Hum Mutat. 2011 Sep 6. doi: 10.1002/humu.21602. [Epub ahead of print]

Rapid and Efficient Human Mutation Detection Using a Bench-Top Next-Generation DNA Sequencer.

Jiang Q, Turner T, Sosa MX, Rakha A, Arnold S, Chakravarti A.

Source

Center for Complex Disease Genomics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Abstract

Next-generation sequencing (NGS) technologies can be a boon to human mutation detection given their high throughput: consequently, many genes and samples may be simultaneously studied with high coverage for accurate detection of heterozygotes. In circumstances requiring the intensive study of a few genes, particularly in clinical applications, a rapid turn-around is another desirable goal. To this end, we assessed the performance of the bench-top 454 GS Junior platform as an optimized solution for mutation detection by amplicon sequencing of three type 3 semaphorin genes SEMA3A, SEMA3C and SEMA3D implicated in Hirschsprung disease (HSCR). We performed mutation detection on 39 PCR amplicons totaling 14,014bp in 47 samples studied in pools of 12 samples. Each 10-hour run was able to generate ∼75,000 reads and ∼28 million high-quality bases at an average read length of 371bp. The overall sequencing error was 0.26 changes per kb at a coverage depth of ≥20 reads. Altogether, 37 sequence variants were found in this study of which 10 were unique to HSCR patients. We identified five missense mutations in these three genes that may potentially be involved in the pathogenesis of HSCR and need to be studied in larger patient samples. ©2011 Wiley-Liss, Inc.
© 2011 Wiley-Liss, Inc.

PMID:

21898659

[PubMed - as supplied by publisher]