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1.  The Human OligoGenome Resource: a database of oligonucleotide capture probes for resequencing target regions across the human genome 
Nucleic Acids Research  2011;40(D1):D1137-D1143.
Recent exponential growth in the throughput of next-generation DNA sequencing platforms has dramatically spurred the use of accessible and scalable targeted resequencing approaches. This includes candidate region diagnostic resequencing and novel variant validation from whole genome or exome sequencing analysis. We have previously demonstrated that selective genomic circularization is a robust in-solution approach for capturing and resequencing thousands of target human genome loci such as exons and regulatory sequences. To facilitate the design and production of customized capture assays for any given region in the human genome, we developed the Human OligoGenome Resource (http://oligogenome.stanford.edu/). This online database contains over 21 million capture oligonucleotide sequences. It enables one to create customized and highly multiplexed resequencing assays of target regions across the human genome and is not restricted to coding regions. In total, this resource provides 92.1% in silico coverage of the human genome. The online server allows researchers to download a complete repository of oligonucleotide probes and design customized capture assays to target multiple regions throughout the human genome. The website has query tools for selecting and evaluating capture oligonucleotides from specified genomic regions.
doi:10.1093/nar/gkr973
PMCID: PMC3245143  PMID: 22102592
2.  A Flexible Approach for Highly Multiplexed Candidate Gene Targeted Resequencing 
PLoS ONE  2011;6(6):e21088.
We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps from the initial capture assay to single nucleotide variant (SNV) discovery. The capture methodology uses in-solution 80-mer oligonucleotides. To provide optimal flexibility in choosing human gene targets, we designed an in silico set of oligonucleotides, the Human OligoExome, that covers the gene exons annotated by the Consensus Coding Sequencing Project (CCDS). This resource is openly available as an Internet accessible database where one can download capture oligonucleotides sequences for any CCDS gene and design custom capture assays. Using this resource, we demonstrated the flexibility of this assay by custom designing capture assays ranging from 10 to over 100 gene targets with total capture sizes from over 100 Kilobases to nearly one Megabase. We established a method to reduce capture variability and incorporated indexing schemes to increase sample throughput. Our approach has multiple applications that include but are not limited to population targeted resequencing studies of specific gene subsets, validation of variants discovered in whole genome sequencing surveys and possible diagnostic analysis of disease gene subsets. We also present a cost analysis demonstrating its cost-effectiveness for large population studies.
doi:10.1371/journal.pone.0021088
PMCID: PMC3127857  PMID: 21738606
3.  Targeted Capture of Homoeologous Coding and Noncoding Sequence in Polyploid Cotton 
G3: Genes|Genomes|Genetics  2012;2(8):921-930.
Targeted sequence capture is a promising technology in many areas in biology. These methods enable efficient and relatively inexpensive sequencing of hundreds to thousands of genes or genomic regions from many more individuals than is practical using whole-genome sequencing approaches. Here, we demonstrate the feasibility of target enrichment using sequence capture in polyploid cotton. To capture and sequence both members of each gene pair (homeologs) of wild and domesticated Gossypium hirsutum, we created custom hybridization probes to target 1000 genes (500 pairs of homeologs) using information from the cotton transcriptome. Two widely divergent samples of G. hirsutum were hybridized to four custom NimbleGen capture arrays containing probes for targeted genes. We show that the two coresident homeologs in the allopolyploid nucleus were efficiently captured with high coverage. The capture efficiency was similar between the two accessions and independent of whether the samples were multiplexed. A significant amount of flanking, nontargeted sequence (untranslated regions and introns) was also captured and sequenced along with the targeted exons. Intraindividual heterozygosity is low in both wild and cultivated Upland cotton, as expected from the high level of inbreeding in natural G. hirsutum and bottlenecks accompanying domestication. In addition, levels of heterozygosity appeared asymmetrical with respect to genome (AT or DT) in cultivated cotton. The approach used here is general, scalable, and may be adapted for many different research inquiries involving polyploid plant genomes.
doi:10.1534/g3.112.003392
PMCID: PMC3411248  PMID: 22908041
Gossypium; allopolyploidy; homoeologs; sequence capture; next-generation sequencing
4.  Genetic variation and linkage disequilibrium in Bacillus anthracis 
Scientific Reports  2011;1:169.
