2015-04-16

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

Calibrating genomic and allelic coverage bias in single-cell sequencing

PubMed Central

Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

2016-01-01

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

Whole genome sequencing data and de novo draft assemblies for 66 teleost species

PubMed Central

Malmstrøm, Martin; Matschiner, Michael; Tørresen, Ole K.; Jakobsen, Kjetill S.; Jentoft, Sissel

2017-01-01

Teleost fishes comprise more than half of all vertebrate species, yet genomic data are only available for 0.2% of their diversity. Here, we present whole genome sequencing data for 66 new species of teleosts, vastly expanding the availability of genomic data for this important vertebrate group. We report on de novo assemblies based on low-coverage (9–39×) sequencing and present detailed methodology for all analyses. To facilitate further utilization of this data set, we present statistical analyses of the gene space completeness and verify the expected phylogenetic position of the sequenced genomes in a large mitogenomic context. We further present a nuclear marker set used for phylogenetic inference and evaluate each gene tree in relation to the species tree to test for homogeneity in the phylogenetic signal. Collectively, these analyses illustrate the robustness of this highly diverse data set and enable extensive reuse of the selected phylogenetic markers and the genomic data in general. This data set covers all major teleost lineages and provides unprecedented opportunities for comparative studies of teleosts. PMID:28094797

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

NASA Astrophysics Data System (ADS)

Weisbrod, Chad R.; Kaiser, Nathan K.; Syka, John E. P.; Early, Lee; Mullen, Christopher; Dunyach, Jean-Jacques; English, A. Michelle; Anderson, Lissa C.; Blakney, Greg T.; Shabanowitz, Jeffrey; Hendrickson, Christopher L.; Marshall, Alan G.; Hunt, Donald F.

2017-09-01

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., 60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. [Figure not available: see fulltext.

High-throughput gene mapping in Caenorhabditis elegans.

PubMed

Swan, Kathryn A; Curtis, Damian E; McKusick, Kathleen B; Voinov, Alexander V; Mapa, Felipa A; Cancilla, Michael R

2002-07-01

Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 +/- 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18.

Immobilized Pepsin Microreactor for Rapid Peptide Mapping with Nanoelectrospray Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Long, Ying; Wood, Troy D.

2015-01-01

Most enzymatic microreactors for protein digestion are based on trypsin, but proteins with hydrophobic segments may be difficult to digest because of the paucity of Arg and Lys residues. Microreactors based on pepsin, which is less specific than trypsin, can overcome this challenge. Here, an integrated immobilized pepsin microreactor (IPMR)/nanoelectrospray emitter is examined for its potential for peptide mapping. For myoglobin, equivalent sequence coverage is obtained in a thousandth the time of solution digestion with better sequence coverage. While sequence coverage of cytochrome c is lesser than solution in this short duration, more highly-charged peptic peptides are produced and a number of peaks are unidentified at low-resolution, suggesting that high-resolution mass spectrometry is needed to take full advantage of integrated IPMR/nanoelectrospray devices.

A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data

PubMed Central

2013-01-01

Background The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Results Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. Conclusions In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data. PMID:23442253

Analysis of quality raw data of second generation sequencers with Quality Assessment Software.

PubMed

Ramos, Rommel Tj; Carneiro, Adriana R; Baumbach, Jan; Azevedo, Vasco; Schneider, Maria Pc; Silva, Artur

2011-04-18

Second generation technologies have advantages over Sanger; however, they have resulted in new challenges for the genome construction process, especially because of the small size of the reads, despite the high degree of coverage. Independent of the program chosen for the construction process, DNA sequences are superimposed, based on identity, to extend the reads, generating contigs; mismatches indicate a lack of homology and are not included. This process improves our confidence in the sequences that are generated. We developed Quality Assessment Software, with which one can review graphs showing the distribution of quality values from the sequencing reads. This software allow us to adopt more stringent quality standards for sequence data, based on quality-graph analysis and estimated coverage after applying the quality filter, providing acceptable sequence coverage for genome construction from short reads. Quality filtering is a fundamental step in the process of constructing genomes, as it reduces the frequency of incorrect alignments that are caused by measuring errors, which can occur during the construction process due to the size of the reads, provoking misassemblies. Application of quality filters to sequence data, using the software Quality Assessment, along with graphing analyses, provided greater precision in the definition of cutoff parameters, which increased the accuracy of genome construction.

14 CFR 205.5 - Minimum coverage.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Minimum coverage. 205.5 Section 205.5... REGULATIONS AIRCRAFT ACCIDENT LIABILITY INSURANCE § 205.5 Minimum coverage. (a) Insurance contracts and self... maintain the following coverage: (1) Third-party aircraft accident liability coverage for bodily injury to...

Deep Space Network and Lunar Network Communication Coverage of the Moon

NASA Technical Reports Server (NTRS)

Lee, Charles H.; Cheung, Kar-Ming

2006-01-01

In this article, we describe the communication coverage analysis for the lunar network and the Earth ground stations. The first part of this article focuses on the direct communication coverage of the Moon from the Earth's ground stations. In particular, we assess the coverage performance of the Moon based on the existing Deep Space Network (DSN) antennas and the complimentary coverage of other potential stations at Hartebeesthoek, South Africa and at Santiago, Chile. We also address the coverage sensitivity based on different DSN antenna scenarios and their capability to provide single and redundant coverage of the Moon. The second part of this article focuses on the framework of the constrained optimization scheme to seek a stable constellation six relay satellites in two planes that not only can provide continuous communication coverage to any users on the Moon surface, but can also deliver data throughput in a highly efficient manner.

MCMC-ODPR: primer design optimization using Markov Chain Monte Carlo sampling.

PubMed

Kitchen, James L; Moore, Jonathan D; Palmer, Sarah A; Allaby, Robin G

2012-11-05

Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base.

MCMC-ODPR: Primer design optimization using Markov Chain Monte Carlo sampling

PubMed Central

2012-01-01

Background Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. Results After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. Conclusions MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base. PMID:23126469

Identification of copy number variants in whole-genome data using Reference Coverage Profiles

PubMed Central

Glusman, Gustavo; Severson, Alissa; Dhankani, Varsha; Robinson, Max; Farrah, Terry; Mauldin, Denise E.; Stittrich, Anna B.; Ament, Seth A.; Roach, Jared C.; Brunkow, Mary E.; Bodian, Dale L.; Vockley, Joseph G.; Shmulevich, Ilya; Niederhuber, John E.; Hood, Leroy

2015-01-01

The identification of DNA copy numbers from short-read sequencing data remains a challenge for both technical and algorithmic reasons. The raw data for these analyses are measured in tens to hundreds of gigabytes per genome; transmitting, storing, and analyzing such large files is cumbersome, particularly for methods that analyze several samples simultaneously. We developed a very efficient representation of depth of coverage (150–1000× compression) that enables such analyses. Current methods for analyzing variants in whole-genome sequencing (WGS) data frequently miss copy number variants (CNVs), particularly hemizygous deletions in the 1–100 kb range. To fill this gap, we developed a method to identify CNVs in individual genomes, based on comparison to joint profiles pre-computed from a large set of genomes. We analyzed depth of coverage in over 6000 high quality (>40×) genomes. The depth of coverage has strong sequence-specific fluctuations only partially explained by global parameters like %GC. To account for these fluctuations, we constructed multi-genome profiles representing the observed or inferred diploid depth of coverage at each position along the genome. These Reference Coverage Profiles (RCPs) take into account the diverse technologies and pipeline versions used. Normalization of the scaled coverage to the RCP followed by hidden Markov model (HMM) segmentation enables efficient detection of CNVs and large deletions in individual genomes. Use of pre-computed multi-genome coverage profiles improves our ability to analyze each individual genome. We make available RCPs and tools for performing these analyses on personal genomes. We expect the increased sensitivity and specificity for individual genome analysis to be critical for achieving clinical-grade genome interpretation. PMID:25741365

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

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

VanBuren, Robert; Bryant, Doug; Edger, Patrick P.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

DOE PAGES

VanBuren, Robert; Bryant, Doug; Edger, Patrick P.; ...

2015-11-11

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less

SpaceX CRS-12 Live Launch Coverage

NASA Image and Video Library

2017-08-14

Live Launch Coverage of the SpaceX Falcon 9 launch vehicle lift off from Launch Complex 39A at NASA's Kennedy Space Center carrying the Dragon resupply spacecraft to the International Space Station. Liftoff was at 12:31 p.m. EDT. On its 12th commercial resupply services mission to the International Space Station, Dragon will bring up more than 6,400 pounds of supplies and new science experiments and equipment for technology research.

14 CFR 440.11 - Duration of coverage for licensed launch, including suborbital launch, or permitted activities...

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Duration of coverage for licensed launch... Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... attach when a licensed launch or permitted activity starts, and remain in full force and effect as...

14 CFR 440.12 - Duration of coverage for licensed reentry; modifications.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Duration of coverage for licensed reentry; modifications. 440.12 Section 440.12 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION..., shall attach upon commencement of licensed reentry, and remain in full force and effect as follows: (1...

14 CFR 440.12 - Duration of coverage for licensed reentry; modifications.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Duration of coverage for licensed reentry; modifications. 440.12 Section 440.12 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION..., shall attach upon commencement of licensed reentry, and remain in full force and effect as follows: (1...

14 CFR 440.11 - Duration of coverage for licensed launch, including suborbital launch, or permitted activities...

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Duration of coverage for licensed launch... Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... attach when a licensed launch or permitted activity starts, and remain in full force and effect as...

14 CFR 440.11 - Duration of coverage for licensed launch, including suborbital launch, or permitted activities...

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 4 2012-01-01 2012-01-01 false Duration of coverage for licensed launch... Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION... attach when a licensed launch or permitted activity starts, and remain in full force and effect as...

14 CFR 440.12 - Duration of coverage for licensed reentry; modifications.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Duration of coverage for licensed reentry; modifications. 440.12 Section 440.12 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION..., shall attach upon commencement of licensed reentry, and remain in full force and effect as follows: (1...

14 CFR 440.12 - Duration of coverage for licensed reentry; modifications.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Duration of coverage for licensed reentry; modifications. 440.12 Section 440.12 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION..., shall attach upon commencement of licensed reentry, and remain in full force and effect as follows: (1...

14 CFR 440.12 - Duration of coverage for licensed reentry; modifications.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 4 2012-01-01 2012-01-01 false Duration of coverage for licensed reentry; modifications. 440.12 Section 440.12 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION..., shall attach upon commencement of licensed reentry, and remain in full force and effect as follows: (1...

MSeq-CNV: accurate detection of Copy Number Variation from Sequencing of Multiple samples.

PubMed

Malekpour, Seyed Amir; Pezeshk, Hamid; Sadeghi, Mehdi

2018-03-05

Currently a few tools are capable of detecting genome-wide Copy Number Variations (CNVs) based on sequencing of multiple samples. Although aberrations in mate pair insertion sizes provide additional hints for the CNV detection based on multiple samples, the majority of the current tools rely only on the depth of coverage. Here, we propose a new algorithm (MSeq-CNV) which allows detecting common CNVs across multiple samples. MSeq-CNV applies a mixture density for modeling aberrations in depth of coverage and abnormalities in the mate pair insertion sizes. Each component in this mixture density applies a Binomial distribution for modeling the number of mate pairs with aberration in the insertion size and also a Poisson distribution for emitting the read counts, in each genomic position. MSeq-CNV is applied on simulated data and also on real data of six HapMap individuals with high-coverage sequencing, in 1000 Genomes Project. These individuals include a CEU trio of European ancestry and a YRI trio of Nigerian ethnicity. Ancestry of these individuals is studied by clustering the identified CNVs. MSeq-CNV is also applied for detecting CNVs in two samples with low-coverage sequencing in 1000 Genomes Project and six samples form the Simons Genome Diversity Project.

Genome Wide Characterization of Simple Sequence Repeats in Cucumber

USDA-ARS?s Scientific Manuscript database

The whole genome sequence of the cucumber cultivar Gy14 was recently sequenced at 15× coverage with the Roche 454 Titanium technology. The microsatellite DNA sequences (simple sequence repeats, SSRs) in the assembled scaffolds were computationally explored and characterized. A total of 112,073 SSRs ...

Two Low Coverage Bird Genomes and a Comparison of Reference-Guided versus De Novo Genome Assemblies

PubMed Central

Card, Daren C.; Schield, Drew R.; Reyes-Velasco, Jacobo; Fujita, Matthew K.; Andrew, Audra L.; Oyler-McCance, Sara J.; Fike, Jennifer A.; Tomback, Diana F.; Ruggiero, Robert P.; Castoe, Todd A.

2014-01-01

As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5–5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies. PMID:25192061

CNNdel: Calling Structural Variations on Low Coverage Data Based on Convolutional Neural Networks

PubMed Central

2017-01-01

Many structural variations (SVs) detection methods have been proposed due to the popularization of next-generation sequencing (NGS). These SV calling methods use different SV-property-dependent features; however, they all suffer from poor accuracy when running on low coverage sequences. The union of results from these tools achieves fairly high sensitivity but still produces low accuracy on low coverage sequence data. That is, these methods contain many false positives. In this paper, we present CNNdel, an approach for calling deletions from paired-end reads. CNNdel gathers SV candidates reported by multiple tools and then extracts features from aligned BAM files at the positions of candidates. With labeled feature-expressed candidates as a training set, CNNdel trains convolutional neural networks (CNNs) to distinguish true unlabeled candidates from false ones. Results show that CNNdel works well with NGS reads from 26 low coverage genomes of the 1000 Genomes Project. The paper demonstrates that convolutional neural networks can automatically assign the priority of SV features and reduce the false positives efficaciously. PMID:28630866

Reproducibility and Consistency of In Vitro Nucleosome Reconstitutions Demonstrated by Invitrosome Isolation and Sequencing

PubMed Central

Kempton, Colton E.; Heninger, Justin R.; Johnson, Steven M.

2014-01-01

Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems. PMID:25093869

Accounting for Errors in Low Coverage High-Throughput Sequencing Data When Constructing Genetic Maps Using Biparental Outcrossed Populations

PubMed Central

Bilton, Timothy P.; Schofield, Matthew R.; Black, Michael A.; Chagné, David; Wilcox, Phillip L.; Dodds, Ken G.

2018-01-01

Next-generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high-density genetic linkage maps, which facilitate the development of nonmodel species’ genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology (e.g., genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sibling family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sibling populations of diploid species, implemented in a package called GUSMap. Our model is based on the Lander–Green hidden Markov model but extended to account for errors present in sequencing data. We were able to obtain accurate estimates of the recombination fractions and overall map distance using GUSMap, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model. PMID:29487138

Accounting for Errors in Low Coverage High-Throughput Sequencing Data When Constructing Genetic Maps Using Biparental Outcrossed Populations.

PubMed

Bilton, Timothy P; Schofield, Matthew R; Black, Michael A; Chagné, David; Wilcox, Phillip L; Dodds, Ken G

2018-05-01

Next-generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high-density genetic linkage maps, which facilitate the development of nonmodel species' genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology ( e.g. , genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sibling family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sibling populations of diploid species, implemented in a package called GUSMap. Our model is based on the Lander-Green hidden Markov model but extended to account for errors present in sequencing data. We were able to obtain accurate estimates of the recombination fractions and overall map distance using GUSMap, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model. Copyright © 2018 Bilton et al.

SpaceX CRS-13 Live Launch Coverage

NASA Image and Video Library

2017-12-15

Live Launch Coverage of the SpaceX Falcon 9 launch vehicle lift off from Space Launch Complex 40 at Cape Canaveral Air Force Station in Florida carrying the Dragon spacecraft filled with research and supplies to the International Space Station. Liftoff was at 10:36 a.m. EST. On its 13th commercial resupply services mission to the International Space Station, Dragon will bring up nearly 4,800 pounds of supplies and new science experiments and equipment for technology research.

Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

PubMed

Shapland, Elaine B; Holmes, Victor; Reeves, Christopher D; Sorokin, Elena; Durot, Maxime; Platt, Darren; Allen, Christopher; Dean, Jed; Serber, Zach; Newman, Jack; Chandran, Sunil

2015-07-17

In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.

The diploid genome sequence of an Asian individual

PubMed Central

Wang, Jun; Wang, Wei; Li, Ruiqiang; Li, Yingrui; Tian, Geng; Goodman, Laurie; Fan, Wei; Zhang, Junqing; Li, Jun; Zhang, Juanbin; Guo, Yiran; Feng, Binxiao; Li, Heng; Lu, Yao; Fang, Xiaodong; Liang, Huiqing; Du, Zhenglin; Li, Dong; Zhao, Yiqing; Hu, Yujie; Yang, Zhenzhen; Zheng, Hancheng; Hellmann, Ines; Inouye, Michael; Pool, John; Yi, Xin; Zhao, Jing; Duan, Jinjie; Zhou, Yan; Qin, Junjie; Ma, Lijia; Li, Guoqing; Yang, Zhentao; Zhang, Guojie; Yang, Bin; Yu, Chang; Liang, Fang; Li, Wenjie; Li, Shaochuan; Li, Dawei; Ni, Peixiang; Ruan, Jue; Li, Qibin; Zhu, Hongmei; Liu, Dongyuan; Lu, Zhike; Li, Ning; Guo, Guangwu; Zhang, Jianguo; Ye, Jia; Fang, Lin; Hao, Qin; Chen, Quan; Liang, Yu; Su, Yeyang; san, A.; Ping, Cuo; Yang, Shuang; Chen, Fang; Li, Li; Zhou, Ke; Zheng, Hongkun; Ren, Yuanyuan; Yang, Ling; Gao, Yang; Yang, Guohua; Li, Zhuo; Feng, Xiaoli; Kristiansen, Karsten; Wong, Gane Ka-Shu; Nielsen, Rasmus; Durbin, Richard; Bolund, Lars; Zhang, Xiuqing; Li, Songgang; Yang, Huanming; Wang, Jian

2009-01-01

Here we present the first diploid genome sequence of an Asian individual. The genome was sequenced to 36-fold average coverage using massively parallel sequencing technology. We aligned the short reads onto the NCBI human reference genome to 99.97% coverage, and guided by the reference genome, we used uniquely mapped reads to assemble a high-quality consensus sequence for 92% of the Asian individual's genome. We identified approximately 3 million single-nucleotide polymorphisms (SNPs) inside this region, of which 13.6% were not in the dbSNP database. Genotyping analysis showed that SNP identification had high accuracy and consistency, indicating the high sequence quality of this assembly. We also carried out heterozygote phasing and haplotype prediction against HapMap CHB and JPT haplotypes (Chinese and Japanese, respectively), sequence comparison with the two available individual genomes (J. D. Watson and J. C. Venter), and structural variation identification. These variations were considered for their potential biological impact. Our sequence data and analyses demonstrate the potential usefulness of next-generation sequencing technologies for personal genomics. PMID:18987735

KSC-99pp0609

NASA Image and Video Library

1999-05-27

A ceremony dedicated the KSC Press Site auditorium as the John Holliman Auditorium to honor the correspondent for his enthusiastic, dedicated coverage of America's space program. The auditorium was built in 1980 and has been the focal point for news coverage of Space Shuttle launches. The ceremony followed the 94th launch of a Space Shuttle, on mission STS-96, earlier this morning

GeneImp: Fast Imputation to Large Reference Panels Using Genotype Likelihoods from Ultralow Coverage Sequencing

PubMed Central

Spiliopoulou, Athina; Colombo, Marco; Orchard, Peter; Agakov, Felix; McKeigue, Paul

2017-01-01

We address the task of genotype imputation to a dense reference panel given genotype likelihoods computed from ultralow coverage sequencing as inputs. In this setting, the data have a high-level of missingness or uncertainty, and are thus more amenable to a probabilistic representation. Most existing imputation algorithms are not well suited for this situation, as they rely on prephasing for computational efficiency, and, without definite genotype calls, the prephasing task becomes computationally expensive. We describe GeneImp, a program for genotype imputation that does not require prephasing and is computationally tractable for whole-genome imputation. GeneImp does not explicitly model recombination, instead it capitalizes on the existence of large reference panels—comprising thousands of reference haplotypes—and assumes that the reference haplotypes can adequately represent the target haplotypes over short regions unaltered. We validate GeneImp based on data from ultralow coverage sequencing (0.5×), and compare its performance to the most recent version of BEAGLE that can perform this task. We show that GeneImp achieves imputation quality very close to that of BEAGLE, using one to two orders of magnitude less time, without an increase in memory complexity. Therefore, GeneImp is the first practical choice for whole-genome imputation to a dense reference panel when prephasing cannot be applied, for instance, in datasets produced via ultralow coverage sequencing. A related future application for GeneImp is whole-genome imputation based on the off-target reads from deep whole-exome sequencing. PMID:28348060

Error and Error Mitigation in Low-Coverage Genome Assemblies

PubMed Central

Hubisz, Melissa J.; Lin, Michael F.; Kellis, Manolis; Siepel, Adam

2011-01-01

The recent release of twenty-two new genome sequences has dramatically increased the data available for mammalian comparative genomics, but twenty of these new sequences are currently limited to ∼2× coverage. Here we examine the extent of sequencing error in these 2× assemblies, and its potential impact in downstream analyses. By comparing 2× assemblies with high-quality sequences from the ENCODE regions, we estimate the rate of sequencing error to be 1–4 errors per kilobase. While this error rate is fairly modest, sequencing error can still have surprising effects. For example, an apparent lineage-specific insertion in a coding region is more likely to reflect sequencing error than a true biological event, and the length distribution of coding indels is strongly distorted by error. We find that most errors are contributed by a small fraction of bases with low quality scores, in particular, by the ends of reads in regions of single-read coverage in the assembly. We explore several approaches for automatic sequencing error mitigation (SEM), making use of the localized nature of sequencing error, the fact that it is well predicted by quality scores, and information about errors that comes from comparisons across species. Our automatic methods for error mitigation cannot replace the need for additional sequencing, but they do allow substantial fractions of errors to be masked or eliminated at the cost of modest amounts of over-correction, and they can reduce the impact of error in downstream phylogenomic analyses. Our error-mitigated alignments are available for download. PMID:21340033

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies.

PubMed

Utturkar, Sagar M; Klingeman, Dawn M; Hurt, Richard A; Brown, Steven D

2017-01-01

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.

SpaceX CRS-14 Live Launch Coverage

NASA Image and Video Library

2018-04-02

Live Launch Coverage of the SpaceX Falcon 9 lift off from Space Launch Complex 40 at Cape Canaveral Air Force Station in Florida carrying the Dragon spacecraft filled with research and supplies to the International Space Station. Liftoff occurred at 4:30 p.m. EDT., on Monday April 2. On its 14th commercial resupply services mission to the International Space Station, Dragon will bring up nearly 5,800 pounds of research, crew supplies, and hardware to the orbiting laboratory.

Misconceptions on Missing Data in RAD-seq Phylogenetics with a Deep-scale Example from Flowering Plants.

PubMed

Eaton, Deren A R; Spriggs, Elizabeth L; Park, Brian; Donoghue, Michael J

2017-05-01

Restriction-site associated DNA (RAD) sequencing and related methods rely on the conservation of enzyme recognition sites to isolate homologous DNA fragments for sequencing, with the consequence that mutations disrupting these sites lead to missing information. There is thus a clear expectation for how missing data should be distributed, with fewer loci recovered between more distantly related samples. This observation has led to a related expectation: that RAD-seq data are insufficiently informative for resolving deeper scale phylogenetic relationships. Here we investigate the relationship between missing information among samples at the tips of a tree and information at edges within it. We re-analyze and review the distribution of missing data across ten RAD-seq data sets and carry out simulations to determine expected patterns of missing information. We also present new empirical results for the angiosperm clade Viburnum (Adoxaceae, with a crown age >50 Ma) for which we examine phylogenetic information at different depths in the tree and with varied sequencing effort. The total number of loci, the proportion that are shared, and phylogenetic informativeness varied dramatically across the examined RAD-seq data sets. Insufficient or uneven sequencing coverage accounted for similar proportions of missing data as dropout from mutation-disruption. Simulations reveal that mutation-disruption, which results in phylogenetically distributed missing data, can be distinguished from the more stochastic patterns of missing data caused by low sequencing coverage. In Viburnum, doubling sequencing coverage nearly doubled the number of parsimony informative sites, and increased by >10X the number of loci with data shared across >40 taxa. Our analysis leads to a set of practical recommendations for maximizing phylogenetic information in RAD-seq studies. [hierarchical redundancy; phylogenetic informativeness; quartet informativeness; Restriction-site associated DNA (RAD) sequencing; sequencing coverage; Viburnum.]. © The authors 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Challenges of coverage policy development for next-generation tumor sequencing panels: experts and payers weigh in.

PubMed

Trosman, Julia R; Weldon, Christine B; Kelley, R Kate; Phillips, Kathryn A

2015-03-01

Next-generation tumor sequencing (NGTS) panels, which include multiple established and novel targets across cancers, are emerging in oncology practice, but lack formal positive coverage by US payers. Lack of coverage may impact access and adoption. This study identified challenges of NGTS coverage by private payers. We conducted semi-structured interviews with 14 NGTS experts on potential NGTS benefits, and with 10 major payers, representing more than 125,000,000 enrollees, on NGTS coverage considerations. We used the framework approach of qualitative research for study design and thematic analyses and simple frequencies to further describe findings. All interviewed payers see potential NGTS benefits, but all noted challenges to formal coverage: 80% state that inherent features of NGTS do not fit the medical necessity definition required for coverage, 70% view NGTS as a bundle of targets versus comprehensive tumor characterization and may evaluate each target individually, and 70% express skepticism regarding new evidence methods proposed for NGTS. Fifty percent of payers expressed sufficient concerns about NGTS adoption and implementation that will preclude their ability to issue positive coverage policies. Payers perceive that NGTS holds significant promise but, in its current form, poses disruptive challenges to coverage policy frameworks. Proactive multidisciplinary efforts to define the direction for NGTS development, evidence generation, and incorporation into coverage policy are necessary to realize its promise and provide patient access. This study contributes to current literature, as possibly the first study to directly interview US payers on NGTS coverage and reimbursement. Copyright © 2015 by the National Comprehensive Cancer Network.

Challenges of Coverage Policy Development for Next-Generation Tumor Sequencing Panels: Experts and Payers Weigh In

PubMed Central

Trosman, Julia R.; Weldon, Christine B.; Kate Kelley, R.; Phillips, Kathryn A.

2015-01-01

Background Next-generation tumor sequencing (NGTS) panels, which include multiple established and novel targets across cancers, are emerging in oncology practice, but lack formal positive coverage by US payers. Lack of coverage may impact access and adoption. This study identified challenges of NGTS coverage by private payers. Methods We conducted semi-structured interviews with 14 NGTS experts on potential NGTS benefits, and with 10 major payers, representing more than 125,000,000 enrollees, on NGTS coverage considerations. We used the framework approach of qualitative research for study design and thematic analyses and simple frequencies to further describe findings. Results All interviewed payers see potential NGTS benefits, but all noted challenges to formal coverage: 80% state that inherent features of NGTS do not fit the medical necessity definition required for coverage, 70% view NGTS as a bundle of targets versus comprehensive tumor characterization and may evaluate each target individually, and 70% express skepticism regarding new evidence methods proposed for NGTS. Fifty percent of payers expressed sufficient concerns about NGTS adoption and implementation that will preclude their ability to issue positive coverage policies. Conclusions Payers perceive that NGTS holds significant promise but, in its current form, poses disruptive challenges to coverage policy frameworks. Proactive multidisciplinary efforts to define the direction for NGTS development, evidence generation, and incorporation into coverage policy are necessary to realize its promise and provide patient access. This study contributes to current literature, as possibly the first study to directly interview US payers on NGTS coverage and reimbursement. PMID:25736008

Press Site Auditorium dedicated to John Holliman

NASA Technical Reports Server (NTRS)

1999-01-01

A ceremony dedicated the KSC Press Site auditorium as the John Holliman Auditorium to honor the correspondent for his enthusiastic, dedicated coverage of America's space program. The auditorium was built in 1980 and has been the focal point for new coverage of Space Shuttle launches. The ceremony followed the 94th launch of a Space Shuttle, on mission STS-96, earlier this morning.

Deep whole-genome sequencing of 90 Han Chinese genomes.

PubMed

Lan, Tianming; Lin, Haoxiang; Zhu, Wenjuan; Laurent, Tellier Christian Asker Melchior; Yang, Mengcheng; Liu, Xin; Wang, Jun; Wang, Jian; Yang, Huanming; Xu, Xun; Guo, Xiaosen

2017-09-01

Next-generation sequencing provides a high-resolution insight into human genetic information. However, the focus of previous studies has primarily been on low-coverage data due to the high cost of sequencing. Although the 1000 Genomes Project and the Haplotype Reference Consortium have both provided powerful reference panels for imputation, low-frequency and novel variants remain difficult to discover and call with accuracy on the basis of low-coverage data. Deep sequencing provides an optimal solution for the problem of these low-frequency and novel variants. Although whole-exome sequencing is also a viable choice for exome regions, it cannot account for noncoding regions, sometimes resulting in the absence of important, causal variants. For Han Chinese populations, the majority of variants have been discovered based upon low-coverage data from the 1000 Genomes Project. However, high-coverage, whole-genome sequencing data are limited for any population, and a large amount of low-frequency, population-specific variants remain uncharacterized. We have performed whole-genome sequencing at a high depth (∼×80) of 90 unrelated individuals of Chinese ancestry, collected from the 1000 Genomes Project samples, including 45 Northern Han Chinese and 45 Southern Han Chinese samples. Eighty-three of these 90 have been sequenced by the 1000 Genomes Project. We have identified 12 568 804 single nucleotide polymorphisms, 2 074 210 short InDels, and 26 142 structural variations from these 90 samples. Compared to the Han Chinese data from the 1000 Genomes Project, we have found 7 000 629 novel variants with low frequency (defined as minor allele frequency < 5%), including 5 813 503 single nucleotide polymorphisms, 1 169 199 InDels, and 17 927 structural variants. Using deep sequencing data, we have built a greatly expanded spectrum of genetic variation for the Han Chinese genome. Compared to the 1000 Genomes Project, these Han Chinese deep sequencing data enhance the characterization of a large number of low-frequency, novel variants. This will be a valuable resource for promoting Chinese genetics research and medical development. Additionally, it will provide a valuable supplement to the 1000 Genomes Project, as well as to other human genome projects. © The Authors 2017. Published by Oxford University Press.

Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys

PubMed Central

Berry, David; Gutierrez, Tony

2017-01-01

Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira, and Thalassolituus, as well as the Alphaproteobacterial genus Thalassospira. Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys. PMID:28567035

Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys.

PubMed

Berry, David; Gutierrez, Tony

2017-01-01

Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira , and Thalassolituus , as well as the Alphaproteobacterial genus Thalassospira . Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys.

Exploiting long read sequencing technologies to establish high quality highly contiguous pig reference genome assemblies

USDA-ARS?s Scientific Manuscript database

The current pig reference genome sequence (Sscrofa10.2) was established using Sanger sequencing and following the clone-by-clone hierarchical shotgun sequencing approach used in the public human genome project. However, as sequence coverage was low (4-6x) the resulting assembly was only of draft qua...

Accurate and exact CNV identification from targeted high-throughput sequence data.

PubMed

Nord, Alex S; Lee, Ming; King, Mary-Claire; Walsh, Tom

2011-04-12

Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data. Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate. Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.

Coastal bacterioplankton community diversity along a latitudinal gradient in Latin America by means of V6 tag pyrosequencing.

PubMed

Thompson, Fabiano L; Bruce, Thiago; Gonzalez, Alessandra; Cardoso, Alexander; Clementino, Maysa; Costagliola, Marcela; Hozbor, Constanza; Otero, Ernesto; Piccini, Claudia; Peressutti, Silvia; Schmieder, Robert; Edwards, Robert; Smith, Mathew; Takiyama, Luis Roberto; Vieira, Ricardo; Paranhos, Rodolfo; Artigas, Luis Felipe

2011-02-01

The bacterioplankton diversity of coastal waters along a latitudinal gradient between Puerto Rico and Argentina was analyzed using a total of 134,197 high-quality sequences from the V6 hypervariable region of the small-subunit ribosomal RNA gene (16S rRNA) (mean length of 60 nt). Most of the OTUs were identified into Proteobacteria, Bacteriodetes, Cyanobacteria, and Actinobacteria, corresponding to approx. 80% of the total number of sequences. The number of OTUs corresponding to species varied between 937 and 1946 in the seven locations. Proteobacteria appeared at high frequency in the seven locations. An enrichment of Cyanobacteria was observed in Puerto Rico, whereas an enrichment of Bacteroidetes was detected in the Argentinian shelf and Uruguayan coastal lagoons. The highest number of sequences of Actinobacteria and Acidobacteria were obtained in the Amazon estuary mouth. The rarefaction curves and Good coverage estimator for species diversity suggested a significant coverage, with values ranging between 92 and 97% for Good coverage. Conserved taxa corresponded to aprox. 52% of all sequences. This study suggests that human-contaminated environments may influence bacterioplankton diversity.

Rare Cell Detection by Single-Cell RNA Sequencing as Guided by Single-Molecule RNA FISH.

PubMed

Torre, Eduardo; Dueck, Hannah; Shaffer, Sydney; Gospocic, Janko; Gupte, Rohit; Bonasio, Roberto; Kim, Junhyong; Murray, John; Raj, Arjun

2018-02-28

Although single-cell RNA sequencing can reliably detect large-scale transcriptional programs, it is unclear whether it accurately captures the behavior of individual genes, especially those that express only in rare cells. Here, we use single-molecule RNA fluorescence in situ hybridization as a gold standard to assess trade-offs in single-cell RNA-sequencing data for detecting rare cell expression variability. We quantified the gene expression distribution for 26 genes that range from ubiquitous to rarely expressed and found that the correspondence between estimates across platforms improved with both transcriptome coverage and increased number of cells analyzed. Further, by characterizing the trade-off between transcriptome coverage and number of cells analyzed, we show that when the number of genes required to answer a given biological question is small, then greater transcriptome coverage is more important than analyzing large numbers of cells. More generally, our report provides guidelines for selecting quality thresholds for single-cell RNA-sequencing experiments aimed at rare cell analyses. Copyright © 2018 Elsevier Inc. All rights reserved.

Temporal Changes in BEXSERO® Antigen Sequence Type Associated with Genetic Lineages of Neisseria meningitidis over a 15-Year Period in Western Australia

PubMed Central

Mowlaboccus, Shakeel; Perkins, Timothy T.; Smith, Helen; Sloots, Theo; Tozer, Sarah; Prempeh, Lydia-Jessica; Tay, Chin Yen; Peters, Fanny; Speers, David; Keil, Anthony D.; Kahler, Charlene M.

2016-01-01

Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). The BEXSERO® vaccine which is used to prevent serogroup B disease is composed of four sub-capsular protein antigens supplemented with an outer membrane vesicle. Since the sub-capsular protein antigens are variably expressed and antigenically variable amongst meningococcal isolates, vaccine coverage can be estimated by the meningococcal antigen typing system (MATS) which measures the propensity of the strain to be killed by vaccinated sera. Whole genome sequencing (WGS) which identifies the alleles of the antigens that may be recognised by the antibody response could represent, in future, an alternative estimate of coverage. In this study, WGS of 278 meningococcal isolates responsible for 62% of IMD in Western Australia from 2000–2014 were analysed for association of genetic lineage (sequence type [ST], clonal complex [cc]) with BEXSERO® antigen sequence type (BAST) and MATS to predict the annual vaccine coverage. A hyper-endemic period of IMD between 2000–05 was caused by cc41/44 with the major sequence type of ST-146 which was not predicted by MATS or BAST to be covered by the vaccine. An increase in serogroup diversity was observed between 2010–14 with the emergence of cc11 serogroup W in the adolescent population and cc23 serogroup Y in the elderly. BASTs were statistically associated with clonal complex although individual antigens underwent antigenic drift from the major type. BAST and MATS predicted an annual range of 44–91% vaccine coverage. Periods of low vaccine coverage in years post-2005 were not a result of the resurgence of cc41/44:ST-146 but were characterised by increased diversity of clonal complexes expressing BASTs which were not predicted by MATS to be covered by the vaccine. The driving force behind the diversity of the clonal complex and BAST during these periods of low vaccine coverage is unknown, but could be due to immune selection and inter-strain competition with carriage of non-disease causing meningococci. PMID:27355628

Non-ECG-gated unenhanced MRA of the carotids: optimization and clinical feasibility.

PubMed

Raoult, H; Gauvrit, J Y; Schmitt, P; Le Couls, V; Bannier, E

2013-11-01

To optimise and assess the clinical feasibility of a carotid non-ECG-gated unenhanced MRA sequence. Sixteen healthy volunteers and 11 patients presenting with internal carotid artery (ICA) disease underwent large field-of-view balanced steady-state free precession (bSSFP) unenhanced MRA at 3T. Sampling schemes acquiring the k-space centre either early (kCE) or late (kCL) in the acquisition window were evaluated. Signal and image quality was scored in comparison to ECG-gated kCE unenhanced MRA and TOF. For patients, computed tomography angiography was used as the reference. In volunteers, kCE sampling yielded higher image quality than kCL and TOF, with fewer flow artefacts and improved signal homogeneity. kCE unenhanced MRA image quality was higher without ECG-gating. Arterial signal and artery/vein contrast were higher with both bSSFP sampling schemes than with TOF. The kCE sequence allowed correct quantification of ten significant stenoses, and it facilitated the identification of an infrapetrous dysplasia, which was outside of the TOF imaging coverage. Non-ECG-gated bSSFP carotid imaging offers high-quality images and is a promising sequence for carotid disease diagnosis in a short acquisition time with high spatial resolution and a large field of view. • Non-ECG-gated unenhanced bSSFP MRA offers high-quality imaging of the carotid arteries. • Sequences using early acquisition of the k-space centre achieve higher image quality. • Non-ECG-gated unenhanced bSSFP MRA allows quantification of significant carotid stenosis. • Short MR acquisition times and ungated sequences are helpful in clinical practice. • High 3D spatial resolution and a large field of view improve diagnostic performance.

Optimization of conditions to sequence long cDNAs from viruses

USDA-ARS?s Scientific Manuscript database

Fourth generation sequencing with the Minion nanopore sequencer provides opportunity to obtain deep coverage and long read for single molecules. This will benefit studies on RNA viruses. In the past, Sanger, Illumina, and Ion Torrent sequencing have been utilized to study RNA viruses. Both technique...

TUMOR HAPLOTYPE ASSEMBLY ALGORITHMS FOR CANCER GENOMICS

PubMed Central

AGUIAR, DEREK; WONG, WENDY S.W.; ISTRAIL, SORIN

2014-01-01

The growing availability of inexpensive high-throughput sequence data is enabling researchers to sequence tumor populations within a single individual at high coverage. But, cancer genome sequence evolution and mutational phenomena like driver mutations and gene fusions are difficult to investigate without first reconstructing tumor haplotype sequences. Haplotype assembly of single individual tumor populations is an exceedingly difficult task complicated by tumor haplotype heterogeneity, tumor or normal cell sequence contamination, polyploidy, and complex patterns of variation. While computational and experimental haplotype phasing of diploid genomes has seen much progress in recent years, haplotype assembly in cancer genomes remains uncharted territory. In this work, we describe HapCompass-Tumor a computational modeling and algorithmic framework for haplotype assembly of copy number variable cancer genomes containing haplotypes at different frequencies and complex variation. We extend our polyploid haplotype assembly model and present novel algorithms for (1) complex variations, including copy number changes, as varying numbers of disjoint paths in an associated graph, (2) variable haplotype frequencies and contamination, and (3) computation of tumor haplotypes using simple cycles of the compass graph which constrain the space of haplotype assembly solutions. The model and algorithm are implemented in the software package HapCompass-Tumor which is available for download from http://www.brown.edu/Research/Istrail_Lab/. PMID:24297529

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.

Architecture Study on Telemetry Coverage for Immediate Post-Separation Phase

NASA Technical Reports Server (NTRS)

Cheung, Kar-Ming; Lee, Charles; Kellogg, Kent; Stocklin, Frank; Zillig, David; Fielhauer, Karl

2008-01-01

This document is the viewgraphs that accompanies a paper that presents the preliminary results of an architecture study that provides continuous telemetry coverage for NASA missions for immediate post-separation phase. After launch when the spacecraft separated from the upper stage, the spacecraft typically executes a number of mission-critical operations prior to the deployment of solar panels and the activation of the primary communication subsystem. JPL, GSFC, and APL have similar design principle statements that require continuous coverage of mission-critical telemetry during the immediate post-separation phase. To conform to these design principles, an architecture that consists of a separate spacecraft transmitter and a robust communication network capable of tracking the spacecraft signals is needed. The main results of this study are as follows: 1) At low altitude (< 10000 km) when most post-separation critical operations are executed, Earth-based network (e.g. Deep Space Network (DSN)) can only provide limited coverage, whereas space-based network (e.g. Space Network (SN)) can provide continuous coverage. 2) Commercial-off-the-shelf SN compatible transmitters are available for small satellite applications. In this paper we present the detailed coverage analysis of Earth-based and Space-based networks. We identify the key functional and performance requirements of the architecture, and describe the proposed selection criteria of the spacecraft transmitter. We conclude the paper with a proposed forward plan.

ACLAME: a CLAssification of Mobile genetic Elements, update 2010.

PubMed

Leplae, Raphaël; Lima-Mendez, Gipsi; Toussaint, Ariane

2010-01-01

The ACLAME database is dedicated to the collection, analysis and classification of sequenced mobile genetic elements (MGEs, in particular phages and plasmids). In addition to providing information on the MGEs content, classifications are available at various levels of organization. At the gene/protein level, families group similar sequences that are expected to share the same function. Families of four or more proteins are manually assigned with a functional annotation using the GeneOntology and the locally developed ontology MeGO dedicated to MGEs. At the genome level, evolutionary cohesive modules group sets of protein families shared among MGEs. At the population level, networks display the reticulate evolutionary relationships among MGEs. To increase the coverage of the phage sequence space, ACLAME version 0.4 incorporates 760 high-quality predicted prophages selected from the Prophinder database. Most of the data can be downloaded from the freely accessible ACLAME web site (http://aclame.ulb.ac.be). The BLAST interface for querying the database has been extended and numerous tools for in-depth analysis of the results have been added.

TDRS-M Live Launch Coverage

NASA Image and Video Library

2017-08-18

Live launch coverage of the Tracking and Data Relay Satellite, TDRS-M, liftoff at 8:39am EDT from Space Launch Complex 41 at Cape Canaveral Air Force Station in Florida. TDRS-M is the latest spacecraft destined for the agency's constellation of communications satellites that allows nearly continuous contact with orbiting spacecraft ranging from the International Space Station and Hubble Space Telescope to the array of scientific observatories.

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

NASA Astrophysics Data System (ADS)

He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

2017-05-01

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative

PubMed Central

Khafizov, Kamil; Madrid-Aliste, Carlos; Almo, Steven C.; Fiser, Andras

2014-01-01

The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins—including proteins for which reliable homology models can be generated—on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long. PMID:24567391

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative.

PubMed

Khafizov, Kamil; Madrid-Aliste, Carlos; Almo, Steven C; Fiser, Andras

2014-03-11

The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins--including proteins for which reliable homology models can be generated--on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Deep whole-genome sequencing of 100 southeast Asian Malays.

PubMed

Wong, Lai-Ping; Ong, Rick Twee-Hee; Poh, Wan-Ting; Liu, Xuanyao; Chen, Peng; Li, Ruoying; Lam, Kevin Koi-Yau; Pillai, Nisha Esakimuthu; Sim, Kar-Seng; Xu, Haiyan; Sim, Ngak-Leng; Teo, Shu-Mei; Foo, Jia-Nee; Tan, Linda Wei-Lin; Lim, Yenly; Koo, Seok-Hwee; Gan, Linda Seo-Hwee; Cheng, Ching-Yu; Wee, Sharon; Yap, Eric Peng-Huat; Ng, Pauline Crystal; Lim, Wei-Yen; Soong, Richie; Wenk, Markus Rene; Aung, Tin; Wong, Tien-Yin; Khor, Chiea-Chuen; Little, Peter; Chia, Kee-Seng; Teo, Yik-Ying

2013-01-10

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Deep Whole-Genome Sequencing of 100 Southeast Asian Malays

PubMed Central

Wong, Lai-Ping; Ong, Rick Twee-Hee; Poh, Wan-Ting; Liu, Xuanyao; Chen, Peng; Li, Ruoying; Lam, Kevin Koi-Yau; Pillai, Nisha Esakimuthu; Sim, Kar-Seng; Xu, Haiyan; Sim, Ngak-Leng; Teo, Shu-Mei; Foo, Jia-Nee; Tan, Linda Wei-Lin; Lim, Yenly; Koo, Seok-Hwee; Gan, Linda Seo-Hwee; Cheng, Ching-Yu; Wee, Sharon; Yap, Eric Peng-Huat; Ng, Pauline Crystal; Lim, Wei-Yen; Soong, Richie; Wenk, Markus Rene; Aung, Tin; Wong, Tien-Yin; Khor, Chiea-Chuen; Little, Peter; Chia, Kee-Seng; Teo, Yik-Ying

2013-01-01

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies. PMID:23290073

Implementation of Whole Genome Sequencing (WGS) for Identification and Characterization of Shiga Toxin-Producing Escherichia coli (STEC) in the United States

PubMed Central

Lindsey, Rebecca L.; Pouseele, Hannes; Chen, Jessica C.; Strockbine, Nancy A.; Carleton, Heather A.

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different databases accurately predicted serotype, virulence, and resistance from WGS data, providing a fast and cheaper alternative to conventional typing techniques. PMID:27242777

Improved Analysis of Nanopore Sequence Data and Scanning Nanopore Techniques

NASA Astrophysics Data System (ADS)

Szalay, Tamas

The field of nanopore research has been driven by the need to inexpensively and rapidly sequence DNA. In order to help realize this goal, this thesis describes the PoreSeq algorithm that identifies and corrects errors in real-world nanopore sequencing data and improves the accuracy of de novo genome assembly with increasing coverage depth. The approach relies on modeling the possible sources of uncertainty that occur as DNA advances through the nanopore and then using this model to find the sequence that best explains multiple reads of the same region of DNA. PoreSeq increases nanopore sequencing read accuracy of M13 bacteriophage DNA from 85% to 99% at 100X coverage. We also use the algorithm to assemble E. coli with 30X coverage and the lambda genome at a range of coverages from 3X to 50X. Additionally, we classify sequence variants at an order of magnitude lower coverage than is possible with existing methods. This thesis also reports preliminary progress towards controlling the motion of DNA using two nanopores instead of one. The speed at which the DNA travels through the nanopore needs to be carefully controlled to facilitate the detection of individual bases. A second nanopore in close proximity to the first could be used to slow or stop the motion of the DNA in order to enable a more accurate readout. The fabrication process for a new pyramidal nanopore geometry was developed in order to facilitate the positioning of the nanopores. This thesis demonstrates that two of them can be placed close enough to interact with a single molecule of DNA, which is a prerequisite for being able to use the driving force of the pores to exert fine control over the motion of the DNA. Another strategy for reading the DNA is to trap it completely with one pore and to move the second nanopore instead. To that end, this thesis also shows that a single strand of immobilized DNA can be captured in a scanning nanopore and examined for a full hour, with data from many scans at many different voltages obtained in order to detect a bound protein placed partway along the molecule.

Implementation of Whole Genome Sequencing (WGS) for Identification and Characterization of Shiga Toxin-Producing Escherichia coli (STEC) in the United States.

PubMed

Lindsey, Rebecca L; Pouseele, Hannes; Chen, Jessica C; Strockbine, Nancy A; Carleton, Heather A

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different databases accurately predicted serotype, virulence, and resistance from WGS data, providing a fast and cheaper alternative to conventional typing techniques.

EXAMINING EVIDENCE IN U.S. PAYER COVERAGE POLICIES FOR MULTI-GENE PANELS AND SEQUENCING TESTS

PubMed Central

Chambers, James D.; Saret, Cayla J.; Anderson, Jordan E.; Deverka, Patricia A.; Douglas, Michael P.; Phillips, Kathryn A.

2017-01-01

Objectives The aim of this study was to examine the evidence payers cited in their coverage policies for multi-gene panels and sequencing tests (panels), and to compare these findings with the evidence payers cited in their coverage policies for other types of medical interventions. Methods We used the University of California at San Francisco TRANSPERS Payer Coverage Registry to identify coverage policies for panels issued by five of the largest US private payers. We reviewed each policy and categorized the evidence cited within as: clinical studies, systematic reviews, technology assessments, cost-effectiveness analyses (CEAs), budget impact studies, and clinical guidelines. We compared the evidence cited in these coverage policies for panels with the evidence cited in policies for other intervention types (pharmaceuticals, medical devices, diagnostic tests and imaging, and surgical interventions) as reported in a previous study. Results Fifty-five coverage policies for panels were included. On average, payers cited clinical guidelines in 84 percent of their coverage policies (range, 73–100 percent), clinical studies in 69 percent (50–87 percent), technology assessments 47 percent (33–86 percent), systematic reviews or meta-analyses 31 percent (7–71 percent), and CEAs 5 percent (0–7 percent). No payers cited budget impact studies in their policies. Payers less often cited clinical studies, systematic reviews, technology assessments, and CEAs in their coverage policies for panels than in their policies for other intervention types. Payers cited clinical guidelines in a comparable proportion of policies for panels and other technology types. Conclusions Payers in our sample less often cited clinical studies and other evidence types in their coverage policies for panels than they did in their coverage policies for other types of medical interventions. PMID:29065945

EXAMINING EVIDENCE IN U.S. PAYER COVERAGE POLICIES FOR MULTI-GENE PANELS AND SEQUENCING TESTS.

