Comprehensive comparison of three commercial human whole-exome capture platforms

BackgroundExome sequencing, which allows the global analysis of protein coding sequences in the human genome, has become an effective and affordable approach to detecting causative genetic mutations in diseases. Currently, there are several commercial human exome capture platforms; however, the relative performances of these have not been characterized sufficiently to know which is best for a particular study.ResultsWe comprehensively compared three platforms: NimbleGen's Sequence Capture Array and SeqCap EZ, and Agilent's SureSelect. We assessed their performance in a variety of ways, including number of genes covered and capture efficacy. Differences that may impact on the choice of platform were that Agilent SureSelect covered approximately 1,100 more genes, while NimbleGen provided better flanking sequence capture. Although all three platforms achieved similar capture specificity of targeted regions, the NimbleGen platforms showed better uniformity of coverage and greater genotype sensitivity at 30- to 100-fold sequencing depth. All three platforms showed similar power in exome SNP calling, including medically relevant SNPs. Compared with genotyping and whole-genome sequencing data, the three platforms achieved a similar accuracy of genotype assignment and SNP detection. Importantly, all three platforms showed similar levels of reproducibility, GC bias and reference allele bias.ConclusionsWe demonstrate key differences between the three platforms, particularly advantages of solutions over array capture and the importance of a large gene target set

Omics-based interpretation of synergism in a soil-derived cellulose-degrading microbial community

Reaching a comprehensive understanding of how nature solves the problem of degrading recalcitrant biomass may eventually allow development of more efficient biorefining processes. Here we interpret genomic and proteomic information generated from a cellulolytic microbial consortium (termed F1RT) enriched from soil. Analyses of reconstructed bacterial draft genomes from all seven uncultured phylotypes in F1RT indicate that its constituent microbes cooperate in both cellulose-degrading and other important metabolic processes. Support for cellulolytic inter-species cooperation came from the discovery of F1RT microbes that encode and express complimentary enzymatic inventories that include both extracellular cellulosomes and secreted free-enzyme systems. Metabolic reconstruction of the seven F1RT phylotypes predicted a wider genomic rationale as to how this particular community functions as well as possible reasons as to why biomass conversion in nature relies on a structured and cooperative microbial community

Identification of Balanced Chromosomal Rearrangements Previously Unknown Among Participants in the 1000 Genomes Project: Implications for Interpretation of Structural Variation in Genomes and the Future of Clinical Cytogenetics

Purpose Recent studies demonstrate that whole-genome sequencing (WGS) enables detection of cryptic rearrangements in apparently balanced chromosomal rearrangements (also known as balanced chromosomal abnormalities, BCAs) previously identified by conventional cytogenetic methods. We aimed to assess our analytical tool for detecting BCAs in The 1000 Genomes Project without knowing affected bands. Methods: The 1000 Genomes Project provides an unprecedented integrated map of structural variants in phenotypically normal subjects, but there is no information on potential inclusion of subjects with apparently BCAs akin to those traditionally detected in diagnostic cytogenetics laboratories. We applied our analytical tool to 1,166 genomes from the 1000 Genomes Project with sufficient physical coverage (8.25-fold). Results: Our approach detected four reciprocal balanced translocations and four inversions ranging in size from 57.9 kb to 13.3 Mb, all of which were confirmed by cytogenetic methods and PCR studies. One of DNAs has a subtle translocation that is not readily identified by chromosome analysis due to similar banding patterns and size of exchanged segments, and another results in disruption of all transcripts of an OMIM gene. Conclusions: Our study demonstrates the extension of utilizing low-coverage WGS for unbiased detection of BCAs including translocations and inversions previously unknown in the 1000 Genomes Project

Two long insertions in YH genome detected by long-range PE.

<p>Mapping the long-range PE reads back to the human genome (NCBI build 37) resulted in the detection of a previously identified ∼8 kb insertion in chromosome 7 (A) and a novel ∼7 kb insertion in chromosome 14 (B) in the YH genome. The abnormally mapped PE reads that supported the insertions by showing unexpected short insert size are shown.</p

Paired-End Sequencing of Long-Range DNA Fragments for <em>De Novo</em> Assembly of Large, Complex Mammalian Genomes by Direct Intra-Molecule Ligation

<div><h3>Background</h3><p>The relatively short read lengths from next generation sequencing (NGS) technologies still pose a challenge for <em>de novo</em> assembly of complex mammal genomes. One important solution is to use paired-end (PE) sequence information experimentally obtained from long-range DNA fragments (>1 kb). Here, we characterize and extend a long-range PE library construction method based on direct intra-molecule ligation (or molecular linker-free circularization) for NGS.</p> <h3>Results</h3><p>We found that the method performs stably for PE sequencing of 2- to 5- kb DNA fragments, and can be extended to 10–20 kb (and even in extremes, up to ∼35 kb). We also characterized the impact of low quality input DNA on the method, and develop a whole-genome amplification (WGA) based protocol using limited input DNA (<1 µg). Using this PE dataset, we accurately assembled the YanHuang (YH) genome, the first sequenced Asian genome, into a scaffold N50 size of >2 Mb, which is over100-times greater than the initial size produced with only small insert PE reads(17 kb). In addition, we mapped two 7- to 8- kb insertions in the YH genome using the larger insert sizes of the long-range PE data.</p> <h3>Conclusions</h3><p>In conclusion, we demonstrate here the effectiveness of this long-range PE sequencing method and its use for the <em>de novo</em> assembly of a large, complex genome using NGS short reads.</p> </div

Summary of <i>de novo</i> YH genome assembly.

<p>The data from the YH project was used for the contig and initial scaffold assembly. Then, the long-range PE data were added step by step for scaffold construction. Genome coverage and gene coverage was calculated using the NCBI build 37 and RefSeq gene set as reference, respectively. The X and Y chromosomes were excluded while calculating genome coverage and gene coverage. For calculation of scaffold N50, N90 and total length, the intra-scaffold gaps were included.</p