We performed whole-genome amplification followed by hybridization of custom-designed resequencing arrays to resequence 303 kb of genomic sequence from a worldwide panel of 39 Bacillus anthracis strains. We used an efficient algorithm contained within a custom software program, UniqueMER, to identify and mask repetitive sequences on the resequencing array to reduce false-positive identification of genetic variation, which can arise from cross-hybridization. We discovered a total of 240 single nucleotide variants (SNVs) and showed that B. anthracis strains have an average of 2.25 differences per 10,000 bases in the region we resequenced. Common SNVs in this region are found to be in complete linkage disequilibrium. These patterns of variation suggest there has been little if any historical recombination among B. anthracis strains since the origin of the pathogen. This pattern of common genetic variation suggests a framework for recognizing new or genetically engineered strains.
doi:10.1038/srep00169
PMCID: PMC3240990  PMID: 22355684
5.  The Application of Targeted and Exome Sequencing for the Identification of Spontaneous and Induced Mutations in Mice 
The Jackson Laboratory has established a large collection of spontaneous and N-ethyl-N-nitrosourea (ENU) induced mouse mutants with a wide variety of medically relevant phenotypes. While spontaneous mutations can be quite complex, including single nucleotide polymorphisms (SNPs), transposon insertions, deletions, or inversions, ENU induced mutations are typically SNPs. The traditional method of identifying the causative mutation through genetic mapping and Sanger sequencing of candidate genes has been effective but is time consuming, requires large populations of mice and can be expensive. In addition, it is particularly challenging when the mutation is not in a coding sequence. With a size of over 3 GB, it is still too expensive to sequence the genomes of the many mutant strains of interest. The combination of array capture for targeted resequencing and/or exome capture followed by high-throughput sequencing on the Illumina GAIIX has greatly accelerated the pace at which mutations have be identified. In deciding what approach should be employed to identify a specific mutation, a number of factors must be considered; 1) Is the mutation spontaneous or ENU induced; 2) Have traditional mapping approaches been exploited, and if not, should they be; 3) If there is mapping data, what is the size of the genetic interval; 4) What data analysis tools will be required; This approach has led to the identification of many mutations that result in disease-relevant phenotypes including craniofacial disorders, neurodegeneration, neuromuscular dysfunctions, cholesterol biosysthesis and reproduction.
PMCID: PMC3186650
6.  Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing 
Nature protocols  2009;4(6):960-974.
Complementary techniques that deepen information content and minimize reagent costs are required to realize the full potential of massively parallel sequencing. Here, we describe a resequencing approach that directs focus to genomic regions of high interest by combining hybridization-based purification of multi-megabase regions with sequencing on the Illumina Genome Analyzer (GA). The capture matrix is created by a microarray on which probes can be programmed as desired to target any non-repeat portion of the genome, while the method requires only a basic familiarity with microarray hybridization. We present a detailed protocol suitable for 1–2 µg of input genomic DNA and highlight key design tips in which high specificity (>65% of reads stem from enriched exons) and high sensitivity (98% targeted base pair coverage) can be achieved. We have successfully applied this to the enrichment of coding regions, in both human and mouse, ranging from 0.5 to 4 Mb in length. From genomic DNA library production to base-called sequences, this procedure takes approximately 9–10 d inclusive of array captures and one Illumina flow cell run.
doi:10.1038/nprot.2009.68
PMCID: PMC2990409  PMID: 19478811
7.  Screening the human exome: a comparison of whole genome and whole transcriptome sequencing 
Genome Biology  2010;11(5):R57.
Background
There is considerable interest in the development of methods to efficiently identify all coding variants present in large sample sets of humans. There are three approaches possible: whole-genome sequencing, whole-exome sequencing using exon capture methods, and RNA-Seq. While whole-genome sequencing is the most complete, it remains sufficiently expensive that cost effective alternatives are important.
Results
Here we provide a systematic exploration of how well RNA-Seq can identify human coding variants by comparing variants identified through high coverage whole-genome sequencing to those identified by high coverage RNA-Seq in the same individual. This comparison allowed us to directly evaluate the sensitivity and specificity of RNA-Seq in identifying coding variants, and to evaluate how key parameters such as the degree of coverage and the expression levels of genes interact to influence performance. We find that although only 40% of exonic variants identified by whole genome sequencing were captured using RNA-Seq; this number rose to 81% when concentrating on genes known to be well-expressed in the source tissue. We also find that a high false positive rate can be problematic when working with RNA-Seq data, especially at higher levels of coverage.