PubMed

Chambers, James D; Saret, Cayla J; Anderson, Jordan E; Deverka, Patricia A; Douglas, Michael P; Phillips, Kathryn A

2017-01-01

The aim of this study was to examine the evidence payers cited in their coverage policies for multi-gene panels and sequencing tests (panels), and to compare these findings with the evidence payers cited in their coverage policies for other types of medical interventions. We used the University of California at San Francisco TRANSPERS Payer Coverage Registry to identify coverage policies for panels issued by five of the largest US private payers. We reviewed each policy and categorized the evidence cited within as: clinical studies, systematic reviews, technology assessments, cost-effectiveness analyses (CEAs), budget impact studies, and clinical guidelines. We compared the evidence cited in these coverage policies for panels with the evidence cited in policies for other intervention types (pharmaceuticals, medical devices, diagnostic tests and imaging, and surgical interventions) as reported in a previous study. Fifty-five coverage policies for panels were included. On average, payers cited clinical guidelines in 84 percent of their coverage policies (range, 73-100 percent), clinical studies in 69 percent (50-87 percent), technology assessments 47 percent (33-86 percent), systematic reviews or meta-analyses 31 percent (7-71 percent), and CEAs 5 percent (0-7 percent). No payers cited budget impact studies in their policies. Payers less often cited clinical studies, systematic reviews, technology assessments, and CEAs in their coverage policies for panels than in their policies for other intervention types. Payers cited clinical guidelines in a comparable proportion of policies for panels and other technology types. Payers in our sample less often cited clinical studies and other evidence types in their coverage policies for panels than they did in their coverage policies for other types of medical interventions.

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.

PubMed

Court, Colin M; Ankeny, Jacob S; Sho, Shonan; Hou, Shuang; Li, Qingyu; Hsieh, Carolyn; Song, Min; Liao, Xinfang; Rochefort, Matthew M; Wainberg, Zev A; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S

2016-09-01

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Space Race Propaganda: U.S. Coverage of the Soviet Sputniks in 1957.

ERIC Educational Resources Information Center

Marlin, Cheryl L.

1987-01-01

Analyzes coverage of the Soviet Sputniks in 1957 by three news magazines--"U.S.News and World Report,""Newsweek," and "Time." Reports that "Time" and "U.S. News" covered the issue in Cold War terms, whereas "Newsweek" put emphasis on the prospects for space exploration. (MM)

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies

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

Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Jr., Richard A.

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted.more » PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Furthermore, our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.« less

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies

DOE PAGES

Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Jr., Richard A.; ...

2017-07-18

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted.more » PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Furthermore, our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.« less

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies

PubMed Central

Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Richard A.; Brown, Steven D.

2017-01-01

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences. PMID:28769883

Expected antenna utilization and overload

NASA Technical Reports Server (NTRS)

Posner, Edward C.

1991-01-01

The trade-offs between the number of antennas at Deep Space Network (DSN) Deep-Space Communications Complex and the fraction of continuous coverage provided to a set of hypothetical spacecraft, assuming random placement of the space craft passes during the day. The trade-offs are fairly robust with respect to the randomness assumption. A sample result is that a three-antenna complex provides an average of 82.6 percent utilization of facilities and coverage of nine spacecraft that each have 8-hour passes, whereas perfect phasing of the passes would yield 100 percent utilization and coverage. One key point is that sometimes fewer than three spacecraft are visible, so an antenna is idle, while at other times, there aren't enough antennas, and some spacecraft do without service. This point of view may be useful in helping to size the network or to develop a normalization for a figure of merit of DSN coverage.

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.

Genomic Sequencing: Assessing The Health Care System, Policy, And Big-Data Implications

PubMed Central

Phillips, Kathryn A.; Trosman, Julia; Kelley, Robin K.; Pletcher, Mark J.; Douglas, Michael P.; Weldon, Christine B.

2014-01-01

New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a “big data” technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. PMID:25006153

Genomic sequencing: assessing the health care system, policy, and big-data implications.

PubMed

Phillips, Kathryn A; Trosman, Julia R; Kelley, Robin K; Pletcher, Mark J; Douglas, Michael P; Weldon, Christine B

2014-07-01

New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a "big data" technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. Project HOPE—The People-to-People Health Foundation, Inc.

High-resolution characterization of a hepatocellular carcinoma genome.

PubMed

Totoki, Yasushi; Tatsuno, Kenji; Yamamoto, Shogo; Arai, Yasuhito; Hosoda, Fumie; Ishikawa, Shumpei; Tsutsumi, Shuichi; Sonoda, Kohtaro; Totsuka, Hirohiko; Shirakihara, Takuya; Sakamoto, Hiromi; Wang, Linghua; Ojima, Hidenori; Shimada, Kazuaki; Kosuge, Tomoo; Okusaka, Takuji; Kato, Kazuto; Kusuda, Jun; Yoshida, Teruhiko; Aburatani, Hiroyuki; Shibata, Tatsuhiro

2011-05-01

Hepatocellular carcinoma, one of the most common virus-associated cancers, is the third most frequent cause of cancer-related death worldwide. By massively parallel sequencing of a primary hepatitis C virus-positive hepatocellular carcinoma (36× coverage) and matched lymphocytes (>28× coverage) from the same individual, we identified more than 11,000 somatic substitutions of the tumor genome that showed predominance of T>C/A>G transition and a decrease of the T>C substitution on the transcribed strand, suggesting preferential DNA repair. Gene annotation enrichment analysis of 63 validated non-synonymous substitutions revealed enrichment of phosphoproteins. We further validated 22 chromosomal rearrangements, generating four fusion transcripts that had altered transcriptional regulation (BCORL1-ELF4) or promoter activity. Whole-exome sequencing at a higher sequence depth (>76× coverage) revealed a TSC1 nonsense substitution in a subpopulation of the tumor cells. This first high-resolution characterization of a virus-associated cancer genome identified previously uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion genes, as well as genetic heterogeneity within the tumor.

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

Molecular Diagnosis of Usher Syndrome: Application of Two Different Next Generation Sequencing-Based Procedures

PubMed Central

Licastro, Danilo; Mutarelli, Margherita; Peluso, Ivana; Neveling, Kornelia; Wieskamp, Nienke; Rispoli, Rossella; Vozzi, Diego; Athanasakis, Emmanouil; D'Eustacchio, Angela; Pizzo, Mariateresa; D'Amico, Francesca; Ziviello, Carmela; Simonelli, Francesca; Fabretto, Antonella; Scheffer, Hans; Gasparini, Paolo; Banfi, Sandro; Nigro, Vincenzo

2012-01-01

Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified. PMID:22952768

Architecture Study on Telemetry Coverage for Immediate Post-Separation Phase

NASA Technical Reports Server (NTRS)

Cheung, Kar-Ming; Lee, Charles H.; Kellogg, Kent H.; Stocklin, Frank J.; Zillig, David J.; Fielhauer, Karl B.

2008-01-01

This paper presents the preliminary results of an architecture study that provides continuous telemetry coverage for NASA missions for immediate post-separation phase. This study is a collaboration effort between Jet Propulsion Laboratory (JPL), Goddard Space Flight Center (GSFC), and Applied Physics Laboratory (APL). After launch when the spacecraft separated from the upper stage, the spacecraft typically executes a number of mission-critical operations prior to the deployment of solar panels and the activation of the primary communication subsystem. JPL, GSFC, and APL have similar design principle statements that require continuous coverage of mission-critical telemetry during the immediate post-separation phase. To conform to these design principles, an architecture that consists of a separate spacecraft transmitter and a robust communication network capable of tracking the spacecraft signals is needed.This paper presents the preliminary results of an architecture study that provides continuous telemetry coverage for NASA missions for immediate post-separation phase. This study is a collaboration effort between Jet Propulsion Laboratory (JPL), Goddard Space Flight Center (GSFC), and Applied Physics Laboratory (APL). After launch when the spacecraft separated from the upper stage, the spacecraft typically executes a number of mission-critical operations prior to the deployment of solar panels and the activation of the primary communication subsystem. JPL, GSFC, and APL have similar design principle statements that require continuous coverage of mission-critical telemetry during the immediate post-separation phase. To conform to these design principles, an architecture that consists of a separate spacecraft transmitter and a robust communication network capable of tracking the spacecraft signals is needed. The main results of this study are as follows: 1) At low altitude (< 10000 km) when most post-separation critical operations are executed, Earth-based network (e.g. Deep Space Network (DSN)) can only provide limited coverage, whereas space-based network (e.g. Space Network (SN)) can provide continuous coverage. 2) Commercial-off-the-shelf SN compatible transmitters are available for small satellite applications. In this paper we present the detailed coverage analysis of Earth-based and Space-based networks. We identify the key functional and performance requirements of the architecture, and describe the proposed selection criteria of the spacecraft transmitter. We conclude the paper with a proposed forward plan.

Facilitated versus Non-Facilitated Online Case Discussions: Comparing Differences in Problem Space Coverage

ERIC Educational Resources Information Center

Ertmer, Peggy A.; Koehler, Adrie A.

2015-01-01

The facilitator plays a key role in guiding students' efforts during case discussions. However, few studies have compared differences in learning outcomes for students participating in facilitated versus non-facilitated discussions. In this research, we used "problem space coverage" as a learning measure to compare outcomes between…

Evaluating imputation algorithms for low-depth genotyping-by-sequencing (GBS) data

USDA-ARS?s Scientific Manuscript database

Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordabl...

ADDITIONAL FOOTAGE FROM COVERAGE OF THE FIRST MEETING OF THE NATIONAL SPACE COUNCIL

NASA Image and Video Library

2017-10-05

Additional footage from coverage of the first meeting of the National Space Council, held on Oct. 5 at the Smithsonian National Air and Space Museum’s Steven F. Udvar-Hazy Center in Chantilly, Virginia. Vice President Mike Pence is the chair of the council. Participants included NASA’s Acting Administrator Robert Lightfoot, as well as a number of Trump Administration cabinet members and senior officials, and aerospace industry leaders.

EEG microstates during resting represent personality differences.

PubMed

Schlegel, Felix; Lehmann, Dietrich; Faber, Pascal L; Milz, Patricia; Gianotti, Lorena R R

2012-01-01

We investigated the spontaneous brain electric activity of 13 skeptics and 16 believers in paranormal phenomena; they were university students assessed with a self-report scale about paranormal beliefs. 33-channel EEG recordings during no-task resting were processed as sequences of momentary potential distribution maps. Based on the maps at peak times of Global Field Power, the sequences were parsed into segments of quasi-stable potential distribution, the 'microstates'. The microstates were clustered into four classes of map topographies (A-D). Analysis of the microstate parameters time coverage, occurrence frequency and duration as well as the temporal sequence (syntax) of the microstate classes revealed significant differences: Believers had a higher coverage and occurrence of class B, tended to decreased coverage and occurrence of class C, and showed a predominant sequence of microstate concatenations from A to C to B to A that was reversed in skeptics (A to B to C to A). Microstates of different topographies, putative "atoms of thought", are hypothesized to represent different types of information processing.The study demonstrates that personality differences can be detected in resting EEG microstate parameters and microstate syntax. Microstate analysis yielded no conclusive evidence for the hypothesized relation between paranormal belief and schizophrenia.

A case of canine borreliosis in Iran caused by Borrelia persica.

PubMed

Shirani, Darush; Rakhshanpoor, Alaleh; Cutler, Sally Jane; Ghazinezhad, Behnaz; Naddaf, Saied Reza

2016-04-01

Tick-borne relapsing fever is an endemic disease in Iran, with most cases attributed to infection by Borrelia persica, which is transmitted by Ornithodoros tholozani soft ticks. Here, we report spirochetemia in blood of a puppy residing in Tehran, Iran. The causative species was identified by use of highly discriminative IGS sequencing; the 489 bp IGS sequence obtained in our study showed 99% identity (100% coverage) when compared with B. persica sequences derived from clinical cases or from O. tholozani ticks. Our IGS sequence also showed 99% similarity over 414 bp (85% coverage) with a strain from a domestic dog, and 96% over 328 bp (69% coverage) with a strain from a domestic cat. Pet-keeping in cosmopolitan cities like Tehran has become increasingly popular in recent years. Animals are often transported into the city in cages or cardboard boxes that might also harbor minute tick larvae and/or early stages of the nymphs bringing them into the urban environment. This may pose a threat to household members who buy and keep these puppies and as a result may come into close contact with infected ticks. Copyright © 2016 Elsevier GmbH. All rights reserved.

ReadXplorer—visualization and analysis of mapped sequences

PubMed Central

Hilker, Rolf; Stadermann, Kai Bernd; Doppmeier, Daniel; Kalinowski, Jörn; Stoye, Jens; Straube, Jasmin; Winnebald, Jörn; Goesmann, Alexander

2014-01-01

Motivation: Fast algorithms and well-arranged visualizations are required for the comprehensive analysis of the ever-growing size of genomic and transcriptomic next-generation sequencing data. Results: ReadXplorer is a software offering straightforward visualization and extensive analysis functions for genomic and transcriptomic DNA sequences mapped on a reference. A unique specialty of ReadXplorer is the quality classification of the read mappings. It is incorporated in all analysis functions and displayed in ReadXplorer's various synchronized data viewers for (i) the reference sequence, its base coverage as (ii) normalizable plot and (iii) histogram, (iv) read alignments and (v) read pairs. ReadXplorer's analysis capability covers RNA secondary structure prediction, single nucleotide polymorphism and deletion–insertion polymorphism detection, genomic feature and general coverage analysis. Especially for RNA-Seq data, it offers differential gene expression analysis, transcription start site and operon detection as well as RPKM value and read count calculations. Furthermore, ReadXplorer can combine or superimpose coverage of different datasets. Availability and implementation: ReadXplorer is available as open-source software at http://www.readxplorer.org along with a detailed manual. Contact: rhilker@mikrobio.med.uni-giessen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24790157

Phylogenomics from Whole Genome Sequences Using aTRAM.

PubMed

Allen, Julie M; Boyd, Bret; Nguyen, Nam-Phuong; Vachaspati, Pranjal; Warnow, Tandy; Huang, Daisie I; Grady, Patrick G S; Bell, Kayce C; Cronk, Quentin C B; Mugisha, Lawrence; Pittendrigh, Barry R; Leonardi, M Soledad; Reed, David L; Johnson, Kevin P

2017-09-01

Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.]. © The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Space Shuttle Underside Astronaut Communications Performance Evaluation

NASA Technical Reports Server (NTRS)

Hwu, Shian U.; Dobbins, Justin A.; Loh, Yin-Chung; Kroll, Quin D.; Sham, Catherine C.

2005-01-01

The Space Shuttle Ultra High Frequency (UHF) communications system is planned to provide Radio Frequency (RF) coverage for astronauts working underside of the Space Shuttle Orbiter (SSO) for thermal tile inspection and repairing. This study is to assess the Space Shuttle UHF communication performance for astronauts in the shadow region without line-of-sight (LOS) to the Space Shuttle and Space Station UHF antennas. To insure the RF coverage performance at anticipated astronaut worksites, the link margin between the UHF antennas and Extravehicular Activity (EVA) Astronauts with significant vehicle structure blockage was analyzed. A series of near-field measurements were performed using the NASA/JSC Anechoic Chamber Antenna test facilities. Computational investigations were also performed using the electromagnetic modeling techniques. The computer simulation tool based on the Geometrical Theory of Diffraction (GTD) was used to compute the signal strengths. The signal strength was obtained by computing the reflected and diffracted fields along the propagation paths between the transmitting and receiving antennas. Based on the results obtained in this study, RF coverage for UHF communication links was determined for the anticipated astronaut worksite in the shadow region underneath the Space Shuttle.

Compressed Sensing for Resolution Enhancement of Hyperpolarized 13C Flyback 3D-MRSI

PubMed Central

Hu, Simon; Lustig, Michael; Chen, Albert P.; Crane, Jason; Kerr, Adam; Kelley, Douglas A.C.; Hurd, Ralph; Kurhanewicz, John; Nelson, Sarah J.; Pauly, John M.; Vigneron, Daniel B.

2008-01-01

High polarization of nuclear spins in liquid state through dynamic nuclear polarization has enabled the direct monitoring of 13C metabolites in vivo at very high signal to noise, allowing for rapid assessment of tissue metabolism. The abundant SNR afforded by this hyperpolarization technique makes high resolution 13C 3D-MRSI feasible. However, the number of phase encodes that can be fit into the short acquisition time for hyperpolarized imaging limits spatial coverage and resolution. To take advantage of the high SNR available from hyperpolarization, we have applied compressed sensing to achieve a factor of 2 enhancement in spatial resolution without increasing acquisition time or decreasing coverage. In this paper, the design and testing of compressed sensing suited for a flyback 13C 3D-MRSI sequence are presented. The key to this design was the undersampling of spectral k-space using a novel blipped scheme, thus taking advantage of the considerable sparsity in typical hyperpolarized 13C spectra. Phantom tests validated the accuracy of the compressed sensing approach and initial mouse experiments demonstrated in vivo feasibility. PMID:18367420

An Empirical Template Library of Stellar Spectra for a Wide Range of Spectral Classes, Luminosity Classes, and Metallicities Using SDSS BOSS Spectra

NASA Astrophysics Data System (ADS)

Kesseli, Aurora Y.; West, Andrew A.; Veyette, Mark; Harrison, Brandon; Feldman, Dan; Bochanski, John J.

2017-06-01

We present a library of empirical stellar spectra created using spectra from the Sloan Digital Sky Survey’s Baryon Oscillation Spectroscopic Survey. The templates cover spectral types O5 through L3, are binned by metallicity from -2.0 dex through +1.0 dex, and are separated into main-sequence (dwarf) stars and giant stars. With recently developed M dwarf metallicity indicators, we are able to extend the metallicity bins down through the spectral subtype M8, making this the first empirical library with this degree of temperature and metallicity coverage. The wavelength coverage for the templates is from 3650 to 10200 Å at a resolution of better than R ˜ 2000. Using the templates, we identify trends in color space with metallicity and surface gravity, which will be useful for analyzing large data sets from upcoming missions like the Large Synoptic Survey Telescope. Along with the templates, we are releasing a code for automatically (and/or visually) identifying the spectral type and metallicity of a star.

An Empirical Template Library of Stellar Spectra for a Wide Range of Spectral Classes, Luminosity Classes, and Metallicities Using SDSS BOSS Spectra

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

Kesseli, Aurora Y.; West, Andrew A.; Veyette, Mark

We present a library of empirical stellar spectra created using spectra from the Sloan Digital Sky Survey’s Baryon Oscillation Spectroscopic Survey. The templates cover spectral types O5 through L3, are binned by metallicity from −2.0 dex through +1.0 dex, and are separated into main-sequence (dwarf) stars and giant stars. With recently developed M dwarf metallicity indicators, we are able to extend the metallicity bins down through the spectral subtype M8, making this the first empirical library with this degree of temperature and metallicity coverage. The wavelength coverage for the templates is from 3650 to 10200 Å at a resolution ofmore » better than R  ∼ 2000. Using the templates, we identify trends in color space with metallicity and surface gravity, which will be useful for analyzing large data sets from upcoming missions like the Large Synoptic Survey Telescope. Along with the templates, we are releasing a code for automatically (and/or visually) identifying the spectral type and metallicity of a star.« less

VizieR Online Data Catalog: 05 through L3 empirical stellar spectra from SDSS (Kesseli+, 2017)

NASA Astrophysics Data System (ADS)

Kesseli, A. Y.; West, A. A.; Veyette, M.; Harrison, B.; Feldman, D.; Bochanski, J. J.

2017-08-01

We present a library of empirical stellar spectra created using spectra from the Sloan Digital Sky Survey's Baryon Oscillation Spectroscopic Survey. The templates cover spectral types O5 through L3, are binned by metallicity from -2.0dex through +1.0dex, and are separated into main-sequence (dwarf) stars and giant stars. With recently developed M dwarf metallicity indicators, we are able to extend the metallicity bins down through the spectral subtype M8, making this the first empirical library with this degree of temperature and metallicity coverage. The wavelength coverage for the templates is from 3650 to 10200Å at a resolution of better than R~2000. Using the templates, we identify trends in color space with metallicity and surface gravity, which will be useful for analyzing large data sets from upcoming missions like the Large Synoptic Survey Telescope. Along with the templates, we are releasing a code for automatically (and/or visually) identifying the spectral type and metallicity of a star. (3 data files).

Candidate mosaic proteins for a pan-filoviral cytotoxic T-Cell lymphocyte vaccine

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

Fenimore, Paul W; Fischer, William M; Kuiken, Carla

The extremely high fatality rates of many filovirus (FILV) strains the recurrent but rarely identified origin of human epidemics, the only partly identified viral reservoirs and the continuing non-human primate epizootics in Africa make a broadly-protective filovirus vaccine highly desirable. Cytotoxic T-cells (CTL) have been shown to be protective in mice, guinea pigs and non-human primates. In murine models the cytotoxic T-cell epitopes that are protective against Ebola virus have been mapped and in non-human primates CTL-mediated protection between viral strains (John Dye: specify) has been demonstrated using two filoviral proteins, nucleoprotein (NP) and glycoprotein (GP). These immunological results suggestmore » that the CTL avenue of immunity deserves consideration for a vaccine. The poorly-understood viral reservoirs means that it is difficult to predict what strains are likely to cause epidemics. Thus, there is a premium on developing a pan-filoviral vaccine. The genetic diversity of FILV is large, roughly the same scale as human immunodeficiency virus (HIV). This presents a serious challenge for the vaccine designer because a traditional vaccine aspiring to pan-filoviral coverage is likely to require the inclusion of many antigenic reagents. A recent method for optimizing cytotoxic T-cell lymphocyte epitope coverage with mosaic antigens was successful in improving potential CTL epitope coverage against HIV and may be useful in the context of very different viruses, such as the filoviruses discussed here. Mosaic proteins are recombinants composed of fragments of wild-type proteins joined at locations resulting in exclusively natural k-mers, 9 {le} k {le} 15, and having approximately the same length as the wild-type proteins. The use of mosaic antigens is motivated by three conjectures: (1) optimizing a mosaic protein to maximize coverage of k-mers found in a set of reference proteins will give better odds of including broadly-protective CTL epitopes in a vaccine than is possible with a wild-type protein, (2) reducing the number of low-prevalence k-mers minimizes the likelihood of undesirable immunodominance, and (3) excluding exogenous k-mers will result in mosaic proteins whose processing for presentation is close to what occurs with wild-type proteins. The first and second applications of the mosaic method were to HIV and Hepatitis C Virus (HCV). HIV is the virus with the largest number of known sequences, and consequently a plethora of information for the CTL vaccine designer to incorporate into their mosaics. Experience with HIV and HCV mosaics supports the validity of the three conjectures above. The available FILV sequences are probably closer to the minimum amount of information needed to make a meaningful mosaic vaccine candidate. There were 532 protein sequences in the National Institutes of Health GenPept database in November 2007 when our reference set was downloaded. These sequences come from both Ebola and Marburg viruses (EBOV and MARV), representing transcripts of all 7 genes. The coverage of viral diversity by the 7 genes is variable, with genes 1 (nucleoprotein, NP), 4 (glycoprotein, GP; soluble glycoprotein, sGP) and 7 (polymerase, L) giving the best coverage. Broadly-protective vaccine candidates for diverse viruses, such as HIV or Hepatitis C virus (HCV) have required pools of antigens. FILV is similar in this regard. While we have designed CTL mosaic proteins using all 7 types of filoviral proteins, only NP, GP and L proteins are reported here. If it were important to include other proteins in a mosaic CTL vaccine, additional sequences would be required to cover the space of known viral diversity.« less

HetMappsS: Heterozygous mapping strategy for high resolution Genotyping-by-Sequencing Markers

USDA-ARS?s Scientific Manuscript database

Reduced representation genotyping approaches, such as genotyping-by-sequencing (GBS), provide opportunities to generate high-resolution genetic maps at a low per-sample cost. However, missing data and non-uniform sequence coverage can complicate map creation in highly heterozygous species. To facili...

Method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation in conjunction with mass spectrometric analysis

DOEpatents

Laskin, Julia [Richland, WA; Futrell, Jean H [Richland, WA

2008-04-29

The invention relates to a method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation (SID) in conjunction with mass spectrometric analysis. Results demonstrate formation of a wide distribution of structure-specific fragments having wide sequence coverage useful for sequencing and identifying the complex molecules.

Whole genome sequence analysis of BT-474 using complete Genomics' standard and long fragment read technologies.

PubMed

Ciotlos, Serban; Mao, Qing; Zhang, Rebecca Yu; Li, Zhenyu; Chin, Robert; Gulbahce, Natali; Liu, Sophie Jia; Drmanac, Radoje; Peters, Brock A

2016-01-01

The cell line BT-474 is a popular cell line for studying the biology of cancer and developing novel drugs. However, there is no complete, published genome sequence for this highly utilized scientific resource. In this study we sought to provide a comprehensive and useful data set for the scientific community by generating a whole genome sequence for BT-474. Five μg of genomic DNA, isolated from an early passage of the BT-474 cell line, was used to generate a whole genome sequence (114X coverage) using Complete Genomics' standard sequencing process. To provide additional variant phasing and structural variation data we also processed and analyzed two separate libraries of 5 and 6 individual cells to depths of 99X and 87X, respectively, using Complete Genomics' Long Fragment Read (LFR) technology. BT-474 is a highly aneuploid cell line with an extremely complex genome sequence. This ~300X total coverage genome sequence provides a more complete understanding of this highly utilized cell line at the genomic level.

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.

Coverage of STS-104 Launch Coverage of Flight Controllers in MCC.

NASA Image and Video Library

2001-07-12

JSC2001-E-21333 (12 July 2001) --- From a familiar setting in the shuttle flight control room (WFCR)at Houston's Mission Control Center (MCC), Wayne Hale, ascent flight director for STS-104, pays close attention to new data related to the Space Shuttle Atlantis and its impending launch from the Kennedy Space Center (KSC) in Florida.

Coverage of STS-104 Launch Coverage of Flight Controllers in MCC.

NASA Image and Video Library

2001-07-12

JSC2001-E-21333 (12 July 2001) --- From a familiar setting in the shuttle flight control room (WFCR) at Houston's Mission Control Center (MCC), Wayne Hale, ascent flight director for STS-104, pays close attention to new data related to the Space Shuttle Atlantis and its impending launch from the Kennedy Space Center (KSC) in Florida.

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.

Structural analysis of a set of proteins resulting from a bacterial genomics project.

PubMed

Badger, J; Sauder, J M; Adams, J M; Antonysamy, S; Bain, K; Bergseid, M G; Buchanan, S G; Buchanan, M D; Batiyenko, Y; Christopher, J A; Emtage, S; Eroshkina, A; Feil, I; Furlong, E B; Gajiwala, K S; Gao, X; He, D; Hendle, J; Huber, A; Hoda, K; Kearins, P; Kissinger, C; Laubert, B; Lewis, H A; Lin, J; Loomis, K; Lorimer, D; Louie, G; Maletic, M; Marsh, C D; Miller, I; Molinari, J; Muller-Dieckmann, H J; Newman, J M; Noland, B W; Pagarigan, B; Park, F; Peat, T S; Post, K W; Radojicic, S; Ramos, A; Romero, R; Rutter, M E; Sanderson, W E; Schwinn, K D; Tresser, J; Winhoven, J; Wright, T A; Wu, L; Xu, J; Harris, T J R

2005-09-01

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. Copyright 2005 Wiley-Liss, Inc.

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

Sequenced sorghum mutant library- an efficient platform for discovery of causal gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. We applied whole-genome sequencing to 256 phenotyped mutant lines of sorghum (Sorghum bicolor L. Moench) to 16x coverage. Comparisons with the reference sequence revealed >1.8 million canonical EMS-induced G/C to A...

Launch mission summary and terminal countdown, Delta 153 Satellite Business Systems satellite (SBS-A)

NASA Technical Reports Server (NTRS)

1980-01-01

A brief summary of the launch vehicle, spacecraft, and mission is contained. Information relative to launch windows, vehicle telemetry coverage, realtime data flow, telemetry coverage by station, selected trajectory information, and a brief sequence of flight events is also included.

Developmental validation of a custom panel including 273 SNPs for forensic application using Ion Torrent PGM.

PubMed

Zhang, Suhua; Bian, Yingnan; Chen, Anqi; Zheng, Hancheng; Gao, Yuzhen; Hou, Yiping; Li, Chengtao

2017-03-01

Utilizing massively parallel sequencing (MPS) technology for SNP testing in forensic genetics is becoming attractive because of the shortcomings of STR markers, such as their high mutation rates and disadvantages associated with the current PCR-CE method as well as its limitations regarding multiplex capabilities. MPS offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. In this study, we designed a customized SNP panel that includes 273 forensically relevant identity SNPs chosen from SNPforID, IISNP, and the HapMap database as well as previously related studies and evaluated the levels of genotyping precision, sequence coverage, sensitivity and SNP performance using the Ion Torrent PGM. In a concordant study of the custom MPS-SNP panel, only four MPS callings were missing due to coverage reads that were too low (<20), whereas the others were fully concordant with Sanger's sequencing results across the two control samples, that is, 9947A and 9948. The analyses indicated a balanced coverage among the included loci, with the exception of the 16 SNPs that were used to detect an inconsistent allele balance and/or lower coverage reads among 50 tested individuals from the Chinese HAN population and the above controls. With the exception of the 16 poorly performing SNPs, the sequence coverage obtained was extensive for the bulk of the SNPs, and only three Y-SNPs (rs16980601, rs11096432, rs3900) showed a mean coverage below 1000. Analyses of the dilution series of control DNA 9948 yielded reproducible results down to 1ng of DNA input. In addition, we provide an analysis tool for automated data quality control and genotyping checks, and we conclude that the SNP targets are polymorphic and independent in the Chinese HAN population. In summary, the evaluation of the sensitivity, accuracy and genotyping performance provides strong support for the application of MPS technology in forensic SNP analysis, and the assay offers a straightforward sample-to-genotype workflow that could be beneficial in forensic casework with respect to both individual identification and complex kinship issues. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Caititu: a tool to graphically represent peptide sequence coverage and domain distribution.

PubMed

Carvalho, Paulo C; Junqueira, Magno; Valente, Richard H; Domont, Gilberto B

2008-10-07

Here we present Caititu, an easy-to-use proteomics software to graphically represent peptide sequence coverage and domain distribution for different correlated samples (e.g. originated from 2D gel spots) relatively to the full-sequence of the known protein they are related to. Although Caititu has a broad applicability, we exemplify its usefulness in Toxinology using snake venom as a model. For example, proteolytic processing may lead to inactivation or loss of domains. Therefore, our proposed graphic representation for peptides identified by two dimensional electrophoresis followed by mass spectrometric identification of excised spots can aid in inferring what kind of processing happened to the toxins, if any. Caititu is freely available to download at: http://pcarvalho.com/things/caititu.

3D Reconstruction of Space Objects from Multi-Views by a Visible Sensor

PubMed Central

Zhang, Haopeng; Wei, Quanmao; Jiang, Zhiguo

2017-01-01

In this paper, a novel 3D reconstruction framework is proposed to recover the 3D structural model of a space object from its multi-view images captured by a visible sensor. Given an image sequence, this framework first estimates the relative camera poses and recovers the depths of the surface points by the structure from motion (SFM) method, then the patch-based multi-view stereo (PMVS) algorithm is utilized to generate a dense 3D point cloud. To resolve the wrong matches arising from the symmetric structure and repeated textures of space objects, a new strategy is introduced, in which images are added to SFM in imaging order. Meanwhile, a refining process exploiting the structural prior knowledge that most sub-components of artificial space objects are composed of basic geometric shapes is proposed and applied to the recovered point cloud. The proposed reconstruction framework is tested on both simulated image datasets and real image datasets. Experimental results illustrate that the recovered point cloud models of space objects are accurate and have a complete coverage of the surface. Moreover, outliers and points with severe noise are effectively filtered out by the refinement, resulting in an distinct improvement of the structure and visualization of the recovered points. PMID:28737675

Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits.

PubMed

Imprialou, Martha; Kahles, André; Steffen, Joshua G; Osborne, Edward J; Gan, Xiangchao; Lempe, Janne; Bhomra, Amarjit; Belfield, Eric; Visscher, Anne; Greenhalgh, Robert; Harberd, Nicholas P; Goram, Richard; Hein, Jotun; Robert-Seilaniantz, Alexandre; Jones, Jonathan; Stegle, Oliver; Kover, Paula; Tsiantis, Miltos; Nordborg, Magnus; Rätsch, Gunnar; Clark, Richard M; Mott, Richard

2017-04-01

To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii , isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions. Copyright © 2017 by the Genetics Society of America.

Draft versus finished sequence data for DNA and protein diagnostic signature development

PubMed Central

Gardner, Shea N.; Lam, Marisa W.; Smith, Jason R.; Torres, Clinton L.; Slezak, Tom R.

2005-01-01

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10−3–10−5 (∼8× coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of ∼1% (3× to 6× coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures. PMID:16243783

Ancestry estimation and control of population stratification for sequence-based association studies.

PubMed

Wang, Chaolong; Zhan, Xiaowei; Bragg-Gresham, Jennifer; Kang, Hyun Min; Stambolian, Dwight; Chew, Emily Y; Branham, Kari E; Heckenlively, John; Fulton, Robert; Wilson, Richard K; Mardis, Elaine R; Lin, Xihong; Swaroop, Anand; Zöllner, Sebastian; Abecasis, Gonçalo R

2014-04-01

Estimating individual ancestry is important in genetic association studies where population structure leads to false positive signals, although assigning ancestry remains challenging with targeted sequence data. We propose a new method for the accurate estimation of individual genetic ancestry, based on direct analysis of off-target sequence reads, and implement our method in the publicly available LASER software. We validate the method using simulated and empirical data and show that the method can accurately infer worldwide continental ancestry when used with sequencing data sets with whole-genome shotgun coverage as low as 0.001×. For estimates of fine-scale ancestry within Europe, the method performs well with coverage of 0.1×. On an even finer scale, the method improves discrimination between exome-sequenced study participants originating from different provinces within Finland. Finally, we show that our method can be used to improve case-control matching in genetic association studies and to reduce the risk of spurious findings due to population structure.

3D Compressed Sensing for Highly Accelerated Hyperpolarized 13C MRSI With In Vivo Applications to Transgenic Mouse Models of Cancer

PubMed Central

Hu, Simon; Lustig, Michael; Balakrishnan, Asha; Larson, Peder E. Z.; Bok, Robert; Kurhanewicz, John; Nelson, Sarah J.; Goga, Andrei; Pauly, John M.; Vigneron, Daniel B.

2010-01-01

High polarization of nuclear spins in liquid state through hyperpolarized technology utilizing dynamic nuclear polarization has enabled the direct monitoring of 13C metabolites in vivo at a high signal-to-noise ratio. Acquisition time limitations due to T1 decay of the hyperpolarized signal require accelerated imaging methods, such as compressed sensing, for optimal speed and spatial coverage. In this paper, the design and testing of a new echo-planar 13C three-dimensional magnetic resonance spectroscopic imaging (MRSI) compressed sensing sequence is presented. The sequence provides up to a factor of 7.53 in acceleration with minimal reconstruction artifacts. The key to the design is employing x and y gradient blips during a fly-back readout to pseudorandomly undersample kf-kx-ky space. The design was validated in simulations and phantom experiments where the limits of undersampling and the effects of noise on the compressed sensing nonlinear reconstruction were tested. Finally, this new pulse sequence was applied in vivo in preclinical studies involving transgenic prostate cancer and transgenic liver cancer murine models to obtain much higher spatial and temporal resolution than possible with conventional echo-planar spectroscopic imaging methods. PMID:20017160

Recovering complete and draft population genomes from metagenome datasets

DOE PAGES

Sangwan, Naseer; Xia, Fangfang; Gilbert, Jack A.

2016-03-08

Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem ofmore » chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution.« less

Recovering complete and draft population genomes from metagenome datasets

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

Sangwan, Naseer; Xia, Fangfang; Gilbert, Jack A.

Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem ofmore » chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution.« less

On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin

NASA Astrophysics Data System (ADS)

Nicolardi, Simone; Giera, Martin; Kooijman, Pieter; Kraj, Agnieszka; Chervet, Jean-Pierre; Deelder, André M.; van der Burgt, Yuri E. M.

2013-12-01

Particularly in the field of middle- and top-down peptide and protein analysis, disulfide bridges can severely hinder fragmentation and thus impede sequence analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS approach, which was applied to the analysis of the primary structure of oxytocin, containing one disulfide bridge, and of hepcidin, containing four disulfide bridges. The presented workflow provided up to 80 % (on-line) conversion of disulfide bonds in both peptides. With minimal sample preparation, such reduction resulted in a higher number of peptide backbone cleavages upon CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle times, including electrode recovery, were rapid and, therefore, might very well be coupled with liquid chromatography for protein or peptide separation, which has great potential for high-throughput analysis.

Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation.

PubMed

Artieri, Carlo G; Fraser, Hunter B

2014-12-01

The recent advent of ribosome profiling-sequencing of short ribosome-bound fragments of mRNA-has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling data set have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling. © 2014 Artieri and Fraser; Published by Cold Spring Harbor Laboratory Press.

New in-depth rainbow trout transcriptome reference and digital atlas of gene expression

USDA-ARS?s Scientific Manuscript database

Sequencing the rainbow trout genome is underway and a transcriptome reference sequence is required to help in genome assembly and gene discovery. Previously, we reported a transcriptome reference sequence using a 19X coverage of 454-pyrosequencing data. Although this work added a great wealth of ann...

Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds

USDA-ARS?s Scientific Manuscript database

Genomic structural variations are an important source of genetic diversity. Copy number variations (CNVs), gains and losses of large regions of genomic sequence between individuals of a species, are known to be associated with both diseases and phenotypic traits. Deeply sequenced genomes are often u...

Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

PubMed

Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

2017-11-06

Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

The study of human Y chromosome variation through ancient DNA.

PubMed

Kivisild, Toomas

2017-05-01

High throughput sequencing methods have completely transformed the study of human Y chromosome variation by offering a genome-scale view on genetic variation retrieved from ancient human remains in context of a growing number of high coverage whole Y chromosome sequence data from living populations from across the world. The ancient Y chromosome sequences are providing us the first exciting glimpses into the past variation of male-specific compartment of the genome and the opportunity to evaluate models based on previously made inferences from patterns of genetic variation in living populations. Analyses of the ancient Y chromosome sequences are challenging not only because of issues generally related to ancient DNA work, such as DNA damage-induced mutations and low content of endogenous DNA in most human remains, but also because of specific properties of the Y chromosome, such as its highly repetitive nature and high homology with the X chromosome. Shotgun sequencing of uniquely mapping regions of the Y chromosomes to sufficiently high coverage is still challenging and costly in poorly preserved samples. To increase the coverage of specific target SNPs capture-based methods have been developed and used in recent years to generate Y chromosome sequence data from hundreds of prehistoric skeletal remains. Besides the prospects of testing directly as how much genetic change in a given time period has accompanied changes in material culture the sequencing of ancient Y chromosomes allows us also to better understand the rate at which mutations accumulate and get fixed over time. This review considers genome-scale evidence on ancient Y chromosome diversity that has recently started to accumulate in geographic areas favourable to DNA preservation. More specifically the review focuses on examples of regional continuity and change of the Y chromosome haplogroups in North Eurasia and in the New World.

Switch from oral to inactivated poliovirus vaccine in Yogyakarta Province, Indonesia: summary of coverage, immunity, and environmental surveillance.

PubMed

Wahjuhono, Gendro; Revolusiana; Widhiastuti, Dyah; Sundoro, Julitasari; Mardani, Tri; Ratih, Woro Umi; Sutomo, Retno; Safitri, Ida; Sampurno, Ondri Dwi; Rana, Bardan; Roivainen, Merja; Kahn, Anna-Lea; Mach, Ondrej; Pallansch, Mark A; Sutter, Roland W

2014-11-01

Inactivated poliovirus vaccine (IPV) is rarely used in tropical developing countries. To generate additional scientific information, especially on the possible emergence of vaccine-derived polioviruses (VDPVs) in an IPV-only environment, we initiated an IPV introduction project in Yogyakarta, an Indonesian province. In this report, we present the coverage, immunity, and VDPV surveillance results. In Yogyakarta, we established environmental surveillance starting in 2004; and conducted routine immunization coverage and seroprevalence surveys before and after a September 2007 switch from oral poliovirus vaccine (OPV) to IPV, using standard coverage and serosurvey methods. Rates and types of polioviruses found in sewage samples were analyzed, and all poliovirus isolates after the switch were sequenced. Vaccination coverage (>95%) and immunity (approximately 100%) did not change substantially before and after the IPV switch. No VDPVs were detected. Before the switch, 58% of environmental samples contained Sabin poliovirus; starting 6 weeks after the switch, Sabin polioviruses were rarely isolated, and if they were, genetic sequencing suggested recent introductions. This project demonstrated that under almost ideal conditions (good hygiene, maintenance of universally high IPV coverage, and corresponding high immunity against polioviruses), no emergence and circulation of VDPV could be detected in a tropical developing country setting. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities.

PubMed

Drury, Suzanne; Williams, Hywel; Trump, Natalie; Boustred, Christopher; Lench, Nicholas; Scott, Richard H; Chitty, Lyn S

2015-10-01

In the absence of aneuploidy or other pathogenic cytogenetic abnormality, fetuses with increased nuchal translucency (NT ≥ 3.5 mm) and/or other sonographic abnormalities have a greater incidence of genetic syndromes, but defining the underlying pathology can be challenging. Here, we investigate the value of whole exome sequencing in fetuses with sonographic abnormalities but normal microarray analysis. Whole exome sequencing was performed on DNA extracted from chorionic villi or amniocytes in 24 fetuses with unexplained ultrasound findings. In the first 14 cases sequencing was initially performed on fetal DNA only. For the remaining 10, the trio of fetus, mother and father was sequenced simultaneously. In 21% (5/24) cases, exome sequencing provided definitive diagnoses (Milroy disease, hypophosphatasia, achondrogenesis type 2, Freeman-Sheldon syndrome and Baraitser-Winter Syndrome). In a further case, a plausible diagnosis of orofaciodigital syndrome type 6 was made. In two others, a single mutation in an autosomal recessive gene was identified, but incomplete sequencing coverage precluded exclusion of the presence of a second mutation. Whole exome sequencing improves prenatal diagnosis in euploid fetuses with abnormal ultrasound scans. In order to expedite interpretation of results, trio sequencing should be employed, but interpretation can still be compromised by incomplete coverage of relevant genes. © 2015 John Wiley & Sons, Ltd.

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

Verifying Digital Components of Physical Systems: Experimental Evaluation of Test Quality

NASA Astrophysics Data System (ADS)

Laputenko, A. V.; López, J. E.; Yevtushenko, N. V.

2018-03-01

This paper continues the study of high quality test derivation for verifying digital components which are used in various physical systems; those are sensors, data transfer components, etc. We have used logic circuits b01-b010 of the package of ITC'99 benchmarks (Second Release) for experimental evaluation which as stated before, describe digital components of physical systems designed for various applications. Test sequences are derived for detecting the most known faults of the reference logic circuit using three different approaches to test derivation. Three widely used fault types such as stuck-at-faults, bridges, and faults which slightly modify the behavior of one gate are considered as possible faults of the reference behavior. The most interesting test sequences are short test sequences that can provide appropriate guarantees after testing, and thus, we experimentally study various approaches to the derivation of the so-called complete test suites which detect all fault types. In the first series of experiments, we compare two approaches for deriving complete test suites. In the first approach, a shortest test sequence is derived for testing each fault. In the second approach, a test sequence is pseudo-randomly generated by the use of an appropriate software for logic synthesis and verification (ABC system in our study) and thus, can be longer. However, after deleting sequences detecting the same set of faults, a test suite returned by the second approach is shorter. The latter underlines the fact that in many cases it is useless to spend `time and efforts' for deriving a shortest distinguishing sequence; it is better to use the test minimization afterwards. The performed experiments also show that the use of only randomly generated test sequences is not very efficient since such sequences do not detect all the faults of any type. After reaching the fault coverage around 70%, saturation is observed, and the fault coverage cannot be increased anymore. For deriving high quality short test suites, the approach that is the combination of randomly generated sequences together with sequences which are aimed to detect faults not detected by random tests, allows to reach the good fault coverage using shortest test sequences.

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.

NASA Provides Coast-to-Coast Coverage of Aug. 21 Solar Eclipse (The International Space Station)

NASA Image and Video Library

2017-08-21

On Monday, Aug. 21, NASA provided coast-to-coast coverage of the solar eclipse across America – featuring views of the phenomenon from unique vantage points, including from the ground, from aircraft, and from spacecraft including the ISS, during a live broadcast seen on NASA Television and the agency’s website. This is footage from The International Space Station.

Deep sequencing with intronic capture enables identification of an APC exon 10 inversion in a patient with polyposis.

PubMed

Shirts, Brian H; Salipante, Stephen J; Casadei, Silvia; Ryan, Shawnia; Martin, Judith; Jacobson, Angela; Vlaskin, Tatyana; Koehler, Karen; Livingston, Robert J; King, Mary-Claire; Walsh, Tom; Pritchard, Colin C

2014-10-01

Single-exon inversions have rarely been described in clinical syndromes and are challenging to detect using Sanger sequencing. We report the case of a 40-year-old woman with adenomatous colon polyps too numerous to count and who had a complex inversion spanning the entire exon 10 in APC (the gene encoding for adenomatous polyposis coli), causing exon skipping and resulting in a frameshift and premature protein truncation. In this study, we employed complete APC gene sequencing using high-coverage next-generation sequencing by ColoSeq, analysis with BreakDancer and SLOPE software, and confirmatory transcript analysis. ColoSeq identified a complex small genomic rearrangement consisting of an inversion that results in translational skipping of exon 10 in the APC gene. This mutation would not have been detected by traditional sequencing or gene-dosage methods. We report a case of adenomatous polyposis resulting from a complex single-exon inversion. Our report highlights the benefits of large-scale sequencing methods that capture intronic sequences with high enough depth of coverage-as well as the use of informatics tools-to enable detection of small pathogenic structural rearrangements.

Sequence Composition and Gene Content of the Short Arm of Rye (Secale cereale) Chromosome 1

PubMed Central

Fluch, Silvia; Kopecky, Dieter; Burg, Kornel; Šimková, Hana; Taudien, Stefan; Petzold, Andreas; Kubaláková, Marie; Platzer, Matthias; Berenyi, Maria; Krainer, Siegfried; Doležel, Jaroslav; Lelley, Tamas

2012-01-01

Background The purpose of the study is to elucidate the sequence composition of the short arm of rye chromosome 1 (Secale cereale) with special focus on its gene content, because this portion of the rye genome is an integrated part of several hundreds of bread wheat varieties worldwide. Methodology/Principal Findings Multiple Displacement Amplification of 1RS DNA, obtained from flow sorted 1RS chromosomes, using 1RS ditelosomic wheat-rye addition line, and subsequent Roche 454FLX sequencing of this DNA yielded 195,313,589 bp sequence information. This quantity of sequence information resulted in 0.43× sequence coverage of the 1RS chromosome arm, permitting the identification of genes with estimated probability of 95%. A detailed analysis revealed that more than 5% of the 1RS sequence consisted of gene space, identifying at least 3,121 gene loci representing 1,882 different gene functions. Repetitive elements comprised about 72% of the 1RS sequence, Gypsy/Sabrina (13.3%) being the most abundant. More than four thousand simple sequence repeat (SSR) sites mostly located in gene related sequence reads were identified for possible marker development. The existence of chloroplast insertions in 1RS has been verified by identifying chimeric chloroplast-genomic sequence reads. Synteny analysis of 1RS to the full genomes of Oryza sativa and Brachypodium distachyon revealed that about half of the genes of 1RS correspond to the distal end of the short arm of rice chromosome 5 and the proximal region of the long arm of Brachypodium distachyon chromosome 2. Comparison of the gene content of 1RS to 1HS barley chromosome arm revealed high conservation of genes related to chromosome 5 of rice. Conclusions The present study revealed the gene content and potential gene functions on this chromosome arm and demonstrated numerous sequence elements like SSRs and gene-related sequences, which can be utilised for future research as well as in breeding of wheat and rye. PMID:22328922

High Throughput Transcriptomics @ USEPA (Toxicology ...

EPA Pesticide Factsheets

The ideal chemical testing approach will provide complete coverage of all relevant toxicological responses. It should be sensitive and specific It should identify the mechanism/mode-of-action (with dose-dependence). It should identify responses relevant to the species of interest. Responses should ideally be translated into tissue-, organ-, and organism-level effects. It must be economical and scalable. Using a High Throughput Transcriptomics platform within US EPA provides broader coverage of biological activity space and toxicological MOAs and helps fill the toxicological data gap. Slide presentation at the 2016 ToxForum on using High Throughput Transcriptomics at US EPA for broader coverage biological activity space and toxicological MOAs.

Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics.

PubMed

Mikkelsen, Martin; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels

2014-09-01

of sequencing of whole mitochondrial genome, HV1 and HV2 DNA with the second generation system (SGS) Roche 454 GS Junior were compared with results of Sanger sequencing and SNP typing with SNaPshot single base extension detected with MALDI-TOF and capillary electrophoresis. We investigated the performance of the software analysis of the data, reproducibility, ability to sequence homopolymeric regions, detection of mixtures and heteroplasmy as well as the implications of the depth of coverage. We found full reproducibility between samples sequenced twice with SGS. We found close to full concordance between the mtDNA sequences of 26 samples obtained with (1) the 454 SGS method using a depth of coverage above 100 and (2) Sanger sequencing and SNP typing. The discrepancies were primarily observed in homopolymeric regions. The 454 SGS method was able to sequence 95% of the reads correctly in homopolymers up to 4 bases, and up to 6 bases could be sequenced with similar success if the results were carefully, visually inspected. The 454 technology was able to detect mixtures or heteroplasmy of approximately 10%. We detected previously unreported heteroplasmy in the GM9947A component of the NIST human mitochondrial DNA SRM-2392 standard reference material. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird.

PubMed

Biedrzycka, Aleksandra; Sebastian, Alvaro; Migalska, Magdalena; Westerdahl, Helena; Radwan, Jacek

2017-07-01

Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage. © 2016 John Wiley & Sons Ltd.

AD-LIBS: inferring ancestry across hybrid genomes using low-coverage sequence data.

PubMed

Schaefer, Nathan K; Shapiro, Beth; Green, Richard E

2017-04-04

Inferring the ancestry of each region of admixed individuals' genomes is useful in studies ranging from disease gene mapping to speciation genetics. Current methods require high-coverage genotype data and phased reference panels, and are therefore inappropriate for many data sets. We present a software application, AD-LIBS, that uses a hidden Markov model to infer ancestry across hybrid genomes without requiring variant calling or phasing. This approach is useful for non-model organisms and in cases of low-coverage data, such as ancient DNA. We demonstrate the utility of AD-LIBS with synthetic data. We then use AD-LIBS to infer ancestry in two published data sets: European human genomes with Neanderthal ancestry and brown bear genomes with polar bear ancestry. AD-LIBS correctly infers 87-91% of ancestry in simulations and produces ancestry maps that agree with published results and global ancestry estimates in humans. In brown bears, we find more polar bear ancestry than has been published previously, using both AD-LIBS and an existing software application for local ancestry inference, HAPMIX. We validate AD-LIBS polar bear ancestry maps by recovering a geographic signal within bears that mirrors what is seen in SNP data. Finally, we demonstrate that AD-LIBS is more effective than HAPMIX at inferring ancestry when preexisting phased reference data are unavailable and genomes are sequenced to low coverage. AD-LIBS is an effective tool for ancestry inference that can be used even when few individuals are available for comparison or when genomes are sequenced to low coverage. AD-LIBS is therefore likely to be useful in studies of non-model or ancient organisms that lack large amounts of genomic DNA. AD-LIBS can therefore expand the range of studies in which admixture mapping is a viable tool.

