Abstract
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.
Highlights
Exome 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
The platforms are based on a chip-hybrid method (NimbleGen Sequence Capture Array) or a solutionhybridization method (NimbleGen SeqCap EZ) with a common set of DNA probes, and a solution hybridization method with RNA probes (Agilent SureSelect)
We constructed a total of six libraries for the three platforms and used the HiSeq2000 to initially produce >30-fold coverage of unique mapped paired-end 90-bp reads (PE90) for each library
Summary
Exome 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. Several platforms for human exome capture for massively parallel sequencing have been developed and marketed to date [11,12,13,14] In principle, these platforms fall into three classes: DNA-chip-based capture [11,12], DNA-probe-based solution hybridization [14], and RNA-probe-based solution hybridization [13]. These platforms fall into three classes: DNA-chip-based capture [11,12], DNA-probe-based solution hybridization [14], and RNA-probe-based solution hybridization [13] These platforms have resulted in great success in pioneering studies hunting for variants causing rare human diseases [11,15,16,17,18,19,20,21], and have been adopted in efforts towards deciphering human common disease and cancer genomes. How many human genes are targeted by each approach and how even is their coverage? How do capture efficacy, technological reproducibility and biases among the different platforms compare? How much input DNA is required and how convenient is each experimentally? How does the cost-effectiveness compare? And what is the power and accuracy of SNP calling, especially for medically important rare SNPs? Up till publicly accessible explorations of methodology have been limited to proof-of-concept studies [11,13,14,22], reviews [23,24], or comparisons carried out on only a subset of genes rather than at the whole-genome level [25]
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