Conclusions
We conclude that as long as a tissue relevant to the trait under study is available and suitable quality control screens are implemented, RNA-Seq is a fast and inexpensive alternative approach for finding coding variants in genes with sufficiently high expression levels.
doi:10.1186/gb-2010-11-5-r57
PMCID: PMC2898068  PMID: 20598109
8.  Targeted enrichment beyond the consensus coding DNA sequence exome reveals exons with higher variant densities 
Genome Biology  2011;12(7):R68.
Background
Enrichment of loci by DNA hybridization-capture, followed by high-throughput sequencing, is an important tool in modern genetics. Currently, the most common targets for enrichment are the protein coding exons represented by the consensus coding DNA sequence (CCDS). The CCDS, however, excludes many actual or computationally predicted coding exons present in other databases, such as RefSeq and Vega, and non-coding functional elements such as untranslated and regulatory regions. The number of variants per base pair (variant density) and our ability to interrogate regions outside of the CCDS regions is consequently less well understood.
Results
We examine capture sequence data from outside of the CCDS regions and find that extremes of GC content that are present in different subregions of the genome can reduce the local capture sequence coverage to less than 50% relative to the CCDS. This effect is due to biases inherent in both the Illumina and SOLiD sequencing platforms that are exacerbated by the capture process. Interestingly, for two subregion types, microRNA and predicted exons, the capture process yields higher than expected coverage when compared to whole genome sequencing. Lastly, we examine the variation present in non-CCDS regions and find that predicted exons, as well as exonic regions specific to RefSeq and Vega, show much higher variant densities than the CCDS.
Conclusions
We show that regions outside of the CCDS perform less efficiently in capture sequence experiments. Further, we show that the variant density in computationally predicted exons is more than 2.5-times higher than that observed in the CCDS.
doi:10.1186/gb-2011-12-7-r68
PMCID: PMC3218830  PMID: 21787409
9.  Gene-Centric Characteristics of Genome-Wide Association Studies 
PLoS ONE  2007;2(12):e1262.
Background
The high-throughput genotyping chips have contributed greatly to genome-wide association (GWA) studies to identify novel disease susceptibility single nucleotide polymorphisms (SNPs). The high-density chips are designed using two different SNP selection approaches, the direct gene-centric approach, and the indirect quasi-random SNPs or linkage disequilibrium (LD)-based tagSNPs approaches. Although all these approaches can provide high genome coverage and ascertain variants in genes, it is not clear to which extent these approaches could capture the common genic variants. It is also important to characterize and compare the differences between these approaches.
Methodology/Principal Findings
In our study, by using both the Phase II HapMap data and the disease variants extracted from OMIM, a gene-centric evaluation was first performed to evaluate the ability of the approaches in capturing the disease variants in Caucasian population. Then the distribution patterns of SNPs were also characterized in genic regions, evolutionarily conserved introns and nongenic regions, ontologies and pathways. The results show that, no mater which SNP selection approach is used, the current high-density SNP chips provide very high coverage in genic regions and can capture most of known common disease variants under HapMap frame. The results also show that the differences between the direct and the indirect approaches are relatively small. Both have similar SNP distribution patterns in these gene-centric characteristics.
Conclusions/Significance
This study suggests that the indirect approaches not only have the advantage of high coverage but also are useful for studies focusing on various functional SNPs either in genes or in the conserved regions that the direct approach supports. The study and the annotation of characteristics will be helpful for designing and analyzing GWA studies that aim to identify genetic risk factors involved in common diseases, especially variants in genes and conserved regions.
doi:10.1371/journal.pone.0001262
PMCID: PMC2092383  PMID: 18060058
10.  Efficient and Cost Effective Population Resequencing by Pooling and In-Solution Hybridization 
PLoS ONE  2011;6(3):e18353.
High-throughput sequencing of targeted genomic loci in large populations is an effective approach for evaluating the contribution of rare variants to disease risk. We evaluated the feasibility of using in-solution hybridization-based target capture on pooled DNA samples to enable cost-efficient population sequencing studies. For this, we performed pooled sequencing of 100 HapMap samples across ∼600 kb of DNA sequence using the Illumina GAIIx. Using our accurate variant calling method for pooled sequence data, we were able to not only identify single nucleotide variants with a low false discovery rate (<1%) but also accurately detect short insertion/deletion variants. In addition, with sufficient coverage per individual in each pool (30-fold) we detected 97.2% of the total variants and 93.6% of variants below 5% in frequency. Finally, allele frequencies for single nucleotide variants (SNVs) estimated from the pooled data and the HapMap genotype data were tightly correlated (correlation coefficient > =  0.995).
doi:10.1371/journal.pone.0018353
PMCID: PMC3068187  PMID: 21479135
11.  Microarray-based resequencing of multiple Bacillus anthracis isolates 
Genome Biology  2004;6(1):R10.