From atoms to layers: in situ gold cluster growth kinetics during sputter deposition

NASA Astrophysics Data System (ADS)

Schwartzkopf, Matthias; Buffet, Adeline; Körstgens, Volker; Metwalli, Ezzeldin; Schlage, Kai; Benecke, Gunthard; Perlich, Jan; Rawolle, Monika; Rothkirch, André; Heidmann, Berit; Herzog, Gerd; Müller-Buschbaum, Peter; Röhlsberger, Ralf; Gehrke, Rainer; Stribeck, Norbert; Roth, Stephan V.

2013-05-01

The adjustment of size-dependent catalytic, electrical and optical properties of gold cluster assemblies is a very significant issue in modern applied nanotechnology. We present a real-time investigation of the growth kinetics of gold nanostructures from small nuclei to a complete gold layer during magnetron sputter deposition with high time resolution by means of in situ microbeam grazing incidence small-angle X-ray scattering (μGISAXS). We specify the four-stage growth including their thresholds with sub-monolayer resolution and identify phase transitions monitored in Yoneda intensity as a material-specific characteristic. An innovative and flexible geometrical model enables the extraction of morphological real space parameters, such as cluster size and shape, correlation distance, layer porosity and surface coverage, directly from reciprocal space scattering data. This approach enables a large variety of future investigations of the influence of different process parameters on the thin metal film morphology. Furthermore, our study allows for deducing the wetting behavior of gold cluster films on solid substrates and provides a better understanding of the growth kinetics in general, which is essential for optimization of manufacturing parameters, saving energy and resources.The adjustment of size-dependent catalytic, electrical and optical properties of gold cluster assemblies is a very significant issue in modern applied nanotechnology. We present a real-time investigation of the growth kinetics of gold nanostructures from small nuclei to a complete gold layer during magnetron sputter deposition with high time resolution by means of in situ microbeam grazing incidence small-angle X-ray scattering (μGISAXS). We specify the four-stage growth including their thresholds with sub-monolayer resolution and identify phase transitions monitored in Yoneda intensity as a material-specific characteristic. An innovative and flexible geometrical model enables the extraction of morphological real space parameters, such as cluster size and shape, correlation distance, layer porosity and surface coverage, directly from reciprocal space scattering data. This approach enables a large variety of future investigations of the influence of different process parameters on the thin metal film morphology. Furthermore, our study allows for deducing the wetting behavior of gold cluster films on solid substrates and provides a better understanding of the growth kinetics in general, which is essential for optimization of manufacturing parameters, saving energy and resources. Electronic supplementary information (ESI) available: The full GISAXS image sequence of the experiment, the model-based IsGISAXS-simulation sequence as movie files for comparison and detailed information about sample cleaning, XRR, FESEM, IsGISAXS, comparison μGIWAXS/μGISAXS, and sampling statistics. See DOI: 10.1039/c3nr34216f

High-Throughput Gene Mapping in Caenorhabditis elegans

PubMed Central

Swan, Kathryn A.; Curtis, Damian E.; McKusick, Kathleen B.; Voinov, Alexander V.; Mapa, Felipa A.; Cancilla, Michael R.

2002-01-01

Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 ± 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18. [The sequence data described in this paper have been submitted to the NCBI dbSNP data library under accession nos. 4388625–4389689 and GenBank dbSTS under accession nos. 973810–974874. The following individuals and institutions kindly provided reagents, samples, or unpublished information as indicated in the paper: The C. elegans Sequencing Consortium and The Caenorhabditis Genetics Center.] PMID:12097347

Simultaneous Multi-Slice fMRI using Spiral Trajectories

PubMed Central

Zahneisen, Benjamin; Poser, Benedikt A.; Ernst, Thomas; Stenger, V. Andrew

2014-01-01

Parallel imaging methods using multi-coil receiver arrays have been shown to be effective for increasing MRI acquisition speed. However parallel imaging methods for fMRI with 2D sequences show only limited improvements in temporal resolution because of the long echo times needed for BOLD contrast. Recently, Simultaneous Multi-Slice (SMS) imaging techniques have been shown to increase fMRI temporal resolution by factors of four and higher. In SMS fMRI multiple slices can be acquired simultaneously using Echo Planar Imaging (EPI) and the overlapping slices are un-aliased using a parallel imaging reconstruction with multiple receivers. The slice separation can be further improved using the “blipped-CAIPI” EPI sequence that provides a more efficient sampling of the SMS 3D k-space. In this paper a blipped-spiral SMS sequence for ultra-fast fMRI is presented. The blipped-spiral sequence combines the sampling efficiency of spiral trajectories with the SMS encoding concept used in blipped-CAIPI EPI. We show that blipped spiral acquisition can achieve almost whole brain coverage at 3 mm isotropic resolution in 168 ms. It is also demonstrated that the high temporal resolution allows for dynamic BOLD lag time measurement using visual/motor and retinotopic mapping paradigms. The local BOLD lag time within the visual cortex following the retinotopic mapping stimulation of expanding flickering rings is directly measured and easily translated into an eccentricity map of the cortex. PMID:24518259

Targeted gene panel sequencing in children with very early onset inflammatory bowel disease--evaluation and prospective analysis.

PubMed

Kammermeier, Jochen; Drury, Suzanne; James, Chela T; Dziubak, Robert; Ocaka, Louise; Elawad, Mamoun; Beales, Philip; Lench, Nicholas; Uhlig, Holm H; Bacchelli, Chiara; Shah, Neil

2014-11-01

Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

A HIGH COVERAGE GENOME SEQUENCE FROM AN ARCHAIC DENISOVAN INDIVIDUAL

PubMed Central

Meyer, Matthias; Kircher, Martin; Gansauge, Marie-Theres; Li, Heng; Racimo, Fernando; Mallick, Swapan; Schraiber, Joshua G.; Jay, Flora; Prüfer, Kay; de Filippo, Cesare; Sudmant, Peter H.; Alkan, Can; Fu, Qiaomei; Do, Ron; Rohland, Nadin; Tandon, Arti; Siebauer, Michael; Green, Richard E.; Bryc, Katarzyna; Briggs, Adrian W.; Stenzel, Udo; Dabney, Jesse; Shendure, Jay; Kitzman, Jacob; Hammer, Michael F.; Shunkov, Michael V.; Derevianko, Anatoli P.; Patterson, Nick; Andrés, Aida M.; Eichler, Evan E.; Slatkin, Montgomery; Reich, David; Kelso, Janet; Pääbo, Svante

2013-01-01

We present a DNA library preparation method that has allowed us to reconstruct a high coverage (30X) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans. PMID:22936568

TU-H-CAMPUS-JeP3-05: Adaptive Determination of Needle Sequence HDR Prostate Brachytherapy with Divergent Needle-By-Needle Delivery

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

Borot de Battisti, M; Maenhout, M; Lagendijk, J J W

Purpose: To develop a new method which adaptively determines the optimal needle insertion sequence for HDR prostate brachytherapy involving divergent needle-by-needle dose delivery by e.g. a robotic device. A needle insertion sequence is calculated at the beginning of the intervention and updated after each needle insertion with feedback on needle positioning errors. Methods: Needle positioning errors and anatomy changes may occur during HDR brachytherapy which can lead to errors in the delivered dose. A novel strategy was developed to calculate and update the needle sequence and the dose plan after each needle insertion with feedback on needle positioning errors. Themore » dose plan optimization was performed by numerical simulations. The proposed needle sequence determination optimizes the final dose distribution based on the dose coverage impact of each needle. This impact is predicted stochastically by needle insertion simulations. HDR procedures were simulated with varying number of needle insertions (4 to 12) using 11 patient MR data-sets with PTV, prostate, urethra, bladder and rectum delineated. Needle positioning errors were modeled by random normally distributed angulation errors (standard deviation of 3 mm at the needle’s tip). The final dose parameters were compared in the situations where the needle with the largest vs. the smallest dose coverage impact was selected at each insertion. Results: Over all scenarios, the percentage of clinically acceptable final dose distribution improved when the needle selected had the largest dose coverage impact (91%) compared to the smallest (88%). The differences were larger for few (4 to 6) needle insertions (maximum difference scenario: 79% vs. 60%). The computation time of the needle sequence optimization was below 60s. Conclusion: A new adaptive needle sequence determination for HDR prostate brachytherapy was developed. Coupled to adaptive planning, the selection of the needle with the largest dose coverage impact increases chances of reaching the clinical constraints. M. Borot de Battisti is funded by Philips Medical Systems Nederland B.V.; M. Moerland is principal investigator on a contract funded by Philips Medical Systems Nederland B.V.; G. Hautvast and D. Binnekamp are fulltime employees of Philips Medical Systems Nederland B.V.« less

Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys.

PubMed

Barouch, Dan H; O'Brien, Kara L; Simmons, Nathaniel L; King, Sharon L; Abbink, Peter; Maxfield, Lori F; Sun, Ying-Hua; La Porte, Annalena; Riggs, Ambryice M; Lynch, Diana M; Clark, Sarah L; Backus, Katherine; Perry, James R; Seaman, Michael S; Carville, Angela; Mansfield, Keith G; Szinger, James J; Fischer, Will; Muldoon, Mark; Korber, Bette

2010-03-01

The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.

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.

Removal of nuisance signals from limited and sparse 1H MRSI data using a union-of-subspaces model.

PubMed

Ma, Chao; Lam, Fan; Johnson, Curtis L; Liang, Zhi-Pei

2016-02-01

To remove nuisance signals (e.g., water and lipid signals) for (1) H MRSI data collected from the brain with limited and/or sparse (k, t)-space coverage. A union-of-subspace model is proposed for removing nuisance signals. The model exploits the partial separability of both the nuisance signals and the metabolite signal, and decomposes an MRSI dataset into several sets of generalized voxels that share the same spectral distributions. This model enables the estimation of the nuisance signals from an MRSI dataset that has limited and/or sparse (k, t)-space coverage. The proposed method has been evaluated using in vivo MRSI data. For conventional chemical shift imaging data with limited k-space coverage, the proposed method produced "lipid-free" spectra without lipid suppression during data acquisition at 130 ms echo time. For sparse (k, t)-space data acquired with conventional pulses for water and lipid suppression, the proposed method was also able to remove the remaining water and lipid signals with negligible residuals. Nuisance signals in (1) H MRSI data reside in low-dimensional subspaces. This property can be utilized for estimation and removal of nuisance signals from (1) H MRSI data even when they have limited and/or sparse coverage of (k, t)-space. The proposed method should prove useful especially for accelerated high-resolution (1) H MRSI of the brain. © 2015 Wiley Periodicals, Inc.

Comparison and quantitative verification of mapping algorithms for whole genome bisulfite sequencing

USDA-ARS?s Scientific Manuscript database

Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitat...

Fast imputation using medium or low-coverage sequence data

USDA-ARS?s Scientific Manuscript database

Accurate genotype imputation can greatly reduce costs and increase benefits by combining whole-genome sequence data of varying read depth and microarray genotypes of varying densities. For large populations, an efficient strategy chooses the two haplotypes most likely to form each genotype and updat...

Coverage of STS-104 Launch Coverage of Flight Controllers in MCC.

NASA Image and Video Library

2001-07-12

JSC2001-E-21338 (12 July 2001) --- Robert Gest (left), with United Space Alliance (USA); Steven A. Hawley, deputy director of flight crew operations; and Alan L. (Lee) Briscoe, chief engineer for the Mission Operations Directorate, watch their monitors at the MOD console in the shuttle flight control room (WFCR) as the external tank oxygen vent hood is raised and retracted minutes prior to the launch of the Space Shuttle Atlantis.

Coverage of STS-104 Launch Coverage of Flight Controllers in MCC.

NASA Image and Video Library

2001-07-12

JSC2001-E-21341 (12 July 2001) --- From a familiar setting near the rear of shuttle flight control room (WFCR) at Houston's Mission Control Center (MCC), Wayne Hale (second left), ascent flight director for STS-104, pays close attention to new data related to the Space Shuttle Atlantis and its impending launch from the Kennedy Space Center (KSC) in Florida. Several other flight controllers are visible in the wide shot.

Role of Mitochondrial Inheritance on Prostate Cancer Outcome in African American Men. Addendum

DTIC Science & Technology

2016-11-01

DNA sequencing technique developed by our collaborator using single amplicon long-range PCR that permits deep coverage (10,000-20,000X on average) of...the mitochondrial genome. We have sequenced 652 samples derived from frozen fully using this technology. The additional DNA samples derived from...paraffin embedded (FFPE) tissue were more challenging, but have now been sequenced . Mapping of DNA variants in our sequenced genomes to mitochondrial

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

PubMed Central

Harris, R. Alan; Wang, Ting; Coarfa, Cristian; Nagarajan, Raman P.; Hong, Chibo; Downey, Sara L.; Johnson, Brett E.; Fouse, Shaun D.; Delaney, Allen; Zhao, Yongjun; Olshen, Adam; Ballinger, Tracy; Zhou, Xin; Forsberg, Kevin J.; Gu, Junchen; Echipare, Lorigail; O’Geen, Henriette; Lister, Ryan; Pelizzola, Mattia; Xi, Yuanxin; Epstein, Charles B.; Bernstein, Bradley E.; Hawkins, R. David; Ren, Bing; Chung, Wen-Yu; Gu, Hongcang; Bock, Christoph; Gnirke, Andreas; Zhang, Michael Q.; Haussler, David; Ecker, Joseph; Li, Wei; Farnham, Peggy J.; Waterland, Robert A.; Meissner, Alexander; Marra, Marco A.; Hirst, Martin; Milosavljevic, Aleksandar; Costello, Joseph F.

2010-01-01

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression. PMID:20852635

The Not So Simple Globular Cluster ω Cen. I. Spatial Distribution of the Multiple Stellar Populations

NASA Astrophysics Data System (ADS)

Calamida, A.; Strampelli, G.; Rest, A.; Bono, G.; Ferraro, I.; Saha, A.; Iannicola, G.; Scolnic, D.; James, D.; Smith, C.; Zenteno, A.

2017-04-01

We present a multi-band photometric catalog of ≈1.7 million cluster members for a field of view of ≈2° × 2° across ω Cen. Photometry is based on images collected with the Dark Energy Camera on the 4 m Blanco telescope and the Advanced Camera for Surveys on the Hubble Space Telescope. The unprecedented photometric accuracy and field coverage allowed us, for the first time, to investigate the spatial distribution of ω Cen multiple populations from the core to the tidal radius, confirming its very complex structure. We found that the frequency of blue main-sequence stars is increasing compared to red main-sequence stars starting from a distance of ≈25‧ from the cluster center. Blue main-sequence stars also show a clumpy spatial distribution, with an excess in the northeast quadrant of the cluster pointing toward the direction of the Galactic center. Stars belonging to the reddest and faintest red-giant branch also show a more extended spatial distribution in the outskirts of ω Cen, a region never explored before. Both these stellar sub-populations, according to spectroscopic measurements, are more metal-rich compared to the cluster main stellar population. These findings, once confirmed, make ω Cen the only stellar system currently known where metal-rich stars have a more extended spatial distribution compared to metal-poor stars. Kinematic and chemical abundance measurements are now needed for stars in the external regions of ω Cen to better characterize the properties of these sub-populations. Based on observations made with the Dark Energy Camera (DECam) on the 4 m Blanco telescope (NOAO) under programs 2014A-0327, 2015A-0151, 2016A-0189, PIs: A. Calamida, A. Rest, and on observations made with the NASA/ESA Hubble Space Telescope, obtained by the Space Telescope Science Institute. STScI is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS 5-26555.

Precipitation measurements for earth-space communications: Accuracy requirements and ground-truth techniques

NASA Technical Reports Server (NTRS)

Ippolito, L. J.; Kaul, R.

1981-01-01

Rainfall which is regarded as one of the more important observations for the measurements of this most variable parameter was made continuously, across large areas and over the sea. Ships could not provide the needed resolution nor could available radars provide the needed breadth of coverage. Microwave observations from the Nimbus-5 satellite offered some hope. Another possibility was suggested by the results of many comparisons between rainfall and the clouds seen in satellite pictures. Sequences of pictures from the first geostationary satellites were employed and a general correspondence between rain and the convective clouds visible in satellite pictures was found. It was demonstrated that the agreement was best for growing clouds. The development methods to infer GATE rainfall from geostationary satellite images are examined.

KSC-99pp0608

NASA Image and Video Library

1999-05-27

NASA Administrator Daniel S. Goldin hands Mrs. Dianne Holliman a plaque honoring her late husband, John Holliman, a CNN national correspondent. Standing behind Goldin is Center Director Roy Bridges. At right is Tom Johnson, news group chairman of CNN. A ceremony dedicated the KSC Press Site auditorium as the John Holliman Auditorium to honor the correspondent for his enthusiastic, dedicated coverage of America's space program. The auditorium was built in 1980 and has been the focal point for new coverage of Space Shuttle launches. The ceremony followed the 94th launch of a Space Shuttle, on mission STS-96, earlier this morning

Press Site Auditorium dedicated to John Holliman

NASA Technical Reports Server (NTRS)

1999-01-01

NASA Administrator Daniel S. Goldin hands Mrs. Dianne Holliman a plaque honoring her late husband, John Holliman, a CNN national correspondent. Standing behind Goldin is Center Director Roy Bridges. At right is Tom Johnson, news group chairman of CNN. A ceremony dedicated the KSC Press Site auditorium as the John Holliman Auditorium to honor the correspondent for his enthusiastic, dedicated coverage of America's space program. The auditorium was built in 1980 and has been the focal point for new coverage of Space Shuttle launches. The ceremony followed the 94th launch of a Space Shuttle, on mission STS-96, earlier this morning.

Increasing the Coverage of Medicinal Chemistry-Relevant Space in Commercial Fragments Screening

PubMed Central

2014-01-01

Analyzing the chemical space coverage in commercial fragment screening collections revealed the overlap between bioactive medicinal chemistry substructures and rule-of-three compliant fragments is only ∼25%. We recommend including these fragments in fragment screening libraries to maximize confidence in discovering hit matter within known bioactive chemical space, while incorporation of nonoverlapping substructures could offer novel hits in screening libraries. Using principal component analysis, polar and three-dimensional substructures display a higher-than-average enrichment of bioactive compounds, indicating increasing representation of these substructures may be beneficial in fragment screening. PMID:24405118

Virome Assembly and Annotation: A Surprise in the Namib Desert

PubMed Central

Hesse, Uljana; van Heusden, Peter; Kirby, Bronwyn M.; Olonade, Israel; van Zyl, Leonardo J.; Trindade, Marla

2017-01-01

Sequencing, assembly, and annotation of environmental virome samples is challenging. Methodological biases and differences in species abundance result in fragmentary read coverage; sequence reconstruction is further complicated by the mosaic nature of viral genomes. In this paper, we focus on biocomputational aspects of virome analysis, emphasizing latent pitfalls in sequence annotation. Using simulated viromes that mimic environmental data challenges we assessed the performance of five assemblers (CLC-Workbench, IDBA-UD, SPAdes, RayMeta, ABySS). Individual analyses of relevant scaffold length fractions revealed shortcomings of some programs in reconstruction of viral genomes with excessive read coverage (IDBA-UD, RayMeta), and in accurate assembly of scaffolds ≥50 kb (SPAdes, RayMeta, ABySS). The CLC-Workbench assembler performed best in terms of genome recovery (including highly covered genomes) and correct reconstruction of large scaffolds; and was used to assemble a virome from a copper rich site in the Namib Desert. We found that scaffold network analysis and cluster-specific read reassembly improved reconstruction of sequences with excessive read coverage, and that strict data filtering for non-viral sequences prior to downstream analyses was essential. In this study we describe novel viral genomes identified in the Namib Desert copper site virome. Taxonomic affiliations of diverse proteins in the dataset and phylogenetic analyses of circovirus-like proteins indicated links to the marine habitat. Considering additional evidence from this dataset we hypothesize that viruses may have been carried from the Atlantic Ocean into the Namib Desert by fog and wind, highlighting the impact of the extended environment on an investigated niche in metagenome studies. PMID:28167933

Coprolites as a source of information on the genome and diet of the cave hyena

PubMed Central

Bon, Céline; Berthonaud, Véronique; Maksud, Frédéric; Labadie, Karine; Poulain, Julie; Artiguenave, François; Wincker, Patrick; Aury, Jean-Marc; Elalouf, Jean-Marc

2012-01-01

We performed high-throughput sequencing of DNA from fossilized faeces to evaluate this material as a source of information on the genome and diet of Pleistocene carnivores. We analysed coprolites derived from the extinct cave hyena (Crocuta crocuta spelaea), and sequenced 90 million DNA fragments from two specimens. The DNA reads enabled a reconstruction of the cave hyena mitochondrial genome with up to a 158-fold coverage. This genome, and those sequenced from extant spotted (Crocuta crocuta) and striped (Hyaena hyaena) hyena specimens, allows for the establishment of a robust phylogeny that supports a close relationship between the cave and the spotted hyena. We also demonstrate that high-throughput sequencing yields data for cave hyena multi-copy and single-copy nuclear genes, and that about 50 per cent of the coprolite DNA can be ascribed to this species. Analysing the data for additional species to indicate the cave hyena diet, we retrieved abundant sequences for the red deer (Cervus elaphus), and characterized its mitochondrial genome with up to a 3.8-fold coverage. In conclusion, we have demonstrated the presence of abundant ancient DNA in the coprolites surveyed. Shotgun sequencing of this material yielded a wealth of DNA sequences for a Pleistocene carnivore and allowed unbiased identification of diet. PMID:22456883

Sequence Analysis of the Genome of Carnation (Dianthus caryophyllus L.)

PubMed Central

Yagi, Masafumi; Kosugi, Shunichi; Hirakawa, Hideki; Ohmiya, Akemi; Tanase, Koji; Harada, Taro; Kishimoto, Kyutaro; Nakayama, Masayoshi; Ichimura, Kazuo; Onozaki, Takashi; Yamaguchi, Hiroyasu; Sasaki, Nobuhiro; Miyahara, Taira; Nishizaki, Yuzo; Ozeki, Yoshihiro; Nakamura, Noriko; Suzuki, Takamasa; Tanaka, Yoshikazu; Sato, Shusei; Shirasawa, Kenta; Isobe, Sachiko; Miyamura, Yoshinori; Watanabe, Akiko; Nakayama, Shinobu; Kishida, Yoshie; Kohara, Mitsuyo; Tabata, Satoshi

2014-01-01

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp. PMID:24344172

The genome sequence of the outbreeding globe artichoke constructed de novo incorporating a phase-aware low-pass sequencing strategy of F1 progeny

PubMed Central

Scaglione, Davide; Reyes-Chin-Wo, Sebastian; Acquadro, Alberto; Froenicke, Lutz; Portis, Ezio; Beitel, Christopher; Tirone, Matteo; Mauro, Rosario; Lo Monaco, Antonino; Mauromicale, Giovanni; Faccioli, Primetta; Cattivelli, Luigi; Rieseberg, Loren; Michelmore, Richard; Lanteri, Sergio

2016-01-01

Globe artichoke (Cynara cardunculus var. scolymus) is an out-crossing, perennial, multi-use crop species that is grown worldwide and belongs to the Compositae, one of the most successful Angiosperm families. We describe the first genome sequence of globe artichoke. The assembly, comprising of 13,588 scaffolds covering 725 of the 1,084 Mb genome, was generated using ~133-fold Illumina sequencing data and encodes 26,889 predicted genes. Re-sequencing (30×) of globe artichoke and cultivated cardoon (C. cardunculus var. altilis) parental genotypes and low-coverage (0.5 to 1×) genotyping-by-sequencing of 163 F1 individuals resulted in 73% of the assembled genome being anchored in 2,178 genetic bins ordered along 17 chromosomal pseudomolecules. This was achieved using a novel pipeline, SOILoCo (Scaffold Ordering by Imputation with Low Coverage), to detect heterozygous regions and assign parental haplotypes with low sequencing read depth and of unknown phase. SOILoCo provides a powerful tool for de novo genome analysis of outcrossing species. Our data will enable genome-scale analyses of evolutionary processes among crops, weeds, and wild species within and beyond the Compositae, and will facilitate the identification of economically important genes from related species. PMID:26786968

On the Power and the Systematic Biases of the Detection of Chromosomal Inversions by Paired-End Genome Sequencing

PubMed Central

Lucas Lledó, José Ignacio; Cáceres, Mario

2013-01-01

One of the most used techniques to study structural variation at a genome level is paired-end mapping (PEM). PEM has the advantage of being able to detect balanced events, such as inversions and translocations. However, inversions are still quite difficult to predict reliably, especially from high-throughput sequencing data. We simulated realistic PEM experiments with different combinations of read and library fragment lengths, including sequencing errors and meaningful base-qualities, to quantify and track down the origin of false positives and negatives along sequencing, mapping, and downstream analysis. We show that PEM is very appropriate to detect a wide range of inversions, even with low coverage data. However, % of inversions located between segmental duplications are expected to go undetected by the most common sequencing strategies. In general, longer DNA libraries improve the detectability of inversions far better than increments of the coverage depth or the read length. Finally, we review the performance of three algorithms to detect inversions —SVDetect, GRIAL, and VariationHunter—, identify common pitfalls, and reveal important differences in their breakpoint precisions. These results stress the importance of the sequencing strategy for the detection of structural variants, especially inversions, and offer guidelines for the design of future genome sequencing projects. PMID:23637806

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

Enhancing genome assemblies by integrating non-sequence based data

PubMed Central

2011-01-01

Introduction Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. Methods The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Results Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. Conclusions We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses. PMID:21554765

Enhancing genome assemblies by integrating non-sequence based data.

PubMed

Heider, Thomas N; Lindsay, James; Wang, Chenwei; O'Neill, Rachel J; Pask, Andrew J

2011-05-28

Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses.

Identifying currents in the gene pool for bacterial populations using an integrative approach.

PubMed

Tang, Jing; Hanage, William P; Fraser, Christophe; Corander, Jukka

2009-08-01

The evolution of bacterial populations has recently become considerably better understood due to large-scale sequencing of population samples. It has become clear that DNA sequences from a multitude of genes, as well as a broad sample coverage of a target population, are needed to obtain a relatively unbiased view of its genetic structure and the patterns of ancestry connected to the strains. However, the traditional statistical methods for evolutionary inference, such as phylogenetic analysis, are associated with several difficulties under such an extensive sampling scenario, in particular when a considerable amount of recombination is anticipated to have taken place. To meet the needs of large-scale analyses of population structure for bacteria, we introduce here several statistical tools for the detection and representation of recombination between populations. Also, we introduce a model-based description of the shape of a population in sequence space, in terms of its molecular variability and affinity towards other populations. Extensive real data from the genus Neisseria are utilized to demonstrate the potential of an approach where these population genetic tools are combined with an phylogenetic analysis. The statistical tools introduced here are freely available in BAPS 5.2 software, which can be downloaded from http://web.abo.fi/fak/mnf/mate/jc/software/baps.html.

The GPS Space Service Volume

NASA Technical Reports Server (NTRS)

Bauer, F. H.; Moreau, M. C.; Dahle-Melsaether, M. E.; Petrofski, W. P.; Stanton, B. J.; Thomason, S.; Harris, G. A.; Sena, R. P.; Temple, L. Parker, III

2006-01-01

Prior to the advent of artificial satellites, the concept of navigating in space and the desire to understand and validate the laws of planetary and satellite motion dates back centuries. At the initiation of orbital flight in 1957, space navigation was dominated by inertial and groundbased tracking methods, underpinned by the laws of planetary motion. It was early in the 1980s that GPS was first explored as a system useful for refining the position, velocity, and timing (PVT) of other spacecraft equipped with GPS receivers. As a result, an entirely new GPS utility was developed beyond its original purpose of providing PVT services for land, maritime, and air applications. Spacecraft both above and below the GPS constellation now receive the GPS signals, including the signals that spill over the limb of the Earth. The use of radionavigation satellite services for space navigation in High Earth Orbits is in fact a capability unique to GPS. Support to GPS space applications is being studied and planned as an important improvement to GPS. This paper discusses the formalization of PVT services in space as part of an overall GPS improvement effort. It describes the GPS Space Service Volume (SSV) and compares it to the Terrestrial Service Volume (TSV). It also discusses SSV coverage with the current GPS constellation, coverage characteristics as a function of altitude, expected power levels, and coverage figures of merit.

Sleeping arrangements and mass distribution of bed nets in six districts in central and northern Mozambique.

PubMed

Plucinski, M M; Chicuecue, S; Macete, E; Chambe, G A; Muguande, O; Matsinhe, G; Colborn, J; Yoon, S S; Doyle, T J; Kachur, S P; Aide, P; Alonso, P L; Guinovart, C; Morgan, J

2015-12-01

Universal coverage with insecticide-treated bed nets is a cornerstone of modern malaria control. Mozambique has developed a novel bed net allocation strategy, where the number of bed nets allocated per household is calculated on the basis of household composition and assumptions about who sleeps with whom. We set out to evaluate the performance of the novel allocation strategy. A total of 1994 households were visited during household surveys following two universal coverage bed net distribution campaigns in Sofala and Nampula provinces in 2010-2013. Each sleeping space was observed for the presence of a bed net, and the sleeping patterns for each household were recorded. The observed coverage and efficiency were compared to a simulated coverage and efficiency had conventional allocation strategies been used. A composite indicator, the product of coverage and efficiency, was calculated. Observed sleeping patterns were compared with the sleeping pattern assumptions. In households reached by the campaign, 93% (95% CI: 93-94%) of sleeping spaces in Sofala and 84% (82-86%) in Nampula were covered by campaign bed nets. The achieved efficiency was high, with 92% (91-93%) of distributed bed nets in Sofala and 93% (91-95%) in Nampula covering a sleeping space. Using the composite indicator, the novel allocation strategy outperformed all conventional strategies in Sofala and was tied for best in Nampula. The sleeping pattern assumptions were completely satisfied in 66% of households in Sofala and 56% of households in Nampula. The most common violation of the sleeping pattern assumptions was that male children 3-10 years of age tended not to share sleeping spaces with female children 3-10 or 10-16 years of age. The sleeping pattern assumptions underlying the novel bed net allocation strategy are generally valid, and net allocation using these assumptions can achieve high coverage and compare favourably with conventional allocation strategies. © 2015 The Authors. Tropical Medicine & International Health Published by John Wiley & Sons Ltd.

Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples

PubMed Central

Martino, Amanda J.; Rhodes, Matthew E.; Biddle, Jennifer F.; Brandt, Leah D.; Tomsho, Lynn P.; House, Christopher H.

2011-01-01

A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples. PMID:22319519

Discovery of Influenza A Virus Sequence Pairs and Their Combinations for Simultaneous Heterosubtypic Targeting that Hedge against Antiviral Resistance

PubMed Central

Lin, Jing; Pramono, Zacharias Aloysius Dwi; Maurer-Stroh, Sebastian

2016-01-01

The multiple circulating human influenza A virus subtypes coupled with the perpetual genomic mutations and segment reassortment events challenge the development of effective therapeutics. The capacity to drug most RNAs motivates the investigation on viral RNA targets. 123,060 segment sequences from 35,938 strains of the most prevalent subtypes also infecting humans–H1N1, 2009 pandemic H1N1, H3N2, H5N1 and H7N9, were used to identify 1,183 conserved RNA target sequences (≥15-mer) in the internal segments. 100% theoretical coverage in simultaneous heterosubtypic targeting is achieved by pairing specific sequences from the same segment (“Duals”) or from two segments (“Doubles”); 1,662 Duals and 28,463 Doubles identified. By combining specific Duals and/or Doubles to form a target graph wherein an edge connecting two vertices (target sequences) represents a Dual or Double, it is possible to hedge against antiviral resistance besides maintaining 100% heterosubtypic coverage. To evaluate the hedging potential, we define the hedge-factor as the minimum number of resistant target sequences that will render the graph to become resistant i.e. eliminate all the edges therein; a target sequence or a graph is considered resistant when it cannot achieve 100% heterosubtypic coverage. In an n-vertices graph (n ≥ 3), the hedge-factor is maximal (= n– 1) when it is a complete graph i.e. every distinct pair in a graph is either a Dual or Double. Computational analyses uncover an extensive number of complete graphs of different sizes. Monte Carlo simulations show that the mutation counts and time elapsed for a target graph to become resistant increase with the hedge-factor. Incidentally, target sequences which were reported to reduce virus titre in experiments are included in our target graphs. The identity of target sequence pairs for heterosubtypic targeting and their combinations for hedging antiviral resistance are useful toolkits to construct target graphs for different therapeutic objectives. PMID:26771381

Framing Space: UK Newspaper Reporting of the Beagle 2 and Cassini-Huygens Space Missions

NASA Astrophysics Data System (ADS)

Jergovic, B.; Miller, S.

2008-05-01

Relatively little scholarly work has been done on looking at the portrayal of astronomy and space science in the media. This short article examines the UK press coverage of two space missions: the Beagle 2 mission to Mars and the Cassini-Huygens mission to Saturn and its moon Titan. In both cases, the leading scientists exerted a strong influence on what journalists reported, to the extent that some journalists appeared to be almost "embedded" in the mission. For the most part the coverage is positive in tone and the loss of the Beagle 2 spacecraft does not reflect badly on the (later) Cassini-Huygens coverage. Most journalists only covered the actual mission events and, in the case of Huygens, did not follow up to cover the peer-reviewed scientific articles that appeared later. Off-the-cuff comments made by scientists at the time of the missions were widely reported. There appears to be an appreciation by journalists and (by inference) their readership that this was science in the making, and that allowances should be made if these comments later turned out to be inaccurate.

Volumetrically-Derived Global Navigation Satellite System Performance Assessment from the Earths Surface through the Terrestrial Service Volume and the Space Service Volume

NASA Technical Reports Server (NTRS)

Welch, Bryan W.

2016-01-01

NASA is participating in the International Committee on Global Navigation Satellite Systems (GNSS) (ICG)'s efforts towards demonstrating the benefits to the space user from the Earth's surface through the Terrestrial Service Volume (TSV) to the edge of the Space Service Volume (SSV), when a multi-GNSS solution space approach is utilized. The ICG Working Group: Enhancement of GNSS Performance, New Services and Capabilities has started a three phase analysis initiative as an outcome of recommendations at the ICG-10 meeting, in preparation for the ICG-11 meeting. The first phase of that increasing complexity and fidelity analysis initiative was recently expanded to compare nadir-facing and zenith-facing user hemispherical antenna coverage with omnidirectional antenna coverage at different distances of 8,000 km altitude and 36,000 km altitude. This report summarizes the performance using these antenna coverage techniques at distances ranging from 100 km altitude to 36,000 km to be all encompassing, as well as the volumetrically-derived system availability metrics.

Newspaper Front Page Coverage of "the Korean Airliner Boeing 747 Massacre" in Six Newspapers.

ERIC Educational Resources Information Center

Kang, Jong Geun

A study investigated three United States and three foreign newspapers to determine the direction or bias of coverage of the 1983 Korean Airline (KAL) incident and any differences in coverage. It was hypothesized (1) that the amount of space allotted to the story in U. S. newspapers would be greater than that in foreign newspapers; (2) that there…

The need for an assembly pilot project

USDA-ARS?s Scientific Manuscript database

Progress has been rapid since the June 2008 start of the cacao genome sequencing project with the completion of the physical map and the accumulation of approximately 10x coverage of the genome with Titanium 454 sequence data of Matina1-6, the highly homozygous Amelonado tree chosen for the project....

Fast imputation using medium- or low-coverage sequence data

USDA-ARS?s Scientific Manuscript database

Direct imputation from raw sequence reads can be more accurate than calling genotypes first and then imputing, especially if read depth is low or error rates high, but different imputation strategies are required than those used for data from genotyping chips. A fast algorithm to impute from lower t...

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...

Coverage of Unification Issue in North and South Korean Papers.

ERIC Educational Resources Information Center

Chang, Won H.

1981-01-01

Concludes that while the North Korean press gives more space to stories concerning the unification of the two Koreas than does the South, it is also more hostile and less varied in its coverage of the issue. (FL)

Alternative Enzymes Lead to Improvements in Sequence Coverage and PTM Analysis

PubMed Central

Hooper, Kyle; Rosenblatt, Michael; Urh, Marjeta; Saveliev, Sergei; Hosfield, Chris; Kobs, Gary; Ford, Michael; Jones, Richard; Amunugama, Ravi; Allen, David; Brazas, Robert

2013-01-01

The profiling of proteins using biological mass spectrometry (bottom up proteomics) most commonly requires trypsin. Trypsin is advantageous in that it produces peptides of optimal charge and size. However, for applications in which the proteins under investigation are part of a complex mixture or not isolated at high levels (i.e. low ng from an immunoprecipitation), sequence coverage is rarely complete. In addition, we have found that in several cases, like phosphorylation, acetylation, and methylation, alternative proteases are required to prepare peptides suitable for MS detection. This poster will provide specific examples which demonstrate this observation. For example, the application of a combined Trypsin/ Lys-C mixture reduces the number of missed cleavages by more than 3-fold producing samples with lower CV's (for biological replicates). The mixture is also well-suited for the complete proteolysis of hydrophobic, compact proteins. The addition of chymotrypsin and elastase has been found to be useful for identifying phosphorylation sites on proteins, especially on sequences where the site of phosphorylation inhibits trypsin (i.e. proximal to K or R). Many epigenetic applications have focused on histone modifications, like lysine acetylation and arginine methylation. Alternative proteases like Asp-N, Glu-C, and chymotrypsin have been especially useful given the fact that the modified K and R residues are resistant to c-terminal cleavage by trypsin. Finally, in the case of serum profiling, the addition of the endoglycosidase, PNGase F has been found to improve sequence coverage due to the removal of N-linked glycans.

ECHO: A reference-free short-read error correction algorithm

PubMed Central

Kao, Wei-Chun; Chan, Andrew H.; Song, Yun S.

2011-01-01

Developing accurate, scalable algorithms to improve data quality is an important computational challenge associated with recent advances in high-throughput sequencing technology. In this study, a novel error-correction algorithm, called ECHO, is introduced for correcting base-call errors in short-reads, without the need of a reference genome. Unlike most previous methods, ECHO does not require the user to specify parameters of which optimal values are typically unknown a priori. ECHO automatically sets the parameters in the assumed model and estimates error characteristics specific to each sequencing run, while maintaining a running time that is within the range of practical use. ECHO is based on a probabilistic model and is able to assign a quality score to each corrected base. Furthermore, it explicitly models heterozygosity in diploid genomes and provides a reference-free method for detecting bases that originated from heterozygous sites. On both real and simulated data, ECHO is able to improve the accuracy of previous error-correction methods by several folds to an order of magnitude, depending on the sequence coverage depth and the position in the read. The improvement is most pronounced toward the end of the read, where previous methods become noticeably less effective. Using a whole-genome yeast data set, it is demonstrated here that ECHO is capable of coping with nonuniform coverage. Also, it is shown that using ECHO to perform error correction as a preprocessing step considerably facilitates de novo assembly, particularly in the case of low-to-moderate sequence coverage depth. PMID:21482625

Low-coverage, whole-genome sequencing of Artocarpus camansi (Moraceae) for phylogenetic marker development and gene discovery1

PubMed Central

Gardner, Elliot M.; Johnson, Matthew G.; Ragone, Diane; Wickett, Norman J.; Zerega, Nyree J. C.

2016-01-01

Premise of the study: We used moderately low-coverage (17×) whole-genome sequencing of Artocarpus camansi (Moraceae) to develop genomic resources for Artocarpus and Moraceae. Methods and Results: A de novo assembly of Illumina short reads (251,378,536 pairs, 2 × 100 bp) accounted for 93% of the predicted genome size. Predicted coding regions were used in a three-way orthology search with published genomes of Morus notabilis and Cannabis sativa. Phylogenetic markers for Moraceae were developed from 333 inferred single-copy exons. Ninety-eight putative MADS-box genes were identified. Analysis of all predicted coding regions resulted in preliminary annotation of 49,089 genes. An analysis of synonymous substitutions for pairs of orthologs (Ks analysis) in M. notabilis and A. camansi strongly suggested a lineage-specific whole-genome duplication in Artocarpus. Conclusions: This study substantially increases the genomic resources available for Artocarpus and Moraceae and demonstrates the value of low-coverage de novo assemblies for nonmodel organisms with moderately large genomes. PMID:27437173

KSC-99pp0607

NASA Image and Video Library

1999-05-27

From left, Center Director Roy Bridges and NASA Administrator Daniel S. Goldin applaud as Jay Holliman, with the help of his mother, Mrs. Dianne Holliman, unveils a plaque honoring his father, the late John Holliman. At right is Tom Johnson, news group chairman of CNN. The occasion was the dedication of the KSC Press Site auditorium as the John Holliman Auditorium to honor the CNN national correspondent for his enthusiastic, dedicated coverage of America's space program. The auditorium was built in 1980 and has been the focal point for new coverage of Space Shuttle launches. The ceremony followed the 94th launch of a Space Shuttle, on mission STS-96, earlier this morning

Demographic history and rare allele sharing among human populations.

PubMed

Gravel, Simon; Henn, Brenna M; Gutenkunst, Ryan N; Indap, Amit R; Marth, Gabor T; Clark, Andrew G; Yu, Fuli; Gibbs, Richard A; Bustamante, Carlos D

2011-07-19

High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2-4× coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after ∼1,000 sequenced chromosomes per population, whereas ∼2,500 chromosomes will be needed for the same number of synonymous variants. Beyond this point, the number of segregating sites in the European and Asian panel populations is expected to overcome that of the African panel because of faster recent population growth. Overall, we find that the majority of human genomic variable sites are rare and exhibit little sharing among diverged populations. Our results emphasize that replication of disease association for specific rare genetic variants across diverged populations must overcome both reduced statistical power because of rarity and higher population divergence.

Demographic history and rare allele sharing among human populations

PubMed Central

Gravel, Simon; Henn, Brenna M.; Gutenkunst, Ryan N.; Indap, Amit R.; Marth, Gabor T.; Clark, Andrew G.; Yu, Fuli; Gibbs, Richard A.; Bustamante, Carlos D.; Altshuler, David L.; Durbin, Richard M.; Abecasis, Gonçalo R.; Bentley, David R.; Chakravarti, Aravinda; Clark, Andrew G.; Collins, Francis S.; De La Vega, Francisco M.; Donnelly, Peter; Egholm, Michael; Flicek, Paul; Gabriel, Stacey B.; Gibbs, Richard A.; Knoppers, Bartha M.; Lander, Eric S.; Lehrach, Hans; Mardis, Elaine R.; McVean, Gil A.; Nickerson, Debbie A.; Peltonen, Leena; Schafer, Alan J.; Sherry, Stephen T.; Wang, Jun; Wilson, Richard K.; Gibbs, Richard A.; Deiros, David; Metzker, Mike; Muzny, Donna; Reid, Jeff; Wheeler, David; Wang, Jun; Li, Jingxiang; Jian, Min; Li, Guoqing; Li, Ruiqiang; Liang, Huiqing; Tian, Geng; Wang, Bo; Wang, Jian; Wang, Wei; Yang, Huanming; Zhang, Xiuqing; Zheng, Huisong; Lander, Eric S.; Altshuler, David L.; Ambrogio, Lauren; Bloom, Toby; Cibulskis, Kristian; Fennell, Tim J.; Gabriel, Stacey B.; Jaffe, David B.; Shefler, Erica; Sougnez, Carrie L.; Bentley, David R.; Gormley, Niall; Humphray, Sean; Kingsbury, Zoya; Koko-Gonzales, Paula; Stone, Jennifer; McKernan, Kevin J.; Costa, Gina L.; Ichikawa, Jeffry K.; Lee, Clarence C.; Sudbrak, Ralf; Lehrach, Hans; Borodina, Tatiana A.; Dahl, Andreas; Davydov, Alexey N.; Marquardt, Peter; Mertes, Florian; Nietfeld, Wilfiried; Rosenstiel, Philip; Schreiber, Stefan; Soldatov, Aleksey V.; Timmermann, Bernd; Tolzmann, Marius; Egholm, Michael; Affourtit, Jason; Ashworth, Dana; Attiya, Said; Bachorski, Melissa; Buglione, Eli; Burke, Adam; Caprio, Amanda; Celone, Christopher; Clark, Shauna; Conners, David; Desany, Brian; Gu, Lisa; Guccione, Lorri; Kao, Kalvin; Kebbel, Andrew; Knowlton, Jennifer; Labrecque, Matthew; McDade, Louise; Mealmaker, Craig; Minderman, Melissa; Nawrocki, Anne; Niazi, Faheem; Pareja, Kristen; Ramenani, Ravi; Riches, David; Song, Wanmin; Turcotte, Cynthia; Wang, Shally; Mardis, Elaine R.; Wilson, Richard K.; Dooling, David; Fulton, Lucinda; Fulton, Robert; Weinstock, George; Durbin, Richard M.; Burton, John; Carter, David M.; Churcher, Carol; Coffey, Alison; Cox, Anthony; Palotie, Aarno; Quail, Michael; Skelly, Tom; Stalker, James; Swerdlow, Harold P.; Turner, Daniel; De Witte, Anniek; Giles, Shane; Gibbs, Richard A.; Wheeler, David; Bainbridge, Matthew; Challis, Danny; Sabo, Aniko; Yu, Fuli; Yu, Jin; Wang, Jun; Fang, Xiaodong; Guo, Xiaosen; Li, Ruiqiang; Li, Yingrui; Luo, Ruibang; Tai, Shuaishuai; Wu, Honglong; Zheng, Hancheng; Zheng, Xiaole; Zhou, Yan; Li, Guoqing; Wang, Jian; Yang, Huanming; Marth, Gabor T.; Garrison, Erik P.; Huang, Weichun; Indap, Amit; Kural, Deniz; Lee, Wan-Ping; Leong, Wen Fung; Quinlan, Aaron R.; Stewart, Chip; Stromberg, Michael P.; Ward, Alistair N.; Wu, Jiantao; Lee, Charles; Mills, Ryan E.; Shi, Xinghua; Daly, Mark J.; DePristo, Mark A.; Altshuler, David L.; Ball, Aaron D.; Banks, Eric; Bloom, Toby; Browning, Brian L.; Cibulskis, Kristian; Fennell, Tim J.; Garimella, Kiran V.; Grossman, Sharon R.; Handsaker, Robert E.; Hanna, Matt; Hartl, Chris; Jaffe, David B.; Kernytsky, Andrew M.; Korn, Joshua M.; Li, Heng; Maguire, Jared R.; McCarroll, Steven A.; McKenna, Aaron; Nemesh, James C.; Philippakis, Anthony A.; Poplin, Ryan E.; Price, Alkes; Rivas, Manuel A.; Sabeti, Pardis C.; Schaffner, Stephen F.; Shefler, Erica; Shlyakhter, Ilya A.; Cooper, David N.; Ball, Edward V.; Mort, Matthew; Phillips, Andrew D.; Stenson, Peter D.; Sebat, Jonathan; Makarov, Vladimir; Ye, Kenny; Yoon, Seungtai C.; Bustamante, Carlos D.; Clark, Andrew G.; Boyko, Adam; Degenhardt, Jeremiah; Gravel, Simon; Gutenkunst, Ryan N.; Kaganovich, Mark; Keinan, Alon; Lacroute, Phil; Ma, Xin; Reynolds, Andy; Clarke, Laura; Flicek, Paul; Cunningham, Fiona; Herrero, Javier; Keenen, Stephen; Kulesha, Eugene; Leinonen, Rasko; McLaren, William M.; Radhakrishnan, Rajesh; Smith, Richard E.; Zalunin, Vadim; Zheng-Bradley, Xiangqun; Korbel, Jan O.; Stütz, Adrian M.; Humphray, Sean; Bauer, Markus; Cheetham, R. Keira; Cox, Tony; Eberle, Michael; James, Terena; Kahn, Scott; Murray, Lisa; Chakravarti, Aravinda; Ye, Kai; De La Vega, Francisco M.; Fu, Yutao; Hyland, Fiona C. L.; Manning, Jonathan M.; McLaughlin, Stephen F.; Peckham, Heather E.; Sakarya, Onur; Sun, Yongming A.; Tsung, Eric F.; Batzer, Mark A.; Konkel, Miriam K.; Walker, Jerilyn A.; Sudbrak, Ralf; Albrecht, Marcus W.; Amstislavskiy, Vyacheslav S.; Herwig, Ralf; Parkhomchuk, Dimitri V.; Sherry, Stephen T.; Agarwala, Richa; Khouri, Hoda M.; Morgulis, Aleksandr O.; Paschall, Justin E.; Phan, Lon D.; Rotmistrovsky, Kirill E.; Sanders, Robert D.; Shumway, Martin F.; Xiao, Chunlin; McVean, Gil A.; Auton, Adam; Iqbal, Zamin; Lunter, Gerton; Marchini, Jonathan L.; Moutsianas, Loukas; Myers, Simon; Tumian, Afidalina; Desany, Brian; Knight, James; Winer, Roger; Craig, David W.; Beckstrom-Sternberg, Steve M.; Christoforides, Alexis; Kurdoglu, Ahmet A.; Pearson, John V.; Sinari, Shripad A.; Tembe, Waibhav D.; Haussler, David; Hinrichs, Angie S.; Katzman, Sol J.; Kern, Andrew; Kuhn, Robert M.; Przeworski, Molly; Hernandez, Ryan D.; Howie, Bryan; Kelley, Joanna L.; Melton, S. Cord; Abecasis, Gonçalo R.; Li, Yun; Anderson, Paul; Blackwell, Tom; Chen, Wei; Cookson, William O.; Ding, Jun; Kang, Hyun Min; Lathrop, Mark; Liang, Liming; Moffatt, Miriam F.; Scheet, Paul; Sidore, Carlo; Snyder, Matthew; Zhan, Xiaowei; Zöllner, Sebastian; Awadalla, Philip; Casals, Ferran; Idaghdour, Youssef; Keebler, John; Stone, Eric A.; Zilversmit, Martine; Jorde, Lynn; Xing, Jinchuan; Eichler, Evan E.; Aksay, Gozde; Alkan, Can; Hajirasouliha, Iman; Hormozdiari, Fereydoun; Kidd, Jeffrey M.; Sahinalp, S. Cenk; Sudmant, Peter H.; Mardis, Elaine R.; Chen, Ken; Chinwalla, Asif; Ding, Li; Koboldt, Daniel C.; McLellan, Mike D.; Dooling, David; Weinstock, George; Wallis, John W.; Wendl, Michael C.; Zhang, Qunyuan; Durbin, Richard M.; Albers, Cornelis A.; Ayub, Qasim; Balasubramaniam, Senduran; Barrett, Jeffrey C.; Carter, David M.; Chen, Yuan; Conrad, Donald F.; Danecek, Petr; Dermitzakis, Emmanouil T.; Hu, Min; Huang, Ni; Hurles, Matt E.; Jin, Hanjun; Jostins, Luke; Keane, Thomas M.; Le, Si Quang; Lindsay, Sarah; Long, Quan; MacArthur, Daniel G.; Montgomery, Stephen B.; Parts, Leopold; Stalker, James; Tyler-Smith, Chris; Walter, Klaudia; Zhang, Yujun; Gerstein, Mark B.; Snyder, Michael; Abyzov, Alexej; Balasubramanian, Suganthi; Bjornson, Robert; Du, Jiang; Grubert, Fabian; Habegger, Lukas; Haraksingh, Rajini; Jee, Justin; Khurana, Ekta; Lam, Hugo Y. K.; Leng, Jing; Mu, Xinmeng Jasmine; Urban, Alexander E.; Zhang, Zhengdong; Li, Yingrui; Luo, Ruibang; Marth, Gabor T.; Garrison, Erik P.; Kural, Deniz; Quinlan, Aaron R.; Stewart, Chip; Stromberg, Michael P.; Ward, Alistair N.; Wu, Jiantao; Lee, Charles; Mills, Ryan E.; Shi, Xinghua; McCarroll, Steven A.; Banks, Eric; DePristo, Mark A.; Handsaker, Robert E.; Hartl, Chris; Korn, Joshua M.; Li, Heng; Nemesh, James C.; Sebat, Jonathan; Makarov, Vladimir; Ye, Kenny; Yoon, Seungtai C.; Degenhardt, Jeremiah; Kaganovich, Mark; Clarke, Laura; Smith, Richard E.; Zheng-Bradley, Xiangqun; Korbel, Jan O.; Humphray, Sean; Cheetham, R. Keira; Eberle, Michael; Kahn, Scott; Murray, Lisa; Ye, Kai; De La Vega, Francisco M.; Fu, Yutao; Peckham, Heather E.; Sun, Yongming A.; Batzer, Mark A.; Konkel, Miriam K.; Walker, Jerilyn A.; Xiao, Chunlin; Iqbal, Zamin; Desany, Brian; Blackwell, Tom; Snyder, Matthew; Xing, Jinchuan; Eichler, Evan E.; Aksay, Gozde; Alkan, Can; Hajirasouliha, Iman; Hormozdiari, Fereydoun; Kidd, Jeffrey M.; Chen, Ken; Chinwalla, Asif; Ding, Li; McLellan, Mike D.; Wallis, John W.; Hurles, Matt E.; Conrad, Donald F.; Walter, Klaudia; Zhang, Yujun; Gerstein, Mark B.; Snyder, Michael; Abyzov, Alexej; Du, Jiang; Grubert, Fabian; Haraksingh, Rajini; Jee, Justin; Khurana, Ekta; Lam, Hugo Y. K.; Leng, Jing; Mu, Xinmeng Jasmine; Urban, Alexander E.; Zhang, Zhengdong; Gibbs, Richard A.; Bainbridge, Matthew; Challis, Danny; Coafra, Cristian; Dinh, Huyen; Kovar, Christie; Lee, Sandy; Muzny, Donna; Nazareth, Lynne; Reid, Jeff; Sabo, Aniko; Yu, Fuli; Yu, Jin; Marth, Gabor T.; Garrison, Erik P.; Indap, Amit; Leong, Wen Fung; Quinlan, Aaron R.; Stewart, Chip; Ward, Alistair N.; Wu, Jiantao; Cibulskis, Kristian; Fennell, Tim J.; Gabriel, Stacey B.; Garimella, Kiran V.; Hartl, Chris; Shefler, Erica; Sougnez, Carrie L.; Wilkinson, Jane; Clark, Andrew G.; Gravel, Simon; Grubert, Fabian; Clarke, Laura; Flicek, Paul; Smith, Richard E.; Zheng-Bradley, Xiangqun; Sherry, Stephen T.; Khouri, Hoda M.; Paschall, Justin E.; Shumway, Martin F.; Xiao, Chunlin; McVean, Gil A.; Katzman, Sol J.; Abecasis, Gonçalo R.; Blackwell, Tom; Mardis, Elaine R.; Dooling, David; Fulton, Lucinda; Fulton, Robert; Koboldt, Daniel C.; Durbin, Richard M.; Balasubramaniam, Senduran; Coffey, Allison; Keane, Thomas M.; MacArthur, Daniel G.; Palotie, Aarno; Scott, Carol; Stalker, James; Tyler-Smith, Chris; Gerstein, Mark B.; Balasubramanian, Suganthi; Chakravarti, Aravinda; Knoppers, Bartha M.; Abecasis, Gonçalo R.; Bustamante, Carlos D.; Gharani, Neda; Gibbs, Richard A.; Jorde, Lynn; Kaye, Jane S.; Kent, Alastair; Li, Taosha; McGuire, Amy L.; McVean, Gil A.; Ossorio, Pilar N.; Rotimi, Charles N.; Su, Yeyang; Toji, Lorraine H.; TylerSmith, Chris; Brooks, Lisa D.; Felsenfeld, Adam L.; McEwen, Jean E.; Abdallah, Assya; Juenger, Christopher R.; Clemm, Nicholas C.; Collins, Francis S.; Duncanson, Audrey; Green, Eric D.; Guyer, Mark S.; Peterson, Jane L.; Schafer, Alan J.; Abecasis, Gonçalo R.; Altshuler, David L.; Auton, Adam; Brooks, Lisa D.; Durbin, Richard M.; Gibbs, Richard A.; Hurles, Matt E.; McVean, Gil A.

