Summary Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

SUMMARYCancer is driven by mutation. Worldwide, tobacco smoking is the major lifestyle exposure that causes cancer, exerting carcinogenicity through >60 chemicals that bind and mutate DNA. Using massively parallel sequencing technology, we sequenced a small cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking. 22,910 somatic substitutions were identified, including 132 in coding exons. Multiple mutation signatures testify to the cocktail of carcinogens in tobacco smoke and their proclivities for particular bases and surrounding sequence context. Effects of transcription-coupled repair and a second, more general expression-linked repair pathway were evident. We identified a tandem duplication that duplicates exons 3-8 of CHD7 in-frame, and another two lines carrying PVT1-CHD7 fusion genes, suggesting that CHD7 may be recurrently rearranged in this disease. These findings illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer.

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)(+) transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160× average target coverage, 93% of nucleotides had at least 20× coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.

Using the massively parallel technique of sequencing by oligonucleotide ligation and detection (SOLiD; Applied Biosystems), we have assessed the in vivo positions of more than 44 million putative nucleosome cores in the multicellular genetic model organism Caenorhabditis elegans. These analyses provide a global view of the chromatin architecture of a multicellular animal at extremely high density and resolution. While we observe some degree of reproducible positioning throughout the genome in our mixed stage population of animals, we note that the major chromatin feature in the worm is a diversity of allowed nucleosome positions at the vast majority of individual loci. While absolute positioning of nucleosomes can vary substantially, relative positioning of nucleosomes (in a repeated array structure likely to be maintained at least in part by steric constraints) appears to be a significant property of chromatin structure. The high density of nucleosomal reads enabled a substantial extension of previous analysis describing the usage of individual oligonucleotide sequences along the span of the nucleosome core and linker. We release this data set, via the UCSC Genome Browser, as a resource for the high-resolution analysis of chromatin conformation and DNA accessibility at individual loci within the C. elegans genome.[Supplemental material is available online at www.genome.org. SOLiD raw sequencing data from this study have been submitted to the Short Read Archive at NCBI under accession no. SRA001023.]The regulation of genetic information within eukaryotic cells involves a high degree of specificity both in the availability of individual DNA-binding factors in individual cells, and in availability in the genome of DNA sequences that are their potential targets. Modulating the accessibility of individual DNA sequences are many complex interactions, the most prevalent of which are the interactions between histone octamers and DNA in compacted chromosomes. Each histone core interacts with 147 bp of DNA, which coil 1.7 times around the histone octamer (Luger et al. 1997;Davey et al. 2002) to form the basic unit of chromatin structure, the nucleosome. Since the first description over three decades ago (Kornberg 1974), the nucleosome and its role in gene regulation has been the subject of intensive study and speculation.As we have an increasingly detailed functional view of the genome, the tools of high-throughput molecular characterization have been used to begin obtaining a genome-wide description of nucleosome positions (Satchwell et al. 1986;Yuan et al. 2005;Johnson et al. 2006;Albert et al. 2007;Lee et al. 2007;Peckham et al. 2007;Schones et al. 2008;Shivaswamy et al. 2008). These data have in turn been used in attempts to reveal nucleosome positioning signals in DNA sequence (Satchwell et al. 1986;Ioshikhes et al. 1996;Segal et al. 2006;Yuan and Liu 2008). Although of great interest and value, sequence-based predictions of nucleosome position have been limited to date in their accuracy and resolution.In the litera...

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding ;183 haploid coverage of aligned sequence and close to 3003 clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed matepaired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.[Supplemental material is available online at

Although histones can form nucleosomes on virtually any genomic sequence, DNA sequences show considerable variability in their binding affinity. We have used DNA sequences of Saccharomyces cerevisiae whose nucleosome binding affinities have been experimentally determined (Yuan et al. 2005) to train a support vector machine to identify the nucleosome formation potential of any given sequence of DNA. The DNA sequences whose nucleosome formation potential are most accurately predicted are those that contain strong nucleosome forming or inhibiting signals and are found within nucleosome length stretches of genomic DNA with continuous nucleosome formation or inhibition signals. We have accurately predicted the experimentally determined nucleosome positions across a well-characterized promoter region of S. cerevisiae and identified strong periodicity within 199 center-aligned mononucleosomes studied recently (Segal et al. 2006) despite there being no periodicity information used to train the support vector machine. Our analysis suggests that only a subset of nucleosomes are likely to be positioned by intrinsic sequence signals. This observation is consistent with the available experimental data and is inconsistent with the proposal of a nucleosome positioning code. Finally, we show that intrinsic nucleosome positioning signals are both more inhibitory and more variable in promoter regions than in open reading frames in S. cerevisiae.

Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain. We studied a mutant strain of Pichia stipitis, a yeast capable of converting xylose to ethanol. This unusually efficient mutant strain was developed through repeated rounds of chemical mutagenesis, strain selection, transformation, and genetic manipulation over a period of seven years. We resequenced this strain on three different sequencing platforms. Surprisingly, we found fewer than a dozen mutations in open reading frames. All three sequencing technologies were able to identify each single nucleotide mutation given at least 10-15-fold nominal sequence coverage. Our results show that detecting mutations in evolved and engineered organisms is rapid and cost-effective at the whole-genome level using new sequencing technologies. Identification of specific mutations in strains with altered phenotypes will add insight into specific gene functions and guide further metabolic engineering efforts.[Supplemental material is available online at www.genome.org. Complete data sets are available at the NCBI Short Read Archive under accession no. SRA 001158 (ftp://ftp.ncbi.nih.gov/pub/TraceDB/ShortRead).]Pichia stipitis (Pignal) is a haploid yeast related to endosymbionts of beetles that degrade rotting wood (Suh et al. 2003). It is an important organism for bioenergy production from lignocellulosic materials because of its high capacity to ferment xylose and cellobiose to ethanol (Parekh et al. 1988). We previously sequenced the reference strain, Pichia stipitis CBS-6054, resulting in a completely characterized genome of eight chromosomes totaling 15.4 Mb of sequence (Jeffries et al. 2007). This strain has been subjected to chemical mutagenesis, phenotypic selection, genetic engineering, and adaptive evolution in order to develop strains improved for ethanol production. Chemical mutagenesis and selection resulted in small improvements in ethanol production attributable in part to carbon catabolite derepression (Supplemental Fig. 1; Methods). Disruption of CYC1 (cyctochrome c, isoform 1) to create strain Shi21 increased the specific ethanol production rate by 50% and the ethanol yield by 10%; however, the nature of additional mutational events leading to this phenotype was uncharacterized.Traditional methods for identifying mutations are laborand time-intensive, so we tested the ability of next-generation sequencing technologies to determine the differences in this improved strain's entire genome relative to the reference strain. We generated high-coverage, whole-genome data sets using single fragment end reads from three next-generation sequencing platforms: 454 Life Sc...