Custom-designed resequencing arrays were used to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains. Sequence quality was shown to be very high by replication and by comparison to independently generated shotgun sequence
We used custom-designed resequencing arrays to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains. Sequence quality was shown to be very high by replication (discrepancy rate of 7.4 × 10-7) and by comparison to independently generated shotgun sequence (discrepancy rate < 2.5 × 10-6). Population genomics studies of microbial pathogens using rapid resequencing technologies such as resequencing arrays are critical for recognizing newly emerging or genetically engineered strains.
doi:10.1186/gb-2004-6-1-r10
PMCID: PMC549062  PMID: 15642093
12.  Exome sequencing: the sweet spot before whole genomes 
Human Molecular Genetics  2010;19(R2):R145-R151.
The development of massively parallel sequencing technologies, coupled with new massively parallel DNA enrichment technologies (genomic capture), has allowed the sequencing of targeted regions of the human genome in rapidly increasing numbers of samples. Genomic capture can target specific areas in the genome, including genes of interest and linkage regions, but this limits the study to what is already known. Exome capture allows an unbiased investigation of the complete protein-coding regions in the genome. Researchers can use exome capture to focus on a critical part of the human genome, allowing larger numbers of samples than are currently practical with whole-genome sequencing. In this review, we briefly describe some of the methodologies currently used for genomic and exome capture and highlight recent applications of this technology.
doi:10.1093/hmg/ddq333
PMCID: PMC2953745  PMID: 20705737
13.  Variant discovery in targeted resequencing using whole genome amplified DNA 
BMC Genomics  2013;14:468.
Background
Next generation sequencing and advances in genomic enrichment technologies have enabled the discovery of the full spectrum of variants from common to rare alleles in the human population. The application of such technologies can be limited by the amount of DNA available. Whole genome amplification (WGA) can overcome such limitations. Here we investigate applicability of using WGA by comparing SNP and INDEL variant calls from a single genomic/WGA sample pair from two capture separate experiments: a 50 Mbp whole exome capture and a custom capture array of 4 Mbp region on chr12.
Results
Our results comparing variant calls derived from genomic and WGA DNA show that the majority of variant SNP and INDEL calls are common to both callsets, both at the site and genotype level and suggest that allele bias plays a minimal role when using WGA DNA in re-sequencing studies.
Conclusions
Although the results of this study are based on a limited sample size, they suggest that using WGA DNA allows the discovery of the vast majority of variants, and achieves high concordance metrics, when comparing to genomic DNA calls.
doi:10.1186/1471-2164-14-468
PMCID: PMC3716764  PMID: 23837845
Whole genome amplified DNA; Capture sequencing; Next generation sequencing; Variant discovery
14.  Enriching targeted sequencing experiments for rare disease alleles 
Bioinformatics  2011;27(15):2112-2118.
Motivation: Next-generation targeted resequencing of genome-wide association study (GWAS)-associated genomic regions is a common approach for follow-up of indirect association of common alleles. However, it is prohibitively expensive to sequence all the samples from a well-powered GWAS study with sufficient depth of coverage to accurately call rare genotypes. As a result, many studies may use next-generation sequencing for single nucleotide polymorphism (SNP) discovery in a smaller number of samples, with the intent to genotype candidate SNPs with rare alleles captured by resequencing. This approach is reasonable, but may be inefficient for rare alleles if samples are not carefully selected for the resequencing experiment.
Results: We have developed a probability-based approach, SampleSeq, to select samples for a targeted resequencing experiment that increases the yield of rare disease alleles substantially over random sampling of cases or controls or sampling based on genotypes at associated SNPs from GWAS data. This technique allows for smaller sample sizes for resequencing experiments, or allows the capture of rarer risk alleles. When following up multiple regions, SampleSeq selects subjects with an even representation of all the regions. SampleSeq also can be used to calculate the sample size needed for the resequencing to increase the chance of successful capture of rare alleles of desired frequencies.