2011-01-01

High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2–4× coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after ∼1,000 sequenced chromosomes per population, whereas ∼2,500 chromosomes will be needed for the same number of synonymous variants. Beyond this point, the number of segregating sites in the European and Asian panel populations is expected to overcome that of the African panel because of faster recent population growth. Overall, we find that the majority of human genomic variable sites are rare and exhibit little sharing among diverged populations. Our results emphasize that replication of disease association for specific rare genetic variants across diverged populations must overcome both reduced statistical power because of rarity and higher population divergence. PMID:21730125

Liver imaging at 3.0 T: diffusion-induced black-blood echo-planar imaging with large anatomic volumetric coverage as an alternative for specific absorption rate-intensive echo-train spin-echo sequences: feasibility study.

PubMed

van den Bos, Indra C; Hussain, Shahid M; Krestin, Gabriel P; Wielopolski, Piotr A

2008-07-01

Institutional Review Board approval and signed informed consent were obtained by all participants for an ongoing sequence optimization project at 3.0 T. The purpose of this study was to evaluate breath-hold diffusion-induced black-blood echo-planar imaging (BBEPI) as a potential alternative for specific absorption rate (SAR)-intensive spin-echo sequences, in particular, the fast spin-echo (FSE) sequences, at 3.0 T. Fourteen healthy volunteers (seven men, seven women; mean age +/- standard deviation, 32.7 years +/- 6.8) were imaged for this purpose. Liver coverage (20 cm, z-axis) was always performed in one 25-second breath hold. Imaging parameters were varied interactively with regard to echo time, diffusion b value, and voxel size. Images were evaluated and compared with fat-suppressed T2-weighted FSE images for image quality, liver delineation, geometric distortions, fat suppression, suppression of the blood signal, contrast-to-noise ratio (CNR), and signal-to-noise ratio (SNR). An optimized short- (25 msec) and long-echo (80 msec) BBEPI provided full anatomic, single breath-hold liver coverage (100 and 50 sections, respectively), with resulting voxel sizes of 3.3 x 2.7 x 2.0 mm and 3.3 x 2.7 x 4.0 mm, respectively. Repetition time was 6300 msec, matrix size was 160 x 192, and an acceleration factor of 2.00 was used. b Values of more than 20 sec/mm(2) showed better suppression of the blood signal but b values of 10 sec/mm(2) provided improved volume coverage and signal consistency. Compared with fat-suppressed T2-weighted FSE, the optimized BBEPI sequence provided (a) comparable image quality and liver delineation, (b) acceptable geometric distortions, (c) improved suppression of fat and blood signals, and (d) high CNR and SNR. BBEPI is feasible for fast, low-SAR, thin-section morphologic imaging of the entire liver in a single breath hold at 3.0 T. (c) RSNA, 2008.

Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing.

PubMed

Zhang, Jin; Ruhlman, Tracey A; Mower, Jeffrey P; Jansen, Robert K

2013-12-29

Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants.

Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing

PubMed Central

2013-01-01

Background Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Results Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. Conclusions The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants. PMID:24373163

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

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

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

DOE PAGES

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; ...

2014-01-01

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Comparison of illumina and 454 deep sequencing in participants failing raltegravir-based antiretroviral therapy.

PubMed

Li, Jonathan Z; Chapman, Brad; Charlebois, Patrick; Hofmann, Oliver; Weiner, Brian; Porter, Alyssa J; Samuel, Reshmi; Vardhanabhuti, Saran; Zheng, Lu; Eron, Joseph; Taiwo, Babafemi; Zody, Michael C; Henn, Matthew R; Kuritzkes, Daniel R; Hide, Winston; Wilson, Cara C; Berzins, Baiba I; Acosta, Edward P; Bastow, Barbara; Kim, Peter S; Read, Sarah W; Janik, Jennifer; Meres, Debra S; Lederman, Michael M; Mong-Kryspin, Lori; Shaw, Karl E; Zimmerman, Louis G; Leavitt, Randi; De La Rosa, Guy; Jennings, Amy

2014-01-01

The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs. A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser. Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454. In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.

LoRTE: Detecting transposon-induced genomic variants using low coverage PacBio long read sequences.

PubMed

Disdero, Eric; Filée, Jonathan

2017-01-01

Population genomic analysis of transposable elements has greatly benefited from recent advances of sequencing technologies. However, the short size of the reads and the propensity of transposable elements to nest in highly repeated regions of genomes limits the efficiency of bioinformatic tools when Illumina or 454 technologies are used. Fortunately, long read sequencing technologies generating read length that may span the entire length of full transposons are now available. However, existing TE population genomic softwares were not designed to handle long reads and the development of new dedicated tools is needed. LoRTE is the first tool able to use PacBio long read sequences to identify transposon deletions and insertions between a reference genome and genomes of different strains or populations. Tested against simulated and genuine Drosophila melanogaster PacBio datasets, LoRTE appears to be a reliable and broadly applicable tool to study the dynamic and evolutionary impact of transposable elements using low coverage, long read sequences. LoRTE is an efficient and accurate tool to identify structural genomic variants caused by TE insertion or deletion. LoRTE is available for download at http://www.egce.cnrs-gif.fr/?p=6422.

A-to-I RNA editing occurs at over a hundred million genomic sites, located in a majority of human genes.

PubMed

Bazak, Lily; Haviv, Ami; Barak, Michal; Jacob-Hirsch, Jasmine; Deng, Patricia; Zhang, Rui; Isaacs, Farren J; Rechavi, Gideon; Li, Jin Billy; Eisenberg, Eli; Levanon, Erez Y

2014-03-01

RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected ∼1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes).

PubMed

Dessimoz, Christophe; Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-09-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes)

PubMed Central

Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-01-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references. PMID:21712341

A massive parallel sequencing workflow for diagnostic genetic testing of mismatch repair genes

PubMed Central

Hansen, Maren F; Neckmann, Ulrike; Lavik, Liss A S; Vold, Trine; Gilde, Bodil; Toft, Ragnhild K; Sjursen, Wenche

2014-01-01

The purpose of this study was to develop a massive parallel sequencing (MPS) workflow for diagnostic analysis of mismatch repair (MMR) genes using the GS Junior system (Roche). A pathogenic variant in one of four MMR genes, (MLH1, PMS2, MSH6, and MSH2), is the cause of Lynch Syndrome (LS), which mainly predispose to colorectal cancer. We used an amplicon-based sequencing method allowing specific and preferential amplification of the MMR genes including PMS2, of which several pseudogenes exist. The amplicons were pooled at different ratios to obtain coverage uniformity and maximize the throughput of a single-GS Junior run. In total, 60 previously identified and distinct variants (substitutions and indels), were sequenced by MPS and successfully detected. The heterozygote detection range was from 19% to 63% and dependent on sequence context and coverage. We were able to distinguish between false-positive and true-positive calls in homopolymeric regions by cross-sample comparison and evaluation of flow signal distributions. In addition, we filtered variants according to a predefined status, which facilitated variant annotation. Our study shows that implementation of MPS in routine diagnostics of LS can accelerate sample throughput and reduce costs without compromising sensitivity, compared to Sanger sequencing. PMID:24689082

Modularity of Protein Folds as a Tool for Template-Free Modeling of Structures.

PubMed

Vallat, Brinda; Madrid-Aliste, Carlos; Fiser, Andras

2015-08-01

Predicting the three-dimensional structure of proteins from their amino acid sequences remains a challenging problem in molecular biology. While the current structural coverage of proteins is almost exclusively provided by template-based techniques, the modeling of the rest of the protein sequences increasingly require template-free methods. However, template-free modeling methods are much less reliable and are usually applicable for smaller proteins, leaving much space for improvement. We present here a novel computational method that uses a library of supersecondary structure fragments, known as Smotifs, to model protein structures. The library of Smotifs has saturated over time, providing a theoretical foundation for efficient modeling. The method relies on weak sequence signals from remotely related protein structures to create a library of Smotif fragments specific to the target protein sequence. This Smotif library is exploited in a fragment assembly protocol to sample decoys, which are assessed by a composite scoring function. Since the Smotif fragments are larger in size compared to the ones used in other fragment-based methods, the proposed modeling algorithm, SmotifTF, can employ an exhaustive sampling during decoy assembly. SmotifTF successfully predicts the overall fold of the target proteins in about 50% of the test cases and performs competitively when compared to other state of the art prediction methods, especially when sequence signal to remote homologs is diminishing. Smotif-based modeling is complementary to current prediction methods and provides a promising direction in addressing the structure prediction problem, especially when targeting larger proteins for modeling.

Comprehensive computational design of ordered peptide macrocycles

PubMed Central

Hosseinzadeh, Parisa; Bhardwaj, Gaurav; Mulligan, Vikram Khipple; Shortridge, Matthew D.; Craven, Timothy W.; Pardo-Avila, Fátima; Rettie, Stephen A.; Kim, David E.; Silva, Daniel-Adriano; Ibrahim, Yehia M.; Webb, Ian K.; Cort, John R.; Adkins, Joshua N.; Varani, Gabriele; Baker, David

2018-01-01

Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods. PMID:29242347

Analyses of Hypomethylated Oil Palm Gene Space

PubMed Central

Jayanthi, Nagappan; Mohd-Amin, Ab Halim; Azizi, Norazah; Chan, Kuang-Lim; Maqbool, Nauman J.; Maclean, Paul; Brauning, Rudi; McCulloch, Alan; Moraga, Roger; Ong-Abdullah, Meilina; Singh, Rajinder

2014-01-01

Demand for palm oil has been increasing by an average of ∼8% the past decade and currently accounts for about 59% of the world's vegetable oil market. This drives the need to increase palm oil production. Nevertheless, due to the increasing need for sustainable production, it is imperative to increase productivity rather than the area cultivated. Studies on the oil palm genome are essential to help identify genes or markers that are associated with important processes or traits, such as flowering, yield and disease resistance. To achieve this, 294,115 and 150,744 sequences from the hypomethylated or gene-rich regions of Elaeis guineensis and E. oleifera genome were sequenced and assembled into contigs. An additional 16,427 shot-gun sequences and 176 bacterial artificial chromosomes (BAC) were also generated to check the quality of libraries constructed. Comparison of these sequences revealed that although the methylation-filtered libraries were sequenced at low coverage, they still tagged at least 66% of the RefSeq supported genes in the BAC and had a filtration power of at least 2.0. A total 33,752 microsatellites and 40,820 high-quality single nucleotide polymorphism (SNP) markers were identified. These represent the most comprehensive collection of microsatellites and SNPs to date and would be an important resource for genetic mapping and association studies. The gene models predicted from the assembled contigs were mined for genes of interest, and 242, 65 and 14 oil palm transcription factors, resistance genes and miRNAs were identified respectively. Examples of the transcriptional factors tagged include those associated with floral development and tissue culture, such as homeodomain proteins, MADS, Squamosa and Apetala2. The E. guineensis and E. oleifera hypomethylated sequences provide an important resource to understand the molecular mechanisms associated with important agronomic traits in oil palm. PMID:24497974

Population-genomic variation within RNA viruses of the Western honey bee, Apis mellifera, inferred from deep sequencing

PubMed Central

2013-01-01

Background Deep sequencing of viruses isolated from infected hosts is an efficient way to measure population-genetic variation and can reveal patterns of dispersal and natural selection. In this study, we mined existing Illumina sequence reads to investigate single-nucleotide polymorphisms (SNPs) within two RNA viruses of the Western honey bee (Apis mellifera), deformed wing virus (DWV) and Israel acute paralysis virus (IAPV). All viral RNA was extracted from North American samples of honey bees or, in one case, the ectoparasitic mite Varroa destructor. Results Coverage depth was generally lower for IAPV than DWV, and marked gaps in coverage occurred in several narrow regions (< 50 bp) of IAPV. These coverage gaps occurred across sequencing runs and were virtually unchanged when reads were re-mapped with greater permissiveness (up to 8% divergence), suggesting a recurrent sequencing artifact rather than strain divergence. Consensus sequences of DWV for each sample showed little phylogenetic divergence, low nucleotide diversity, and strongly negative values of Fu and Li’s D statistic, suggesting a recent population bottleneck and/or purifying selection. The Kakugo strain of DWV fell outside of all other DWV sequences at 100% bootstrap support. IAPV consensus sequences supported the existence of multiple clades as had been previously reported, and Fu and Li’s D was closer to neutral expectation overall, although a sliding-window analysis identified a significantly positive D within the protease region, suggesting selection maintains diversity in that region. Within-sample mean diversity was comparable between the two viruses on average, although for both viruses there was substantial variation among samples in mean diversity at third codon positions and in the number of high-diversity sites. FST values were bimodal for DWV, likely reflecting neutral divergence in two low-diversity populations, whereas IAPV had several sites that were strong outliers with very low FST. Conclusions This initial survey of genetic variation within honey bee RNA viruses suggests future directions for studies examining the underlying causes of population-genetic structure in these economically important pathogens. PMID:23497218

Population-genomic variation within RNA viruses of the Western honey bee, Apis mellifera, inferred from deep sequencing.

PubMed

Cornman, Robert Scott; Boncristiani, Humberto; Dainat, Benjamin; Chen, Yanping; vanEngelsdorp, Dennis; Weaver, Daniel; Evans, Jay D

2013-03-07

Deep sequencing of viruses isolated from infected hosts is an efficient way to measure population-genetic variation and can reveal patterns of dispersal and natural selection. In this study, we mined existing Illumina sequence reads to investigate single-nucleotide polymorphisms (SNPs) within two RNA viruses of the Western honey bee (Apis mellifera), deformed wing virus (DWV) and Israel acute paralysis virus (IAPV). All viral RNA was extracted from North American samples of honey bees or, in one case, the ectoparasitic mite Varroa destructor. Coverage depth was generally lower for IAPV than DWV, and marked gaps in coverage occurred in several narrow regions (< 50 bp) of IAPV. These coverage gaps occurred across sequencing runs and were virtually unchanged when reads were re-mapped with greater permissiveness (up to 8% divergence), suggesting a recurrent sequencing artifact rather than strain divergence. Consensus sequences of DWV for each sample showed little phylogenetic divergence, low nucleotide diversity, and strongly negative values of Fu and Li's D statistic, suggesting a recent population bottleneck and/or purifying selection. The Kakugo strain of DWV fell outside of all other DWV sequences at 100% bootstrap support. IAPV consensus sequences supported the existence of multiple clades as had been previously reported, and Fu and Li's D was closer to neutral expectation overall, although a sliding-window analysis identified a significantly positive D within the protease region, suggesting selection maintains diversity in that region. Within-sample mean diversity was comparable between the two viruses on average, although for both viruses there was substantial variation among samples in mean diversity at third codon positions and in the number of high-diversity sites. FST values were bimodal for DWV, likely reflecting neutral divergence in two low-diversity populations, whereas IAPV had several sites that were strong outliers with very low FST. This initial survey of genetic variation within honey bee RNA viruses suggests future directions for studies examining the underlying causes of population-genetic structure in these economically important pathogens.

Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

PubMed

Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

2018-03-19

To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test

PubMed Central

Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

2018-01-01

Purpose Genetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use. Methods We prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing. Results WGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24% P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A. Conclusion WGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort. PMID:28771251

Sequence analysis of the genome of carnation (Dianthus caryophyllus L.).

PubMed

Yagi, Masafumi; Kosugi, Shunichi; Hirakawa, Hideki; Ohmiya, Akemi; Tanase, Koji; Harada, Taro; Kishimoto, Kyutaro; Nakayama, Masayoshi; Ichimura, Kazuo; Onozaki, Takashi; Yamaguchi, Hiroyasu; Sasaki, Nobuhiro; Miyahara, Taira; Nishizaki, Yuzo; Ozeki, Yoshihiro; Nakamura, Noriko; Suzuki, Takamasa; Tanaka, Yoshikazu; Sato, Shusei; Shirasawa, Kenta; Isobe, Sachiko; Miyamura, Yoshinori; Watanabe, Akiko; Nakayama, Shinobu; Kishida, Yoshie; Kohara, Mitsuyo; Tabata, Satoshi

2014-06-01

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. 'Francesco' was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568,887,315 bp, consisting of 45,088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16,644 bp and 60,737 bp, respectively, and the longest scaffold was 1,287,144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼ 98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp. © The Author 2013. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Comparison of single cell sequencing data between two whole genome amplification methods on two sequencing platforms.

PubMed

Chen, DaYang; Zhen, HeFu; Qiu, Yong; Liu, Ping; Zeng, Peng; Xia, Jun; Shi, QianYu; Xie, Lin; Zhu, Zhu; Gao, Ya; Huang, GuoDong; Wang, Jian; Yang, HuanMing; Chen, Fang

2018-03-21

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

PubMed

Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

2018-04-01

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

Comparison of the Equine Reference Sequence with Its Sanger Source Data and New Illumina Reads

PubMed Central

Rebolledo-Mendez, Jovan; Hestand, Matthew S.; Coleman, Stephen J.; Zeng, Zheng; Orlando, Ludovic; MacLeod, James N.; Kalbfleisch, Ted

2015-01-01

The reference assembly for the domestic horse, EquCab2, published in 2009, was built using approximately 30 million Sanger reads from a Thoroughbred mare named Twilight. Contiguity in the assembly was facilitated using nearly 315 thousand BAC end sequences from Twilight’s half brother Bravo. Since then, it has served as the foundation for many genome-wide analyses that include not only the modern horse, but ancient horses and other equid species as well. As data mapped to this reference has accumulated, consistent variation between mapped datasets and the reference, in terms of regions with no read coverage, single nucleotide variants, and small insertions/deletions have become apparent. In many cases, it is not clear whether these differences are the result of true sequence variation between the research subjects’ and Twilight’s genome or due to errors in the reference. EquCab2 is regarded as “The Twilight Assembly.” The objective of this study was to identify inconsistencies between the EquCab2 assembly and the source Twilight Sanger data used to build it. To that end, the original Sanger and BAC end reads have been mapped back to this equine reference and assessed with the addition of approximately 40X coverage of new Illumina Paired-End sequence data. The resulting mapped datasets identify those regions with low Sanger read coverage, as well as variation in genomic content that is not consistent with either the original Twilight Sanger data or the new genomic sequence data generated from Twilight on the Illumina platform. As the haploid EquCab2 reference assembly was created using Sanger reads derived largely from a single individual, the vast majority of variation detected in a mapped dataset comprised of those same Sanger reads should be heterozygous. In contrast, homozygous variations would represent either errors in the reference or contributions from Bravo's BAC end sequences. Our analysis identifies 720,843 homozygous discrepancies between new, high throughput genomic sequence data generated for Twilight and the EquCab2 reference assembly. Most of these represent errors in the assembly, while approximately 10,000 are demonstrated to be contributions from another horse. Other results are presented that include the binary alignment map file of the mapped Sanger reads, a list of variants identified as discrepancies between the source data and resulting reference, and a BED annotation file that lists the regions of the genome whose consensus was likely derived from low coverage alignments. PMID:26107638

Low coverage sequencing of three echinoderm genomes: the brittle star Ophionereis fasciata, the sea star Patiriella regularis, and the sea cucumber Australostichopus mollis.

PubMed

Long, Kyle A; Nossa, Carlos W; Sewell, Mary A; Putnam, Nicholas H; Ryan, Joseph F

2016-01-01

There are five major extant groups of Echinodermata: Crinoidea (feather stars and sea lillies), Ophiuroidea (brittle stars and basket stars), Asteroidea (sea stars), Echinoidea (sea urchins, sea biscuits, and sand dollars), and Holothuroidea (sea cucumbers). These animals are known for their pentaradial symmetry as adults, unique water vascular system, mutable collagenous tissues, and endoskeletons of high magnesium calcite. To our knowledge, the only echinoderm species with a genome sequence available to date is Strongylocentrotus pupuratus (Echinoidea). The availability of additional echinoderm genome sequences is crucial for understanding the biology of these animals. Here we present assembled draft genomes of the brittle star Ophionereis fasciata, the sea star Patiriella regularis, and the sea cucumber Australostichopus mollis from Illumina sequence data with coverages of 12.5x, 22.5x, and 21.4x, respectively. These data provide a resource for mining gene superfamilies, identifying non-coding RNAs, confirming gene losses, and designing experimental constructs. They will be important comparative resources for future genomic studies in echinoderms.

Three-dimensional sampling perfection with application-optimised contrasts using a different flip angle evolutions sequence for routine imaging of the spine: preliminary experience

PubMed Central

Tins, B; Cassar-Pullicino, V; Haddaway, M; Nachtrab, U

2012-01-01

Objectives The bulk of spinal imaging is still performed with conventional two-dimensional sequences. This study assesses the suitability of three-dimensional sampling perfection with application-optimised contrasts using a different flip angle evolutions (SPACE) sequence for routine spinal imaging. Methods 62 MRI examinations of the spine were evaluated by 2 examiners in consensus for the depiction of anatomy and presence of artefact. We noted pathologies that might be missed using the SPACE sequence only or the SPACE and a sagittal T1 weighted sequence. The reference standards were sagittal and axial T1 weighted and T2 weighted sequences. At a later date the evaluation was repeated by one of the original examiners and an additional examiner. Results There was good agreement of the single evaluations and consensus evaluation for the conventional sequences: κ>0.8, confidence interval (CI)>0.6–1.0. For the SPACE sequence, depiction of anatomy was very good for 84% of cases, with high interobserver agreement, but there was poor interobserver agreement for other cases. For artefact assessment of SPACE, κ=0.92, CI=0.92–1.0. The SPACE sequence was superior to conventional sequences for depiction of anatomy and artefact resistance. The SPACE sequence occasionally missed bone marrow oedema. In conjunction with sagittal T1 weighted sequences, no abnormality was missed. The isotropic SPACE sequence was superior to conventional sequences in imaging difficult anatomy such as in scoliosis and spondylolysis. Conclusion The SPACE sequence allows excellent assessment of anatomy owing to high spatial resolution and resistance to artefact. The sensitivity for bone marrow abnormalities is limited. PMID:22374284

Three-dimensional sampling perfection with application-optimised contrasts using a different flip angle evolutions sequence for routine imaging of the spine: preliminary experience.

PubMed

Tins, B; Cassar-Pullicino, V; Haddaway, M; Nachtrab, U

2012-08-01

The bulk of spinal imaging is still performed with conventional two-dimensional sequences. This study assesses the suitability of three-dimensional sampling perfection with application-optimised contrasts using a different flip angle evolutions (SPACE) sequence for routine spinal imaging. 62 MRI examinations of the spine were evaluated by 2 examiners in consensus for the depiction of anatomy and presence of artefact. We noted pathologies that might be missed using the SPACE sequence only or the SPACE and a sagittal T(1) weighted sequence. The reference standards were sagittal and axial T(1) weighted and T(2) weighted sequences. At a later date the evaluation was repeated by one of the original examiners and an additional examiner. There was good agreement of the single evaluations and consensus evaluation for the conventional sequences: κ>0.8, confidence interval (CI)>0.6-1.0. For the SPACE sequence, depiction of anatomy was very good for 84% of cases, with high interobserver agreement, but there was poor interobserver agreement for other cases. For artefact assessment of SPACE, κ=0.92, CI=0.92-1.0. The SPACE sequence was superior to conventional sequences for depiction of anatomy and artefact resistance. The SPACE sequence occasionally missed bone marrow oedema. In conjunction with sagittal T(1) weighted sequences, no abnormality was missed. The isotropic SPACE sequence was superior to conventional sequences in imaging difficult anatomy such as in scoliosis and spondylolysis. The SPACE sequence allows excellent assessment of anatomy owing to high spatial resolution and resistance to artefact. The sensitivity for bone marrow abnormalities is limited.

How the Networks Cover Education: Schools Are not the Media's Pet.

ERIC Educational Resources Information Center

Robinson, Michael J.

1984-01-01

An analysis of network news coverage of public education from April 1982 to April 1983 showed that such coverage was overwhelmingly negative, with almost four times as much news space devoted to "bad news" about public education as to "good news." (CMG)

Sequence space coverage, entropy of genomes and the potential to detect non-human DNA in human samples

PubMed Central

Liu, Zhandong; Venkatesh, Santosh S; Maley, Carlo C

2008-01-01

Background Genomes store information for building and maintaining organisms. Complete sequencing of many genomes provides the opportunity to study and compare global information properties of those genomes. Results We have analyzed aspects of the information content of Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli (K-12) genomes. Virtually all possible (> 98%) 12 bp oligomers appear in vertebrate genomes while < 2% of 19 bp oligomers are present. Other species showed different ranges of > 98% to < 2% of possible oligomers in D. melanogaster (12–17 bp), C. elegans (11–17 bp), A. thaliana (11–17 bp), S. cerevisiae (10–16 bp) and E. coli (9–15 bp). Frequencies of unique oligomers in the genomes follow similar patterns. We identified a set of 2.6 M 15-mers that are more than 1 nucleotide different from all 15-mers in the human genome and so could be used as probes to detect microbes in human samples. In a human sample, these probes would detect 100% of the 433 currently fully sequenced prokaryotes and 75% of the 3065 fully sequenced viruses. The human genome is significantly more compact in sequence space than a random genome. We identified the most frequent 5- to 20-mers in the human genome, which may prove useful as PCR primers. We also identified a bacterium, Anaeromyxobacter dehalogenans, which has an exceptionally low diversity of oligomers given the size of its genome and its GC content. The entropy of coding regions in the human genome is significantly higher than non-coding regions and chromosomes. However chromosomes 1, 2, 9, 12 and 14 have a relatively high proportion of coding DNA without high entropy, and chromosome 20 is the opposite with a low frequency of coding regions but relatively high entropy. Conclusion Measures of the frequency of oligomers are useful for designing PCR assays and for identifying chromosomes and organisms with hidden structure that had not been previously recognized. This information may be used to detect novel microbes in human tissues. PMID:18973670

Sequence space coverage, entropy of genomes and the potential to detect non-human DNA in human samples.

PubMed

Liu, Zhandong; Venkatesh, Santosh S; Maley, Carlo C

2008-10-30

Genomes store information for building and maintaining organisms. Complete sequencing of many genomes provides the opportunity to study and compare global information properties of those genomes. We have analyzed aspects of the information content of Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli (K-12) genomes. Virtually all possible (> 98%) 12 bp oligomers appear in vertebrate genomes while < 2% of 19 bp oligomers are present. Other species showed different ranges of > 98% to < 2% of possible oligomers in D. melanogaster (12-17 bp), C. elegans (11-17 bp), A. thaliana (11-17 bp), S. cerevisiae (10-16 bp) and E. coli (9-15 bp). Frequencies of unique oligomers in the genomes follow similar patterns. We identified a set of 2.6 M 15-mers that are more than 1 nucleotide different from all 15-mers in the human genome and so could be used as probes to detect microbes in human samples. In a human sample, these probes would detect 100% of the 433 currently fully sequenced prokaryotes and 75% of the 3065 fully sequenced viruses. The human genome is significantly more compact in sequence space than a random genome. We identified the most frequent 5- to 20-mers in the human genome, which may prove useful as PCR primers. We also identified a bacterium, Anaeromyxobacter dehalogenans, which has an exceptionally low diversity of oligomers given the size of its genome and its GC content. The entropy of coding regions in the human genome is significantly higher than non-coding regions and chromosomes. However chromosomes 1, 2, 9, 12 and 14 have a relatively high proportion of coding DNA without high entropy, and chromosome 20 is the opposite with a low frequency of coding regions but relatively high entropy. Measures of the frequency of oligomers are useful for designing PCR assays and for identifying chromosomes and organisms with hidden structure that had not been previously recognized. This information may be used to detect novel microbes in human tissues.

CMsearch: simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction.

PubMed

Cui, Xuefeng; Lu, Zhiwu; Wang, Sheng; Jing-Yan Wang, Jim; Gao, Xin

2016-06-15

Protein homology detection, a fundamental problem in computational biology, is an indispensable step toward predicting protein structures and understanding protein functions. Despite the advances in recent decades on sequence alignment, threading and alignment-free methods, protein homology detection remains a challenging open problem. Recently, network methods that try to find transitive paths in the protein structure space demonstrate the importance of incorporating network information of the structure space. Yet, current methods merge the sequence space and the structure space into a single space, and thus introduce inconsistency in combining different sources of information. We present a novel network-based protein homology detection method, CMsearch, based on cross-modal learning. Instead of exploring a single network built from the mixture of sequence and structure space information, CMsearch builds two separate networks to represent the sequence space and the structure space. It then learns sequence-structure correlation by simultaneously taking sequence information, structure information, sequence space information and structure space information into consideration. We tested CMsearch on two challenging tasks, protein homology detection and protein structure prediction, by querying all 8332 PDB40 proteins. Our results demonstrate that CMsearch is insensitive to the similarity metrics used to define the sequence and the structure spaces. By using HMM-HMM alignment as the sequence similarity metric, CMsearch clearly outperforms state-of-the-art homology detection methods and the CASP-winning template-based protein structure prediction methods. Our program is freely available for download from http://sfb.kaust.edu.sa/Pages/Software.aspx : xin.gao@kaust.edu.sa Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

First-pass myocardial perfusion imaging with whole-heart coverage using L1-SPIRiT accelerated variable density spiral trajectories

PubMed Central

Yang, Yang; Kramer, Christopher M.; Shaw, Peter W.; Meyer, Craig H.; Salerno, Michael

2015-01-01

Purpose To design and evaluate 2D L1-SPIRiT accelerated spiral pulse sequences for first-pass myocardial perfusion imaging with whole heart coverage capable of measuring 8 slices at 2 mm in-plane resolution at heart rates up to 125 beats per minute (BPM). Methods Combinations of 5 different spiral trajectories and 4 k-t sampling patterns were retrospectively simulated in 25 fully sampled datasets and reconstructed with L1-SPIRiT to determine the best combination of parameters. Two candidate sequences were prospectively evaluated in 34 human subjects to assess in-vivo performance. Results A dual density broad transition spiral trajectory with either angularly uniform or golden angle in time k-t sampling pattern had the largest structural similarity (SSIM) and smallest root mean square error (RMSE) from the retrospective simulation, and the L1-SPIRiT reconstruction had well-preserved temporal dynamics. In vivo data demonstrated that both of the sampling patterns could produce high quality perfusion images with whole-heart coverage. Conclusion First-pass myocardial perfusion imaging using accelerated spirals with optimized trajectory and k-t sampling pattern can produce high quality 2D-perfusion images with wholeheart coverage at the heart rates up to 125 BPM. PMID:26538511

Transfer of genetic therapy across human populations: molecular targets for increasing patient coverage in repeat expansion diseases

PubMed Central

Varela, Miguel A; Curtis, Helen J; Douglas, Andrew GL; Hammond, Suzan M; O'Loughlin, Aisling J; Sobrido, Maria J; Scholefield, Janine; Wood, Matthew JA

2016-01-01

Allele-specific gene therapy aims to silence expression of mutant alleles through targeting of disease-linked single-nucleotide polymorphisms (SNPs). However, SNP linkage to disease varies between populations, making such molecular therapies applicable only to a subset of patients. Moreover, not all SNPs have the molecular features necessary for potent gene silencing. Here we provide knowledge to allow the maximisation of patient coverage by building a comprehensive understanding of SNPs ranked according to their predicted suitability toward allele-specific silencing in 14 repeat expansion diseases: amyotrophic lateral sclerosis and frontotemporal dementia, dentatorubral-pallidoluysian atrophy, myotonic dystrophy 1, myotonic dystrophy 2, Huntington's disease and several spinocerebellar ataxias. Our systematic analysis of DNA sequence variation shows that most annotated SNPs are not suitable for potent allele-specific silencing across populations because of suboptimal sequence features and low variability (>97% in HD). We suggest maximising patient coverage by selecting SNPs with high heterozygosity across populations, and preferentially targeting SNPs that lead to purine:purine mismatches in wild-type alleles to obtain potent allele-specific silencing. We therefore provide fundamental knowledge on strategies for optimising patient coverage of therapeutics for microsatellite expansion disorders by linking analysis of population genetic variation to the selection of molecular targets. PMID:25990798

Transfer of genetic therapy across human populations: molecular targets for increasing patient coverage in repeat expansion diseases.

PubMed

Varela, Miguel A; Curtis, Helen J; Douglas, Andrew G L; Hammond, Suzan M; O'Loughlin, Aisling J; Sobrido, Maria J; Scholefield, Janine; Wood, Matthew J A

2016-02-01

Allele-specific gene therapy aims to silence expression of mutant alleles through targeting of disease-linked single-nucleotide polymorphisms (SNPs). However, SNP linkage to disease varies between populations, making such molecular therapies applicable only to a subset of patients. Moreover, not all SNPs have the molecular features necessary for potent gene silencing. Here we provide knowledge to allow the maximisation of patient coverage by building a comprehensive understanding of SNPs ranked according to their predicted suitability toward allele-specific silencing in 14 repeat expansion diseases: amyotrophic lateral sclerosis and frontotemporal dementia, dentatorubral-pallidoluysian atrophy, myotonic dystrophy 1, myotonic dystrophy 2, Huntington's disease and several spinocerebellar ataxias. Our systematic analysis of DNA sequence variation shows that most annotated SNPs are not suitable for potent allele-specific silencing across populations because of suboptimal sequence features and low variability (>97% in HD). We suggest maximising patient coverage by selecting SNPs with high heterozygosity across populations, and preferentially targeting SNPs that lead to purine:purine mismatches in wild-type alleles to obtain potent allele-specific silencing. We therefore provide fundamental knowledge on strategies for optimising patient coverage of therapeutics for microsatellite expansion disorders by linking analysis of population genetic variation to the selection of molecular targets.

Tracking motions from satellite water vapor imagery: Quantitative applications to hurricane track forecasting

NASA Technical Reports Server (NTRS)

Velden, Christopher; Nieman, Steve; Aberson, Sim; Franklin, James

1993-01-01

Water vapor imagery from GOES satellites has been available for over a decade. These data are used extensively, mainly in a qualitative mode, by forecasters in the United States (Weldon and Holmes, 1991). Some attempts have been made at quantifying the data by tracking features in time sequences of the imagery (Stewart et al., 1985; Hayden and Stewart, 1987). For a variety of reasons, applications of this approach have produced marginal results (Velden, 1990). Recently, METEOSAT-3 (M-3) was repositioned at 50W by the European Space Agency, in order to provide complete coverage of the Atlantic Ocean. Data from this satellite are being transmitted to the U.S. for operational use. Compared with the GOES satellite, the M-3 has a superior resolution and signal-to-noise ratio in its water vapor channel, which translates into improved automated tracking capabilities. During a period in 1992 which included the Atlantic hurricane season, water vapor tracking algorithms were applied to the M-3 data in order to evaluate the coverage, accuracy and model impact of the derived vectors. Data sets were produced during several tropical cyclone cases, including Hurricane Andrew. In this paper, the M-3 water vapor wind sets are assessed, and their impact on a hurricane track forecast model is examined.

Global Precipitation Measurement - Report 9 Core Coverage Trade Space Analysis

NASA Technical Reports Server (NTRS)

Mailhe, Laurie; Schiff, Conrad; Mendelsohn, Chad; Everett, David; Folta, David

2002-01-01

This paper summarizes the GPM-Core coverage trade space analysis. The goal of this analysis was to determine the GPM-Core sensitivity to changes in altitude and inclination for the three onboard instruments: the radiometer, the KU band radar and the KA band radar. This study will enable a better choice of the nominal GPM-Core orbit as well as the optimal size of the maintenance box (+/-1 km, +/-5 km..). For this work, we used two different figures-of-merit: (1) the time required to cover 100% of the +/-65 deg latitude band and (2) the coverage obtained for a given propagation time (7 days and 30 days). The first figure-of-merit is used for the radiometer as it has a sensor cone half-angle between 3 to 5 times bigger than the radars. Thus, we anticipate that for this instrument the period of the orbit (i.e. altitude) will be the main driver and that the 100% coverage value will be reached within less than a week. The second figure-of-merit is used for the radar instruments as they have small sensor cone half-angle and will, in some cases, never reach the 100% coverage threshold point.

ISS Mini AERCam Radio Frequency (RF) Coverage Analysis Using iCAT Development Tool

NASA Technical Reports Server (NTRS)

Bolen, Steve; Vazquez, Luis; Sham, Catherine; Fredrickson, Steven; Fink, Patrick; Cox, Jan; Phan, Chau; Panneton, Robert

2003-01-01

The long-term goals of the National Aeronautics and Space Administration's (NASA's) Human Exploration and Development of Space (HEDS) enterprise may require the development of autonomous free-flier (FF) robotic devices to operate within the vicinity of low-Earth orbiting spacecraft to supplement human extravehicular activities (EVAs) in space. Future missions could require external visual inspection of the spacecraft that would be difficult, or dangerous, for humans to perform. Under some circumstance, it may be necessary to employ an un-tethered communications link between the FF and the users. The interactive coverage analysis tool (ICAT) is a software tool that has been developed to perform critical analysis of the communications link performance for a FF operating in the vicinity of the International Space Station (ISS) external environment. The tool allows users to interactively change multiple parameters of the communications link parameters to efficiently perform systems engineering trades on network performance. These trades can be directly translated into design and requirements specifications. This tool significantly reduces the development time in determining a communications network topology by allowing multiple parameters to be changed, and the results of link coverage to be statistically characterized and plotted interactively.

NASA Tech Briefs, July 1998. Volume 22, No. 7

NASA Technical Reports Server (NTRS)

1998-01-01

Topics include: special coverage on graphics and simulation, electronic components and circuits, electronic systems, materials, a second special coverage on mechanical technology, machinery/automation, and a special section of electronics Tech Briefs. There is also a section devoted to the technology of the International Space Station.

Press Site Auditorium dedicated to John Holliman

NASA Technical Reports Server (NTRS)

1999-01-01

From left, Center Director Roy Bridges and NASA Administrator Daniel S. Goldin applaud as Jay Holliman, with the help of his mother, Mrs. Dianne Holliman, unveils a plaque honoring his father, the late John Holliman. At right is Tom Johnson, news group chairman of CNN. The occasion was the dedication of the KSC Press Site auditorium as the John Holliman Auditorium to honor the CNN national correspondent for his enthusiastic, dedicated coverage of America's space program. The auditorium was built in 1980 and has been the focal point for new coverage of Space Shuttle launches. The ceremony followed the 94th launch of a Space Shuttle, on mission STS-96, earlier this morning.

14 CFR 1253.200 - Application.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Application. 1253.200 Section 1253.200 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION NONDISCRIMINATION ON THE BASIS OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Coverage § 1253.200 Application...

Acceleration techniques and their impact on arterial input function sampling: Non-accelerated versus view-sharing and compressed sensing sequences.

PubMed

Benz, Matthias R; Bongartz, Georg; Froehlich, Johannes M; Winkel, David; Boll, Daniel T; Heye, Tobias

2018-07-01

The aim was to investigate the variation of the arterial input function (AIF) within and between various DCE MRI sequences. A dynamic flow-phantom and steady signal reference were scanned on a 3T MRI using fast low angle shot (FLASH) 2d, FLASH3d (parallel imaging factor (P) = P0, P2, P4), volumetric interpolated breath-hold examination (VIBE) (P = P0, P3, P2 × 2, P2 × 3, P3 × 2), golden-angle radial sparse parallel imaging (GRASP), and time-resolved imaging with stochastic trajectories (TWIST). Signal over time curves were normalized and quantitatively analyzed by full width half maximum (FWHM) measurements to assess variation within and between sequences. The coefficient of variation (CV) for the steady signal reference ranged from 0.07-0.8%. The non-accelerated gradient echo FLASH2d, FLASH3d, and VIBE sequences showed low within sequence variation with 2.1%, 1.0%, and 1.6%. The maximum FWHM CV was 3.2% for parallel imaging acceleration (VIBE P2 × 3), 2.7% for GRASP and 9.1% for TWIST. The FWHM CV between sequences ranged from 8.5-14.4% for most non-accelerated/accelerated gradient echo sequences except 6.2% for FLASH3d P0 and 0.3% for FLASH3d P2; GRASP FWHM CV was 9.9% versus 28% for TWIST. MRI acceleration techniques vary in reproducibility and quantification of the AIF. Incomplete coverage of the k-space with TWIST as a representative of view-sharing techniques showed the highest variation within sequences and might be less suited for reproducible quantification of the AIF. Copyright © 2018 Elsevier B.V. All rights reserved.

Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia

PubMed Central

Carninci, Piero; Waki, Kazunori; Shiraki, Toshiyuki; Konno, Hideaki; Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Arakawa, Takahiro; Ishii, Yoshiyuki; Sasaki, Daisuke; Bono, Hidemasa; Kondo, Shinji; Sugahara, Yuichi; Saito, Rintaro; Osato, Naoki; Fukuda, Shiro; Sato, Kenjiro; Watahiki, Akira; Hirozane-Kishikawa, Tomoko; Nakamura, Mari; Shibata, Yuko; Yasunishi, Ayako; Kikuchi, Noriko; Yoshiki, Atsushi; Kusakabe, Moriaki; Gustincich, Stefano; Beisel, Kirk; Pavan, William; Aidinis, Vassilis; Nakagawara, Akira; Held, William A.; Iwata, Hiroo; Kono, Tomohiro; Nakauchi, Hiromitsu; Lyons, Paul; Wells, Christine; Hume, David A.; Fagiolini, Michela; Hensch, Takao K.; Brinkmeier, Michelle; Camper, Sally; Hirota, Junji; Mombaerts, Peter; Muramatsu, Masami; Okazaki, Yasushi; Kawai, Jun; Hayashizaki, Yoshihide

2003-01-01

We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3′-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5′ end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5′-end clusters identify regions that are potential promoters for 8637 known genes and 5′-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete. PMID:12819125

The Genome Sequence of Taurine Cattle: A Window to Ruminant Biology and Evolution

USDA-ARS?s Scientific Manuscript database

As a major step toward understanding the biology and evolution of ruminants, the cattle genome was sequenced to ~7x coverage using a combined whole genome shotgun and BAC skim approach. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs found in seven mammalian...

Len Gen: The international lentil genome sequencing project

USDA-ARS?s Scientific Manuscript database

We have been sequencing CDC Redberry using NGS of paired-end and mate-pair libraries over a wide range of sizes and technologies. The most recent draft (v0.7) of approximately 150x coverage produced scaffolds covering over half the genome (2.7 Gb of the expected 4.3 Gb). Long reads from PacBio sequ...

Complete mitochondrial genome of Helicoverpa zea (Boddie) and expression profiles of mitochondrial-encoded genes in early and late embryos

USDA-ARS?s Scientific Manuscript database

The mitochondrial genome of the bollworm, Helicoverpa zea, was assembled using paired-end nucleotide sequence reads generated with a next-generation sequencing platform. Assembly resulted in a mitogenome of 15,348 bp with greater than 17,000-fold average coverage. Organization of the H. zea mitogen...

An integrated genetic linkage map of watermelon and genetic diversity based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers

USDA-ARS?s Scientific Manuscript database

Watermelon (Citrullus lanatus var. lanatus) is an important vegetable fruit throughout the world. A high number of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers should provide large coverage of the watermelon genome and high phylogenetic resolution of germplasm acces...

Drug release through liposome pores.

PubMed

Dan, Nily

2015-02-01

Electrical, ultrasound and other types of external fields are known to induce the formation of pores in cellular and model membranes. This paper examines drug release through field induced liposome pores using Monte Carlo simulations. We find that drug release rates vary as a function of pore size and spacing, as well as the overall fraction of surface area covered by pores: The rate of release from liposomes is found to increase rapidly with pore surface coverage, approaching that of the fully ruptured liposome at fractional pore areas. For a given pore surface coverage, the pore size affects the release rate in the limit of low coverage, but not when the pores cover a relatively high fraction of the liposome surface area. On the other hand, for a given pore size and surface coverage, the distribution of pores significantly affects the release in the limit of high surface coverage: The rate of release from a liposome covered with a regularly spaced array of pores is, in this limit, higher than the release rate from (most) systems where the pores are distributed randomly on the liposome surface. In contrast, there is little effect of the pore distribution on release when the pore surface coverage is low. The simulation results are in good agreement with the predictions of detailed diffusion models. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of active transposable elements in Arabidopsis thaliana using Oxford Nanopore Sequencing technology.

