Dataset Information

A systematic evaluation of hybridization-based mouse exome capture system.

ABSTRACT: BACKGROUND: Exome sequencing is increasingly used to search for phenotypically-relevant sequence variants in the mouse genome. All of the current hybridization-based mouse exome capture systems are designed based on the genome reference sequences of the C57BL/6 J strain. Given that the substantial sequence divergence exists between C57BL/6 J and other distantly-related strains, the impact of sequence divergence on the efficiency of such capture systems needs to be systematically evaluated before they can be widely applied to the study of those strains. RESULTS: Using the Agilent SureSelect mouse exome capture system, we performed exome sequencing on F1 generation hybrid mice that were derived by crossing two divergent strains, C57BL/6 J and SPRET/EiJ. Our results showed that the C57BL/6 J-based probes captured the sequences derived from C57BL/6 J alleles more efficiently and that the bias was higher for the target regions with greater sequence divergence. At low sequencing depths, the bias also affected the efficiency of variant detection. However, the effects became negligible when sufficient sequencing depth was achieved. CONCLUSION: Sufficient sequence depth needs to be planned to match the sequence divergence between C57BL/6 J and the strain to be studied, when the C57BL/6 J-based Agilent SureSelect exome capture system is to be used.

SUBMITTER: Gao Q

PROVIDER: S-EPMC3722074 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A systematic evaluation of hybridization-based mouse exome capture system.

Gao Qingsong Q Sun Wei W You Xintian X Froehler Sebastian S Chen Wei W

BMC genomics 20130721

<h4>Background</h4>Exome sequencing is increasingly used to search for phenotypically-relevant sequence variants in the mouse genome. All of the current hybridization-based mouse exome capture systems are designed based on the genome reference sequences of the C57BL/6 J strain. Given that the substantial sequence divergence exists between C57BL/6 J and other distantly-related strains, the impact of sequence divergence on the efficiency of such capture systems needs to be systematically evaluated ...[more]

PMID: 23870319

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Project description: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.

Dataset Information

A systematic evaluation of hybridization-based mouse exome capture system.

Publications

A systematic evaluation of hybridization-based mouse exome capture system.

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