Software: http://biostat.mc.vanderbilt.edu/SampleSeq
Contact: chun.li@vanderbilt.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr324
PMCID: PMC3137214  PMID: 21700677
15.  Multiplex target capture with double-stranded DNA probes 
Genome Medicine  2013;5(5):50.
Target enrichment technologies utilize single-stranded oligonucleotide probes to capture candidate genomic regions from a DNA sample before sequencing. We describe target capture using double-stranded probes, which consist of single-stranded, complementary long padlock probes (cLPPs), each selectively capturing one strand of a genomic target through circularization. Using two probes per target increases sensitivity for variant detection and cLPPs are easily produced by PCR at low cost. Additionally, we introduce an approach for generating capture libraries with uniformly randomized template orientations. This facilitates bidirectional sequencing of both the sense and antisense template strands during one paired-end read, which maximizes target coverage.
doi:10.1186/gm454
PMCID: PMC3706973  PMID: 23718862
16.  Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in middle eastern families 
Genome Biology  2011;12(9):R89.
Background
Identification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity.
Results
A custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands co-segregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p.S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population.
Conclusions
Critical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss.
doi:10.1186/gb-2011-12-9-r89
PMCID: PMC3308052  PMID: 21917145
17.  Enhancer Chip: Detecting Human Copy Number Variations in Regulatory Elements 
PLoS ONE  2012;7(12):e52264.
Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.
doi:10.1371/journal.pone.0052264
PMCID: PMC3527541  PMID: 23284961
18.  High-resolution autosomal radiation hybrid maps of the pig genome and their contribution to the genome sequence assembly 
BMC Genomics  2012;13:585.
Background
The release of the porcine genome sequence offers great perspectives for Pig genetics and genomics, and more generally will contribute to the understanding of mammalian genome biology and evolution. The process of producing a complete genome sequence of high quality, while facilitated by high-throughput sequencing technologies, remains a difficult task. The porcine genome was sequenced using a combination of a hierarchical shotgun strategy and data generated with whole genome shotgun. In addition to the BAC contig map used for the clone-by-clone approach, genomic mapping resources for the pig include two radiation hybrid (RH) panels at two different resolutions. These two panels have been used extensively for the physical mapping of pig genes and markers prior to the availability of the pig genome sequence.
Results
In order to contribute to the assembly of the pig genome, we genotyped the two radiation hybrid (RH) panels with a SNP array (the Illumina porcineSNP60 array) and produced high density physical RH maps for each pig autosome. We first present the methods developed to obtain high density RH maps with 38,379 SNPs from the SNP array genotyping. We then show how they were useful to identify problems in a draft of the pig genome assembly, and how the RH maps enabled the problems to be corrected in the porcine genome sequence. Finally, we used the RH maps to predict the position of 2,703 SNPs and 1,328 scaffolds currently unplaced on the porcine genome assembly.
Conclusions
A complete process, from genotyping of a high density SNP array on RH panels, to the construction of genome-wide high density RH maps, and finally their exploitation for validating and improving a genome assembly is presented here. The study includes the cross-validation of RH based findings with independent information from genetic data and comparative mapping with the Human genome. Several additional resources are also provided, in particular the predicted genomic location of currently unplaced SNPs and associated scaffolds summing up to a total of 72 megabases, that can be useful for the exploitation of the pig genome assembly.
doi:10.1186/1471-2164-13-585
PMCID: PMC3499281  PMID: 23153393
19.  Improving mapping and SNP-calling performance in multiplexed targeted next-generation sequencing 
BMC Genomics  2012;13:417.
Background
Compared to classical genotyping, targeted next-generation sequencing (tNGS) can be custom-designed to interrogate entire genomic regions of interest, in order to detect novel as well as known variants. To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity (E. coli genes, multiplexed E. coli, and multiplexed HapMap BRCA1/2 regions).
Results
We successfully enriched barcoded NGS libraries instead of genomic DNA, achieving reproducible coverage profiles (Pearson correlation coefficients of up to 0.99) across multiplexed samples, with <10% strand bias. However, the SNP calling quality was substantially affected by the choice of tools and mapping strategy. With the aim of reducing computational requirements, we compared conventional whole-genome mapping and SNP-calling with a new faster approach: target-region mapping with subsequent ‘read-backmapping’ to the whole genome to reduce the false detection rate. Consequently, we developed a combined mapping pipeline, which includes standard tools (BWA, SAMtools, etc.), and tested it on public HiSeq2000 exome data from the 1000 Genomes Project. Our pipeline saved 12 hours of run time per Hiseq2000 exome sample and detected ~5% more SNPs than the conventional whole genome approach. This suggests that more potential novel SNPs may be discovered using both approaches than with just the conventional approach.