PubMed

Debladis, Emilie; Llauro, Christel; Carpentier, Marie-Christine; Mirouze, Marie; Panaud, Olivier

2017-07-17

Transposables elements (TEs) contribute to both structural and functional dynamics of most eukaryotic genomes. Because of their propensity to densely populate plant and animal genomes, the precise estimation of the impact of transposition on genomic diversity has been considered as one of the main challenges of today's genomics. The recent development of NGS (next generation sequencing) technologies has open new perspectives in population genomics by providing new methods for high throughput detection of Transposable Elements-associated Structural Variants (TEASV). However, these have relied on Illumina platform that generates short reads (up to 350 nucleotides). This limitation in size of sequence reads can cause high false discovery rate (FDR) and therefore limit the power of detection of TEASVs, especially in the case of large, complex genomes. The newest sequencing technologies, such as Oxford Nanopore Technologies (ONT) can generate kilobases-long reads thus representing a promising tool for TEASV detection in plant and animals. We present the results of a pilot experiment for TEASV detection on the model plant species Arabidopsis thaliana using ONT sequencing and show that it can be used efficiently to detect TE movements. We generated a ~0.8X genome coverage of a met1-derived epigenetic recombinant inbred line (epiRIL) using a MinIon device with R7 chemistry. We were able to detect nine new copies of the LTR-retrotransposon Evadé (EVD). We also evidenced the activity of the DNA transposon CACTA, CAC1. Even at a low sequence coverage (0.8X), ONT sequencing allowed us to reliably detect several TE insertions in Arabidopsis thaliana genome. The long read length allowed a precise and un-ambiguous mapping of the structural variations caused by the activity of TEs. This suggests that the trade-off between read length and genome coverage for TEASV detection may be in favor of the former. Should the technology be further improved both in terms of lower error rate and operation costs, it could be efficiently used in diversity studies at population level.

GapFiller: a de novo assembly approach to fill the gap within paired reads

PubMed Central

2012-01-01

Background Next Generation Sequencing technologies are able to provide high genome coverages at a relatively low cost. However, due to limited reads' length (from 30 bp up to 200 bp), specific bioinformatics problems have become even more difficult to solve. De novo assembly with short reads, for example, is more complicated at least for two reasons: first, the overall amount of "noisy" data to cope with increased and, second, as the reads' length decreases the number of unsolvable repeats grows. Our work's aim is to go at the root of the problem by providing a pre-processing tool capable to produce (in-silico) longer and highly accurate sequences from a collection of Next Generation Sequencing reads. Results In this paper a seed-and-extend local assembler is presented. The kernel algorithm is a loop that, starting from a read used as seed, keeps extending it using heuristics whose main goal is to produce a collection of error-free and longer sequences. In particular, GapFiller carefully detects reliable overlaps and operates clustering similar reads in order to reconstruct the missing part between the two ends of the same insert. Our tool's output has been validated on 24 experiments using both simulated and real paired reads datasets. The output sequences are declared correct when the seed-mate is found. In the experiments performed, GapFiller was able to extend high percentages of the processed seeds and find their mates, with a false positives rate that turned out to be nearly negligible. Conclusions GapFiller, starting from a sufficiently high short reads coverage, is able to produce high coverages of accurate longer sequences (from 300 bp up to 3500 bp). The procedure to perform safe extensions, together with the mate-found check, turned out to be a powerful criterion to guarantee contigs' correctness. GapFiller has further potential, as it could be applied in a number of different scenarios, including the post-processing validation of insertions/deletions detection pipelines, pre-processing routines on datasets for de novo assembly pipelines, or in any hierarchical approach designed to assemble, analyse or validate pools of sequences. PMID:23095524

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

Ruggles, Kelly V.; Tang, Zuojian; Wang, Xuya

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations and splice variants identified in cancer cells are translated. Herein we therefore describe a proteogenomic data integration tool (QUILTS) and illustrate its application to whole genome, transcriptome and global MS peptide sequence datasets generated from a pair of luminal and basal-like breast cancer patient derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS process replicates. Despite over thirty sample replicates, only about 10% of all SNV (somatic andmore » germline) were detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNV without a detectable mRNA transcript were also observed demonstrating the transcriptome coverage was also incomplete (~80%). In contrast to germ-line variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than the luminal tumor raising the possibility of differential translation or protein degradation effects. In conclusion, the QUILTS program integrates DNA, RNA and peptide sequencing to assess the degree to which somatic mutations are translated and therefore biologically active. By identifying gaps in sequence coverage QUILTS benchmarks current technology and assesses progress towards whole cancer proteome and transcriptome analysis.« less

Spaces of ideal convergent sequences.

PubMed

Mursaleen, M; Sharma, Sunil K

2014-01-01

In the present paper, we introduce some sequence spaces using ideal convergence and Musielak-Orlicz function ℳ = (M(k)). We also examine some topological properties of the resulting sequence spaces.

Whole-Genome Sequencing and Assembly with High-Throughput, Short-Read Technologies

PubMed Central

Sundquist, Andreas; Ronaghi, Mostafa; Tang, Haixu; Pevzner, Pavel; Batzoglou, Serafim

2007-01-01

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology. PMID:17534434

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.

A sequence of 'factishes': the media-metaphorical knowledge dynamics structuring the German press coverage of the human genome.

PubMed

Doring, Martin

2005-12-01

This article deals with the cultural framing of the near sequencing of the human genome and its impact on the media coverage in Germany. It investigates in particular the way in which the weekly journal Die Zeit and the daily newspaper Frankfurter Rundschau reported this media event and its aftermath between June 2000 and June 2001. Both newspapers are quality papers that played an essential role in framing the human genome debate--alongside the Frankfurter Allgemeine Zeitung--which became the most prominent genomic forum. The decoding of the human genome prompted a huge controversy concerning the ethics of human engineering, research on stem cells and Preimplantation Genetic Diagnosis. The main aim of this article is to show how this controversy was structured by metaphor. The media coverage of the genome generated DNA-factishes--a neologism designating the ambivalence of something as fact (fait) and as a fetish (fetiche)--that mostly propagated images of a new DNA-scienticism or biological determinism. Mediated by cultural experiences, the human genome became a highly artificial and social construct of a 'NatureCulture'.

Efficient Genome-Wide Sequencing and Low-Coverage Pedigree Analysis from Noninvasively Collected Samples

PubMed Central

Snyder-Mackler, Noah; Majoros, William H.; Yuan, Michael L.; Shaver, Amanda O.; Gordon, Jacob B.; Kopp, Gisela H.; Schlebusch, Stephen A.; Wall, Jeffrey D.; Alberts, Susan C.; Mukherjee, Sayan; Zhou, Xiang; Tung, Jenny

2016-01-01

Research on the genetics of natural populations was revolutionized in the 1990s by methods for genotyping noninvasively collected samples. However, these methods have remained largely unchanged for the past 20 years and lag far behind the genomics era. To close this gap, here we report an optimized laboratory protocol for genome-wide capture of endogenous DNA from noninvasively collected samples, coupled with a novel computational approach to reconstruct pedigree links from the resulting low-coverage data. We validated both methods using fecal samples from 62 wild baboons, including 48 from an independently constructed extended pedigree. We enriched fecal-derived DNA samples up to 40-fold for endogenous baboon DNA and reconstructed near-perfect pedigree relationships even with extremely low-coverage sequencing. We anticipate that these methods will be broadly applicable to the many research systems for which only noninvasive samples are available. The lab protocol and software (“WHODAD”) are freely available at www.tung-lab.org/protocols-and-software.html and www.xzlab.org/software.html, respectively. PMID:27098910

Shifted Transversal Design smart-pooling for high coverage interactome mapping

PubMed Central

Xin, Xiaofeng; Rual, Jean-François; Hirozane-Kishikawa, Tomoko; Hill, David E.; Vidal, Marc; Boone, Charles; Thierry-Mieg, Nicolas

2009-01-01

“Smart-pooling,” in which test reagents are multiplexed in a highly redundant manner, is a promising strategy for achieving high efficiency, sensitivity, and specificity in systems-level projects. However, previous applications relied on low redundancy designs that do not leverage the full potential of smart-pooling, and more powerful theoretical constructions, such as the Shifted Transversal Design (STD), lack experimental validation. Here we evaluate STD smart-pooling in yeast two-hybrid (Y2H) interactome mapping. We employed two STD designs and two established methods to perform ORFeome-wide Y2H screens with 12 baits. We found that STD pooling achieves similar levels of sensitivity and specificity as one-on-one array-based Y2H, while the costs and workloads are divided by three. The screening-sequencing approach is the most cost- and labor-efficient, yet STD identifies about twofold more interactions. Screening-sequencing remains an appropriate method for quickly producing low-coverage interactomes, while STD pooling appears as the method of choice for obtaining maps with higher coverage. PMID:19447967

Genome analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of the etiologic agent of tuberculosis

PubMed Central

Supply, Philip; Marceau, Michael; Mangenot, Sophie; Roche, David; Rouanet, Carine; Khanna, Varun; Majlessi, Laleh; Criscuolo, Alexis; Tap, Julien; Pawlik, Alexandre; Fiette, Laurence; Orgeur, Mickael; Fabre, Michel; Parmentier, Cécile; Frigui, Wafa; Simeone, Roxane; Boritsch, Eva C.; Debrie, Anne-Sophie; Willery, Eve; Walker, Danielle; Quail, Michael A.; Ma, Laurence; Bouchier, Christiane; Salvignol, Grégory; Sayes, Fadel; Cascioferro, Alessandro; Seemann, Torsten; Barbe, Valérie; Locht, Camille; Gutierrez, Maria-Cristina; Leclerc, Claude; Bentley, Stephen; Stinear, Timothy P.; Brisse, Sylvain; Médigue, Claudine; Parkhill, Julian; Cruveiller, Stéphane; Brosch, Roland

2013-01-01

Global spread and genetic monomorphism are hallmarks of Mycobacterium tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii, and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology, are restricted to East-Africa. Here, we sequenced and analyzed the genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5x coverage), 454/Roche (13-18x coverage) and/or Illumina DNA sequencing (45-105x coverage). We show that STB are highly recombinogenic and evolutionary early-branching, with larger genome sizes, 25-fold more SNPs, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse-infection experiments revealed that STB are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral, STB-like pool of mycobacteria by gain of persistence and virulence mechanisms and we provide genome-wide insights into the molecular events involved. PMID:23291586

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

A user's guide to quantitative and comparative analysis of metagenomic datasets.

PubMed

Luo, Chengwei; Rodriguez-R, Luis M; Konstantinidis, Konstantinos T

2013-01-01

Metagenomics has revolutionized microbiological studies during the past decade and provided new insights into the diversity, dynamics, and metabolic potential of natural microbial communities. However, metagenomics still represents a field in development, and standardized tools and approaches to handle and compare metagenomes have not been established yet. An important reason accounting for the latter is the continuous changes in the type of sequencing data available, for example, long versus short sequencing reads. Here, we provide a guide to bioinformatic pipelines developed to accomplish the following tasks, focusing primarily on those developed by our team: (i) assemble a metagenomic dataset; (ii) determine the level of sequence coverage obtained and the amount of sequencing required to obtain complete coverage; (iii) identify the taxonomic affiliation of a metagenomic read or assembled contig; and (iv) determine differentially abundant genes, pathways, and species between different datasets. Most of these pipelines do not depend on the type of sequences available or can be easily adjusted to fit different types of sequences, and are freely available (for instance, through our lab Web site: http://www.enve-omics.gatech.edu/). The limitations of current approaches, as well as the computational aspects that can be further improved, will also be briefly discussed. The work presented here provides practical guidelines on how to perform metagenomic analysis of microbial communities characterized by varied levels of diversity and establishes approaches to handle the resulting data, independent of the sequencing platform employed. © 2013 Elsevier Inc. All rights reserved.

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

PubMed Central

Li, Linlin; Deng, Xutao; Mee, Edward T.; Collot-Teixeira, Sophie; Anderson, Rob; Schepelmann, Silke; Minor, Philip D.; Delwart, Eric

2014-01-01

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries on the efficiency of viral detection and virus genome coverage were compared. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces. PMID:25497414

Acoustic-noise-optimized diffusion-weighted imaging.

PubMed

Ott, Martin; Blaimer, Martin; Grodzki, David M; Breuer, Felix A; Roesch, Julie; Dörfler, Arnd; Heismann, Björn; Jakob, Peter M

2015-12-01

This work was aimed at reducing acoustic noise in diffusion-weighted MR imaging (DWI) that might reach acoustic noise levels of over 100 dB(A) in clinical practice. A diffusion-weighted readout-segmented echo-planar imaging (EPI) sequence was optimized for acoustic noise by utilizing small readout segment widths to obtain low gradient slew rates and amplitudes instead of faster k-space coverage. In addition, all other gradients were optimized for low slew rates. Volunteer and patient imaging experiments were conducted to demonstrate the feasibility of the method. Acoustic noise measurements were performed and analyzed for four different DWI measurement protocols at 1.5T and 3T. An acoustic noise reduction of up to 20 dB(A) was achieved, which corresponds to a fourfold reduction in acoustic perception. The image quality was preserved at the level of a standard single-shot (ss)-EPI sequence, with a 27-54% increase in scan time. The diffusion-weighted imaging technique proposed in this study allowed a substantial reduction in the level of acoustic noise compared to standard single-shot diffusion-weighted EPI. This is expected to afford considerably more patient comfort, but a larger study would be necessary to fully characterize the subjective changes in patient experience.

Sensitivity of the orbiting JEM-EUSO mission to large-scale anisotropies

NASA Astrophysics Data System (ADS)

Weiler, Thomas; Anchordoqui, Luis; Denton, Peter

2013-04-01

Uniform sky coverage and very large apertures are advantages of future extreme-energy, space-based cosmic-ray observatories. In this talk we will quantify the advantage of an all-sky/4pi observatory such as JEM-EUSO over the one to two steradian coverage of a ground-based observatory such as Auger. We exploit the availability of spherical harmonics in the case of 4pi coverage. The resulting Y(lm) coefficients will likely become a standard analysis tool for near-future, space-based, cosmic-ray astronomy. We demonstrate the use of Y(lm)'s with extractions of simulated dipole and quadrupole anisotropies. (A dipole anisotropy is expected if a single source-region such as Cen A dominates the sky, while a quadrupole moment is expected if a 2D source region such as the Supergalactic Plane dominates the sky.)

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.

Quick, sensitive and specific detection and evaluation of quantification of minor variants by high-throughput sequencing.

PubMed

Leung, Ross Ka-Kit; Dong, Zhi Qiang; Sa, Fei; Chong, Cheong Meng; Lei, Si Wan; Tsui, Stephen Kwok-Wing; Lee, Simon Ming-Yuen

2014-02-01

Minor variants have significant implications in quasispecies evolution, early cancer detection and non-invasive fetal genotyping but their accurate detection by next-generation sequencing (NGS) is hampered by sequencing errors. We generated sequencing data from mixtures at predetermined ratios in order to provide insight into sequencing errors and variations that can arise for which simulation cannot be performed. The information also enables better parameterization in depth of coverage, read quality and heterogeneity, library preparation techniques, technical repeatability for mathematical modeling, theory development and simulation experimental design. We devised minor variant authentication rules that achieved 100% accuracy in both testing and validation experiments. The rules are free from tedious inspection of alignment accuracy, sequencing read quality or errors introduced by homopolymers. The authentication processes only require minor variants to: (1) have minimum depth of coverage larger than 30; (2) be reported by (a) four or more variant callers, or (b) DiBayes or LoFreq, plus SNVer (or BWA when no results are returned by SNVer), and with the interassay coefficient of variation (CV) no larger than 0.1. Quantification accuracy undermined by sequencing errors could neither be overcome by ultra-deep sequencing, nor recruiting more variant callers to reach a consensus, such that consistent underestimation and overestimation (i.e. low CV) were observed. To accommodate stochastic error and adjust the observed ratio within a specified accuracy, we presented a proof of concept for the use of a double calibration curve for quantification, which provides an important reference towards potential industrial-scale fabrication of calibrants for NGS.

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.

Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample.

PubMed

Luo, Chengwei; Tsementzi, Despina; Kyrpides, Nikos; Read, Timothy; Konstantinidis, Konstantinos T

2012-01-01

Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.

Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait.

PubMed

Madi, Nada; Al-Nakib, Widad; Mustafa, Abu Salim; Habibi, Nazima

2018-03-01

A metagenomic approach based on target independent next-generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next-generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non-respiratory viruses were detected in 14% of the clinical samples, while sequences of non-human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real-Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections. © 2017 Wiley Periodicals, Inc.

Systematic Evaluation of the Dependence of Deoxyribozyme Catalysis on Random Region Length

PubMed Central

Velez, Tania E.; Singh, Jaydeep; Xiao, Ying; Allen, Emily C.; Wong, On Yi; Chandra, Madhavaiah; Kwon, Sarah C.; Silverman, Scott K.

2012-01-01

Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N20 through N60) for two particular deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20, N30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable. PMID:23088677

LightAssembler: fast and memory-efficient assembly algorithm for high-throughput sequencing reads.

PubMed

El-Metwally, Sara; Zakaria, Magdi; Hamza, Taher

2016-11-01

The deluge of current sequenced data has exceeded Moore's Law, more than doubling every 2 years since the next-generation sequencing (NGS) technologies were invented. Accordingly, we will able to generate more and more data with high speed at fixed cost, but lack the computational resources to store, process and analyze it. With error prone high throughput NGS reads and genomic repeats, the assembly graph contains massive amount of redundant nodes and branching edges. Most assembly pipelines require this large graph to reside in memory to start their workflows, which is intractable for mammalian genomes. Resource-efficient genome assemblers combine both the power of advanced computing techniques and innovative data structures to encode the assembly graph efficiently in a computer memory. LightAssembler is a lightweight assembly algorithm designed to be executed on a desktop machine. It uses a pair of cache oblivious Bloom filters, one holding a uniform sample of [Formula: see text]-spaced sequenced [Formula: see text]-mers and the other holding [Formula: see text]-mers classified as likely correct, using a simple statistical test. LightAssembler contains a light implementation of the graph traversal and simplification modules that achieves comparable assembly accuracy and contiguity to other competing tools. Our method reduces the memory usage by [Formula: see text] compared to the resource-efficient assemblers using benchmark datasets from GAGE and Assemblathon projects. While LightAssembler can be considered as a gap-based sequence assembler, different gap sizes result in an almost constant assembly size and genome coverage. https://github.com/SaraEl-Metwally/LightAssembler CONTACT: sarah_almetwally4@mans.edu.egSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Iterative Correction of Reference Nucleotides (iCORN) using second generation sequencing technology.

PubMed

Otto, Thomas D; Sanders, Mandy; Berriman, Matthew; Newbold, Chris

2010-07-15

The accuracy of reference genomes is important for downstream analysis but a low error rate requires expensive manual interrogation of the sequence. Here, we describe a novel algorithm (Iterative Correction of Reference Nucleotides) that iteratively aligns deep coverage of short sequencing reads to correct errors in reference genome sequences and evaluate their accuracy. Using Plasmodium falciparum (81% A + T content) as an extreme example, we show that the algorithm is highly accurate and corrects over 2000 errors in the reference sequence. We give examples of its application to numerous other eukaryotic and prokaryotic genomes and suggest additional applications. The software is available at http://icorn.sourceforge.net

Photographic coverage of STS-108 Philippe Perrin

NASA Image and Video Library

2002-02-20

JSC2002-E-08241 (20 February 2002) --- Astronaut Philippe Perrin, STS-111 mission specialist representing CNES, the French Space Agency, is photographed during an Extravehicular Mobility Unit (EMU) fit check in a Space Station Airlock Test Article (SSATA) in the Crew Systems Laboratory at the Johnson Space Center (JSC).

Photographic coverage of STS-108 Philippe Perrin

NASA Image and Video Library

2002-02-20

JSC2002-E-08243 (20 February 2002) --- Astronaut Philippe Perrin, STS-111 mission specialist representing CNES, the French Space Agency, is photographed during an Extravehicular Mobility Unit (EMU) fit check in a Space Station Airlock Test Article (SSATA) in the Crew Systems Laboratory at the Johnson Space Center (JSC).

First-pass myocardial perfusion imaging with whole-heart coverage using L1-SPIRiT accelerated variable density spiral trajectories.

PubMed

Yang, Yang; Kramer, Christopher M; Shaw, Peter W; Meyer, Craig H; Salerno, Michael

2016-11-01

To design and evaluate two-dimensional (2D) L1-SPIRiT accelerated spiral pulse sequences for first-pass myocardial perfusion imaging with whole heart coverage capable of measuring eight slices at 2 mm in-plane resolution at heart rates up to 125 beats per minute (BPM). Combinations of five different spiral trajectories and four k-t sampling patterns were retrospectively simulated in 25 fully sampled datasets and reconstructed with L1-SPIRiT to determine the best combination of parameters. Two candidate sequences were prospectively evaluated in 34 human subjects to assess in vivo performance. A dual density broad transition spiral trajectory with either angularly uniform or golden angle in time k-t sampling pattern had the largest structural similarity and smallest root mean square error from the retrospective simulation, and the L1-SPIRiT reconstruction had well-preserved temporal dynamics. In vivo data demonstrated that both of the sampling patterns could produce high quality perfusion images with whole-heart coverage. First-pass myocardial perfusion imaging using accelerated spirals with optimized trajectory and k-t sampling pattern can produce high quality 2D perfusion images with whole-heart coverage at the heart rates up to 125 BPM. Magn Reson Med 76:1375-1387, 2016. © 2015 International Society for Magnetic Resonance in Medicine. © 2015 International Society for Magnetic Resonance in Medicine.

Analysis of delay reducing and fuel saving sequencing and spacing algorithms for arrival traffic

NASA Technical Reports Server (NTRS)

Neuman, Frank; Erzberger, Heinz

1991-01-01

The air traffic control subsystem that performs sequencing and spacing is discussed. The function of the sequencing and spacing algorithms is to automatically plan the most efficient landing order and to assign optimally spaced landing times to all arrivals. Several algorithms are described and their statistical performance is examined. Sequencing brings order to an arrival sequence for aircraft. First-come-first-served sequencing (FCFS) establishes a fair order, based on estimated times of arrival, and determines proper separations. Because of the randomness of the arriving traffic, gaps will remain in the sequence of aircraft. Delays are reduced by time-advancing the leading aircraft of each group while still preserving the FCFS order. Tightly spaced groups of aircraft remain with a mix of heavy and large aircraft. Spacing requirements differ for different types of aircraft trailing each other. Traffic is reordered slightly to take advantage of this spacing criterion, thus shortening the groups and reducing average delays. For heavy traffic, delays for different traffic samples vary widely, even when the same set of statistical parameters is used to produce each sample. This report supersedes NASA TM-102795 on the same subject. It includes a new method of time-advance as well as an efficient method of sequencing and spacing for two dependent runways.

Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

PubMed

Oyola, Samuel O; Otto, Thomas D; Gu, Yong; Maslen, Gareth; Manske, Magnus; Campino, Susana; Turner, Daniel J; Macinnis, Bronwyn; Kwiatkowski, Dominic P; Swerdlow, Harold P; Quail, Michael A

2012-01-03

Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

Identification and correction of systematic error in high-throughput sequence data

PubMed Central

2011-01-01

Background A feature common to all DNA sequencing technologies is the presence of base-call errors in the sequenced reads. The implications of such errors are application specific, ranging from minor informatics nuisances to major problems affecting biological inferences. Recently developed "next-gen" sequencing technologies have greatly reduced the cost of sequencing, but have been shown to be more error prone than previous technologies. Both position specific (depending on the location in the read) and sequence specific (depending on the sequence in the read) errors have been identified in Illumina and Life Technology sequencing platforms. We describe a new type of systematic error that manifests as statistically unlikely accumulations of errors at specific genome (or transcriptome) locations. Results We characterize and describe systematic errors using overlapping paired reads from high-coverage data. We show that such errors occur in approximately 1 in 1000 base pairs, and that they are highly replicable across experiments. We identify motifs that are frequent at systematic error sites, and describe a classifier that distinguishes heterozygous sites from systematic error. Our classifier is designed to accommodate data from experiments in which the allele frequencies at heterozygous sites are not necessarily 0.5 (such as in the case of RNA-Seq), and can be used with single-end datasets. Conclusions Systematic errors can easily be mistaken for heterozygous sites in individuals, or for SNPs in population analyses. Systematic errors are particularly problematic in low coverage experiments, or in estimates of allele-specific expression from RNA-Seq data. Our characterization of systematic error has allowed us to develop a program, called SysCall, for identifying and correcting such errors. We conclude that correction of systematic errors is important to consider in the design and interpretation of high-throughput sequencing experiments. PMID:22099972

Space technology and the optical sciences.

PubMed

Yates, H W

1982-01-15

The earth-orbiting satellites and the deep-space probes have provided for the optical sciences platforms from which to study the earth, the solar system, and the universe with truly revolutionary capability. For the terrestrial sciences the orbiting platforms for optical measurements in both low and geostationary orbits have given us a view of our planet and a global coverage never before possible. For the astronomical applications of optical instruments that "cataract of the telescopic eye," the atmosphere of the earth has been left behind and through proximity, including actual contact, we now have resolution and spectral coverage limited only by money and motive.

Ranking viruses: measures of positional importance within networks define core viruses for rational polyvalent vaccine development.

PubMed

Anderson, Tavis K; Laegreid, William W; Cerutti, Francesco; Osorio, Fernando A; Nelson, Eric A; Christopher-Hennings, Jane; Goldberg, Tony L

2012-06-15

The extraordinary genetic and antigenic variability of RNA viruses is arguably the greatest challenge to the development of broadly effective vaccines. No single viral variant can induce sufficiently broad immunity, and incorporating all known naturally circulating variants into one multivalent vaccine is not feasible. Furthermore, no objective strategies currently exist to select actual viral variants that should be included or excluded in polyvalent vaccines. To address this problem, we demonstrate a method based on graph theory that quantifies the relative importance of viral variants. We demonstrate our method through application to the envelope glycoprotein gene of a particularly diverse RNA virus of pigs: porcine reproductive and respiratory syndrome virus (PRRSV). Using distance matrices derived from sequence nucleotide difference, amino acid difference and evolutionary distance, we constructed viral networks and used common network statistics to assign each sequence an objective ranking of relative 'importance'. To validate our approach, we use an independent published algorithm to score our top-ranked wild-type variants for coverage of putative T-cell epitopes across the 9383 sequences in our dataset. Top-ranked viruses achieve significantly higher coverage than low-ranked viruses, and top-ranked viruses achieve nearly equal coverage as a synthetic mosaic protein constructed in silico from the same set of 9383 sequences. Our approach relies on the network structure of PRRSV but applies to any diverse RNA virus because it identifies subsets of viral variants that are most important to overall viral diversity. We suggest that this method, through the objective quantification of variant importance, provides criteria for choosing viral variants for further characterization, diagnostics, surveillance and ultimately polyvalent vaccine development.

Determining the specific microbial populations and their spatial distribution within the stromatolite ecosystem of Shark Bay.

PubMed

Goh, Falicia; Allen, Michelle A; Leuko, Stefan; Kawaguchi, Tomohiro; Decho, Alan W; Burns, Brendan P; Neilan, Brett A

2009-04-01

The stromatolites at Shark Bay, Western Australia, are analogues of some of the oldest evidence of life on Earth. The aim of this study was to identify and spatially characterize the specific microbial communities associated with Shark Bay intertidal columnar stromatolites. Conventional culturing methods and construction of 16S rDNA clone libraries from community genomic DNA with both universal and specific PCR primers were employed. The estimated coverage, richness and diversity of stromatolite microbial populations were compared with earlier studies on these ecosystems. The estimated coverage for all clone libraries indicated that population coverage was comprehensive. Phylogenetic analyses of stromatolite and surrounding seawater sequences were performed in ARB with the Greengenes database of full-length non-chimaeric 16S rRNA genes. The communities identified exhibited extensive diversity. The most abundant sequences from the stromatolites were alpha- and gamma-proteobacteria (58%), whereas the cyanobacterial community was characterized by sequences related to the genera Euhalothece, Gloeocapsa, Gloeothece, Chroococcidiopsis, Dermocarpella, Acaryochloris, Geitlerinema and Schizothrix. All clones from the archaeal-specific clone libraries were related to the halophilic archaea; however, no archaeal sequence was identified from the surrounding seawater. Fluorescence in situ hybridization also revealed stromatolite surfaces to be dominated by unicellular cyanobacteria, in contrast to the sub-surface archaea and sulphate-reducing bacteria. This study is the first to compare the microbial composition of morphologically similar stromatolites over time and examine the spatial distribution of specific microorganismic groups in these intertidal structures and the surrounding seawater at Shark Bay. The results provide a platform for identifying the key microbial physiology groups and their potential roles in modern stromatolite morphogenesis and ecology.

Non-invasive prenatal testing for fetal chromosomal abnormalities by low-coverage whole-genome sequencing of maternal plasma DNA: review of 1982 consecutive cases in a single center.

PubMed

Lau, T K; Cheung, S W; Lo, P S S; Pursley, A N; Chan, M K; Jiang, F; Zhang, H; Wang, W; Jong, L F J; Yuen, O K C; Chan, H Y C; Chan, W S K; Choy, K W

2014-03-01

To review the performance of non-invasive prenatal testing (NIPT) by low-coverage whole-genome sequencing of maternal plasma DNA at a single center. The NIPT result and pregnancy outcome of 1982 consecutive cases were reviewed. NIPT was based on low coverage (0.1×) whole-genome sequencing of maternal plasma DNA. All subjects were contacted for pregnancy and fetal outcome. Of the 1982 NIPT tests, a repeat blood sample was required in 23 (1.16%). In one case, a conclusive report could not be issued, probably because of an abnormal vanished twin fetus. NIPT was positive for common trisomies in 29 cases (23 were trisomy 21, four were trisomy 18 and two were trisomy 13); all were confirmed by prenatal karyotyping (specificity=100%). In addition, 11 cases were positive for sex-chromosomal abnormalities (SCA), and nine cases were positive for other aneuploidies or deletion/duplication. Fourteen of these 20 subjects agreed to undergo further investigations, and the abnormality was found to be of fetal origin in seven, confined placental mosaicism (CPM) in four, of maternal origin in two and not confirmed in one. Overall, 85.7% of the NIPT-suspected SCA were of fetal origin, and 66.7% of the other abnormalities were caused by CPM. Two of the six cases suspected or confirmed to have CPM were complicated by early-onset growth restriction requiring delivery before 34 weeks. Fetal outcome of the NIPT-negative cases was ascertained in 1645 (85.15%). Three chromosomal abnormalities were not detected by NIPT, including one case each of a balanced translocation, unbalanced translocation and triploidy. There were no known false negatives involving the common trisomies (sensitivity=100%). Low-coverage whole-genome sequencing of maternal plasma DNA was highly accurate in detecting common trisomies. It also enabled the detection of other aneuploidies and structural chromosomal abnormalities with high positive predictive value. Copyright © 2013 ISUOG. Published by John Wiley & Sons Ltd.

Lightweight Shield Against Space Debris

NASA Technical Reports Server (NTRS)

Redmon, John W., Jr.; Lawson, Bobby E.; Miller, Andre E.; Cobb, W. E.

1992-01-01

Report presents concept for lightweight, deployable shield protecting orbiting spacecraft against meteoroids and debris, and functions as barrier to conductive and radiative losses of heat. Shield made in four segments providing 360 degree coverage of cylindrical space-station module.

Biocrusts role on nitrogen cycle and microbial communities from underlying soils in drylands

NASA Astrophysics Data System (ADS)

Anguita-Maeso, Manuel; Miralles*, Isabel; van Wesemael, Bas; Lázaro, Roberto; Ortega, Raúl; Garcia-Salcedo, José Antonio; Soriano**, Miguel

2017-04-01

Biocrusts are distributed in arid areas widely covering most of the soil surface and playing an essential role in the functioning of nitrogen cycle. The absence of biocrust coverage might affect the soil nitrogen content and the quantity and diversity of microbial communities in underlying biocrust soils. To analyse this mater, we have collected three underlying soils biocrusts samples dominated by the lichen Diploschistes diacapsis and Squamarina lentigera from Tabernas desert (southeast of Spain) at two extremes of its spatial distribution range: one with a high percentage of biocrust coverage and other with a huge degradation and low percentage of biocrust coverage in order to determine differences on the total nitrogen content and microbial communities from these underlying soils. DNA from these samples was isolated though a commercial kit and it was used as template for metagenomic analysis. We accomplished a sequencing of the amplicons V4-V5 of the 16S rRNA gene with Next-Generation Sequencing (NGS) Illumina MiSeq platform and a relative quantity of bacteria (rRNA 16S) and fungi (ITS1-5.8S) were conducted by quantitative qPCR. Total nitrogen was measured by the Kjeldahl method. Statistical analyses were based on ANOVAs, heatmap and Generalized Linear Models (GLM). The results showed 1.89E+09 bacteria per gram of soil in the high biocrust coverage position while 6.98E+08 microorganisms per gram of soil were found in the less favourable position according to the lower percentage of biocrust coverage. Similarly, 1.19E+12 was the amount of fungi per gram of soil located in the favourable position with higher biocrust coverage and 7.62E+11 was found in the unfavourable position. Furthermore, the soil under high percentage of biocrust coverage showed the greatest total nitrogen content (1.1 g kg-1) whereas the soil sampled under depressed percentage of biocrust coverage displayed the fewest quantity of total nitrogen content (0.9 g kg-1). Metagenomic and statistical analysis exhibited different bacteria communities according to underlying soils with unlike percentage of biocrust coverage. Opitutus and Adhaeribacter predominated in soil under high biocrust coverage percentage whereas Chelatococcus was found as prevalent bacteria community in soils under low biocrust coverage percentage. Our data illustrate that the percentage of biocrust coverage influence the total nitrogen content in underlying biocrust soils and also affects the amount and the variety of bacteria communities in these underlying soils. (*) Financial support by Marie Curie Intra-European Fellowship (FP7-577 PEOPLE-2013-IEF, Proposal n° 623393) and (**) by the Ministerio de Economía y Competitividad (MINECO) cofinanced with FEDER funds (project CGL2015-71709-R) is acknowledged.

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

PubMed

Ramos, Enrique; Levinson, Benjamin T; Chasnoff, Sara; Hughes, Andrew; Young, Andrew L; Thornton, Katherine; Li, Allie; Vallania, Francesco L M; Province, Michael; Druley, Todd E

2012-12-06

Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

Novel regenerative large-volume immobilized enzyme reactor: preparation, characterization and application.

PubMed

Ruan, Guihua; Wei, Meiping; Chen, Zhengyi; Su, Rihui; Du, Fuyou; Zheng, Yanjie

2014-09-15

A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared with organic-inorganic hybrid silica particles and applied for fast (10 min) and oriented digestion of protein. At first, a thin enzyme support layer was formed in the bottom of the small column by polymerization with α-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol-gel method with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 particles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability of large-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c (Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) and Cyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at 101-105, 156-160, 205-209, 212-218, 229-232, 257-263 and 473-451 of the amino sequence of BSA when digesting 1mg/mL BSA. Eight of common peptides were observed during each of the ten runs of large-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the -C=N- bond by 0.01 M HCl. The sequence coverage of BSA from regenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides. accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When the IMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo, ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective digestion property of the large-volume IMER indicated that the regenerative IMER could be tentatively used for the production of potential bioactive peptides and the study of oriented protein digestion. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid molecular diagnostics of severe primary immunodeficiency determined by using targeted next-generation sequencing.

PubMed

Yu, Hui; Zhang, Victor Wei; Stray-Pedersen, Asbjørg; Hanson, Imelda Celine; Forbes, Lisa R; de la Morena, M Teresa; Chinn, Ivan K; Gorman, Elizabeth; Mendelsohn, Nancy J; Pozos, Tamara; Wiszniewski, Wojciech; Nicholas, Sarah K; Yates, Anne B; Moore, Lindsey E; Berge, Knut Erik; Sorte, Hanne; Bayer, Diana K; ALZahrani, Daifulah; Geha, Raif S; Feng, Yanming; Wang, Guoli; Orange, Jordan S; Lupski, James R; Wang, Jing; Wong, Lee-Jun

2016-10-01

Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of T cells, B cells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management. We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting. The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions. Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7. High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

MeCorS: Metagenome-enabled error correction of single cell sequencing reads

DOE PAGES

Bremges, Andreas; Singer, Esther; Woyke, Tanja; ...

2016-03-15

Here we present a new tool, MeCorS, to correct chimeric reads and sequencing errors in Illumina data generated from single amplified genomes (SAGs). It uses sequence information derived from accompanying metagenome sequencing to accurately correct errors in SAG reads, even from ultra-low coverage regions. In evaluations on real data, we show that MeCorS outperforms BayesHammer, the most widely used state-of-the-art approach. MeCorS performs particularly well in correcting chimeric reads, which greatly improves both accuracy and contiguity of de novo SAG assemblies.

A space-efficient algorithm for local similarities.

PubMed

Huang, X Q; Hardison, R C; Miller, W

1990-10-01

Existing dynamic-programming algorithms for identifying similar regions of two sequences require time and space proportional to the product of the sequence lengths. Often this space requirement is more limiting than the time requirement. We describe a dynamic-programming local-similarity algorithm that needs only space proportional to the sum of the sequence lengths. The method can also find repeats within a single long sequence. To illustrate the algorithm's potential, we discuss comparison of a 73,360 nucleotide sequence containing the human beta-like globin gene cluster and a corresponding 44,594 nucleotide sequence for rabbit, a problem well beyond the capabilities of other dynamic-programming software.

Watchdog activity monitor (WAM) for use wth high coverage processor self-test

NASA Technical Reports Server (NTRS)

Tulpule, Bhalchandra R. (Inventor); Crosset, III, Richard W. (Inventor); Versailles, Richard E. (Inventor)

1988-01-01

A high fault coverage, instruction modeled self-test for a signal processor in a user environment is disclosed. The self-test executes a sequence of sub-tests and issues a state transition signal upon the execution of each sub-test. The self-test may be combined with a watchdog activity monitor (WAM) which provides a test-failure signal in the presence of a counted number of state transitions not agreeing with an expected number. An independent measure of time may be provided in the WAM to increase fault coverage by checking the processor's clock. Additionally, redundant processor systems are protected from inadvertent unsevering of a severed processor using a unique unsever arming technique and apparatus.

Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

PubMed

Desai, Aarti; Marwah, Veer Singh; Yadav, Akshay; Jha, Vineet; Dhaygude, Kishor; Bangar, Ujwala; Kulkarni, Vivek; Jere, Abhay

2013-01-01

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

Advanced Optical Technologies in NASA's Space Communication Program: Status, Challenges, and Future Plans

NASA Technical Reports Server (NTRS)

Pouch, John

2004-01-01

A goal of the NASA Space Communications Project is to enable broad coverage for high-data-rate delivery to the users by means of ground, air, and space-based assets. The NASA Enterprise need will be reviewed. A number of optical space communications technologies being developed by NASA will be described, and the prospective applications will be discussed.

Radiation Environment Modeling for Spacecraft Design: New Model Developments

NASA Technical Reports Server (NTRS)

Barth, Janet; Xapsos, Mike; Lauenstein, Jean-Marie; Ladbury, Ray

2006-01-01

A viewgraph presentation on various new space radiation environment models for spacecraft design is described. The topics include: 1) The Space Radiatio Environment; 2) Effects of Space Environments on Systems; 3) Space Radiatio Environment Model Use During Space Mission Development and Operations; 4) Space Radiation Hazards for Humans; 5) "Standard" Space Radiation Environment Models; 6) Concerns about Standard Models; 7) Inadequacies of Current Models; 8) Development of New Models; 9) New Model Developments: Proton Belt Models; 10) Coverage of New Proton Models; 11) Comparison of TPM-1, PSB97, AP-8; 12) New Model Developments: Electron Belt Models; 13) Coverage of New Electron Models; 14) Comparison of "Worst Case" POLE, CRESELE, and FLUMIC Models with the AE-8 Model; 15) New Model Developments: Galactic Cosmic Ray Model; 16) Comparison of NASA, MSU, CIT Models with ACE Instrument Data; 17) New Model Developmemts: Solar Proton Model; 18) Comparison of ESP, JPL91, KIng/Stassinopoulos, and PSYCHIC Models; 19) New Model Developments: Solar Heavy Ion Model; 20) Comparison of CREME96 to CREDO Measurements During 2000 and 2002; 21) PSYCHIC Heavy ion Model; 22) Model Standardization; 23) Working Group Meeting on New Standard Radiation Belt and Space Plasma Models; and 24) Summary.

Launch mission summary: INTELSAT 5 (F4) ATLAS/CENTAUR-58

NASA Technical Reports Server (NTRS)

1982-01-01

The launch vehicle, spacecraft, and mission are described. Information relative to launch windows, flight plan, trajectory, and radar and telemetry coverage are included with brief sequence of flight events.

Depicting the semicircular canals with inner-ear MRI: a comparison of the SPACE and TrueFISP sequences.

PubMed

Kojima, Shinya; Suzuki, Kazufumi; Hirata, Masami; Shinohara, Hiroyuki; Ueno, Eiko

2013-03-01

To assess the ability of magnetic resonance imaging (MRI) to depict the semicircular canals of the inner ear by comparing results from the sampling perfection with application-optimized contrasts by using different flip angle evolutions (SPACE) sequence with those from the true free induction with steady precession (TrueFISP) sequence. A 1.5-T MRI system was used to perform an in vivo study of 10 healthy volunteers and 17 patients. A three-point visual score was employed for assessing the depiction of the semicircular canals and facial and vestibulocochlear nerves and the contrast-to-noise ratio (CNR) was computed for the vestibule and pons on images with the SPACE and TrueFIPS sequences. There were no susceptibility artifact-related filling defects with the SPACE sequence. However, the TrueFISP sequence showed filling defects for at least one semicircular canal on both sides in seven cases for healthy subjects and in 10 cases for patients. The CNR with the SPACE sequence was significantly higher than with the TrueFISP sequence (P < 0.05). There was no statistically significant difference in depicting the facial and the vestibulocochlear nerves (P = 0.32). For the depiction of the semicircular canal, the SPACE sequence is superior to the TrueFISP sequence. Copyright © 2012 Wiley Periodicals, Inc.

Single molecule sequencing of the M13 virus genome without amplification

PubMed Central

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X.; Yan, Qin; Deem, Michael W.; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias. PMID:29253901

Single molecule sequencing of the M13 virus genome without amplification.

PubMed

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X; Yan, Qin; Deem, Michael W; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

Fault model of the 2014 Cephalonia seismic sequence - Evidence of spatiotemporal fault segmentation along the NW edge of Aegean Arc

NASA Astrophysics Data System (ADS)

Saltogianni, Vasso; Moschas, Fanis; Stiros, Stathis

2017-04-01

Finite fault models (FFM) are presented for the two main shocks of the 2014 Cephalonia (Ionian Sea, Greece) seismic sequence (M 6.0) which produced extreme peak ground accelerations ( 0.7g) in the west edge of the Aegean Arc, an area in which the poor coverage by seismological and GPS/INSAR data makes FFM a real challenge. Modeling was based on co-seismic GPS data and on the recently introduced TOPological INVersion algorithm. The latter is a novel uniform grid search-based technique in n-dimensional spaces, is based on the concept of stochastic variables and which can identify multiple unconstrained ("free") solutions in a specified search space. Derived FFMs for the 2014 earthquakes correspond to an essentially strike slip fault and of part of a shallow thrust, the surface projection of both of which run roughly along the west coast of Cephalonia. Both faults correlate with pre-existing faults. The 2014 faults, in combination with the faults of the 2003 and 2015 Leucas earthquakes farther NE, form a string of oblique slip, partly overlapping fault segments with variable geometric and kinematic characteristics along the NW edge of the Aegean Arc. This composite fault, usually regarded as the Cephalonia Transform Fault, accommodates shear along this part of the Arc. Because of the highly fragmented crust, dominated by major thrusts in this area, fault activity is associated with 20km long segments and magnitude 6.0-6.5 earthquakes recurring in intervals of a few seconds to 10 years.

A DETAILED FAR-ULTRAVIOLET SPECTRAL ATLAS OF MAIN-SEQUENCE B STARS

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

Smith, Myron A.

2010-02-01

We have constructed a detailed spectral atlas covering the wavelength region 930-1225 A for 10 sharp-lined B0-B9 stars near the main sequence. Most of the spectra we assembled are from the archives of the Far Ultraviolet Spectroscopic Explorer satellite, but for nine stars, wavelength coverage above 1188 A was taken from high-resolution International Ultraviolet Explorer or echelle Hubble Space Telescope/Space Telescope Imaging Spectrograph spectra. To represent the tenth star at type B0.2 V, we used the Copernicus atlas of {tau} Sco. We made extensive line identifications in the region 949-1225 A of all atomic features having published oscillator strengths atmore » types B0, B2, and B8. These are provided as a supplementary data product-hence the term detailed atlas. Our list of found features totals 2288, 1612, and 2469 lines, respectively. We were able to identify 92%, 98%, and 98% of these features with known atomic transitions with varying degrees of certainty in these spectra. The remaining lines do not have published oscillator strengths. Photospheric lines account for 94%, 87%, and 91%, respectively, of all our identifications, with the remainder being due to interstellar (usually molecular H{sub 2}) lines. We also discuss the numbers of lines with respect to the distributions of various ions for these three most studied spectral subtypes. A table is also given of 162 least blended lines that can be used as possible diagnostics of physical conditions in B star atmospheres.« less

Comparison of next generation sequencing technologies for transcriptome characterization

PubMed Central

2009-01-01

Background We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis. Results The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics. Conclusion NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms. PMID:19646272

Illuminating choices for library prep: a comparison of library preparation methods for whole genome sequencing of Cryptococcus neoformans using Illumina HiSeq.

PubMed

Rhodes, Johanna; Beale, Mathew A; Fisher, Matthew C

2014-01-01

The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.

An efficient study design to test parent-of-origin effects in family trios.

PubMed

Yu, Xiaobo; Chen, Gao; Feng, Rui

2017-11-01

Increasing evidence has shown that genes may cause prenatal, neonatal, and pediatric diseases depending on their parental origins. Statistical models that incorporate parent-of-origin effects (POEs) can improve the power of detecting disease-associated genes and help explain the missing heritability of diseases. In many studies, children have been sequenced for genome-wide association testing. But it may become unaffordable to sequence their parents and evaluate POEs. Motivated by the reality, we proposed a budget-friendly study design of sequencing children and only genotyping their parents through single nucleotide polymorphism array. We developed a powerful likelihood-based method, which takes into account both sequence reads and linkage disequilibrium to infer the parental origins of children's alleles and estimate their POEs on the outcome. We evaluated the performance of our proposed method and compared it with an existing method using only genotypes, through extensive simulations. Our method showed higher power than the genotype-based method. When either the mean read depth or the pair-end length was reasonably large, our method achieved ideal power. When single parents' genotypes were unavailable or parental genotypes at the testing locus were not typed, both methods lost power compared with when complete data were available; but the power loss from our method was smaller than the genotype-based method. We also extended our method to accommodate mixed genotype, low-, and high-coverage sequence data from children and their parents. At presence of sequence errors, low-coverage parental sequence data may lead to lower power than parental genotype data. © 2017 WILEY PERIODICALS, INC.

Rapid and accurate pyrosequencing of angiosperm plastid genomes

PubMed Central

Moore, Michael J; Dhingra, Amit; Soltis, Pamela S; Shaw, Regina; Farmerie, William G; Folta, Kevin M; Soltis, Douglas E

2006-01-01

Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae). Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid genome sequence was generated for a significant reduction in time and cost over traditional shotgun-based genome sequencing techniques, although with approximately half the coverage of previously reported GS 20 de novo genome sequence. The GS 20 should be broadly applicable to angiosperm plastid genome sequencing, and therefore promises to expand the scale of plant genetic and phylogenetic research dramatically. PMID:16934154

A parametric study of rate of advance and area coverage rate performance of synthetic aperture radar.

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

Raynal, Ann Marie; William H. Hensley, Jr.; Burns, Bryan L.

2014-11-01

The linear ground distance per unit time and ground area covered per unit time of producing synthetic aperture radar (SAR) imagery, termed rate of advance (ROA) and area coverage rate (ACR), are important metrics for platform and radar performance in surveillance applications. These metrics depend on many parameters of a SAR system such as wavelength, aircraft velocity, resolution, antenna beamwidth, imaging mode, and geometry. Often the effects of these parameters on rate of advance and area coverage rate are non-linear. This report addresses the impact of different parameter spaces as they relate to rate of advance and area coverage ratemore » performance.« less

WEED SURVEYING OF PHACELIA (PHACELIA TANACETIFOLIA L.) AND EVALUATING THE EFFICIENCY OF THE WEED CONTROL.

PubMed

Horváth, E; Szabó, R

2014-01-01

The experiment was set up in an area of 9 ha that was split into 4 plots: in plot 1 the row spacing was 12 cm and the seeding rate was 10 kg; in plot 2 the row spacing was 24 cm and the seeding rate was 10 kg; in plot 3 the row spacing was 24 cm and the seeding rate was 8 kg; in plot 4 the row spacing was 12 cm and the seeding rate was 8 kg. After the weed surveying, the total weed coverage was established as follows: in plot 1 the total weed coverage was 11.34%, in plot 2 it was 12.3%, in plot 3 it was 18%, and in plot 4 the total weed coverage was 15%. Based on the weed survey, on the test area the following dicotyledon weeds belonging to the T4 Raunkiaer plant life-form category occupied the highest percentage: heal-all, black-bindweed, goosefoot. The proportion of the perennial dicotyledons: field bindweed (G3), tuberous pea (G1), white campion (H3) was negligible. In all four cases the weed control was executed using the same herbicide in the same doses and with regard to the weed species it showed the same level of efficiency. The smaller row spacing and higher seeding rate has a beneficial effect on the weed suppressing capacity of the crop, the crop's weed suppressing capacity is better and the development of the weeds becomes worse.

Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™.

PubMed

Luck, Ashley N; Slatko, Barton E; Foster, Jeremy M

2017-01-01

Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA. Here, we report use of Cappable-seq™ to specifically enrich, in a single step, Wolbachia endobacterial mRNA transcripts from total RNA prepared from the parasitic filarial nematode, Brugia malayi. The obligate Wolbachia endosymbiont is a proven drug target for many human filarial infections, yet the precise nature of its symbiosis with the nematode host is poorly understood. Insightful analysis of the expression levels of Wolbachia genes predicted to underpin the mutualistic association and of known drug target genes at different life cycle stages or in response to drug treatments is typically challenged by low transcriptomic coverage. Cappable-seq resulted in up to ~ 5-fold increase in the number of reads mapping to Wolbachia. On average, coverage of Wolbachia transcripts from B. malayi microfilariae was enriched ~40-fold by Cappable-seq. Additionally, this method has an additional benefit of selectively removing abundant prokaryotic ribosomal RNAs.The deeper microbial transcriptome sequencing afforded by Cappable-seq facilitates more detailed characterization of gene expression levels of pathogens and symbionts present in animal tissues.

Diversity arrays technology (DArT) markers in apple for genetic linkage maps.

PubMed

Schouten, Henk J; van de Weg, W Eric; Carling, Jason; Khan, Sabaz Ali; McKay, Steven J; van Kaauwen, Martijn P W; Wittenberg, Alexander H J; Koehorst-van Putten, Herma J J; Noordijk, Yolanda; Gao, Zhongshan; Rees, D Jasper G; Van Dyk, Maria M; Jaccoud, Damian; Considine, Michael J; Kilian, Andrzej

2012-03-01

Diversity Arrays Technology (DArT) provides a high-throughput whole-genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. The work presented here details the development and performance of a DArT genotyping array for apple. This is the first paper on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high-throughput method for obtaining accurate and reproducible marker data, despite the low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. The standard complexity reduction method, based on the methylation-sensitive PstI restriction enzyme, resulted in a high frequency of markers, although there was 52-54% redundancy due to the repeated sampling of highly similar sequences. Sequencing of the marker clones showed that they are significantly enriched for low-copy, genic regions. The genome coverage using the standard method was 55-76%. For improved genome coverage, an alternative complexity reduction method was examined, which resulted in less redundancy and additional segregating markers. The DArT markers proved to be of high quality and were very suitable for genetic mapping at low cost for the apple, providing moderate genome coverage. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9579-5) contains supplementary material, which is available to authorized users.

Systematic evaluation of the impact of ChIP-seq read designs on genome coverage, peak identification, and allele-specific binding detection.

PubMed

Zhang, Qi; Zeng, Xin; Younkin, Sam; Kawli, Trupti; Snyder, Michael P; Keleş, Sündüz

2016-02-24

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments revolutionized genome-wide profiling of transcription factors and histone modifications. Although maturing sequencing technologies allow these experiments to be carried out with short (36-50 bps), long (75-100 bps), single-end, or paired-end reads, the impact of these read parameters on the downstream data analysis are not well understood. In this paper, we evaluate the effects of different read parameters on genome sequence alignment, coverage of different classes of genomic features, peak identification, and allele-specific binding detection. We generated 101 bps paired-end ChIP-seq data for many transcription factors from human GM12878 and MCF7 cell lines. Systematic evaluations using in silico variations of these data as well as fully simulated data, revealed complex interplay between the sequencing parameters and analysis tools, and indicated clear advantages of paired-end designs in several aspects such as alignment accuracy, peak resolution, and most notably, allele-specific binding detection. Our work elucidates the effect of design on the downstream analysis and provides insights to investigators in deciding sequencing parameters in ChIP-seq experiments. We present the first systematic evaluation of the impact of ChIP-seq designs on allele-specific binding detection and highlights the power of pair-end designs in such studies.

Reforming Central Government Health Scheme into a 'Universal Health Coverage' model.

PubMed

Sarwal, Rakesh

2015-01-01

Universal Health Coverage (UHC) is now recognized as a goal of all health systems, irrespective of income levels. In the absence of a one-size solution, each country has to develop strategies suited to its circumstances. How does the Central Government Health Scheme (CGHS) stand up to the goals and global experience of UHC, and what can be done to make it a model? I relied on publicly available documents to identify key features of UHC, and relate it to the architecture of and practices in CGHS. Combining WHO's framework of health systems functions with log frame approach, I constructed a 'UHC status tool' of key elements and expected norms of UHC. CGHS has been performing strongly on financing function and for the range of services covered. It has performed rather poorly on all other elements of UHC. I build the argument for continued public provision of health, as opposed to insurance, on grounds of cost, public good nature of outpatient care and public health services. I suggest and strategize a sequence of reforms in CGHS anchored in health system strengthening, governance and financing, comprehensive primary care and client participation. It is both possible and desirable to transform CGHS into a UHC model within the same fiscal space. Merger of finance pools of centrally administered health schemes is suggested as a step towards UHC in India. Copyright 2015, NMJI.

Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDI-TOF.

PubMed Central

Tolson, D A; Nicholson, N H

1998-01-01

The determination of DNA sequences by partial exonuclease digestion followed by Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF) is a well established method. When the same procedure is applied to RNA, difficulties arise due to the small (1 Da) mass difference between the nucleotides U and C, which makes unambiguous assignment difficult using a MALDI-TOF instrument. Here we report our experiences with sequence specific endonucleases and chemical methods followed by MALDI-TOF to resolve these sequence ambiguities. We have found chemical methods superior to endonucleases both in terms of correct specificity and extent of sequence coverage. This methodology can be used in combination with exonuclease digestion to rapidly assign RNA sequences. PMID:9421498

Cost analysis of whole genome sequencing in German clinical practice.

PubMed

Plöthner, Marika; Frank, Martin; von der Schulenburg, J-Matthias Graf

2017-06-01

Whole genome sequencing (WGS) is an emerging tool in clinical diagnostics. However, little has been said about its procedure costs, owing to a dearth of related cost studies. This study helps fill this research gap by analyzing the execution costs of WGS within the setting of German clinical practice. First, to estimate costs, a sequencing process related to clinical practice was undertaken. Once relevant resources were identified, a quantification and monetary evaluation was conducted using data and information from expert interviews with clinical geneticists, and personnel at private enterprises and hospitals. This study focuses on identifying the costs associated with the standard sequencing process, and the procedure costs for a single WGS were analyzed on the basis of two sequencing platforms-namely, HiSeq 2500 and HiSeq Xten, both by Illumina, Inc. In addition, sensitivity analyses were performed to assess the influence of various uses of sequencing platforms and various coverage values on a fixed-cost degression. In the base case scenario-which features 80 % utilization and 30-times coverage-the cost of a single WGS analysis with the HiSeq 2500 was estimated at €3858.06. The cost of sequencing materials was estimated at €2848.08; related personnel costs of €396.94 and acquisition/maintenance costs (€607.39) were also found. In comparison, the cost of sequencing that uses the latest technology (i.e., HiSeq Xten) was approximately 63 % cheaper, at €1411.20. The estimated costs of WGS currently exceed the prediction of a 'US$1000 per genome', by more than a factor of 3.8. In particular, the material costs in themselves exceed this predicted cost.

The American cranberry: first insights into the whole genome of a species adapted to bog habitat.

PubMed

Polashock, James; Zelzion, Ehud; Fajardo, Diego; Zalapa, Juan; Georgi, Laura; Bhattacharya, Debashish; Vorsa, Nicholi

2014-06-13

The American cranberry (Vaccinium macrocarpon Ait.) is one of only three widely-cultivated fruit crops native to North America- the other two are blueberry (Vaccinium spp.) and native grape (Vitis spp.). In terms of taxonomy, cranberries are in the core Ericales, an order for which genome sequence data are currently lacking. In addition, cranberries produce a host of important polyphenolic secondary compounds, some of which are beneficial to human health. Whereas next-generation sequencing technology is allowing the advancement of whole-genome sequencing, one major obstacle to the successful assembly from short-read sequence data of complex diploid (and higher ploidy) organisms is heterozygosity. Cranberry has the advantage of being diploid (2n = 2x = 24) and self-fertile. To minimize the issue of heterozygosity, we sequenced the genome of a fifth-generation inbred genotype (F ≥ 0.97) derived from five generations of selfing originating from the cultivar Ben Lear. The genome size of V. macrocarpon has been estimated to be about 470 Mb. Genomic sequences were assembled into 229,745 scaffolds representing 420 Mbp (N50 = 4,237 bp) with 20X average coverage. The number of predicted genes was 36,364 and represents 17.7% of the assembled genome. Of the predicted genes, 30,090 were assigned to candidate genes based on homology. Genes supported by transcriptome data totaled 13,170 (36%). Shotgun sequencing of the cranberry genome, with an average sequencing coverage of 20X, allowed efficient assembly and gene calling. The candidate genes identified represent a useful collection to further study important biochemical pathways and cellular processes and to use for marker development for breeding and the study of horticultural characteristics, such as disease resistance.

The American cranberry: first insights into the whole genome of a species adapted to bog habitat

PubMed Central

2014-01-01

Background The American cranberry (Vaccinium macrocarpon Ait.) is one of only three widely-cultivated fruit crops native to North America- the other two are blueberry (Vaccinium spp.) and native grape (Vitis spp.). In terms of taxonomy, cranberries are in the core Ericales, an order for which genome sequence data are currently lacking. In addition, cranberries produce a host of important polyphenolic secondary compounds, some of which are beneficial to human health. Whereas next-generation sequencing technology is allowing the advancement of whole-genome sequencing, one major obstacle to the successful assembly from short-read sequence data of complex diploid (and higher ploidy) organisms is heterozygosity. Cranberry has the advantage of being diploid (2n = 2x = 24) and self-fertile. To minimize the issue of heterozygosity, we sequenced the genome of a fifth-generation inbred genotype (F ≥ 0.97) derived from five generations of selfing originating from the cultivar Ben Lear. Results The genome size of V. macrocarpon has been estimated to be about 470 Mb. Genomic sequences were assembled into 229,745 scaffolds representing 420 Mbp (N50 = 4,237 bp) with 20X average coverage. The number of predicted genes was 36,364 and represents 17.7% of the assembled genome. Of the predicted genes, 30,090 were assigned to candidate genes based on homology. Genes supported by transcriptome data totaled 13,170 (36%). Conclusions Shotgun sequencing of the cranberry genome, with an average sequencing coverage of 20X, allowed efficient assembly and gene calling. The candidate genes identified represent a useful collection to further study important biochemical pathways and cellular processes and to use for marker development for breeding and the study of horticultural characteristics, such as disease resistance. PMID:24927653

Matrix Transformations between Certain Sequence Spaces over the Non-Newtonian Complex Field

PubMed Central

Efe, Hakan

2014-01-01

In some cases, the most general linear operator between two sequence spaces is given by an infinite matrix. So the theory of matrix transformations has always been of great interest in the study of sequence spaces. In the present paper, we introduce the matrix transformations in sequence spaces over the field ℂ* and characterize some classes of infinite matrices with respect to the non-Newtonian calculus. Also we give the necessary and sufficient conditions on an infinite matrix transforming one of the classical sets over ℂ* to another one. Furthermore, the concept for sequence-to-sequence and series-to-series methods of summability is given with some illustrated examples. PMID:25110740

Comparing an accelerated 3D fast spin-echo sequence (CS-SPACE) for knee 3-T magnetic resonance imaging with traditional 3D fast spin-echo (SPACE) and routine 2D sequences.

PubMed

Altahawi, Faysal F; Blount, Kevin J; Morley, Nicholas P; Raithel, Esther; Omar, Imran M

2017-01-01

To compare a faster, new, high-resolution accelerated 3D-fast-spin-echo (3D-FSE) acquisition sequence (CS-SPACE) to traditional 2D and high-resolution 3D sequences for knee 3-T magnetic resonance imaging (MRI). Twenty patients received knee MRIs that included routine 2D (T1, PD ± FS, T2-FS; 0.5 × 0.5 × 3 mm 3 ; ∼10 min), traditional 3D FSE (SPACE-PD-FS; 0.5 × 0.5 × 0.5 mm 3 ; ∼7.5 min), and accelerated 3D-FSE prototype (CS-SPACE-PD-FS; 0.5 × 0.5 × 0.5 mm 3 ; ∼5 min) acquisitions on a 3-T MRI system (Siemens MAGNETOM Skyra). Three musculoskeletal radiologists (MSKRs) prospectively and independently reviewed the studies with graded surveys comparing image and diagnostic quality. Tissue-specific signal-to-noise ratios (SNR) and contrast-to-noise ratios (CNR) were also compared. MSKR-perceived diagnostic quality of cartilage was significantly higher for CS-SPACE than for SPACE and 2D sequences (p < 0.001). Assessment of diagnostic quality of menisci and synovial fluid was higher for CS-SPACE than for SPACE (p < 0.001). CS-SPACE was not significantly different from SPACE but had lower assessments than 2D sequences for evaluation of bones, ligaments, muscles, and fat (p ≤ 0.004). 3D sequences had higher spatial resolution, but lower overall assessed contrast (p < 0.001). Overall image quality from CS-SPACE was assessed as higher than SPACE (p = 0.007), but lower than 2D sequences (p < 0.001). Compared to SPACE, CS-SPACE had higher fluid SNR and CNR against all other tissues (all p < 0.001). The CS-SPACE prototype allows for faster isotropic acquisitions of knee MRIs over currently used protocols. High fluid-to-cartilage CNR and higher spatial resolution over routine 2D sequences may present a valuable role for CS-SPACE in the evaluation of cartilage and menisci.

[Green space vegetation quantity in workshop area of Wuhan Iron and Steel Company].

PubMed

Chen, Fang; Zhou, Zhixiang; Wang, Pengcheng; Li, Haifang; Zhong, Yingfei

2006-04-01

Aimed at the complex community structure and higher fragmentation of urban green space, and based on the investigation of synusia structure and its coverage, this paper studied the vegetation quantity of ornamental green space in the workshop area of Wuhan Iron and Steel Company, with the help of GIS. The results showed that different life forms of ornamental plants in this area had a greater difference in their single leaf area and leaf area index (LAI), and the LAI was not only depended on single leaf area, but also governed by the shape of tree crown and the intensive degree of branches and leaves. The total vegetation quantity was 1 694.2 hm2, with the average LAI being 7.75, and the vegetation quantity of arbor-shrub-herb and arbor-shrub communities accounted for 79.7% and 92.3% of the total, respectively, reflecting that the green space structure was dominated by arbor species and by arbor-shrub-herb and arbor-shrub community types. Single layer-structured lawn had a less percentage, while the vegetation quantity of herb synusia accounted for 22.9% of the total, suggesting an afforestation characteristic of "making use of every bit of space" in the workshop area. The vegetation quantity of urban ornamental green space depended on the area of green space, its synusia structure, and the LAI and coverage of ornamental plants. In enlarging urban green space, ornamental plant species with high LAI should be selected, and community structure should be improved to have a higher vegetation quantity in urban area. To quantify the vegetation quantity of urban ornamental green space more accurately, synusia should be taken as the unit to measure the LAI of typical species, and the synusia structure and its coverage of different community types should be investigated with the help of remote sensing images and GIS.

Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA.

PubMed

Weiberg, Arne; Pöhler, Dirk; Morgenstern, Burkhard; Karlovsky, Petr

2008-10-13

cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively. With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation) was written in Perl. cDNA-AFLP protocols described in the literature and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set. Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each of the marking enzymes is recommended.

Neo-sex Chromosomes in the Monarch Butterfly, Danaus plexippus

PubMed Central

Mongue, Andrew J.; Nguyen, Petr; Voleníková, Anna; Walters, James R.

2017-01-01

We report the discovery of a neo-sex chromosome in the monarch butterfly, Danaus plexippus, and several of its close relatives. Z-linked scaffolds in the D. plexippus genome assembly were identified via sex-specific differences in Illumina sequencing coverage. Additionally, a majority of the D. plexippus genome assembly was assigned to chromosomes based on counts of one-to-one orthologs relative to the butterfly Melitaea cinxia (with replication using two other lepidopteran species), in which genome scaffolds have been mapped to linkage groups. Sequencing coverage-based assessments of Z linkage combined with homology-based chromosomal assignments provided strong evidence for a Z-autosome fusion in the Danaus lineage, involving the autosome homologous to chromosome 21 in M. cinxia. Coverage analysis also identified three notable assembly errors resulting in chimeric Z-autosome scaffolds. Cytogenetic analysis further revealed a large W chromosome that is partially euchromatic, consistent with being a neo-W chromosome. The discovery of a neo-Z and the provisional assignment of chromosome linkage for >90% of D. plexippus genes lays the foundation for novel insights concerning sex chromosome evolution in this female-heterogametic model species for functional and evolutionary genomics. PMID:28839116

Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin.

PubMed

Floris, Federico; Chiron, Lionel; Lynch, Alice M; Barrow, Mark P; Delsuc, Marc-André; O'Connor, Peter B

2018-06-04

Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%. Graphical Abstract Two-dimensional mass spectrometry, when applied to primary fragment ions from the source, allows deep-sequencing of the protein calmodulin.

Comparative evaluation of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) and high-pH reversed phase (Hp-RP) chromatography in profiling of rat kidney proteome.

PubMed

Hao, Piliang; Ren, Yan; Dutta, Bamaprasad; Sze, Siu Kwan

2013-04-26

ERLIC and high-pH RP (Hp-RP) have been reported to be promising alternatives to strong cation exchange (SCX) in proteome fractionation. Here we compared the performance of ERLIC, concatenated ERLIC and concatenated Hp-RP in proteome profiling. The protein identification is comparable in these three strategies, but significantly more unique peptides are identified by the two concatenation methods, resulting in a significant increase of the average protein sequence coverage. The pooling of fractions from spaced intervals results in more uniform distribution of peptides in each fraction compared with the chromatogram-based pooling of adjacent fractions. ERLIC fractionates peptides according to their pI and GRAVY values. These properties remains but becomes less remarkable in concatenated ERLIC. In contrast, the average pI and GRAVY values of the peptides are comparable in each fraction in concatenated Hp-RP. ERLIC performs the best in identifying peptides with pI>9 among the three strategies, while concatenated Hp-RP is good at identifying peptides with pI<4. These advantages are useful when either basic or acidic peptides/proteins are analytical targets. The power of ERLIC in identification of basic peptides seems to be due to their efficient separation from acidic peptides. This study facilitates the choice of proper fractionation strategies based on specific objectives. For in-depth proteomic analysis of a cell, tissue and plasma, multidimensional liquid chromatography (MDLC) is still necessary to reduce sample complexity for improving analytical dynamic range and proteome coverage. This work conducts a direct comparison of three promising first-dimensional proteome fractionation methods. They are comparable in identifying proteins, but concatenated ERLIC and concatenated Hp-RP identify significantly more unique peptides than ERLIC. ERLIC is good at analyzing basic peptides, while concatenated Hp-RP performs the best in analyzing acidic peptides with pI<4. This will facilitate the choice of the proper peptide fractionation strategy based on a specific need. A combination of different fractionation strategies can be used to increase the sequence coverage and number of protein identification due to the complementary effect between different methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Enhancing Protein Disulfide Bond Cleavage by UV Excitation and Electron Capture Dissociation for Top-Down Mass Spectrometry.

PubMed

Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R Ogorzalek; Julian, Ryan R; Loo, Joseph A

2015-11-15

The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.

Overview of recurrent chromosomal losses in retinoblastoma detected by low coverage next generation sequencing

PubMed Central

García-Chequer, A.J.; Méndez-Tenorio, A.; Olguín-Ruiz, G.; Sánchez-Vallejo, C.; Isa, P.; Arias, C.F.; Torres, J.; Hernández-Angeles, A.; Ramírez-Ortiz, M.A.; Lara, C.; Cabrera-Muñoz, M.L.; Sadowinski-Pine, S.; Bravo-Ortiz, J.C.; Ramón-García, G.; Diegopérez-Ramírez, J.; Ramírez-Reyes, G.; Casarrubias-Islas, R.; Ramírez, J.; Orjuela, M.A.; Ponce-Castañeda, M.V.

2016-01-01

Genes are frequently lost or gained in malignant tumors and the analysis of these changes can be informative about the underlying tumor biology. Retinoblastoma is a pediatric intraocular malignancy, and since deletions in chromosome 13 have been described in this tumor, we performed genome wide sequencing with the Illumina platform to test whether recurrent losses could be detected in low coverage data from DNA pools of Rb cases. An in silico reference profile for each pool was created from the human genome sequence GRCh37p5; a chromosome integrity score and a graphics 40 Kb window analysis approach, allowed us to identify with high resolution previously reported non random recurrent losses in all chromosomes of these tumors. We also found a pattern of gains and losses associated to clear and dark cytogenetic bands respectively. We further analyze a pool of medulloblastoma and found a more stable genomic profile and previously reported losses in this tumor. This approach facilitates identification of recurrent deletions from many patients that may be biological relevant for tumor development. PMID:26883451

Experimental investigation of an RNA sequence space

NASA Technical Reports Server (NTRS)

Lee, Youn-Hyung; Dsouza, Lisa; Fox, George E.

1993-01-01

Modern rRNAs are the historic consequence of an ongoing evolutionary exploration of a sequence space. These extant sequences belong to a special subset of the sequence space that is comprised only of those primary sequences that can validly perform the biological function(s) required of the particular RNA. If it were possible to readily identify all such valid sequences, stochastic predictions could be made about the relative likelihood of various evolutionary pathways available to an RNA. Herein an experimental system which can assess whether a particular sequence is likely to have validity as a eubacterial 5S rRNA is described. A total of ten naturally occurring, and hence known to be valid, sequences and two point mutants of unknown validity were used to test the usefulness of the approach. Nine of the ten valid sequences tested positive whereas both mutants tested as clearly defective. The tenth valid sequence gave results that would be interpreted as reflecting a borderline status were the answer not known. These results demonstrate that it is possible to experimentally determine which sequences in local regions of the sequence space are potentially valid 5S rRNAs.

Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species

PubMed Central

Wu, Chengcang; Proestou, Dina; Carter, Dorothy; Nicholson, Erica; Santos, Filippe; Zhao, Shaying; Zhang, Hong-Bin; Goldsmith, Marian R

2009-01-01

Background Manduca sexta, Heliothis virescens, and Heliconius erato represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date. Results We report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152–175 kb. We estimated that the genome coverage of each library ranged from 6–9 ×, with the two combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6~8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of ~200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the H. erato genome is somewhat more abundant in repeat elements and simple repeats than those of M. sexta and H. virescens. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of H. virescens being the most conserved as a typical lepidopteran, whereas both genomes of H. erato and M. sexta appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences. Conclusion The high-quality and large-insert BAC libraries of the insects, together with the identified BACs containing genes of interest, provide valuable information, resources and tools for comprehensive understanding and studies of the insect genomes and for addressing many fundamental questions in Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and evolution of the insect genomes. PMID:19558662

Genomic Characterization of DArT Markers Based on High-Density Linkage Analysis and Physical Mapping to the Eucalyptus Genome

PubMed Central

Petroli, César D.; Sansaloni, Carolina P.; Carling, Jason; Steane, Dorothy A.; Vaillancourt, René E.; Myburg, Alexander A.; da Silva, Orzenil Bonfim; Pappas, Georgios Joannis; Kilian, Andrzej; Grattapaglia, Dario

2012-01-01

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference genome is yet available to allow such detailed characterization. PMID:22984541

Ammonium sulfate and MALDI in-source decay: a winning combination for sequencing peptides

PubMed Central

Delvolve, Alice; Woods, Amina S.

2009-01-01

In previous papers we highlighted the role of ammonium sulfate in increasing peptide fragmentation by in source decay (ISD). The current work systematically investigated effects of MALDI extraction delay, peptide amino acid composition, matrix and ammonium sulfate concentration on peptides ISD fragmentation. The data confirmed that ammonium sulfate increased peptides signal to noise ratio as well as their in source fragmentation resulting in complete sequence coverage regardless of the amino acid composition. This method is easy, inexpensive and generates the peptides sequence instantly. PMID:19877641

Metagenomic ventures into outer sequence space.

PubMed

Dutilh, Bas E

Sequencing DNA or RNA directly from the environment often results in many sequencing reads that have no homologs in the database. These are referred to as "unknowns," and reflect the vast unexplored microbial sequence space of our biosphere, also known as "biological dark matter." However, unknowns also exist because metagenomic datasets are not optimally mined. There is a pressure on researchers to publish and move on, and the unknown sequences are often left for what they are, and conclusions drawn based on reads with annotated homologs. This can cause abundant and widespread genomes to be overlooked, such as the recently discovered human gut bacteriophage crAssphage. The unknowns may be enriched for bacteriophage sequences, the most abundant and genetically diverse component of the biosphere and of sequence space. However, it remains an open question, what is the actual size of biological sequence space? The de novo assembly of shotgun metagenomes is the most powerful tool to address this question.

An efficient genetic algorithm for maximum coverage deployment in wireless sensor networks.

PubMed

Yoon, Yourim; Kim, Yong-Hyuk

2013-10-01

Sensor networks have a lot of applications such as battlefield surveillance, environmental monitoring, and industrial diagnostics. Coverage is one of the most important performance metrics for sensor networks since it reflects how well a sensor field is monitored. In this paper, we introduce the maximum coverage deployment problem in wireless sensor networks and analyze the properties of the problem and its solution space. Random deployment is the simplest way to deploy sensor nodes but may cause unbalanced deployment and therefore, we need a more intelligent way for sensor deployment. We found that the phenotype space of the problem is a quotient space of the genotype space in a mathematical view. Based on this property, we propose an efficient genetic algorithm using a novel normalization method. A Monte Carlo method is adopted to design an efficient evaluation function, and its computation time is decreased without loss of solution quality using a method that starts from a small number of random samples and gradually increases the number for subsequent generations. The proposed genetic algorithms could be further improved by combining with a well-designed local search. The performance of the proposed genetic algorithm is shown by a comparative experimental study. When compared with random deployment and existing methods, our genetic algorithm was not only about twice faster, but also showed significant performance improvement in quality.

Identification of novel antibody-reactive detection sites for comprehensive gluten monitoring.

PubMed

Röckendorf, Niels; Meckelein, Barbara; Scherf, Katharina A; Schalk, Kathrin; Koehler, Peter; Frey, Andreas

2017-01-01

Certain cereals like wheat, rye or barley contain gluten, a protein mixture that can trigger celiac disease (CD). To make gluten-free diets available for affected individuals the gluten content of foodstuff must be monitored. For this purpose, antibody-based assays exist which rely on the recognition of certain linear gluten sequence motifs. Yet, not all CD-active gluten constituents and fragments formed during food processing/fermentation may be covered by those tests. In this study, we therefore assayed the coverage of reportedly CD-active gluten components by currently available detection antibodies and determined the antibody-inducing capacity of wheat gluten constituents in order to provide novel diagnostic targets for comprehensive gluten quantitation. Immunizations of outbred mice with purified gliadins and glutenins were conducted and the linear target recognition profile of the sera was recorded using synthetic peptide arrays that covered the sequence space of gluten constituents present in those preparations. The resulting murine immunorecognition profile of gluten demonstrated that further linear binding sites beyond those recognized by the monoclonal antibodies α20, R5 and G12 exist and may be exploitable as diagnostic targets. We conclude that the safety of foodstuffs for CD patients can be further improved by complementing current tests with antibodies directed against additional CD-active gluten components. Currently unrepresented linear gluten detection sites in glutenins and α-gliadins suggest sequences QQQYPS, PQQSFP, QPGQGQQG and QQPPFS as novel targets for antibody generation.

Identification of novel antibody-reactive detection sites for comprehensive gluten monitoring

PubMed Central

Röckendorf, Niels; Meckelein, Barbara; Scherf, Katharina A.; Schalk, Kathrin; Koehler, Peter

2017-01-01

Certain cereals like wheat, rye or barley contain gluten, a protein mixture that can trigger celiac disease (CD). To make gluten-free diets available for affected individuals the gluten content of foodstuff must be monitored. For this purpose, antibody-based assays exist which rely on the recognition of certain linear gluten sequence motifs. Yet, not all CD-active gluten constituents and fragments formed during food processing/fermentation may be covered by those tests. In this study, we therefore assayed the coverage of reportedly CD-active gluten components by currently available detection antibodies and determined the antibody-inducing capacity of wheat gluten constituents in order to provide novel diagnostic targets for comprehensive gluten quantitation. Immunizations of outbred mice with purified gliadins and glutenins were conducted and the linear target recognition profile of the sera was recorded using synthetic peptide arrays that covered the sequence space of gluten constituents present in those preparations. The resulting murine immunorecognition profile of gluten demonstrated that further linear binding sites beyond those recognized by the monoclonal antibodies α20, R5 and G12 exist and may be exploitable as diagnostic targets. We conclude that the safety of foodstuffs for CD patients can be further improved by complementing current tests with antibodies directed against additional CD-active gluten components. Currently unrepresented linear gluten detection sites in glutenins and α-gliadins suggest sequences QQQYPS, PQQSFP, QPGQGQQG and QQPPFS as novel targets for antibody generation. PMID:28759621

Toward Prostate Cancer Contouring Guidelines on Magnetic Resonance Imaging: Dominant Lesion Gross and Clinical Target Volume Coverage Via Accurate Histology Fusion

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

Gibson, Eli; Biomedical Engineering, University of Western Ontario, London, Ontario; Centre for Medical Image Computing, University College London, London

Purpose: Defining prostate cancer (PCa) lesion clinical target volumes (CTVs) for multiparametric magnetic resonance imaging (mpMRI) could support focal boosting or treatment to improve outcomes or lower morbidity, necessitating appropriate CTV margins for mpMRI-defined gross tumor volumes (GTVs). This study aimed to identify CTV margins yielding 95% coverage of PCa tumors for prospective cases with high likelihood. Methods and Materials: Twenty-five men with biopsy-confirmed clinical stage T1 or T2 PCa underwent pre-prostatectomy mpMRI, yielding T2-weighted, dynamic contrast-enhanced, and apparent diffusion coefficient images. Digitized whole-mount histology was contoured and registered to mpMRI scans (error ≤2 mm). Four observers contoured lesion GTVs onmore » each mpMRI scan. CTVs were defined by isotropic and anisotropic expansion from these GTVs and from multiparametric (unioned) GTVs from 2 to 3 scans. Histologic coverage (proportions of tumor area on co-registered histology inside the CTV, measured for Gleason scores [GSs] ≥6 and ≥7) and prostate sparing (proportions of prostate volume outside the CTV) were measured. Nonparametric histologic-coverage prediction intervals defined minimal margins yielding 95% coverage for prospective cases with 78% to 92% likelihood. Results: On analysis of 72 true-positive tumor detections, 95% coverage margins were 9 to 11 mm (GS ≥ 6) and 8 to 10 mm (GS ≥ 7) for single-sequence GTVs and were 8 mm (GS ≥ 6) and 6 mm (GS ≥ 7) for 3-sequence GTVs, yielding CTVs that spared 47% to 81% of prostate tissue for the majority of tumors. Inclusion of T2-weighted contours increased sparing for multiparametric CTVs with 95% coverage margins for GS ≥6, and inclusion of dynamic contrast-enhanced contours increased sparing for GS ≥7. Anisotropic 95% coverage margins increased the sparing proportions to 71% to 86%. Conclusions: Multiparametric magnetic resonance imaging–defined GTVs expanded by appropriate margins may support focal boosting or treatment of PCa; however, these margins, accounting for interobserver and intertumoral variability, may preclude highly conformal CTVs. Multiparametric GTVs and anisotropic margins may reduce the required margins and improve prostate sparing.« less

Online Case-Based Discussions: Examining Coverage of the Afforded Problem Space

ERIC Educational Resources Information Center

Ertmer, Peggy A.; Koehler, Adrie A.

2014-01-01

Case studies hold great potential for engaging students in disciplinary content. However, little is known about the extent to which students actually cover the problem space afforded by a particular case study. In this research, we compared the problem space afforded by an instructional design case study with the actual content covered by 16…

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

PubMed

Nacheva, Elizabeth; Mokretar, Katya; Soenmez, Aynur; Pittman, Alan M; Grace, Colin; Valli, Roberto; Ejaz, Ayesha; Vattathil, Selina; Maserati, Emanuela; Houlden, Henry; Taanman, Jan-Willem; Schapira, Anthony H; Proukakis, Christos

2017-01-01

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

PubMed Central

Nacheva, Elizabeth; Mokretar, Katya; Soenmez, Aynur; Pittman, Alan M.; Grace, Colin; Valli, Roberto; Ejaz, Ayesha; Vattathil, Selina; Maserati, Emanuela; Houlden, Henry; Taanman, Jan-Willem; Schapira, Anthony H.

2017-01-01

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance. PMID:28683077

Predicted cycloartenol synthase protein from Kandelia obovata and Rhizophora stylosa using online software of Phyre2 and Swiss-model

NASA Astrophysics Data System (ADS)

Basyuni, M.; Sulistiyono, N.; Wati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

2018-03-01

Cloning of Kandelia obovata KcCAS gene (previously known as Kandelia candel) and Rhizophora stylosa RsCAS have already have been reported and encoded cycloartenol synthases. In this study, the predicted KcCAS and RsCAS protein were analyzed using online software of Phyre2 and Swiss-model. The protein modelling for KcCAS and RsCAS cycloartenol synthases was determined using Pyre2 had similar results with slightly different in sequence identity. By contrast, the Swiss-model for KcCAS slightly had higher sequence identity (47.31%) and Qmean (0.70) compared to RsCAS. No difference of ligands binding site which is considered as modulators for both cycloartenol synthases. The range of predicted protein derived from 91-757 amino acid residues with coverage sequence similarities 0.86, respectively from template model of lanosterol synthase from the human. Homology modelling revealed that 706 residues (93% of the amino acid sequence) had been modelled with 100.0% confidence by the single highest scoring template for both KcCAS and RsCAS using Phyre2. This coverage was more elevated than swiss-model predicted (86%). The present study suggested that both genes are responsible for the genesis of cycloartenol in these mangrove plants.

High-throughput amplicon sequencing and stream benthic bacteria: identifying the best taxonomic level for multiple-stressor research

PubMed Central

Salis, R. K.; Bruder, A.; Piggott, J. J.; Summerfield, T. C.; Matthaei, C. D.

2017-01-01

Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Advances in molecular techniques allow studying microbial communities in situ and with high taxonomic resolution. However, the taxonomic level which provides the best trade-off between our ability to detect multiple-stressor effects versus the goal of studying entire communities remains unknown. We used outdoor mesocosms simulating small streams to investigate the effects of four agricultural stressors (nutrient enrichment, the nitrification inhibitor dicyandiamide (DCD), fine sediment and flow velocity reduction) on stream bacteria (phyla, orders, genera, and species represented by Operational Taxonomic Units with 97% sequence similarity). Community composition was assessed using amplicon sequencing (16S rRNA gene, V3-V4 region). DCD was the most pervasive stressor, affecting evenness and most abundant taxa, followed by sediment and flow velocity. Stressor pervasiveness was similar across taxonomic levels and lower levels did not perform better in detecting stressor effects. Community coverage decreased from 96% of all sequences for abundant phyla to 28% for species. Order-level responses were generally representative of responses of corresponding genera and species, suggesting that this level may represent the best compromise between stressor sensitivity and coverage of bacterial communities. PMID:28327636

Evaluating Variant Calling Tools for Non-Matched Next-Generation Sequencing Data

NASA Astrophysics Data System (ADS)

Sandmann, Sarah; de Graaf, Aniek O.; Karimi, Mohsen; van der Reijden, Bert A.; Hellström-Lindberg, Eva; Jansen, Joop H.; Dugas, Martin

2017-02-01

Valid variant calling results are crucial for the use of next-generation sequencing in clinical routine. However, there are numerous variant calling tools that usually differ in algorithms, filtering strategies, recommendations and thus, also in the output. We evaluated eight open-source tools regarding their ability to call single nucleotide variants and short indels with allelic frequencies as low as 1% in non-matched next-generation sequencing data: GATK HaplotypeCaller, Platypus, VarScan, LoFreq, FreeBayes, SNVer, SAMtools and VarDict. We analysed two real datasets from patients with myelodysplastic syndrome, covering 54 Illumina HiSeq samples and 111 Illumina NextSeq samples. Mutations were validated by re-sequencing on the same platform, on a different platform and expert based review. In addition we considered two simulated datasets with varying coverage and error profiles, covering 50 samples each. In all cases an identical target region consisting of 19 genes (42,322 bp) was analysed. Altogether, no tool succeeded in calling all mutations. High sensitivity was always accompanied by low precision. Influence of varying coverages- and background noise on variant calling was generally low. Taking everything into account, VarDict performed best. However, our results indicate that there is a need to improve reproducibility of the results in the context of multithreading.

A high-coverage draft genome of the mycalesine butterfly Bicyclus anynana.

PubMed

Nowell, Reuben W; Elsworth, Ben; Oostra, Vicencio; Zwaan, Bas J; Wheat, Christopher W; Saastamoinen, Marjo; Saccheri, Ilik J; Van't Hof, Arjen E; Wasik, Bethany R; Connahs, Heidi; Aslam, Muhammad L; Kumar, Sujai; Challis, Richard J; Monteiro, Antónia; Brakefield, Paul M; Blaxter, Mark

2017-07-01

The mycalesine butterfly Bicyclus anynana, the "Squinting bush brown," is a model organism in the study of lepidopteran ecology, development, and evolution. Here, we present a draft genome sequence for B. anynana to serve as a genomics resource for current and future studies of this important model species. Seven libraries with insert sizes ranging from 350 bp to 20 kb were constructed using DNA from an inbred female and sequenced using both Illumina and PacBio technology; 128 Gb of raw Illumina data was filtered to 124 Gb and assembled to a final size of 475 Mb (∼×260 assembly coverage). Contigs were scaffolded using mate-pair, transcriptome, and PacBio data into 10 800 sequences with an N50 of 638 kb (longest scaffold 5 Mb). The genome is comprised of 26% repetitive elements and encodes a total of 22 642 predicted protein-coding genes. Recovery of a BUSCO set of core metazoan genes was almost complete (98%). Overall, these metrics compare well with other recently published lepidopteran genomes. We report a high-quality draft genome sequence for Bicyclus anynana. The genome assembly and annotated gene models are available at LepBase (http://ensembl.lepbase.org/index.html). © The Authors 2017. Published by Oxford University Press.

A high-coverage draft genome of the mycalesine butterfly Bicyclus anynana

PubMed Central

Elsworth, Ben; Oostra, Vicencio; Zwaan, Bas J.; Wheat, Christopher W.; Saastamoinen, Marjo; Saccheri, Ilik J.; van’t Hof, Arjen E.; Wasik, Bethany R.; Connahs, Heidi; Aslam, Muhammad L.; Kumar, Sujai; Challis, Richard J.; Monteiro, Antónia; Brakefield, Paul M.

2017-01-01

Abstract The mycalesine butterfly Bicyclus anynana, the “Squinting bush brown,” is a model organism in the study of lepidopteran ecology, development, and evolution. Here, we present a draft genome sequence for B. anynana to serve as a genomics resource for current and future studies of this important model species. Seven libraries with insert sizes ranging from 350 bp to 20 kb were constructed using DNA from an inbred female and sequenced using both Illumina and PacBio technology; 128 Gb of raw Illumina data was filtered to 124 Gb and assembled to a final size of 475 Mb (∼×260 assembly coverage). Contigs were scaffolded using mate-pair, transcriptome, and PacBio data into 10 800 sequences with an N50 of 638 kb (longest scaffold 5 Mb). The genome is comprised of 26% repetitive elements and encodes a total of 22 642 predicted protein-coding genes. Recovery of a BUSCO set of core metazoan genes was almost complete (98%). Overall, these metrics compare well with other recently published lepidopteran genomes. We report a high-quality draft genome sequence for Bicyclus anynana. The genome assembly and annotated gene models are available at LepBase (http://ensembl.lepbase.org/index.html). PMID:28486658

Fast inner-volume imaging of the lumbar spine with a spatially focused excitation using a 3D-TSE sequence.

PubMed

Riffel, Philipp; Michaely, Henrik J; Morelli, John N; Paul, Dominik; Kannengiesser, Stephan; Schoenberg, Stefan O; Haneder, Stefan

2015-04-01

The purpose of this study was to evaluate the feasibility and technical quality of a zoomed three-dimensional (3D) turbo spin-echo (TSE) sampling perfection with application optimized contrasts using different flip-angle evolutions (SPACE) sequence of the lumbar spine. In this prospective feasibility study, nine volunteers underwent a 3-T magnetic resonance examination of the lumbar spine including 1) a conventional 3D T2-weighted (T2w) SPACE sequence with generalized autocalibrating partially parallel acquisition technique acceleration factor 2 and 2) a zoomed 3D T2w SPACE sequence with a reduced field of view (reduction factor 2). Images were evaluated with regard to image sharpness, signal homogeneity, and the presence of artifacts by two experienced radiologists. For quantitative analysis, signal-to-noise ratio (SNR) values were calculated. Image sharpness of anatomic structures was statistically significantly greater with zoomed SPACE (P < .0001), whereas the signal homogeneity was statistically significantly greater with conventional SPACE (cSPACE; P = .0003). There were no statistically significant differences in extent of artifacts. Acquisition times were 8:20 minutes for cSPACE and 6:30 minutes for zoomed SPACE. Readers 1 and 2 selected zSPACE as the preferred sequence in five of nine cases. In two of nine cases, both sequences were rated as equally preferred by both the readers. SNR values were statistically significantly greater with cSPACE. In comparison to a cSPACE sequences, zoomed SPACE imaging of the lumbar spine provides sharper images in conjunction with a 25% reduction in acquisition time. Copyright © 2015 AUR. Published by Elsevier Inc. All rights reserved.

Use case scenarios - space based receiver assessment : GPS Adjacent Band Compatibility Assessment Workshop VI.

DOT National Transportation Integrated Search

2017-03-30

This workshop presentation discusses space based receiver applications, NASA developed receivers, the TriG receiver, on-orbit assessment parameters, the TriG GNSS-RO antenna gain pattern, the GNSS-RO antenna beam Earth grazing coverage area, assessme...

Coverage Bias and Sensitivity of Variant Calling for Four Whole-genome Sequencing Technologies

PubMed Central

Lasitschka, Bärbel; Jones, David; Northcott, Paul; Hutter, Barbara; Jäger, Natalie; Kool, Marcel; Taylor, Michael; Lichter, Peter; Pfister, Stefan; Wolf, Stephan; Brors, Benedikt; Eils, Roland

2013-01-01

The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina’s HiSeq2000, Life Technologies’ SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics’ technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms with Sanger sequencing, which is prohibitively expensive for whole genome studies. Here we present a detailed comparison of the performance of all currently available whole genome sequencing platforms, especially regarding their ability to call SNVs and to evenly cover the genome and specific genomic regions. Unlike earlier studies, we base our comparison on four different samples, allowing us to assess the between-sample variation of the platforms. We find a pronounced GC bias in GC-rich regions for Life Technologies’ platforms, with Complete Genomics performing best here, while we see the least bias in GC-poor regions for HiSeq2000 and 5500xl. HiSeq2000 gives the most uniform coverage and displays the least sample-to-sample variation. In contrast, Complete Genomics exhibits by far the smallest fraction of bases not covered, while the SOLiD platforms reveal remarkable shortcomings, especially in covering CpG islands. When comparing the performance of the four platforms for calling SNPs, HiSeq2000 and Complete Genomics achieve the highest sensitivity, while the SOLiD platforms show the lowest false positive rate. Finally, we find that integrating sequencing data from different platforms offers the potential to combine the strengths of different technologies. In summary, our results detail the strengths and weaknesses of all four whole-genome sequencing platforms. It indicates application areas that call for a specific sequencing platform and disallow other platforms. This helps to identify the proper sequencing platform for whole genome studies with different application scopes. PMID:23776689

Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens.

PubMed

Staats, Martijn; Erkens, Roy H J; van de Vossenberg, Bart; Wieringa, Jan J; Kraaijeveld, Ken; Stielow, Benjamin; Geml, József; Richardson, James E; Bakker, Freek T

2013-01-01

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.

Analysis of BAC end sequences in oak, a keystone forest tree species, providing insight into the composition of its genome

PubMed Central

2011-01-01

Background One of the key goals of oak genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of forests to increase their health and productivity. Deep-coverage large-insert genomic libraries are a crucial tool for attaining this objective. We report herein the construction of a BAC library for Quercus robur, its characterization and an analysis of BAC end sequences. Results The EcoRI library generated consisted of 92,160 clones, 7% of which had no insert. Levels of chloroplast and mitochondrial contamination were below 3% and 1%, respectively. Mean clone insert size was estimated at 135 kb. The library represents 12 haploid genome equivalents and, the likelihood of finding a particular oak sequence of interest is greater than 99%. Genome coverage was confirmed by PCR screening of the library with 60 unique genetic loci sampled from the genetic linkage map. In total, about 20,000 high-quality BAC end sequences (BESs) were generated by sequencing 15,000 clones. Roughly 5.88% of the combined BAC end sequence length corresponded to known retroelements while ab initio repeat detection methods identified 41 additional repeats. Collectively, characterized and novel repeats account for roughly 8.94% of the genome. Further analysis of the BESs revealed 1,823 putative genes suggesting at least 29,340 genes in the oak genome. BESs were aligned with the genome sequences of Arabidopsis thaliana, Vitis vinifera and Populus trichocarpa. One putative collinear microsyntenic region encoding an alcohol acyl transferase protein was observed between oak and chromosome 2 of V. vinifera. Conclusions This BAC library provides a new resource for genomic studies, including SSR marker development, physical mapping, comparative genomics and genome sequencing. BES analysis provided insight into the structure of the oak genome. These sequences will be used in the assembly of a future genome sequence for oak. PMID:21645357

LSST Operations Simulator

NASA Astrophysics Data System (ADS)

Cook, K. H.; Delgado, F.; Miller, M.; Saha, A.; Allsman, R.; Pinto, P.; Gee, P. A.

2005-12-01

We have developed an operations simulator for LSST and used it to explore design and operations parameter space for this large etendue telescope and its ten year survey mission. The design is modular, with separate science programs coded in separate modules. There is a sophisticated telescope module with all motions parametrized for ease of testing different telescope capabilities, e.g. effect of acceleration capabilities of various motors on science output. Sky brightness is calculated as a function of moon phase and separation. A sophisticated exposure time calculator has been developed for LSST which is being incorporated into the simulator to allow specification of S/N requirements. All important parameters for the telescope, the site and the science programs are easily accessible in configuration files. Seeing and cloud data from the three candidate LSST sites are used for our simulations. The simulator has two broad categories of science proposals: sky coverage and transient events. Sky coverage proposals base their observing priorities on a required number of observations for each field in a particular filter with specified conditions (maximum seeing, sky brightness, etc) and one is used for a weak lensing investigation. Transient proposals are highly configurable. A transient proposal can require sequential, multiple exposures in various filters with a specified sequence of filters, and require a particular cadence for multiple revisits to complete an observation sequence. Each science proposal ranks potential observations based upon the internal logic of that proposal. We present the results of a variety of mixed science program observing simulations, showing how varied programs can be carried out simultaneously, with many observations serving multiple science goals. The simulator has shown that LSST can carry out its multiple missions under a variety of conditions. KHC's work was performed under the auspices of the US DOE, NNSA by the Univ. of California, LLNL under contract No. W-7405-Eng-48.

Fast lossless compression via cascading Bloom filters

PubMed Central

2014-01-01

Background Data from large Next Generation Sequencing (NGS) experiments present challenges both in terms of costs associated with storage and in time required for file transfer. It is sometimes possible to store only a summary relevant to particular applications, but generally it is desirable to keep all information needed to revisit experimental results in the future. Thus, the need for efficient lossless compression methods for NGS reads arises. It has been shown that NGS-specific compression schemes can improve results over generic compression methods, such as the Lempel-Ziv algorithm, Burrows-Wheeler transform, or Arithmetic Coding. When a reference genome is available, effective compression can be achieved by first aligning the reads to the reference genome, and then encoding each read using the alignment position combined with the differences in the read relative to the reference. These reference-based methods have been shown to compress better than reference-free schemes, but the alignment step they require demands several hours of CPU time on a typical dataset, whereas reference-free methods can usually compress in minutes. Results We present a new approach that achieves highly efficient compression by using a reference genome, but completely circumvents the need for alignment, affording a great reduction in the time needed to compress. In contrast to reference-based methods that first align reads to the genome, we hash all reads into Bloom filters to encode, and decode by querying the same Bloom filters using read-length subsequences of the reference genome. Further compression is achieved by using a cascade of such filters. Conclusions Our method, called BARCODE, runs an order of magnitude faster than reference-based methods, while compressing an order of magnitude better than reference-free methods, over a broad range of sequencing coverage. In high coverage (50-100 fold), compared to the best tested compressors, BARCODE saves 80-90% of the running time while only increasing space slightly. PMID:25252952

Fast lossless compression via cascading Bloom filters.

PubMed

Rozov, Roye; Shamir, Ron; Halperin, Eran

2014-01-01

Data from large Next Generation Sequencing (NGS) experiments present challenges both in terms of costs associated with storage and in time required for file transfer. It is sometimes possible to store only a summary relevant to particular applications, but generally it is desirable to keep all information needed to revisit experimental results in the future. Thus, the need for efficient lossless compression methods for NGS reads arises. It has been shown that NGS-specific compression schemes can improve results over generic compression methods, such as the Lempel-Ziv algorithm, Burrows-Wheeler transform, or Arithmetic Coding. When a reference genome is available, effective compression can be achieved by first aligning the reads to the reference genome, and then encoding each read using the alignment position combined with the differences in the read relative to the reference. These reference-based methods have been shown to compress better than reference-free schemes, but the alignment step they require demands several hours of CPU time on a typical dataset, whereas reference-free methods can usually compress in minutes. We present a new approach that achieves highly efficient compression by using a reference genome, but completely circumvents the need for alignment, affording a great reduction in the time needed to compress. In contrast to reference-based methods that first align reads to the genome, we hash all reads into Bloom filters to encode, and decode by querying the same Bloom filters using read-length subsequences of the reference genome. Further compression is achieved by using a cascade of such filters. Our method, called BARCODE, runs an order of magnitude faster than reference-based methods, while compressing an order of magnitude better than reference-free methods, over a broad range of sequencing coverage. In high coverage (50-100 fold), compared to the best tested compressors, BARCODE saves 80-90% of the running time while only increasing space slightly.

Launch mission summary: Intelsat 5 (F3) Atlas/Centaur-55

NASA Technical Reports Server (NTRS)

1981-01-01

Intelsat 5 (F3) spacecraft, launch vehicle, and mission are described. Information relative to launch windows, flight plan, radar and telemetry coverage, selected trajectory information, and a brief sequence of flight events is provided.

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

Determination of a Screening Metric for High Diversity DNA Libraries.