Conclusions
We recommend applying our general ‘two-step’ mapping approach for more efficient SNP discovery in tNGS. Our study has also shown the benefit of computing inter-sample SNP-concordances and inspecting read alignments in order to attain more confident results.
doi:10.1186/1471-2164-13-417
PMCID: PMC3563481  PMID: 22913592
Two-stage mapping; Read-backmapping; Software performance; SNP discovery; Multiplexed targeted next-generation sequencing
20.  Timing of human protein evolution as revealed by massively parallel capture of Neandertal nuclear DNA sequences 
Science (New York, N.Y.)  2010;328(5979):723-725.
Whole genome shotgun sequencing is now possible for extinct organisms, as well as the targeted capture of specific regions. However, targeted resequencing of megabase sized parts of nuclear genomes has yet to be demonstrated for ancient DNA. Here we show that hybridization capture on microarrays can be used to generate large scale targeted data from Neandertal DNA even in the presence of ~99.8% microbial DNA. It is thus now possible to generate high quality data from large regions of the nuclear genome from Neandertals and other extinct organisms. Using this approach we have sequenced ~14,000 protein coding positions that have been inferred to have changed on the human lineage since the last common ancestor shared with chimpanzees. We identify 88 amino acid substitutions that have become fixed in all humans since the divergence from the Neandertals.
doi:10.1126/science.1188046
PMCID: PMC3140021  PMID: 20448179
21.  Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales 
BMC Genomics  2012;13:403.
Background
To date, exon capture has largely been restricted to species with fully sequenced genomes, which has precluded its application to lineages that lack high quality genomic resources. We developed a novel strategy for designing array-based exon capture in chipmunks (Tamias) based on de novo transcriptome assemblies. We evaluated the performance of our approach across specimens from four chipmunk species.
Results
We selectively targeted 11,975 exons (~4 Mb) on custom capture arrays, and enriched over 99% of the targets in all libraries. The percentage of aligned reads was highly consistent (24.4-29.1%) across all specimens, including in multiplexing up to 20 barcoded individuals on a single array. Base coverage among specimens and within targets in each species library was uniform, and the performance of targets among independent exon captures was highly reproducible. There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions. We did observe a decline in capture performance of a subset of targets designed from a much more divergent ground squirrel genome (30 My), however, over 90% of the targets were also recovered. Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified.
Conclusions
Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life.
doi:10.1186/1471-2164-13-403
PMCID: PMC3472323  PMID: 22900609
Microarray-based exon capture; Phylogenetics; Population genomics; SNP identification; Tamias; Target enrichment
22.  Experiences with array-based sequence capture; toward clinical applications 
Although sequencing of a human genome gradually becomes an option, zooming in on the region of interest remains attractive and cost saving. We performed array-based sequence capture using 385K Roche NimbleGen, Inc. arrays to zoom in on the protein-coding and immediate intron-flanking sequences of 112 genes, potentially involved in mental retardation and congenital malformation. Captured material was sequenced using Illumina technology. A data analysis pipeline was built that detects sequence variants, positions them in relation to the gene, checks for presence in databases (eg, db single-nucleotide polymorphism (SNP)) and predicts the potential consequences at the level of RNA splicing and protein translation. In the samples analyzed, all known variants were reliably detected, including pathogenic variants from control cases and SNPs derived from array experiments. Although overall coverage varied considerably, it was reproducible per region and facilitated the detection of large deletions and duplications (copy number variations), including a partial deletion in the B3GALTL gene from a patient sample. For ultimate diagnostic application, overall results need to be improved. Future arrays should contain probes from both DNA strands, and to obtain a more even coverage, one could add fewer probes from densely and more probes from sparsely covered regions.
doi:10.1038/ejhg.2010.145
PMCID: PMC3039511  PMID: 21102627
capture array; heterogeneous disorders; sequencing
23.  Detection of copy number variation from array intensity and sequencing read depth using a stepwise Bayesian model 
BMC Bioinformatics  2010;11:539.
Background
Copy number variants (CNVs) have been demonstrated to occur at a high frequency and are now widely believed to make a significant contribution to the phenotypic variation in human populations. Array-based comparative genomic hybridization (array-CGH) and newly developed read-depth approach through ultrahigh throughput genomic sequencing both provide rapid, robust, and comprehensive methods to identify CNVs on a whole-genome scale.
Results
We developed a Bayesian statistical analysis algorithm for the detection of CNVs from both types of genomic data. The algorithm can analyze such data obtained from PCR-based bacterial artificial chromosome arrays, high-density oligonucleotide arrays, and more recently developed high-throughput DNA sequencing. Treating parameters--e.g., the number of CNVs, the position of each CNV, and the data noise level--that define the underlying data generating process as random variables, our approach derives the posterior distribution of the genomic CNV structure given the observed data. Sampling from the posterior distribution using a Markov chain Monte Carlo method, we get not only best estimates for these unknown parameters but also Bayesian credible intervals for the estimates. We illustrate the characteristics of our algorithm by applying it to both synthetic and experimental data sets in comparison to other segmentation algorithms.
Conclusions
In particular, the synthetic data comparison shows that our method is more sensitive than other approaches at low false positive rates. Furthermore, given its Bayesian origin, our method can also be seen as a technique to refine CNVs identified by fast point-estimate methods and also as a framework to integrate array-CGH and sequencing data with other CNV-related biological knowledge, all through informative priors.
doi:10.1186/1471-2105-11-539
PMCID: PMC2992546  PMID: 21034510
24.  Enrichment of sequencing targets from the human genome by solution hybridization 
Genome Biology  2009;10(10):R116.
A method for target sequence enrichment from the human genome is described. This hybridization-based approach using oligonucleotide probes in solution has excellent sensitivity and accuracy for calling SNPs
To exploit fully the potential of current sequencing technologies for population-based studies, one must enrich for loci from the human genome. Here we evaluate the hybridization-based approach by using oligonucleotide capture probes in solution to enrich for approximately 3.9 Mb of sequence target. We demonstrate that the tiling probe frequency is important for generating sequence data with high uniform coverage of targets. We obtained 93% sensitivity to detect SNPs, with a calling accuracy greater than 99%.
doi:10.1186/gb-2009-10-10-r116
PMCID: PMC2784331  PMID: 19835619
25.  Targeted Re-Sequencing Identified rs3106189 at the 5′ UTR of TAPBP and rs1052918 at the 3′ UTR of TCF3 to Be Associated with the Overall Survival of Colorectal Cancer Patients 
PLoS ONE  2013;8(8):e70307.
Recent studies have demonstrated the power of deep re-sequencing of the whole genome or exome in understanding cancer genomes. However, targeted capture of selected genomic whole gene-body regions, rather than the whole exome, have several advantages: 1) the genes can be selected based on biology or a hypothesis; 2) mutations in promoter and intronic regions, which have important regulatory roles, can be investigated; and 3) less expensive than whole genome or whole exome sequencing. Therefore, we designed custom high-density oligonucleotide microarrays (NimbleGen Inc.) to capture approximately 1.7 Mb target regions comprising the genomic regions of 28 genes related to colorectal cancer including genes belonging to the WNT signaling pathway, as well as important transcription factors or colon-specific genes that are over expressed in colorectal cancer (CRC). The 1.7 Mb targeted regions were sequenced with a coverage ranged from 32× to 45× for the 28 genes. We identified a total of 2342 sequence variations in the CRC and corresponding adjacent normal tissues. Among them, 738 were novel sequence variations based on comparisons with the SNP database (dbSNP135). We validated 56 of 66 SNPs in a separate cohort of 30 CRC tissues using Sequenom MassARRAY iPLEX Platform, suggesting a validation rate of at least 85% (56/66). We found 15 missense mutations among the exonic variations, 21 synonymous SNPs that were predicted to change the exonic splicing motifs, 31 UTR SNPs that were predicted to occur at the transcription factor binding sites, 20 intronic SNPs located near the splicing sites, 43 SNPs in conserved transcription factor binding sites and 32 in CpG islands. Finally, we determined that rs3106189, localized to the 5′ UTR of antigen presenting tapasin binding protein (TAPBP), and rs1052918, localized to the 3′ UTR of transcription factor 3 (TCF3), were associated with overall survival of CRC patients.
doi:10.1371/journal.pone.0070307
PMCID: PMC3734069  PMID: 23940558

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