PubMed

Guido, Nicholas J; Handerson, Steven; Joseph, Elaine M; Leake, Devin; Kung, Li A

2016-01-01

The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synthetic biology allows for libraries with billions of variants, pushing the limits of researchers' ability to qualify libraries for screening by measuring the traditional quality metrics of fidelity and diversity of variants. Instead, when screening variant libraries, researchers typically use a generic, and often insufficient, oversampling rate based on a common rule-of-thumb. We have developed methods to calculate a library-specific oversampling metric, based on fidelity, diversity, and representation of variants, which informs researchers, prior to screening the library, of the amount of oversampling required to ensure that the desired fraction of variant molecules will be sampled. To derive this oversampling metric, we developed a novel alignment tool to efficiently measure frequency counts of individual nucleotide variant positions using next-generation sequencing data. Next, we apply a method based on the "coupon collector" probability theory to construct a curve of upper bound estimates of the sampling size required for any desired variant coverage. The calculated oversampling metric will guide researchers to maximize their efficiency in using highly variant libraries.

Equally parsimonious pathways through an RNA sequence space are not equally likely

NASA Technical Reports Server (NTRS)

Lee, Y. H.; DSouza, L. M.; Fox, G. E.

1997-01-01

An experimental system for determining the potential ability of sequences resembling 5S ribosomal RNA (rRNA) to perform as functional 5S rRNAs in vivo in the Escherichia coli cellular environment was devised previously. Presumably, the only 5S rRNA sequences that would have been fixed by ancestral populations are ones that were functionally valid, and hence the actual historical paths taken through RNA sequence space during 5S rRNA evolution would have most likely utilized valid sequences. Herein, we examine the potential validity of all sequence intermediates along alternative equally parsimonious trajectories through RNA sequence space which connect two pairs of sequences that had previously been shown to behave as valid 5S rRNAs in E. coli. The first trajectory requires a total of four changes. The 14 sequence intermediates provide 24 apparently equally parsimonious paths by which the transition could occur. The second trajectory involves three changes, six intermediate sequences, and six potentially equally parsimonious paths. In total, only eight of the 20 sequence intermediates were found to be clearly invalid. As a consequence of the position of these invalid intermediates in the sequence space, seven of the 30 possible paths consisted of exclusively valid sequences. In several cases, the apparent validity/invalidity of the intermediate sequences could not be anticipated on the basis of current knowledge of the 5S rRNA structure. This suggests that the interdependencies in RNA sequence space may be more complex than currently appreciated. If ancestral sequences predicted by parsimony are to be regarded as actual historical sequences, then the present results would suggest that they should also satisfy a validity requirement and that, in at least limited cases, this conjecture can be tested experimentally.

Detecting false positive sequence homology: a machine learning approach.

PubMed

Fujimoto, M Stanley; Suvorov, Anton; Jensen, Nicholas O; Clement, Mark J; Bybee, Seth M

2016-02-24

Accurate detection of homologous relationships of biological sequences (DNA or amino acid) amongst organisms is an important and often difficult task that is essential to various evolutionary studies, ranging from building phylogenies to predicting functional gene annotations. There are many existing heuristic tools, most commonly based on bidirectional BLAST searches that are used to identify homologous genes and combine them into two fundamentally distinct classes: orthologs and paralogs. Due to only using heuristic filtering based on significance score cutoffs and having no cluster post-processing tools available, these methods can often produce multiple clusters constituting unrelated (non-homologous) sequences. Therefore sequencing data extracted from incomplete genome/transcriptome assemblies originated from low coverage sequencing or produced by de novo processes without a reference genome are susceptible to high false positive rates of homology detection. In this paper we develop biologically informative features that can be extracted from multiple sequence alignments of putative homologous genes (orthologs and paralogs) and further utilized in context of guided experimentation to verify false positive outcomes. We demonstrate that our machine learning method trained on both known homology clusters obtained from OrthoDB and randomly generated sequence alignments (non-homologs), successfully determines apparent false positives inferred by heuristic algorithms especially among proteomes recovered from low-coverage RNA-seq data. Almost ~42 % and ~25 % of predicted putative homologies by InParanoid and HaMStR respectively were classified as false positives on experimental data set. Our process increases the quality of output from other clustering algorithms by providing a novel post-processing method that is both fast and efficient at removing low quality clusters of putative homologous genes recovered by heuristic-based approaches.

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

Synthesis of Volumetric Ring Antenna Array for Terrestrial Coverage Pattern

PubMed Central

Reyna, Alberto; Panduro, Marco A.; Del Rio Bocio, Carlos

2014-01-01

This paper presents a synthesis of a volumetric ring antenna array for a terrestrial coverage pattern. This synthesis regards the spacing among the rings on the planes X-Y, the positions of the rings on the plane X-Z, and uniform and concentric excitations. The optimization is carried out by implementing the particle swarm optimization. The synthesis is compared with previous designs by resulting with proper performance of this geometry to provide an accurate coverage to be applied in satellite applications with a maximum reduction of the antenna hardware as well as the side lobe level reduction. PMID:24701150

Next Generation Fast Neutron Detector for Space Exploration (Mini-FND)

NASA Astrophysics Data System (ADS)

Hassler, D. M.; Ehresmann, B.

2018-02-01

SwRI has developed a miniature Fast Neutron Detector (mini-FND), for use in the Deep Space Gateway, to characterize the neutron albedo radiation. Mini-FND will provide coverage of the biologically relevant neutrons at energies of 500 keV and greater.

The whole genome sequences and experimentally phased haplotypes of over 100 personal genomes.

PubMed

Mao, Qing; Ciotlos, Serban; Zhang, Rebecca Yu; Ball, Madeleine P; Chin, Robert; Carnevali, Paolo; Barua, Nina; Nguyen, Staci; Agarwal, Misha R; Clegg, Tom; Connelly, Abram; Vandewege, Ward; Zaranek, Alexander Wait; Estep, Preston W; Church, George M; Drmanac, Radoje; Peters, Brock A

2016-10-11

Since the completion of the Human Genome Project in 2003, it is estimated that more than 200,000 individual whole human genomes have been sequenced. A stunning accomplishment in such a short period of time. However, most of these were sequenced without experimental haplotype data and are therefore missing an important aspect of genome biology. In addition, much of the genomic data is not available to the public and lacks phenotypic information. As part of the Personal Genome Project, blood samples from 184 participants were collected and processed using Complete Genomics' Long Fragment Read technology. Here, we present the experimental whole genome haplotyping and sequencing of these samples to an average read coverage depth of 100X. This is approximately three-fold higher than the read coverage applied to most whole human genome assemblies and ensures the highest quality results. Currently, 114 genomes from this dataset are freely available in the GigaDB repository and are associated with rich phenotypic data; the remaining 70 should be added in the near future as they are approved through the PGP data release process. For reproducibility analyses, 20 genomes were sequenced at least twice using independent LFR barcoded libraries. Seven genomes were also sequenced using Complete Genomics' standard non-barcoded library process. In addition, we report 2.6 million high-quality, rare variants not previously identified in the Single Nucleotide Polymorphisms database or the 1000 Genomes Project Phase 3 data. These genomes represent a unique source of haplotype and phenotype data for the scientific community and should help to expand our understanding of human genome evolution and function.

A map of human genome variation from population-scale sequencing.

PubMed

Abecasis, Gonçalo R; Altshuler, David; Auton, Adam; Brooks, Lisa D; Durbin, Richard M; Gibbs, Richard A; Hurles, Matt E; McVean, Gil A

2010-10-28

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data set. On average, each person is found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. We demonstrate how these results can be used to inform association and functional studies. From the two trios, we directly estimate the rate of de novo germline base substitution mutations to be approximately 10(-8) per base pair per generation. We explore the data with regard to signatures of natural selection, and identify a marked reduction of genetic variation in the neighbourhood of genes, due to selection at linked sites. These methods and public data will support the next phase of human genetic research.

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

DOE PAGES

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa; ...

2014-09-01

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

Genotype calling from next-generation sequencing data using haplotype information of reads

PubMed Central

Zhi, Degui; Wu, Jihua; Liu, Nianjun; Zhang, Kui

2012-01-01

Motivation: Low coverage sequencing provides an economic strategy for whole genome sequencing. When sequencing a set of individuals, genotype calling can be challenging due to low sequencing coverage. Linkage disequilibrium (LD) based refinement of genotyping calling is essential to improve the accuracy. Current LD-based methods use read counts or genotype likelihoods at individual potential polymorphic sites (PPSs). Reads that span multiple PPSs (jumping reads) can provide additional haplotype information overlooked by current methods. Results: In this article, we introduce a new Hidden Markov Model (HMM)-based method that can take into account jumping reads information across adjacent PPSs and implement it in the HapSeq program. Our method extends the HMM in Thunder and explicitly models jumping reads information as emission probabilities conditional on the states of adjacent PPSs. Our simulation results show that, compared to Thunder, HapSeq reduces the genotyping error rate by 30%, from 0.86% to 0.60%. The results from the 1000 Genomes Project show that HapSeq reduces the genotyping error rate by 12 and 9%, from 2.24% and 2.76% to 1.97% and 2.50% for individuals with European and African ancestry, respectively. We expect our program can improve genotyping qualities of the large number of ongoing and planned whole genome sequencing projects. Contact: dzhi@ms.soph.uab.edu; kzhang@ms.soph.uab.edu Availability: The software package HapSeq and its manual can be found and downloaded at www.ssg.uab.edu/hapseq/. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22285565

A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases.

PubMed

Wilson, Kitchener D; Shen, Peidong; Fung, Eula; Karakikes, Ioannis; Zhang, Angela; InanlooRahatloo, Kolsoum; Odegaard, Justin; Sallam, Karim; Davis, Ronald W; Lui, George K; Ashley, Euan A; Scharfe, Curt; Wu, Joseph C

2015-09-11

Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming. We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost. We used double-stranded probes (complementary long padlock probes), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample. Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery. © 2015 American Heart Association, Inc.

Use of an informed search space maximizes confidence of site-specific assignment of glycoprotein glycosylation.

PubMed

Khatri, Kshitij; Klein, Joshua A; Zaia, Joseph

2017-01-01

In order to interpret glycopeptide tandem mass spectra, it is necessary to estimate the theoretical glycan compositions and peptide sequences, known as the search space. The simplest way to do this is to build a naïve search space from sets of glycan compositions from public databases and to assume that the target glycoprotein is pure. Often, however, purified glycoproteins contain co-purified glycoprotein contaminants that have the potential to confound assignment of tandem mass spectra based on naïve assumptions. In addition, there is increasing need to characterize glycopeptides from complex biological mixtures. Fortunately, liquid chromatography-mass spectrometry (LC-MS) methods for glycomics and proteomics are now mature and accessible. We demonstrate the value of using an informed search space built from measured glycomes and proteomes to define the search space for interpretation of glycoproteomics data. We show this using α-1-acid glycoprotein (AGP) mixed into a set of increasingly complex matrices. As the mixture complexity increases, the naïve search space balloons and the ability to assign glycopeptides with acceptable confidence diminishes. In addition, it is not possible to identify glycopeptides not foreseen as part of the naïve search space. A search space built from released glycan glycomics and proteomics data is smaller than its naïve counterpart while including the full range of proteins detected in the mixture. This maximizes the ability to assign glycopeptide tandem mass spectra with confidence. As the mixture complexity increases, the number of tandem mass spectra per glycopeptide precursor ion decreases, resulting in lower overall scores and reduced depth of coverage for the target glycoprotein. We suggest use of α-1-acid glycoprotein as a standard to gauge effectiveness of analytical methods and bioinformatics search parameters for glycoproteomics studies. Graphical Abstract Assignment of site specific glycosylation from LC-tandemMS data.

Influenza Research Database: an integrated bioinformatics resource for influenza research and surveillance

PubMed Central

Squires, R. Burke; Noronha, Jyothi; Hunt, Victoria; García‐Sastre, Adolfo; Macken, Catherine; Baumgarth, Nicole; Suarez, David; Pickett, Brett E.; Zhang, Yun; Larsen, Christopher N.; Ramsey, Alvin; Zhou, Liwei; Zaremba, Sam; Kumar, Sanjeev; Deitrich, Jon; Klem, Edward; Scheuermann, Richard H.

2012-01-01

Please cite this paper as: Squires et al. (2012) Influenza research database: an integrated bioinformatics resource for influenza research and surveillance. Influenza and Other Respiratory Viruses 6(6), 404–416. Background  The recent emergence of the 2009 pandemic influenza A/H1N1 virus has highlighted the value of free and open access to influenza virus genome sequence data integrated with information about other important virus characteristics. Design  The Influenza Research Database (IRD, http://www.fludb.org) is a free, open, publicly‐accessible resource funded by the U.S. National Institute of Allergy and Infectious Diseases through the Bioinformatics Resource Centers program. IRD provides a comprehensive, integrated database and analysis resource for influenza sequence, surveillance, and research data, including user‐friendly interfaces for data retrieval, visualization and comparative genomics analysis, together with personal log in‐protected ‘workbench’ spaces for saving data sets and analysis results. IRD integrates genomic, proteomic, immune epitope, and surveillance data from a variety of sources, including public databases, computational algorithms, external research groups, and the scientific literature. Results  To demonstrate the utility of the data and analysis tools available in IRD, two scientific use cases are presented. A comparison of hemagglutinin sequence conservation and epitope coverage information revealed highly conserved protein regions that can be recognized by the human adaptive immune system as possible targets for inducing cross‐protective immunity. Phylogenetic and geospatial analysis of sequences from wild bird surveillance samples revealed a possible evolutionary connection between influenza virus from Delaware Bay shorebirds and Alberta ducks. Conclusions  The IRD provides a wealth of integrated data and information about influenza virus to support research of the genetic determinants dictating virus pathogenicity, host range restriction and transmission, and to facilitate development of vaccines, diagnostics, and therapeutics. PMID:22260278

Eclipse Photo/Video Coverage

NASA Image and Video Library

2017-08-21

On Monday, Aug. 21, NASA provided coast-to-coast coverage of the solar eclipse across America – featuring views of the phenomenon from unique vantage points, including from the ground, from aircraft, and from spacecraft including the ISS, during a live broadcast seen on NASA Television and the agency’s website. This is footage from the Kennedy Space Center Visitor Complex, KARS Park at Kennedy, and the Vehicle Assembly Building.

Gene and translation initiation site prediction in metagenomic sequences

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

Hyatt, Philip Douglas; LoCascio, Philip F; Hauser, Loren John

2012-01-01

Gene prediction in metagenomic sequences remains a difficult problem. Current sequencing technologies do not achieve sufficient coverage to assemble the individual genomes in a typical sample; consequently, sequencing runs produce a large number of short sequences whose exact origin is unknown. Since these sequences are usually smaller than the average length of a gene, algorithms must make predictions based on very little data. We present MetaProdigal, a metagenomic version of the gene prediction program Prodigal, that can identify genes in short, anonymous coding sequences with a high degree of accuracy. The novel value of the method consists of enhanced translationmore » initiation site identification, ability to identify sequences that use alternate genetic codes and confidence values for each gene call. We compare the results of MetaProdigal with other methods and conclude with a discussion of future improvements.« less

Influence of Layup Sequence on the Surface Accuracy of Carbon Fiber Composite Space Mirrors

NASA Astrophysics Data System (ADS)

Yang, Zhiyong; Liu, Qingnian; Zhang, Boming; Xu, Liang; Tang, Zhanwen; Xie, Yongjie

2018-04-01

Layup sequence is directly related to stiffness and deformation resistance of the composite space mirror, and error caused by layup sequence can affect the surface precision of composite mirrors evidently. Variation of layup sequence with the same total thickness of composite space mirror changes surface form of the composite mirror, which is the focus of our study. In our research, the influence of varied quasi-isotropic stacking sequences and random angular deviation on the surface accuracy of composite space mirrors was investigated through finite element analyses (FEA). We established a simulation model for the studied concave mirror with 500 mm diameter, essential factors of layup sequences and random angular deviations on different plies were discussed. Five guiding findings were described in this study. Increasing total plies, optimizing stacking sequence and keeping consistency of ply alignment in ply placement are effective to improve surface accuracy of composite mirror.

Footprint Database and web services for the Herschel space observatory

NASA Astrophysics Data System (ADS)

Verebélyi, Erika; Dobos, László; Kiss, Csaba

2015-08-01

Using all telemetry and observational meta-data, we created a searchable database of Herschel observation footprints. Data from the Herschel space observatory is freely available for everyone but no uniformly processed catalog of all observations has been published yet. As a first step, we unified the data model for all three Herschel instruments in all observation modes and compiled a database of sky coverage information. As opposed to methods using a pixellation of the sphere, in our database, sky coverage is stored in exact geometric form allowing for precise area calculations. Indexing of the footprints allows for very fast search among observations based on pointing, time, sky coverage overlap and meta-data. This enables us, for example, to find moving objects easily in Herschel fields. The database is accessible via a web site and also as a set of REST web service functions which makes it usable from program clients like Python or IDL scripts. Data is available in various formats including Virtual Observatory standards.

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.

An all-reflective wide-angle flat-field telescope for space

NASA Technical Reports Server (NTRS)

Hallam, K. L.; Howell, B. J.; Wilson, M. E.

1984-01-01

An all-reflective wide-angle flat-field telescope (WAFFT) designed and built at Goddard Space Flight Center demonstrates the markedly improved wide-angle imaging capability which can be achieved with a design based on a recently announced class of unobscured 3-mirror optical systems. Astronomy and earth observation missions in space dictate the necessity or preference for wide-angle all-reflective systems which can provide UV through IR wavelength coverage and tolerate the space environment. An initial prototype unit has been designed to meet imaging requirements suitable for monitoring the ultraviolet sky from space. The unobscured f/4, 36 mm efl system achieves a full 20 x 30 deg field of view with resolution over a flat focal surface that is well matched for use with advanced ultraviolet image array detectors. Aspects of the design and fabrication approach, which have especially important bearing on the system solution, are reviewed; and test results are compared with the analytic performance predictions. Other possible applications of the WAFFT class of imaging system are briefly discussed. The exceptional wide-angle, high quality resolution, and very wide spectral coverage of the WAFFT-type optical system could make it a very important tool for future space research.

Space Materials Handbook. 3rd; ed.

NASA Technical Reports Server (NTRS)

Rittenhouse, John B.; Singletary, John B.

1969-01-01

This edition is the result of an extensive revision and reworking of the second edition of the Space Materials Handbook along with the incorporation of entirely new subject matter coverage and new materials data. All of the most significant material, phenomena, properties, and principles covered in the original Handbook are presented and expanded in this revised and updated version. However, treatment of theoretical aspects has been condensed in order that more emphasis could be placed on the extensive new materials knowledge and data obtained from the design and successful launching of a wide variety of space systems. The handbook is organized into four parts, namely: space environment, effect of space environment on materials, materials in space, and biological interaction with spacecraft materials. Information on mechanical, physical, and chemical properties and characteristics is given for a wide variety of metallic and nonmetallic materials. The effects of natural and induced environments on materials are appraised. Materials categories include coverage of thermal control materials, optical materials, adhesives, organic structural materials, inorganic structural materials, electronic components and materials, materials for sealing applications, and lubrication materials. In addition, a comprehensive multiple citation index is incorporated which gives ready access to information on specific subject areas with regard to their locations within the Handbook.

[Microclimate and comfortable degree of Shanghai urban open spaces in summer].

PubMed

Cao, Dan; Zhou, Li-chen; Mao, Yi-wei; Li, Yin; Liu, Yi-ning; Wang, Tian-hou

2008-08-01

Based on the observation data of air temperature, relative humidity, wind speed, and solar radiation from May to August 2006, the regulation effects of five types of open spaces (square, fountain, grassplot, corridor, and woodland) in Shanghai urban districts on the microclimate were analyzed, and discomfort index (DI) was introduced to evaluate the effects of these five types of open spaces on human body' s comfortable degree. The results showed that there existed definite differences in the air temperature and relative humidity among the open spaces, with the mean temperature decreased in the order of square > grassplot > fountain > corridor > woodland, and the mean relative humidity decreased in the order of woodland > corridor > fountain > grassplot > square. The area of the square, the wind speed and direction near the fountain, the grass species on the grass-plot, the width and tree coverage of the corridor, and the tree coverage and canopy height of the woodland had significant correlations with the microclimate parameters of corresponding open spaces. Comparing with other three types of open spaces, woodland and corridor had better regulation effects on the microclimate via shading, decreasing air temperature, and increasing relative humidity.

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC

PubMed Central

Walter, Vonn; Patel, Nirali M.; Eberhard, David A.; Hayward, Michele C.; Salazar, Ashley H.; Jo, Heejoon; Soloway, Matthew G.; Wilkerson, Matthew D.; Parker, Joel S.; Yin, Xiaoying; Zhang, Guosheng; Siegel, Marni B.; Rosson, Gary B.; Earp, H. Shelton; Sharpless, Norman E.; Gulley, Margaret L.; Weck, Karen E.

2015-01-01

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion. PMID:26076459

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

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

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.

Choice of Reference Sequence and Assembler for Alignment of Listeria monocytogenes Short-Read Sequence Data Greatly Influences Rates of Error in SNP Analyses

PubMed Central

Pightling, Arthur W.; Petronella, Nicholas; Pagotto, Franco

2014-01-01

The wide availability of whole-genome sequencing (WGS) and an abundance of open-source software have made detection of single-nucleotide polymorphisms (SNPs) in bacterial genomes an increasingly accessible and effective tool for comparative analyses. Thus, ensuring that real nucleotide differences between genomes (i.e., true SNPs) are detected at high rates and that the influences of errors (such as false positive SNPs, ambiguously called sites, and gaps) are mitigated is of utmost importance. The choices researchers make regarding the generation and analysis of WGS data can greatly influence the accuracy of short-read sequence alignments and, therefore, the efficacy of such experiments. We studied the effects of some of these choices, including: i) depth of sequencing coverage, ii) choice of reference-guided short-read sequence assembler, iii) choice of reference genome, and iv) whether to perform read-quality filtering and trimming, on our ability to detect true SNPs and on the frequencies of errors. We performed benchmarking experiments, during which we assembled simulated and real Listeria monocytogenes strain 08-5578 short-read sequence datasets of varying quality with four commonly used assemblers (BWA, MOSAIK, Novoalign, and SMALT), using reference genomes of varying genetic distances, and with or without read pre-processing (i.e., quality filtering and trimming). We found that assemblies of at least 50-fold coverage provided the most accurate results. In addition, MOSAIK yielded the fewest errors when reads were aligned to a nearly identical reference genome, while using SMALT to align reads against a reference sequence that is ∼0.82% distant from 08-5578 at the nucleotide level resulted in the detection of the greatest numbers of true SNPs and the fewest errors. Finally, we show that whether read pre-processing improves SNP detection depends upon the choice of reference sequence and assembler. In total, this study demonstrates that researchers should test a variety of conditions to achieve optimal results. PMID:25144537

Ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry for peptide de novo amino acid sequencing for a seven-protein mixture by paired single-residue transposed Lys-N and Lys-C digestion.

PubMed

Guan, Xiaoyan; Brownstein, Naomi C; Young, Nicolas L; Marshall, Alan G

2017-01-30

Bottom-up tandem mass spectrometry (MS/MS) is regularly used in proteomics to identify proteins from a sequence database. De novo sequencing is also available for sequencing peptides with relatively short sequence lengths. We recently showed that paired Lys-C and Lys-N proteases produce peptides of identical mass and similar retention time, but different tandem mass spectra. Such parallel experiments provide complementary information, and allow for up to 100% MS/MS sequence coverage. Here, we report digestion by paired Lys-C and Lys-N proteases of a seven-protein mixture: human hemoglobin alpha, bovine carbonic anhydrase 2, horse skeletal muscle myoglobin, hen egg white lysozyme, bovine pancreatic ribonuclease, bovine rhodanese, and bovine serum albumin, followed by reversed-phase nanoflow liquid chromatography, collision-induced dissociation, and 14.5 T Fourier transform ion cyclotron resonance mass spectrometry. Matched pairs of product peptide ions of equal precursor mass and similar retention times from each digestion are compared, leveraging single-residue transposed information with independent interferences to confidently identify fragment ion types, residues, and peptides. Selected pairs of product ion mass spectra for de novo sequenced protein segments from each member of the mixture are presented. Pairs of the transposed product ions as well as complementary information from the parallel experiments allow for both high MS/MS coverage for long peptide sequences and high confidence in the amino acid identification. Moreover, the parallel experiments in the de novo sequencing reduce false-positive matches of product ions from the single-residue transposed peptides from the same segment, and thereby further improve the confidence in protein identification. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Value of a single-shot turbo spin-echo pulse sequence for assessing the architecture of the subarachnoid space and the constitutive nature of cerebrospinal fluid.

PubMed

Pease, Anthony; Sullivan, Stacey; Olby, Natasha; Galano, Heather; Cerda-Gonzalez, Sophia; Robertson, Ian D; Gavin, Patrick; Thrall, Donald

2006-01-01

Three case history reports are presented to illustrate the value of the single-shot turbo spin-echo pulse sequence for assessment of the subarachnoid space. The use of the single-shot turbo spin-echo pulse sequence, which is a heavily T2-weighted sequence, allows for a rapid, noninvasive evaluation of the subarachnoid space by using the high signal from cerebrospinal fluid. This sequence can be completed in seconds rather than the several minutes required for a T2-fast spin-echo sequence. Unlike the standard T2-fast spin-echo sequence, a single-shot turbo spin-echo pulse sequence also provides qualitative information about the protein and the cellular content of the cerebrospinal fluid, such as in patients with inflammatory debris or hemorrhage in the cerebrospinal fluid. Although the resolution of the single-shot turbo spin-echo pulse sequence images is relatively poor compared with more conventional sequences, the qualitative information about the subarachnoid space and cerebrospinal fluid and the rapid acquisition time, make it a useful sequence to include in standard protocols of spinal magnetic resonance imaging.

Maintaining and Enhancing Diversity of Sampled Protein Conformations in Robotics-Inspired Methods.

PubMed

Abella, Jayvee R; Moll, Mark; Kavraki, Lydia E

2018-01-01

The ability to efficiently sample structurally diverse protein conformations allows one to gain a high-level view of a protein's energy landscape. Algorithms from robot motion planning have been used for conformational sampling, and several of these algorithms promote diversity by keeping track of "coverage" in conformational space based on the local sampling density. However, large proteins present special challenges. In particular, larger systems require running many concurrent instances of these algorithms, but these algorithms can quickly become memory intensive because they typically keep previously sampled conformations in memory to maintain coverage estimates. In addition, robotics-inspired algorithms depend on defining useful perturbation strategies for exploring the conformational space, which is a difficult task for large proteins because such systems are typically more constrained and exhibit complex motions. In this article, we introduce two methodologies for maintaining and enhancing diversity in robotics-inspired conformational sampling. The first method addresses algorithms based on coverage estimates and leverages the use of a low-dimensional projection to define a global coverage grid that maintains coverage across concurrent runs of sampling. The second method is an automatic definition of a perturbation strategy through readily available flexibility information derived from B-factors, secondary structure, and rigidity analysis. Our results show a significant increase in the diversity of the conformations sampled for proteins consisting of up to 500 residues when applied to a specific robotics-inspired algorithm for conformational sampling. The methodologies presented in this article may be vital components for the scalability of robotics-inspired approaches.

Evaluation of Two Highly-Multiplexed Custom Panels for Massively Parallel Semiconductor Sequencing on Paraffin DNA

PubMed Central

Kotoula, Vassiliki; Lyberopoulou, Aggeliki; Papadopoulou, Kyriaki; Charalambous, Elpida; Alexopoulou, Zoi; Gakou, Chryssa; Lakis, Sotiris; Tsolaki, Eleftheria; Lilakos, Konstantinos; Fountzilas, George

2015-01-01

Background—Aim Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA. Methods Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible. Results Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman’s rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8–99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1–95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3–76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality. Conclusions Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed. PMID:26039550

Data mining of enzymes using specific peptides

PubMed Central

2009-01-01

Background Predicting the function of a protein from its sequence is a long-standing challenge of bioinformatic research, typically addressed using either sequence-similarity or sequence-motifs. We employ the novel motif method that consists of Specific Peptides (SPs) that are unique to specific branches of the Enzyme Commission (EC) functional classification. We devise the Data Mining of Enzymes (DME) methodology that allows for searching SPs on arbitrary proteins, determining from its sequence whether a protein is an enzyme and what the enzyme's EC classification is. Results We extract novel SP sets from Swiss-Prot enzyme data. Using a training set of July 2006, and test sets of July 2008, we find that the predictive power of SPs, both for true-positives (enzymes) and true-negatives (non-enzymes), depends on the coverage length of all SP matches (the number of amino-acids matched on the protein sequence). DME is quite different from BLAST. Comparing the two on an enzyme test set of July 2008, we find that DME has lower recall. On the other hand, DME can provide predictions for proteins regarded by BLAST as having low homologies with known enzymes, thus supplying complementary information. We test our method on a set of proteins belonging to 10 bacteria, dated July 2008, establishing the usefulness of the coverage-length cutoff to determine true-negatives. Moreover, sifting through our predictions we find that some of them have been substantiated by Swiss-Prot annotations by July 2009. Finally we extract, for production purposes, a novel SP set trained on all Swiss-Prot enzymes as of July 2009. This new set increases considerably the recall of DME. The new SP set is being applied to three metagenomes: Sargasso Sea with over 1,000,000 proteins, producing predictions of over 220,000 enzymes, and two human gut metagenomes. The outcome of these analyses can be characterized by the enzymatic profile of the metagenomes, describing the relative numbers of enzymes observed for different EC categories. Conclusions Employing SPs for predicting enzymatic activity of proteins works well once one utilizes coverage-length criteria. In our analysis, L ≥ 7 has led to highly accurate results. PMID:20034383

A second-generation anchored genetic linkage map of the tammar wallaby (Macropus eugenii)

PubMed Central

2011-01-01

Background The tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species. Results A second-generation anchored tammar wallaby genetic linkage map has been constructed based on a total of 148 loci. The linkage map contains the original 64 loci included in the first-generation map, plus an additional 84 microsatellite loci that were chosen specifically to increase coverage and assist with the anchoring and orientation of linkage groups to chromosomes. These additional loci were derived from (a) sequenced BAC clones that had been previously mapped to tammar wallaby chromosomes by fluorescence in situ hybridization (FISH), (b) End sequence from BACs subsequently FISH-mapped to tammar wallaby chromosomes, and (c) tammar wallaby genes orthologous to opossum genes predicted to fill gaps in the tammar wallaby linkage map as well as three X-linked markers from a published study. Based on these 148 loci, eight linkage groups were formed. These linkage groups were assigned (via FISH-mapped markers) to all seven autosomes and the X chromosome. The sex-pooled map size is 1402.4 cM, which is estimated to provide 82.6% total coverage of the genome, with an average interval distance of 10.9 cM between adjacent markers. The overall ratio of female/male map length is 0.84, which is comparable to the ratio of 0.78 obtained for the first-generation map. Conclusions Construction of this second-generation genetic linkage map is a significant step towards complete coverage of the tammar wallaby genome and considerably extends that of the first-generation map. It will be a valuable resource for ongoing tammar wallaby genetic research and assembling the genome sequence. The sex-pooled map is available online at http://compldb.angis.org.au/. PMID:21854616

A second-generation anchored genetic linkage map of the tammar wallaby (Macropus eugenii).

PubMed

Wang, Chenwei; Webley, Lee; Wei, Ke-jun; Wakefield, Matthew J; Patel, Hardip R; Deakin, Janine E; Alsop, Amber; Marshall Graves, Jennifer A; Cooper, Desmond W; Nicholas, Frank W; Zenger, Kyall R

2011-08-19

The tammar wallaby, Macropus eugenii, a small kangaroo used for decades for studies of reproduction and metabolism, is the model Australian marsupial for genome sequencing and genetic investigations. The production of a more comprehensive cytogenetically-anchored genetic linkage map will significantly contribute to the deciphering of the tammar wallaby genome. It has great value as a resource to identify novel genes and for comparative studies, and is vital for the ongoing genome sequence assembly and gene ordering in this species. A second-generation anchored tammar wallaby genetic linkage map has been constructed based on a total of 148 loci. The linkage map contains the original 64 loci included in the first-generation map, plus an additional 84 microsatellite loci that were chosen specifically to increase coverage and assist with the anchoring and orientation of linkage groups to chromosomes. These additional loci were derived from (a) sequenced BAC clones that had been previously mapped to tammar wallaby chromosomes by fluorescence in situ hybridization (FISH), (b) End sequence from BACs subsequently FISH-mapped to tammar wallaby chromosomes, and (c) tammar wallaby genes orthologous to opossum genes predicted to fill gaps in the tammar wallaby linkage map as well as three X-linked markers from a published study. Based on these 148 loci, eight linkage groups were formed. These linkage groups were assigned (via FISH-mapped markers) to all seven autosomes and the X chromosome. The sex-pooled map size is 1402.4 cM, which is estimated to provide 82.6% total coverage of the genome, with an average interval distance of 10.9 cM between adjacent markers. The overall ratio of female/male map length is 0.84, which is comparable to the ratio of 0.78 obtained for the first-generation map. Construction of this second-generation genetic linkage map is a significant step towards complete coverage of the tammar wallaby genome and considerably extends that of the first-generation map. It will be a valuable resource for ongoing tammar wallaby genetic research and assembling the genome sequence. The sex-pooled map is available online at http://compldb.angis.org.au/.

Genomic DNA sequence and cytosine methylation changes of adult rice leaves after seeds space flight

NASA Astrophysics Data System (ADS)

Shi, Jinming

In this study, cytosine methylation on CCGG site and genomic DNA sequence changes of adult leaves of rice after seeds space flight were detected by methylation-sensitive amplification polymorphism (MSAP) and Amplified fragment length polymorphism (AFLP) technique respectively. Rice seeds were planted in the trial field after 4 days space flight on the shenzhou-6 Spaceship of China. Adult leaves of space-treated rice including 8 plants chosen randomly and 2 plants with phenotypic mutation were used for AFLP and MSAP analysis. Polymorphism of both DNA sequence and cytosine methylation were detected. For MSAP analysis, the average polymorphic frequency of the on-ground controls, space-treated plants and mutants are 1.3%, 3.1% and 11% respectively. For AFLP analysis, the average polymorphic frequencies are 1.4%, 2.9%and 8%respectively. Total 27 and 22 polymorphic fragments were cloned sequenced from MSAP and AFLP analysis respectively. Nine of the 27 fragments from MSAP analysis show homology to coding sequence. For the 22 polymorphic fragments from AFLP analysis, no one shows homology to mRNA sequence and eight fragments show homology to repeat region or retrotransposon sequence. These results suggest that although both genomic DNA sequence and cytosine methylation status can be effected by space flight, the genomic region homology to the fragments from genome DNA and cytosine methylation analysis were different.

Global ice and land climate studies using scatterometer image data

NASA Astrophysics Data System (ADS)

Long, David G.; Drinkwater, Mark R.; Holt, Benjamin; Saatchi, Sasan; Bertoia, Cheryl

Scatterometers have provided continuous synoptic microwave radar coverage of the Earth from space for nearly a decade. NASA launched three scatterometers: the current SeaWinds scatterometer onboard QuikSCAT (QSCAT, 13.4 GHz) launched in 1999; the NASA scatterometer (NSCAT, 14.0 GHz), which flew on the Japanese Space Agency's ADEOS-1 platform during 1996-1997 and the Seasat-A scatterometer system (SASS, 14.6 GHz), which flew in 1978. The European Space Agency's (ESA) 5.3-GHz scatterometer (ESCAT) has been carried onboard both the ERS-1 and ERS-2 satellites since 1991.properties, including the phase state, of a particular surface type. Varying response from the surface also results from different polarizations, viewing angles and orientations, and radar frequencies. The wide swath of scatterometers provides near daily global coverage at intrinsic sensor resolutions that are generally between 25-50 km.

Laboratory test data on the stability of the STIS MAMAs

NASA Technical Reports Server (NTRS)

Joseph, Charles L.

1997-01-01

STIS has two MAMA detectors systems with distinctly different tube configurations. The first (designated BAND 1) has an opaque CsI photocathode deposited on the microchannel plate (MCP) providing wavelength coverage from 1150A to 1700A. The other MAMA (designated BAND 2) has a semitransparent CS2Te photocathode deposited on the faceplate in close proximity to the input of the MCP. It covers the 1650A to 3100A bandpass and serves as a backup for the short wavelength detector. Laboratory test data indicate that both of these detectors have good sensitivity, have good uniformity and provide stable response, making each capable of collecting data with a signal-to-noise ratio in excess of 100 per Space Telescope Imaging Spectrograph (STIS) optical resolution element. Over a multiyear development effort, a substantial body of laboratory test data (more than 6 GBytes spanning more than 6 years of collection) has accumulated on more than a dozen fabricated tubes. These tests even included a few destructive evaluations to examine the limitations and operating life. In addition, analyses where conducted regarding impact caused by the specified electronic tolerances and expected changes in the Hubble Space Telescope (HST) thermal environment. Perhaps the simplest test of stability is to collect a sequence of images, each with a uniform illumination, and use these individual "flat fields" to remove the pixel-to-pixel sensitivity in the other flat fields. These sequences typically spanned 3-5 weeks of time. The detectors are very stable, allowing the pixel-to-pixel sensitivity to be removed with good precision. The STIS specification for stability is 1% (sufficient for data with a S/N = 100) over a 1 week period and 2% over 30 days. All Engineering Model Units as well as Flight Detectors tested exceeded this specification.

Abnormal Cone Structure in Foveal Schisis Cavities in X-Linked Retinoschisis from Mutations in Exon 6 of the RS1 Gene

PubMed Central

Ratnam, Kavitha; Birch, David G.; Sundquist, Sanna M.; Lucero, Anna S.; Zhang, Yuhua; Meltzer, Meira; Smaoui, Nizar; Roorda, Austin

2011-01-01

Purpose. To evaluate macular cone structure in patients with X-linked retinoschisis (XLRS) caused by mutations in exon 6 of the RS1 gene. Methods. High-resolution macular images were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography (SD-OCT) in two patients with XLRS and 27 age-similar healthy subjects. Retinal structure was correlated with best-corrected visual acuity, kinetic and static perimetry, fundus-guided microperimetry, full-field electroretinography (ERG), and multifocal ERG. The six coding exons and the flanking intronic regions of the RS1 gene were sequenced in each patient. Results. Two unrelated males, ages 14 and 29, with visual acuity ranging from 20/32 to 20/63, had macular schisis with small relative central scotomas in each eye. The mixed scotopic ERG b-wave was reduced more than the a-wave. SD-OCT showed schisis cavities in the outer and inner nuclear and plexiform layers. Cone spacing was increased within the largest foveal schisis cavities but was normal elsewhere. In each patient, a mutation in exon 6 of the RS1 gene was identified and was predicted to change the amino acid sequence in the discoidin domain of the retinoschisin protein. Conclusions. AOSLO images of two patients with molecularly characterized XLRS revealed increased cone spacing and abnormal packing in the macula of each patient, but cone coverage and function were near normal outside the central foveal schisis cavities. Although cone density is reduced, the preservation of wave-guiding cones at the fovea and eccentric macular regions has prognostic and therapeutic implications for XLRS patients with foveal schisis. (Clinical Trials.gov number, NCT00254605.) PMID:22110067

Applications of Single-Cell Sequencing for Multiomics.

PubMed

Xu, Yungang; Zhou, Xiaobo

2018-01-01

Single-cell sequencing interrogates the sequence or chromatin information from individual cells with advanced next-generation sequencing technologies. It provides a higher resolution of cellular differences and a better understanding of the underlying genetic and epigenetic mechanisms of an individual cell in the context of its survival and adaptation to microenvironment. However, it is more challenging to perform single-cell sequencing and downstream data analysis, owing to the minimal amount of starting materials, sample loss, and contamination. In addition, due to the picogram level of the amount of nucleic acids used, heavy amplification is often needed during sample preparation of single-cell sequencing, resulting in the uneven coverage, noise, and inaccurate quantification of sequencing data. All these unique properties raise challenges in and thus high demands for computational methods that specifically fit single-cell sequencing data. We here comprehensively survey the current strategies and challenges for multiple single-cell sequencing, including single-cell transcriptome, genome, and epigenome, beginning with a brief introduction to multiple sequencing techniques for single cells.

Contrast-enhanced 3-dimensional SPACE versus MP-RAGE for the detection of brain metastases: considerations with a 32-channel head coil.

PubMed

Reichert, Miriam; Morelli, John N; Runge, Val M; Tao, Ai; von Ritschl, Ruediger; von Ritschl, Andreas; Padua, Abraham; Dix, James E; Marra, Michael J; Schoenberg, Stefan O; Attenberger, Ulrike I

2013-01-01

The aim of this study was to compare the detection of brain metastases at 3 T using a 32-channel head coil with 2 different 3-dimensional (3D) contrast-enhanced sequences, a T1-weighted fast spin-echo-based (SPACE; sampling perfection with application-optimized contrasts using different flip angle evolutions) sequence and a conventional magnetization-prepared rapid gradient-echo (MP-RAGE) sequence. Seventeen patients with 161 brain metastases were examined prospectively using both SPACE and MP-RAGE sequences on a 3-T magnetic resonance system. Eight healthy volunteers were similarly examined for determination of signal-to-noise ratio (SNR) values. Parameters were adjusted to equalize acquisition times between the sequences (3 minutes and 30 seconds). The order in which sequences were performed was randomized. Two blinded board-certified neuroradiologists evaluated the number of detectable metastatic lesions with each sequence relative to a criterion standard reading conducted at the Gamma Knife facility by a neuroradiologist with access to all clinical and imaging data. In the volunteer assessment with SPACE and MP-RAGE, SNR (10.3 ± 0.8 vs 7.7 ± 0.7) and contrast-to-noise ratio (0.8 ± 0.2 vs 0.5 ± 0.1) were statistically significantly greater with the SPACE sequence (P < 0.05). Overall, lesion detection was markedly improved with the SPACE sequence (99.1% of lesions for reader 1 and 96.3% of lesions for reader 2) compared with the MP-RAGE sequence (73.6% of lesions for reader 1 and 68.5% of lesions for reader 2; P < 0.01). A 3D T1-weighted fast spin echo sequence (SPACE) improves detection of metastatic lesions relative to 3D T1-weighted gradient-echo-based scan (MP-RAGE) imaging when implemented with a 32-channel head coil at identical scan acquisition times (3 minutes and 30 seconds).

Unexpected series of regular frequency spacing of δ Scuti stars in the non-asymptotic regime - I. The methodology

DOE PAGES

Paparo, M.; Benko, J. M.; Hareter, M.; ...

2016-05-11

In this study, a sequence search method was developed to search the regular frequency spacing in δ Scuti stars through visual inspection and an algorithmic search. We searched for sequences of quasi-equally spaced frequencies, containing at least four members per sequence, in 90 δ Scuti stars observed by CoRoT. We found an unexpectedly large number of independent series of regular frequency spacing in 77 δ Scuti stars (from one to eight sequences) in the non-asymptotic regime. We introduce the sequence search method presenting the sequences and echelle diagram of CoRoT 102675756 and the structure of the algorithmic search. Four sequencesmore » (echelle ridges) were found in the 5–21 d –1 region where the pairs of the sequences are shifted (between 0.5 and 0.59 d –1) by twice the value of the estimated rotational splitting frequency (0.269 d –1). The general conclusions for the whole sample are also presented in this paper. The statistics of the spacings derived by the sequence search method, by FT (Fourier transform of the frequencies), and the statistics of the shifts are also compared. In many stars more than one almost equally valid spacing appeared. The model frequencies of FG Vir and their rotationally split components were used to formulate the possible explanation that one spacing is the large separation while the other is the sum of the large separation and the rotational frequency. In CoRoT 102675756, the two spacings (2.249 and 1.977 d –1) are in better agreement with the sum of a possible 1.710 d –1 large separation and two or one times, respectively, the value of the rotational frequency.« less

Unexpected series of regular frequency spacing of δ Scuti stars in the non-asymptotic regime - I. The methodology

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

Paparo, M.; Benko, J. M.; Hareter, M.

In this study, a sequence search method was developed to search the regular frequency spacing in δ Scuti stars through visual inspection and an algorithmic search. We searched for sequences of quasi-equally spaced frequencies, containing at least four members per sequence, in 90 δ Scuti stars observed by CoRoT. We found an unexpectedly large number of independent series of regular frequency spacing in 77 δ Scuti stars (from one to eight sequences) in the non-asymptotic regime. We introduce the sequence search method presenting the sequences and echelle diagram of CoRoT 102675756 and the structure of the algorithmic search. Four sequencesmore » (echelle ridges) were found in the 5–21 d –1 region where the pairs of the sequences are shifted (between 0.5 and 0.59 d –1) by twice the value of the estimated rotational splitting frequency (0.269 d –1). The general conclusions for the whole sample are also presented in this paper. The statistics of the spacings derived by the sequence search method, by FT (Fourier transform of the frequencies), and the statistics of the shifts are also compared. In many stars more than one almost equally valid spacing appeared. The model frequencies of FG Vir and their rotationally split components were used to formulate the possible explanation that one spacing is the large separation while the other is the sum of the large separation and the rotational frequency. In CoRoT 102675756, the two spacings (2.249 and 1.977 d –1) are in better agreement with the sum of a possible 1.710 d –1 large separation and two or one times, respectively, the value of the rotational frequency.« less

Graph-based optimization of epitope coverage for vaccine antigen design

DOE PAGES

Theiler, James Patrick; Korber, Bette Tina Marie

2017-01-29

Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapidmore » characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. Lastly, we also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications.« less

Graph-based optimization of epitope coverage for vaccine antigen design

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

Theiler, James Patrick; Korber, Bette Tina Marie

Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapidmore » characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. Lastly, we also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications.« less

The Profile Envision and Splicing Tool (PRESTO): Developing an Atmospheric Wind Analysis Tool for Space Launch Vehicles Using Python

NASA Technical Reports Server (NTRS)

Orcutt, John M.; Barbre, Robert E., Jr.; Brenton, James C.; Decker, Ryan K.

2017-01-01

Launch vehicle programs require vertically complete atmospheric profiles. Many systems at the ER to make the necessary measurements, but all have different EVR, vertical coverage, and temporal coverage. MSFC Natural Environments Branch developed a tool to create a vertically complete profile from multiple inputs using Python. Forward work: Finish Formal Testing Acceptance Testing, End-to-End Testing. Formal Release

Coverage of STS-104 Launch Coverage of Flight Controllers in MCC.

NASA Image and Video Library

2001-07-12

JSC2001-E-21337 (12 July 2001) --- Rob Navias (right), at the Public Affairs Officer (PAO) console in Houston's Mission Control Center (MCC), awaits to do his commentary as he views the firing of Space Shuttle Atlantis' main engines on his monitor just seconds before launch and the beginning of the STS-104 mission. Daniel K. Carpenter, director of the Public Affairs Office, looks on.

Optimal Partitioning of a Surveillance Space for Persistent Coverage Using Multiple Autonomous Unmanned Aerial Vehicles: An Integer Programming Approach

DTIC Science & Technology

2014-03-27

asymptotically equal. Carlsson shows that the problem is solved by treating each subregion Ri as a traveling salesman problem (TSP) with a set of points that...terminal state to the goal. If no- travel zones are repre- sented as the union of regions Akx > Bk, the coverage problem can be expressed as an IP [14...3 1.2 Problem Statement

Urbanization reduces and homogenizes trait diversity in stream macroinvertebrate communities.

PubMed

Barnum, Thomas R; Weller, Donald E; Williams, Meghan

2017-12-01

More than one-half of the world's population lives in urban areas, so quantifying the effects of urbanization on ecological communities is important for understanding whether anthropogenic stressors homogenize communities across environmental and climatic gradients. We examined the relationship of impervious surface coverage (a marker of urbanization) and the structure of stream macroinvertebrate communities across the state of Maryland and within each of Maryland's three ecoregions: Coastal Plain, Piedmont, and Appalachian, which differ in stream geomorphology and community composition. We considered three levels of trait organization: individual traits, unique combinations of traits, and community metrics (functional richness, functional evenness, and functional divergence) and three levels of impervious surface coverage (low [<2.5%], medium [2.5% to 10%], and high [>10%]). The prevalence of an individual trait differed very little between low impervious surface and high impervious surface sites. The arrangement of trait combinations in community trait space for each ecoregion differed when impervious surface coverage was low, but the arrangement became more similar among ecoregions as impervious surface coverage increased. Furthermore, trait combinations that occurred only at low or medium impervious surface coverage were clustered in a subset of the community trait space, indicating that impervious surface affected the presence of only a subset of trait combinations. Functional richness declined with increasing impervious surface, providing evidence for environmental filtering. Community metrics that include abundance were also sensitive to increasing impervious surface coverage: functional divergence decreased while functional evenness increased. These changes demonstrate that increasing impervious surface coverage homogenizes the trait diversity of macroinvertebrate communities in streams, despite differences in initial community composition and stream geomorphology among ecoregions. Community metrics were also more sensitive to changes in the abundance rather than the gain or loss of trait combinations, showing the potential for trait-based approaches to serve as early warning indicators of environmental stress for monitoring and biological assessment programs. © 2017 by the Ecological Society of America.

Tibial coverage, meniscus position, size and damage in knees discordant for joint space narrowing – data from the Osteoarthritis Initiative

PubMed Central

Bloecker, K.; Guermazi, A.; Wirth, W.; Benichou, O.; Kwoh, C.K.; Hunter, D.J.; Englund, M.; Resch, H.; Eckstein, F.

2013-01-01

SUMMARY Introduction Meniscal extrusion is thought to be associated with less meniscus coverage of the tibial surface, but the association of radiographic disease stage with quantitative measures of tibial plateau coverage is unknown. We therefore compared quantitative and semi-quantitative measures of meniscus position and morphology in individuals with bilateral painful knees discordant on medial joint space narrowing (mJSN). Methods A sample of 60 participants from the first half (2,678 cases) of the Osteoarthritis Initiative cohort fulfilled the inclusion criteria: bilateral frequent pain, Osteoarthritis Research Society International (OARSI) mJSN grades 1–3 in one, no-JSN in the contra-lateral (CL), and no lateral JSN in either knee (43 unilateral mJSN1; 17 mJSN2/3; 22 men, 38 women, body mass index (BMI) 31.3 ± 3.9 kg/m2). Segmentation and three-dimensional quantitative analysis of the tibial plateau and meniscus, and semi-quantitative evaluation of meniscus damage (magnetic resonance imaging (MRI) osteoarthritis knee score – MOAKS) was performed using coronal 3T MR images (MPR DESSwe and intermediate-weighted turbo spin echo (IW-TSE) images). CL knees were compared using paired t-tests (between-knee, within-person design). Results Medial tibial plateau coverage was 36 ± 9% in mJSN1 vs 45 ± 8% in CL no-JSN knees, and was 31 ± 9% in mJSN2/3 vs 46 ± 6% in no-JSN knees (both P < 0.001). mJSN knees showed greater meniscus extrusion and damage (MOAKS), but no significant difference in meniscus volume. No significant differences in lateral tibial coverage, lateral meniscus morphology or position were observed. Conclusions Knees with medial JSN showed substantially less medial tibial plateau coverage by the meniscus. We suggest that the less meniscal coverage, i.e., less mechanical protection may be a reason for greater rates of cartilage loss observed in JSN knees. PMID:23220556

Nasa Program Plan

NASA Technical Reports Server (NTRS)

1980-01-01

Major facts are given for NASA'S planned FY-1981 through FY-1985 programs in aeronautics, space science, space and terrestrial applications, energy technology, space technology, space transportation systems, space tracking and data systems, and construction of facilities. Competition and cooperation, reimbursable launchings, schedules and milestones, supporting research and technology, mission coverage, and required funding are considered. Tables and graphs summarize new initiatives, significant events, estimates of space shuttle flights, and major missions in astrophysics, planetary exploration, life sciences, environmental and resources observation, and solar terrestrial investigations. The growth in tracking and data systems capabilities is also depicted.

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias.

PubMed

Clarke, Laurence J; Soubrier, Julien; Weyrich, Laura S; Cooper, Alan

2014-11-01

Studies of insect assemblages are suited to the simultaneous DNA-based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR-amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR-amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR-amplified the blend using five primer sets, targeting either COI or 16S, with high-throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers. © 2014 John Wiley & Sons Ltd.

Genome-wide ancestry and divergence patterns from low-coverage sequencing data reveal a complex history of admixture in wild baboons

PubMed Central

Wall, Jeffrey D; Schlebusch, Stephen A; Alberts, Susan C; Cox, Laura A; Snyder-Mackler, Noah; Nevonen, Kimberly; Carbone, Lucia; Tung, Jenny

2017-01-01

Naturally occurring admixture has now been documented in every major primate lineage, suggesting its key role in primate evolutionary history. Active primate hybrid zones can provide valuable insight into this process. Here, we investigate the history of admixture in one of the best-studied natural primate hybrid zones, between yellow baboons (Papio cynocephalus) and anubis baboons (Papio anubis) in the Amboseli ecosystem of Kenya. We generated a new genome assembly for yellow baboon and low coverage genome-wide resequencing data from yellow baboons, anubis baboons, and known hybrids (n=44). Using a novel composite likelihood method for estimating local ancestry from low coverage data, we found high levels of genetic diversity and genetic differentiation between the parent taxa, and excellent agreement between genome-scale ancestry estimates and a priori pedigree, life history, and morphology-based estimates (r2=0.899). However, even putatively unadmixed Amboseli yellow individuals carried a substantial proportion of anubis ancestry, presumably due to historical admixture. Further, the distribution of shared versus fixed differences between a putatively unadmixed Amboseli yellow baboon and an unadmixed anubis baboon, both sequenced at high coverage, are inconsistent with simple isolation-migration or equilibrium migration models. Our findings suggest a complex process of intermittent contact that has occurred multiple times in baboon evolutionary history, despite no obvious fitness costs to hybrids or major geographic or behavioral barriers. In combination with the extensive phenotypic data available for baboon hybrids, our results provide valuable context for understanding the history of admixture in primates, including in our own lineage. PMID:27145036

Genome sequencing in microfabricated high-density picolitre reactors.

PubMed

Margulies, Marcel; Egholm, Michael; Altman, William E; Attiya, Said; Bader, Joel S; Bemben, Lisa A; Berka, Jan; Braverman, Michael S; Chen, Yi-Ju; Chen, Zhoutao; Dewell, Scott B; Du, Lei; Fierro, Joseph M; Gomes, Xavier V; Godwin, Brian C; He, Wen; Helgesen, Scott; Ho, Chun Heen; Ho, Chun He; Irzyk, Gerard P; Jando, Szilveszter C; Alenquer, Maria L I; Jarvie, Thomas P; Jirage, Kshama B; Kim, Jong-Bum; Knight, James R; Lanza, Janna R; Leamon, John H; Lefkowitz, Steven M; Lei, Ming; Li, Jing; Lohman, Kenton L; Lu, Hong; Makhijani, Vinod B; McDade, Keith E; McKenna, Michael P; Myers, Eugene W; Nickerson, Elizabeth; Nobile, John R; Plant, Ramona; Puc, Bernard P; Ronan, Michael T; Roth, George T; Sarkis, Gary J; Simons, Jan Fredrik; Simpson, John W; Srinivasan, Maithreyan; Tartaro, Karrie R; Tomasz, Alexander; Vogt, Kari A; Volkmer, Greg A; Wang, Shally H; Wang, Yong; Weiner, Michael P; Yu, Pengguang; Begley, Richard F; Rothberg, Jonathan M

2005-09-15

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Using Poisson mixed-effects model to quantify transcript-level gene expression in RNA-Seq.

PubMed

Hu, Ming; Zhu, Yu; Taylor, Jeremy M G; Liu, Jun S; Qin, Zhaohui S

2012-01-01

RNA sequencing (RNA-Seq) is a powerful new technology for mapping and quantifying transcriptomes using ultra high-throughput next-generation sequencing technologies. Using deep sequencing, gene expression levels of all transcripts including novel ones can be quantified digitally. Although extremely promising, the massive amounts of data generated by RNA-Seq, substantial biases and uncertainty in short read alignment pose challenges for data analysis. In particular, large base-specific variation and between-base dependence make simple approaches, such as those that use averaging to normalize RNA-Seq data and quantify gene expressions, ineffective. In this study, we propose a Poisson mixed-effects (POME) model to characterize base-level read coverage within each transcript. The underlying expression level is included as a key parameter in this model. Since the proposed model is capable of incorporating base-specific variation as well as between-base dependence that affect read coverage profile throughout the transcript, it can lead to improved quantification of the true underlying expression level. POME can be freely downloaded at http://www.stat.purdue.edu/~yuzhu/pome.html. yuzhu@purdue.edu; zhaohui.qin@emory.edu Supplementary data are available at Bioinformatics online.

Ultrathin type-II GaSb/GaAs quantum wells grown by OMVPE

NASA Astrophysics Data System (ADS)

Pitts, O. J.; Watkins, S. P.; Wang, C. X.; Stotz, J. A. H.; Meyer, T. A.; Thewalt, M. L. W.

2004-09-01

Heterostructures containing monolayer (ML) and submonolayer GaSb insertions in GaAs were grown using organometallic vapour phase epitaxy. At the GaAs-on-GaSb interface, strong intermixing occurs due to the surface segregation of Sb. To form structures with relatively abrupt interfaces, a flashoff growth sequence, in which growth interruptions are employed to desorb Sb from the surface, was introduced. Reflectance-difference spectroscopy and high-resolution X-ray diffraction data demonstrate that interfacial grading is strongly reduced by this procedure. For layer structures grown with the flashoff sequence, a GaSb coverage up to 1 ML can be obtained in the two-dimensional (2D) growth mode. For uncapped GaSb layers, on the other hand, atomic force microscope images show that the 2D-3D growth mode transition occurs at a submonolayer coverage between 0.3 and 0.5 ML. Low-temperature photoluminescence spectra of multiple quantum well samples grown using the flashoff sequence show a strong quantum well-related peak which shifts to lower energies as the amount of Sb incorporated increases. The PL peak energies are consistent with a type-II band lineup at the GaAs/GaSb interface.

Insights from Human/Mouse genome comparisons

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

Pennacchio, Len A.

2003-03-30

Large-scale public genomic sequencing efforts have provided a wealth of vertebrate sequence data poised to provide insights into mammalian biology. These include deep genomic sequence coverage of human, mouse, rat, zebrafish, and two pufferfish (Fugu rubripes and Tetraodon nigroviridis) (Aparicio et al. 2002; Lander et al. 2001; Venter et al. 2001; Waterston et al. 2002). In addition, a high-priority has been placed on determining the genomic sequence of chimpanzee, dog, cow, frog, and chicken (Boguski 2002). While only recently available, whole genome sequence data have provided the unique opportunity to globally compare complete genome contents. Furthermore, the shared evolutionary ancestrymore » of vertebrate species has allowed the development of comparative genomic approaches to identify ancient conserved sequences with functionality. Accordingly, this review focuses on the initial comparison of available mammalian genomes and describes various insights derived from such analysis.« less

Comparison of post-contrast 3D-T1-MPRAGE, 3D-T1-SPACE and 3D-T2-FLAIR MR images in evaluation of meningeal abnormalities at 3-T MRI.

PubMed

Jeevanandham, Balaji; Kalyanpur, Tejas; Gupta, Prashant; Cherian, Mathew

2017-06-01

This study was to assess the usefulness of newer three-dimensional (3D)-T 1 sampling perfection with application optimized contrast using different flip-angle evolutions (SPACE) and 3D-T 2 fluid-attenuated inversion recovery (FLAIR) sequences in evaluation of meningeal abnormalities. 78 patients who presented with high suspicion of meningeal abnormalities were evaluated using post-contrast 3D-T 2 -FLAIR, 3D-T 1 magnetization-prepared rapid gradient-echo (MPRAGE) and 3D-T 1 -SPACE sequences. The images were evaluated independently by two radiologists for cortical gyral, sulcal space, basal cisterns and dural enhancement. The diagnoses were confirmed by further investigations including histopathology. Post-contrast 3D-T 1 -SPACE and 3D-T 2 -FLAIR images yielded significantly more information than MPRAGE images (p < 0.05 for both SPACE and FLAIR images) in detection of meningeal abnormalities. SPACE images best demonstrated abnormalities in dural and sulcal spaces, whereas FLAIR was useful for basal cisterns enhancement. Both SPACE and FLAIR performed equally well in detection of gyral enhancement. In all 10 patients, where both SPACE and T 2 -FLAIR images failed to demonstrate any abnormality, further analysis was also negative. The 3D-T 1 -SPACE sequence best demonstrated abnormalities in dural and sulcal spaces, whereas FLAIR was useful for abnormalities in basal cisterns. Both SPACE and FLAIR performed holds good for detection of gyral enhancement. Post-contrast SPACE and FLAIR sequences are superior to the MPRAGE sequence for evaluation of meningeal abnormalities and when used in combination have the maximum sensitivity for leptomeningeal abnormalities. The negative-predictive value is nearly 100%, where no leptomeningeal abnormality was detected on these sequences. Advances in knowledge: Post-contrast 3D-T 1 -SPACE and 3D-T 2 -FLAIR images are more useful than 3D-T 1 -MPRAGE images in evaluation of meningeal abnormalities.

The spatial alignment of time: Differences in alignment of deictic and sequence time along the sagittal and lateral axes.

PubMed

Walker, Esther J; Bergen, Benjamin K; Núñez, Rafael

2017-04-01

People use space in a variety of ways to structure their thoughts about time. The present report focuses on the different ways that space is employed when reasoning about deictic (past/future relationships) and sequence (earlier/later relationships) time. In the first study, we show that deictic and sequence time are aligned along the lateral axis in a manner consistent with previous work, with past and earlier events associated with left space and future and later events associated with right space. However, the alignment of time with space is different along the sagittal axis. Participants associated future events and earlier events-not later events-with the space in front of their body and past and later events with the space behind, consistent with the sagittal spatial terms (e.g., ahead, in front of) that we use to talk about deictic and sequence time. In the second study, we show that these associations between sequence time and sagittal space are sensitive to person-perspective. This suggests that the particular space-time associations observed in English speakers are influenced by a variety of different spatial properties, including spatial location and perspective. Copyright © 2016. Published by Elsevier B.V.

The complete mitogenome of the river blackfish, Gadopsis marmoratus (Richardson, 1848) (Teleostei: Percichthyidae).

PubMed

Gan, Han Ming; Tan, Mun Hua; Lee, Yin Peng; Austin, Christopher M

2016-05-01

The mitogenome of the Australian freshwater blackfish, Gadopsis marmoratus was recovered coverage by genome skimming using the MiSeq sequencer (GenBank Accession Number: NC_024436). The blackfish mitogenome has 16,407 base pairs made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a 819 bp non-coding AT-rich region. This is the 5th mitogenome sequence to be reported for the family Percichthyidae.

Petite Amateur Navy Satellite (PANSAT)

NASA Technical Reports Server (NTRS)

1989-01-01

The subsystem and structural design of the Naval Post Graduate School's Petite Amateur Navy Satellite (PANSAT) is described. The objectives of PANSAT are: (1) to provide an ideal educational tool for officer students; (2) to provide digital store-and-forward communications, or packet radio, for the amateur radio community; and (3) to provide a low-cost space-based platform for small experiments. PANSAT will be launched from the Shuttle at a nominal altitude of 200 nmi. and an inclination of at least 37 deg. Since there is no attitude control, eight dipole whip antennas will be used to provide isotropic ground coverage for communications. FM digital communications will be used with up-link and down-link on a single frequency in the amateur band of 144 to 146 MHz or 437 to 438 MHz. The satellite's communications subsystem, data processor and sequencer, power subsystem, structure subsystem, and experiment payload are described. The major experiment being considered will test the on-orbit annealing of radiation damaged solar cells.

Dfam: a database of repetitive DNA based on profile hidden Markov models.

PubMed

Wheeler, Travis J; Clements, Jody; Eddy, Sean R; Hubley, Robert; Jones, Thomas A; Jurka, Jerzy; Smit, Arian F A; Finn, Robert D

2013-01-01

We present a database of repetitive DNA elements, called Dfam (http://dfam.janelia.org). Many genomes contain a large fraction of repetitive DNA, much of which is made up of remnants of transposable elements (TEs). Accurate annotation of TEs enables research into their biology and can shed light on the evolutionary processes that shape genomes. Identification and masking of TEs can also greatly simplify many downstream genome annotation and sequence analysis tasks. The commonly used TE annotation tools RepeatMasker and Censor depend on sequence homology search tools such as cross_match and BLAST variants, as well as Repbase, a collection of known TE families each represented by a single consensus sequence. Dfam contains entries corresponding to all Repbase TE entries for which instances have been found in the human genome. Each Dfam entry is represented by a profile hidden Markov model, built from alignments generated using RepeatMasker and Repbase. When used in conjunction with the hidden Markov model search tool nhmmer, Dfam produces a 2.9% increase in coverage over consensus sequence search methods on a large human benchmark, while maintaining low false discovery rates, and coverage of the full human genome is 54.5%. The website provides a collection of tools and data views to support improved TE curation and annotation efforts. Dfam is also available for download in flat file format or in the form of MySQL table dumps.

Strain/species identification in metagenomes using genome-specific markers

PubMed Central

Tu, Qichao; He, Zhili; Zhou, Jizhong

2014-01-01

Shotgun metagenome sequencing has become a fast, cheap and high-throughput technology for characterizing microbial communities in complex environments and human body sites. However, accurate identification of microorganisms at the strain/species level remains extremely challenging. We present a novel k-mer-based approach, termed GSMer, that identifies genome-specific markers (GSMs) from currently sequenced microbial genomes, which were then used for strain/species-level identification in metagenomes. Using 5390 sequenced microbial genomes, 8 770 321 50-mer strain-specific and 11 736 360 species-specific GSMs were identified for 4088 strains and 2005 species (4933 strains), respectively. The GSMs were first evaluated against mock community metagenomes, recently sequenced genomes and real metagenomes from different body sites, suggesting that the identified GSMs were specific to their targeting genomes. Sensitivity evaluation against synthetic metagenomes with different coverage suggested that 50 GSMs per strain were sufficient to identify most microbial strains with ≥0.25× coverage, and 10% of selected GSMs in a database should be detected for confident positive callings. Application of GSMs identified 45 and 74 microbial strains/species significantly associated with type 2 diabetes patients and obese/lean individuals from corresponding gastrointestinal tract metagenomes, respectively. Our result agreed with previous studies but provided strain-level information. The approach can be directly applied to identify microbial strains/species from raw metagenomes, without the effort of complex data pre-processing. PMID:24523352

Press coverage of AIDS in Zimbabwe: a five-year review.

PubMed

Pitts, M; Jackson, H

1993-01-01

Five years of newspaper coverage of HIV and AIDS in Zimbabwe is examined. Both the number of items and the amount of space devoted to the topics has increased steadily over the 5 years. The nature and content of the items show a continuing bias towards issues more closely associated with western patterns of the epidemic, and comparative neglect of personal stories, local issues and items with a counselling focus. Language is also examined and found to reflect a victim and war imagery. The implications for health education in the country are considered and specific recommendations for more constructive media coverage are made.

UNEXPECTED SERIES OF REGULAR FREQUENCY SPACING OF δ SCUTI STARS IN THE NON-ASYMPTOTIC REGIME. I. THE METHODOLOGY

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

Paparó, M.; Benkő, J. M.; Hareter, M.

A sequence search method was developed to search the regular frequency spacing in δ Scuti stars through visual inspection and an algorithmic search. We searched for sequences of quasi-equally spaced frequencies, containing at least four members per sequence, in 90 δ Scuti stars observed by CoRoT . We found an unexpectedly large number of independent series of regular frequency spacing in 77 δ Scuti stars (from one to eight sequences) in the non-asymptotic regime. We introduce the sequence search method presenting the sequences and echelle diagram of CoRoT 102675756 and the structure of the algorithmic search. Four sequences (echelle ridges)more » were found in the 5–21 d{sup −1} region where the pairs of the sequences are shifted (between 0.5 and 0.59 d{sup −1}) by twice the value of the estimated rotational splitting frequency (0.269 d{sup −1}). The general conclusions for the whole sample are also presented in this paper. The statistics of the spacings derived by the sequence search method, by FT (Fourier transform of the frequencies), and the statistics of the shifts are also compared. In many stars more than one almost equally valid spacing appeared. The model frequencies of FG Vir and their rotationally split components were used to formulate the possible explanation that one spacing is the large separation while the other is the sum of the large separation and the rotational frequency. In CoRoT 102675756, the two spacings (2.249 and 1.977 d{sup −1}) are in better agreement with the sum of a possible 1.710 d{sup −1} large separation and two or one times, respectively, the value of the rotational frequency.« less

NASA Scientific and Technical Publications: A Catalog of Special Publications, Reference Publications, Conference Publications, and Technical Papers 1991-1992

DTIC Science & Technology

1993-02-01

Scientific and Technical Information EXOBIOLOGY. HEALTH. MICROBIOLOGY . MICROOR- System during September 1990. Subject coverage includes: GANISMS...Houston. TX N91-24731 National Aeronautics and Space Administration. MICROBIOLOGY ON SPACE STATION FREEDOM Washington, DCr DUANE L. PIERSON, ed...and solution INASA-SP-7011(345)) p 37 N91-16547 Beyond the Baseline 1991ý Proceedings of the Space [NASA-TP-3242) p 43 N92-33483 Microbiology on Space

A draft sequence of the rice genome (Oryza sativa L. ssp. indica).

PubMed

Yu, Jun; Hu, Songnian; Wang, Jun; Wong, Gane Ka-Shu; Li, Songgang; Liu, Bin; Deng, Yajun; Dai, Li; Zhou, Yan; Zhang, Xiuqing; Cao, Mengliang; Liu, Jing; Sun, Jiandong; Tang, Jiabin; Chen, Yanjiong; Huang, Xiaobing; Lin, Wei; Ye, Chen; Tong, Wei; Cong, Lijuan; Geng, Jianing; Han, Yujun; Li, Lin; Li, Wei; Hu, Guangqiang; Huang, Xiangang; Li, Wenjie; Li, Jian; Liu, Zhanwei; Li, Long; Liu, Jianping; Qi, Qiuhui; Liu, Jinsong; Li, Li; Li, Tao; Wang, Xuegang; Lu, Hong; Wu, Tingting; Zhu, Miao; Ni, Peixiang; Han, Hua; Dong, Wei; Ren, Xiaoyu; Feng, Xiaoli; Cui, Peng; Li, Xianran; Wang, Hao; Xu, Xin; Zhai, Wenxue; Xu, Zhao; Zhang, Jinsong; He, Sijie; Zhang, Jianguo; Xu, Jichen; Zhang, Kunlin; Zheng, Xianwu; Dong, Jianhai; Zeng, Wanyong; Tao, Lin; Ye, Jia; Tan, Jun; Ren, Xide; Chen, Xuewei; He, Jun; Liu, Daofeng; Tian, Wei; Tian, Chaoguang; Xia, Hongai; Bao, Qiyu; Li, Gang; Gao, Hui; Cao, Ting; Wang, Juan; Zhao, Wenming; Li, Ping; Chen, Wei; Wang, Xudong; Zhang, Yong; Hu, Jianfei; Wang, Jing; Liu, Song; Yang, Jian; Zhang, Guangyu; Xiong, Yuqing; Li, Zhijie; Mao, Long; Zhou, Chengshu; Zhu, Zhen; Chen, Runsheng; Hao, Bailin; Zheng, Weimou; Chen, Shouyi; Guo, Wei; Li, Guojie; Liu, Siqi; Tao, Ming; Wang, Jian; Zhu, Lihuang; Yuan, Longping; Yang, Huanming

2002-04-05

We have produced a draft sequence of the rice genome for the most widely cultivated subspecies in China, Oryza sativa L. ssp. indica, by whole-genome shotgun sequencing. The genome was 466 megabases in size, with an estimated 46,022 to 55,615 genes. Functional coverage in the assembled sequences was 92.0%. About 42.2% of the genome was in exact 20-nucleotide oligomer repeats, and most of the transposons were in the intergenic regions between genes. Although 80.6% of predicted Arabidopsis thaliana genes had a homolog in rice, only 49.4% of predicted rice genes had a homolog in A. thaliana. The large proportion of rice genes with no recognizable homologs is due to a gradient in the GC content of rice coding sequences.

Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing.

PubMed

Watanabe, Manabu; Kusano, Junko; Ohtaki, Shinsaku; Ishikura, Takashi; Katayama, Jin; Koguchi, Akira; Paumen, Michael; Hayashi, Yoshiharu

2014-09-01

Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line) were used as a model. Single-cell capture was performed using laser capture microdissection (LCM) with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈ 10(6) cells) were subjected to whole genome amplification (WGA). For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel) was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 10(31-35). For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100 × were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100 × were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

Computationally mapping sequence space to understand evolutionary protein engineering.

PubMed

Armstrong, Kathryn A; Tidor, Bruce

2008-01-01

Evolutionary protein engineering has been dramatically successful, producing a wide variety of new proteins with altered stability, binding affinity, and enzymatic activity. However, the success of such procedures is often unreliable, and the impact of the choice of protein, engineering goal, and evolutionary procedure is not well understood. We have created a framework for understanding aspects of the protein engineering process by computationally mapping regions of feasible sequence space for three small proteins using structure-based design protocols. We then tested the ability of different evolutionary search strategies to explore these sequence spaces. The results point to a non-intuitive relationship between the error-prone PCR mutation rate and the number of rounds of replication. The evolutionary relationships among feasible sequences reveal hub-like sequences that serve as particularly fruitful starting sequences for evolutionary search. Moreover, genetic recombination procedures were examined, and tradeoffs relating sequence diversity and search efficiency were identified. This framework allows us to consider the impact of protein structure on the allowed sequence space and therefore on the challenges that each protein presents to error-prone PCR and genetic recombination procedures.

The European sea bass Dicentrarchus labrax genome puzzle: comparative BAC-mapping and low coverage shotgun sequencing

PubMed Central

2010-01-01

Background Food supply from the ocean is constrained by the shortage of domesticated and selected fish. Development of genomic models of economically important fishes should assist with the removal of this bottleneck. European sea bass Dicentrarchus labrax L. (Moronidae, Perciformes, Teleostei) is one of the most important fishes in European marine aquaculture; growing genomic resources put it on its way to serve as an economic model. Results End sequencing of a sea bass genomic BAC-library enabled the comparative mapping of the sea bass genome using the three-spined stickleback Gasterosteus aculeatus genome as a reference. BAC-end sequences (102,690) were aligned to the stickleback genome. The number of mappable BACs was improved using a two-fold coverage WGS dataset of sea bass resulting in a comparative BAC-map covering 87% of stickleback chromosomes with 588 BAC-contigs. The minimum size of 83 contigs covering 50% of the reference was 1.2 Mbp; the largest BAC-contig comprised 8.86 Mbp. More than 22,000 BAC-clones aligned with both ends to the reference genome. Intra-chromosomal rearrangements between sea bass and stickleback were identified. Size distributions of mapped BACs were used to calculate that the genome of sea bass may be only 1.3 fold larger than the 460 Mbp stickleback genome. Conclusions The BAC map is used for sequencing single BACs or BAC-pools covering defined genomic entities by second generation sequencing technologies. Together with the WGS dataset it initiates a sea bass genome sequencing project. This will allow the quantification of polymorphisms through resequencing, which is important for selecting highly performing domesticated fish. PMID:20105308

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates

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

Gallaher, Sean D.; Fitz-Gibbon, Sorel T.; Strenkert, Daniela

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Gen-ome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genomemore » and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar tran-scripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3’-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellargenomes, which is 1/10th as much genetic diversity as is found in the nucleus.« less

Detection of Somatic Mutations in Gastroenteropancreatic Neuroendocrine Tumors Using Targeted Deep Sequencing.

PubMed

Backman, Samuel; Norlén, Olov; Eriksson, Barbro; Skogseid, Britt; Stålberg, Peter; Crona, Joakim

2017-02-01

Mutations affecting the mechanistic target of rapamycin (MTOR) signalling pathway are frequent in human cancer and have been identified in up to 15% of pancreatic neuroendocrine tumours (NETs). Grade A evidence supports the efficacy of MTOR inhibition with everolimus in pancreatic NETs. Although a significant proportion of patients experience disease stabilization, only a minority will show objective tumour responses. It has been proposed that genomic mutations resulting in activation of MTOR signalling could be used to predict sensitivity to everolimus. Patients with NETs that underwent treatment with everolimus at our Institution were identified and those with available tumour tissue were selected for further analysis. Targeted next-generation sequencing (NGS) was used to re-sequence 22 genes that were selected on the basis of documented involvement in the MTOR signalling pathway or in the tumourigenesis of gastroenterpancreatic NETs. Radiological responses were documented using Response Evaluation Criteria in Solid Tumours. Six patients were identified, one had a partial response and four had stable disease. Sequencing of tumour tissue resulted in a median sequence depth of 667.1 (range=404-1301) with 1-fold coverage of 95.9-96.5% and 10-fold coverage of 87.6-92.2%. A total of 494 genetic variants were discovered, four of which were identified as pathogenic. All pathogenic variants were validated using Sanger sequencing and were found exclusively in menin 1 (MEN1) and death domain associated protein (DAXX) genes. No mutations in the MTOR pathway-related genes were observed. Targeted NGS is a feasible method with high diagnostic yield for genetic characterization of pancreatic NETs. A potential association between mutations in NETs and response to everolimus should be investigated by future studies. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

SeqHBase: a big data toolset for family based sequencing data analysis.

PubMed

He, Min; Person, Thomas N; Hebbring, Scott J; Heinzen, Ethan; Ye, Zhan; Schrodi, Steven J; McPherson, Elizabeth W; Lin, Simon M; Peissig, Peggy L; Brilliant, Murray H; O'Rawe, Jason; Robison, Reid J; Lyon, Gholson J; Wang, Kai

2015-04-01

Whole-genome sequencing (WGS) and whole-exome sequencing (WES) technologies are increasingly used to identify disease-contributing mutations in human genomic studies. It can be a significant challenge to process such data, especially when a large family or cohort is sequenced. Our objective was to develop a big data toolset to efficiently manipulate genome-wide variants, functional annotations and coverage, together with conducting family based sequencing data analysis. Hadoop is a framework for reliable, scalable, distributed processing of large data sets using MapReduce programming models. Based on Hadoop and HBase, we developed SeqHBase, a big data-based toolset for analysing family based sequencing data to detect de novo, inherited homozygous, or compound heterozygous mutations that may contribute to disease manifestations. SeqHBase takes as input BAM files (for coverage at every site), variant call format (VCF) files (for variant calls) and functional annotations (for variant prioritisation). We applied SeqHBase to a 5-member nuclear family and a 10-member 3-generation family with WGS data, as well as a 4-member nuclear family with WES data. Analysis times were almost linearly scalable with number of data nodes. With 20 data nodes, SeqHBase took about 5 secs to analyse WES familial data and approximately 1 min to analyse WGS familial data. These results demonstrate SeqHBase's high efficiency and scalability, which is necessary as WGS and WES are rapidly becoming standard methods to study the genetics of familial disorders. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

High Slew-Rate Head-Only Gradient for Improving Distortion in Echo Planar Imaging: Preliminary Experience

PubMed Central

Tan, Ek T.; Lee, Seung-Kyun; Weavers, Paul T.; Graziani, Dominic; Piel, Joseph E.; Shu, Yunhong; Huston, John; Bernstein, Matt A.; Foo, Thomas K.F.

2016-01-01

Purpose To investigate the effects on echo planar imaging (EPI) distortion of using high gradient slew rates (SR) of up to 700 T/m/s for in-vivo human brain imaging, with a dedicated, head-only gradient coil. Materials and Methods Simulation studies were first performed to determine the expected echo spacing and distortion reduction in EPI. A head gradient of 42-cm inner diameter and with asymmetric transverse coils was then installed in a whole-body, conventional 3T MRI system. Human subject imaging was performed on five subjects to determine the effects of EPI on echo spacing and signal dropout at various gradient slew rates. The feasibility of whole-brain imaging at 1.5 mm-isotropic spatial resolution was demonstrated with gradient-echo and spin-echo diffusion-weighted EPI. Results As compared to a whole-body gradient coil, the EPI echo spacing in the head-only gradient coil was reduced by 48%. Simulation and in vivo results, respectively, showed up to 25-26% and 19% improvement in signal dropout. Whole-brain imaging with EPI at 1.5 mm spatial resolution provided good whole-brain coverage, spatial linearity, and low spatial distortion effects. Conclusion Our results of human brain imaging with EPI using the compact head gradient coil at slew rates higher than in conventional whole-body MR systems demonstrate substantially improved image distortion, and point to a potential for benefits to non-EPI pulse sequences. PMID:26921117

High-throughput Database Search and Large-scale Negative Polarity Liquid Chromatography–Tandem Mass Spectrometry with Ultraviolet Photodissociation for Complex Proteomic Samples*

PubMed Central

Madsen, James A.; Xu, Hua; Robinson, Michelle R.; Horton, Andrew P.; Shaw, Jared B.; Giles, David K.; Kaoud, Tamer S.; Dalby, Kevin N.; Trent, M. Stephen; Brodbelt, Jennifer S.

2013-01-01

The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome. To facilitate interpretation and assignment of the resulting UVPD mass spectra of peptide anions, the MassMatrix database search algorithm was modified to allow automated analysis of negative polarity MS/MS spectra. The new UVPD algorithms were developed based on the MassMatrix database search engine by adding specific fragmentation pathways for UVPD. The new UVPD fragmentation pathways in MassMatrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS1 and MS2 data acquired on an Orbitrap mass spectrometer for complex Halobacterium and HeLa proteome samples. Negative mode UVPD led to the identification of 3663 and 2350 peptides for the Halo and HeLa tryptic digests, respectively, corresponding to 655 and 645 peptides that were unique when compared with electron transfer dissociation (ETD), higher energy collision-induced dissociation, and collision-induced dissociation results for the same digests analyzed in the positive mode. In sum, 805 and 619 proteins were identified via UVPD for the Halobacterium and HeLa samples, respectively, with 49 and 50 unique proteins identified in contrast to the more conventional MS/MS methods. The algorithm also features automated charge determination for low mass accuracy data, precursor filtering (including intact charge-reduced peaks), and the ability to combine both positive and negative MS/MS spectra into a single search, and it is freely open to the public. The accuracy and specificity of the MassMatrix UVPD search algorithm was also assessed for low resolution, low mass accuracy data on a linear ion trap. Analysis of a known mixture of three mitogen-activated kinases yielded similar sequence coverage percentages for UVPD of peptide anions versus conventional collision-induced dissociation of peptide cations, and when these methods were combined into a single search, an increase of up to 13% sequence coverage was observed for the kinases. The ability to sequence peptide anions and cations in alternating scans in the same chromatographic run was also demonstrated. Because ETD has a significant bias toward identifying highly basic peptides, negative UVPD was used to improve the identification of the more acidic peptides in conjunction with positive ETD for the more basic species. In this case, tryptic peptides from the cytosolic section of HeLa cells were analyzed by polarity switching nanoLC-MS/MS utilizing ETD for cation sequencing and UVPD for anion sequencing. Relative to searching using ETD alone, positive/negative polarity switching significantly improved sequence coverages across identified proteins, resulting in a 33% increase in unique peptide identifications and more than twice the number of peptide spectral matches. PMID:23695934

A sequencing-based linkage map of cucumber

USDA-ARS?s Scientific Manuscript database

Genetic maps are important tools for molecular breeding, gene cloning, and study of meiotic recombination. In cucumber (Cucumis sativus L.), the marker density, resolution and genome coverage of previously developed genetic maps using PCR-based molecular markers are relatively low. In this study we ...

cn.MOPS: mixture of Poissons for discovering copy number variations in next-generation sequencing data with a low false discovery rate

PubMed Central

Klambauer, Günter; Schwarzbauer, Karin; Mayr, Andreas; Clevert, Djork-Arné; Mitterecker, Andreas; Bodenhofer, Ulrich; Hochreiter, Sepp

2012-01-01

Quantitative analyses of next-generation sequencing (NGS) data, such as the detection of copy number variations (CNVs), remain challenging. Current methods detect CNVs as changes in the depth of coverage along chromosomes. Technological or genomic variations in the depth of coverage thus lead to a high false discovery rate (FDR), even upon correction for GC content. In the context of association studies between CNVs and disease, a high FDR means many false CNVs, thereby decreasing the discovery power of the study after correction for multiple testing. We propose ‘Copy Number estimation by a Mixture Of PoissonS’ (cn.MOPS), a data processing pipeline for CNV detection in NGS data. In contrast to previous approaches, cn.MOPS incorporates modeling of depths of coverage across samples at each genomic position. Therefore, cn.MOPS is not affected by read count variations along chromosomes. Using a Bayesian approach, cn.MOPS decomposes variations in the depth of coverage across samples into integer copy numbers and noise by means of its mixture components and Poisson distributions, respectively. The noise estimate allows for reducing the FDR by filtering out detections having high noise that are likely to be false detections. We compared cn.MOPS with the five most popular methods for CNV detection in NGS data using four benchmark datasets: (i) simulated data, (ii) NGS data from a male HapMap individual with implanted CNVs from the X chromosome, (iii) data from HapMap individuals with known CNVs, (iv) high coverage data from the 1000 Genomes Project. cn.MOPS outperformed its five competitors in terms of precision (1–FDR) and recall for both gains and losses in all benchmark data sets. The software cn.MOPS is publicly available as an R package at http://www.bioinf.jku.at/software/cnmops/ and at Bioconductor. PMID:22302147

cn.MOPS: mixture of Poissons for discovering copy number variations in next-generation sequencing data with a low false discovery rate.

PubMed

Klambauer, Günter; Schwarzbauer, Karin; Mayr, Andreas; Clevert, Djork-Arné; Mitterecker, Andreas; Bodenhofer, Ulrich; Hochreiter, Sepp

2012-05-01

Quantitative analyses of next-generation sequencing (NGS) data, such as the detection of copy number variations (CNVs), remain challenging. Current methods detect CNVs as changes in the depth of coverage along chromosomes. Technological or genomic variations in the depth of coverage thus lead to a high false discovery rate (FDR), even upon correction for GC content. In the context of association studies between CNVs and disease, a high FDR means many false CNVs, thereby decreasing the discovery power of the study after correction for multiple testing. We propose 'Copy Number estimation by a Mixture Of PoissonS' (cn.MOPS), a data processing pipeline for CNV detection in NGS data. In contrast to previous approaches, cn.MOPS incorporates modeling of depths of coverage across samples at each genomic position. Therefore, cn.MOPS is not affected by read count variations along chromosomes. Using a Bayesian approach, cn.MOPS decomposes variations in the depth of coverage across samples into integer copy numbers and noise by means of its mixture components and Poisson distributions, respectively. The noise estimate allows for reducing the FDR by filtering out detections having high noise that are likely to be false detections. We compared cn.MOPS with the five most popular methods for CNV detection in NGS data using four benchmark datasets: (i) simulated data, (ii) NGS data from a male HapMap individual with implanted CNVs from the X chromosome, (iii) data from HapMap individuals with known CNVs, (iv) high coverage data from the 1000 Genomes Project. cn.MOPS outperformed its five competitors in terms of precision (1-FDR) and recall for both gains and losses in all benchmark data sets. The software cn.MOPS is publicly available as an R package at http://www.bioinf.jku.at/software/cnmops/ and at Bioconductor.

EARLY EVALUATION OF NEW HEALTH TECHNOLOGIES: THE CASE FOR PREMARKET STUDIES THAT HARMONIZE REGULATORY AND COVERAGE PERSPECTIVES.

PubMed

Levin, Leslie

2015-01-01

With an increasing awareness that active engagement between policy decision makers, HTA agencies, regulators and payers with industry in the premarket space is needed, a disruptive comprehensive approach is described which moves the evidentiary process exclusively into this space. Single harmonized studies pre-market to address regulatory and coverage needs and expectations are more likely to be efficient and less costly and position evidence to drive rather than test innovation. An example of such a process through the MaRS EXCITE program in Ontario, Canada, now undergoing proof of concept, is briefly discussed. Other examples of dialogue between decision makers and industry pre-market are provided though these are less robust than a comprehensive evidentiary approach.

A privacy-preserving solution for compressed storage and selective retrieval of genomic data.

PubMed

Huang, Zhicong; Ayday, Erman; Lin, Huang; Aiyar, Raeka S; Molyneaux, Adam; Xu, Zhenyu; Fellay, Jacques; Steinmetz, Lars M; Hubaux, Jean-Pierre

2016-12-01

In clinical genomics, the continuous evolution of bioinformatic algorithms and sequencing platforms makes it beneficial to store patients' complete aligned genomic data in addition to variant calls relative to a reference sequence. Due to the large size of human genome sequence data files (varying from 30 GB to 200 GB depending on coverage), two major challenges facing genomics laboratories are the costs of storage and the efficiency of the initial data processing. In addition, privacy of genomic data is becoming an increasingly serious concern, yet no standard data storage solutions exist that enable compression, encryption, and selective retrieval. Here we present a privacy-preserving solution named SECRAM (Selective retrieval on Encrypted and Compressed Reference-oriented Alignment Map) for the secure storage of compressed aligned genomic data. Our solution enables selective retrieval of encrypted data and improves the efficiency of downstream analysis (e.g., variant calling). Compared with BAM, the de facto standard for storing aligned genomic data, SECRAM uses 18% less storage. Compared with CRAM, one of the most compressed nonencrypted formats (using 34% less storage than BAM), SECRAM maintains efficient compression and downstream data processing, while allowing for unprecedented levels of security in genomic data storage. Compared with previous work, the distinguishing features of SECRAM are that (1) it is position-based instead of read-based, and (2) it allows random querying of a subregion from a BAM-like file in an encrypted form. Our method thus offers a space-saving, privacy-preserving, and effective solution for the storage of clinical genomic data. © 2016 Huang et al.; Published by Cold Spring Harbor Laboratory Press.

A privacy-preserving solution for compressed storage and selective retrieval of genomic data

PubMed Central

Huang, Zhicong; Ayday, Erman; Lin, Huang; Aiyar, Raeka S.; Molyneaux, Adam; Xu, Zhenyu; Hubaux, Jean-Pierre

2016-01-01

In clinical genomics, the continuous evolution of bioinformatic algorithms and sequencing platforms makes it beneficial to store patients’ complete aligned genomic data in addition to variant calls relative to a reference sequence. Due to the large size of human genome sequence data files (varying from 30 GB to 200 GB depending on coverage), two major challenges facing genomics laboratories are the costs of storage and the efficiency of the initial data processing. In addition, privacy of genomic data is becoming an increasingly serious concern, yet no standard data storage solutions exist that enable compression, encryption, and selective retrieval. Here we present a privacy-preserving solution named SECRAM (Selective retrieval on Encrypted and Compressed Reference-oriented Alignment Map) for the secure storage of compressed aligned genomic data. Our solution enables selective retrieval of encrypted data and improves the efficiency of downstream analysis (e.g., variant calling). Compared with BAM, the de facto standard for storing aligned genomic data, SECRAM uses 18% less storage. Compared with CRAM, one of the most compressed nonencrypted formats (using 34% less storage than BAM), SECRAM maintains efficient compression and downstream data processing, while allowing for unprecedented levels of security in genomic data storage. Compared with previous work, the distinguishing features of SECRAM are that (1) it is position-based instead of read-based, and (2) it allows random querying of a subregion from a BAM-like file in an encrypted form. Our method thus offers a space-saving, privacy-preserving, and effective solution for the storage of clinical genomic data. PMID:27789525

A real negative selection algorithm with evolutionary preference for anomaly detection

NASA Astrophysics Data System (ADS)

Yang, Tao; Chen, Wen; Li, Tao

2017-04-01

Traditional real negative selection algorithms (RNSAs) adopt the estimated coverage (c0) as the algorithm termination threshold, and generate detectors randomly. With increasing dimensions, the data samples could reside in the low-dimensional subspace, so that the traditional detectors cannot effectively distinguish these samples. Furthermore, in high-dimensional feature space, c0 cannot exactly reflect the detectors set coverage rate for the nonself space, and it could lead the algorithm to be terminated unexpectedly when the number of detectors is insufficient. These shortcomings make the traditional RNSAs to perform poorly in high-dimensional feature space. Based upon "evolutionary preference" theory in immunology, this paper presents a real negative selection algorithm with evolutionary preference (RNSAP). RNSAP utilizes the "unknown nonself space", "low-dimensional target subspace" and "known nonself feature" as the evolutionary preference to guide the generation of detectors, thus ensuring the detectors can cover the nonself space more effectively. Besides, RNSAP uses redundancy to replace c0 as the termination threshold, in this way RNSAP can generate adequate detectors under a proper convergence rate. The theoretical analysis and experimental result demonstrate that, compared to the classical RNSA (V-detector), RNSAP can achieve a higher detection rate, but with less detectors and computing cost.

Dynamics of distribution and density of phreatophytes and other arid land plant communities

NASA Technical Reports Server (NTRS)

Turner, R. M. (Principal Investigator)

1973-01-01

The author has identified the following significant results. Ground truth measurements of plant coverage on six satellite overflight dates reveal unique trends in coverage for the five desert or semi-desert communities selected. Densitometry and multispectral additive color viewing were used in a preliminary analysis of imagery using the electronic satellite image analyzer console at Stanford Research Institute. The densitometric analysis shows promise for mapping boundaries between plant communities. Color additive viewing of a chronologic sequence of the same scene shown in rapid order will provide a method for mapping phreatophyte communities.

PWHATSHAP: efficient haplotyping for future generation sequencing.

PubMed

Bracciali, Andrea; Aldinucci, Marco; Patterson, Murray; Marschall, Tobias; Pisanti, Nadia; Merelli, Ivan; Torquati, Massimo

2016-09-22

Haplotype phasing is an important problem in the analysis of genomics information. Given a set of DNA fragments of an individual, it consists of determining which one of the possible alleles (alternative forms of a gene) each fragment comes from. Haplotype information is relevant to gene regulation, epigenetics, genome-wide association studies, evolutionary and population studies, and the study of mutations. Haplotyping is currently addressed as an optimisation problem aiming at solutions that minimise, for instance, error correction costs, where costs are a measure of the confidence in the accuracy of the information acquired from DNA sequencing. Solutions have typically an exponential computational complexity. WHATSHAP is a recent optimal approach which moves computational complexity from DNA fragment length to fragment overlap, i.e., coverage, and is hence of particular interest when considering sequencing technology's current trends that are producing longer fragments. Given the potential relevance of efficient haplotyping in several analysis pipelines, we have designed and engineered PWHATSHAP, a parallel, high-performance version of WHATSHAP. PWHATSHAP is embedded in a toolkit developed in Python and supports genomics datasets in standard file formats. Building on WHATSHAP, PWHATSHAP exhibits the same complexity exploring a number of possible solutions which is exponential in the coverage of the dataset. The parallel implementation on multi-core architectures allows for a relevant reduction of the execution time for haplotyping, while the provided results enjoy the same high accuracy as that provided by WHATSHAP, which increases with coverage. Due to its structure and management of the large datasets, the parallelisation of WHATSHAP posed demanding technical challenges, which have been addressed exploiting a high-level parallel programming framework. The result, PWHATSHAP, is a freely available toolkit that improves the efficiency of the analysis of genomics information.

Photographic coverage of STS-115 Egress Training. Bldg.9NW, CTT

NASA Image and Video Library

2002-12-03

JSC2002-02132 (3 December 2002) --- Astronaut Daniel C. Burbank, STS-115 mission specialist, uses the Sky-genie to lower himself from a simulated trouble-plagued shuttle in an emergency egress training session in the Space Vehicle Mockup Facility at the Johnson Space Center (JSC). Burbank is wearing a training version of the shuttle launch and entry suit. United Space Alliance (USA) crew trainer David Pogue assisted Burbank.

Preflight coverage of STS-114 & Expedition 7 Crews, Emergency Egress Training

NASA Image and Video Library

2002-09-12

JSC2002-01659 (12 September 2002) --- The STS-114 crewmembers pose for a group photo prior to a training session in the Space Vehicle Mockup Facility at the Johnson Space Center (JSC). From the left are astronauts Soichi Noguchi, mission specialist; Eileen M. Collins, mission commander; Stephen K. Robinson, mission specialist; and James M. Kelly, pilot. Noguchi represents Japan’s National Space Development Agency (NASDA).

Quantifying the impact of cross coverage on physician's workload and performance in radiation oncology.

PubMed

Mosaly, Prithima R; Mazur, Lukasz M; Jones, Ellen L; Hoyle, Lesley; Zagar, Timothy; Chera, Bhishamjit S; Marks, Lawrence B

2013-01-01

To quantitatively assess the difference in workload and performance of radiation oncology physicians during radiation therapy treatment planning tasks under the conditions of "cross coverage" versus planning a patient with whom they were familiar. Eight physicians (3 experienced faculty physicians and 5 physician residents) performed 2 cases. The first case represented a "cross-coverage" scenario where the physicians had no prior information about the case to be planned. The second exposure represented a "regular-coverage" scenario where the physicians were familiar with the patient case to be planned. Each case involved 3 tasks to be completed systematically. Workload was assessed both subjectively (perceived) using National Aeronautics and Space Administration-Task Load Index (NASA-TLX), and objectively (physiological) throughout the task using eye data (via monitoring pupil size and blink rate). Performance of each task and the case was measured using completion time. Subjective willingness to approve or disapprove the generated plan was obtained after completion of the case only. Forty-eight perceived and 48 physiological workload assessments were obtained. Overall, results revealed a significant increase in perceived workload (high NASA-TLX score) and decrease in performance (longer completion time and reduced approval rate) during cross coverage. There were nonsignificant increases in pupil diameter and decreases in the blink rate during cross-coverage versus regular-coverage scenario. In both cross-coverage and regular-coverage scenarios the level of experience did not affect workload and performance. The cross-coverage scenario significantly increases perceived workload and degrades performance versus regular coverage. Hence, to improve patient safety, efforts must be made to develop policies, standard operating procedures, and usability improvements to electronic medical record and treatment planning systems for "easier" information processing to deal with cross coverage, while recognizing strengths and limitations of human performance. Published by Elsevier Inc.

Public Attitudes toward the Apollo Space Program, 1965-1975.

ERIC Educational Resources Information Center

Krugman, Herbert E.

1977-01-01

Analyzes the decline in public support for the Apollo Space Program from 1965 to 1975 in spite of generally positive media coverage. Using data from 31 telephone surveys during the period, concludes that the Moon landing polarized both opponents and proponents and increased opposition because "there was nothing more to be done." (JMF)

Actionable mutations in canine hemangiosarcoma

PubMed Central

Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A.; Wojcik, John; Durham, Amy C.; Mason, Nicola J.

2017-01-01

Background Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Methods Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Results and conclusions Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease. PMID:29190660

Actionable mutations in canine hemangiosarcoma.

PubMed

Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A; Wojcik, John; Durham, Amy C; Mason, Nicola J; Roth, David B

2017-01-01

Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

Circulation of Endemic Type 2 Vaccine-Derived Poliovirus in Egypt from 1983 to 1993

PubMed Central

Yang, Chen-Fu; Naguib, Tary; Yang, Su-Ju; Nasr, Eman; Jorba, Jaume; Ahmed, Nahed; Campagnoli, Ray; van der Avoort, Harrie; Shimizu, Hiroyuki; Yoneyama, Tetsuo; Miyamura, Tatsuo; Pallansch, Mark; Kew, Olen

2003-01-01

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5′ untranslated region (5′ UTR) and noncapsid- 3′ UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide. PMID:12857906

Enhanced Methods for Local Ancestry Assignment in Sequenced Admixed Individuals

PubMed Central

Brown, Robert; Pasaniuc, Bogdan

2014-01-01

Inferring the ancestry at each locus in the genome of recently admixed individuals (e.g., Latino Americans) plays a major role in medical and population genetic inferences, ranging from finding disease-risk loci, to inferring recombination rates, to mapping missing contigs in the human genome. Although many methods for local ancestry inference have been proposed, most are designed for use with genotyping arrays and fail to make use of the full spectrum of data available from sequencing. In addition, current haplotype-based approaches are very computationally demanding, requiring large computational time for moderately large sample sizes. Here we present new methods for local ancestry inference that leverage continent-specific variants (CSVs) to attain increased performance over existing approaches in sequenced admixed genomes. A key feature of our approach is that it incorporates the admixed genomes themselves jointly with public datasets, such as 1000 Genomes, to improve the accuracy of CSV calling. We use simulations to show that our approach attains accuracy similar to widely used computationally intensive haplotype-based approaches with large decreases in runtime. Most importantly, we show that our method recovers comparable local ancestries, as the 1000 Genomes consensus local ancestry calls in the real admixed individuals from the 1000 Genomes Project. We extend our approach to account for low-coverage sequencing and show that accurate local ancestry inference can be attained at low sequencing coverage. Finally, we generalize CSVs to sub-continental population-specific variants (sCSVs) and show that in some cases it is possible to determine the sub-continental ancestry for short chromosomal segments on the basis of sCSVs. PMID:24743331