KNAW Repository

Accurate SNP and mutation detection by targeted custom microarray-based genomic enrichment of short-fragment sequencing libraries

Mokry, M. and Feitsma, H. and Nijman, I.J. and Bruijn de, E. and Zaag van der, P. J. and Guryev, V. and Cuppen, E. (2010) Accurate SNP and mutation detection by targeted custom microarray-based genomic enrichment of short-fragment sequencing libraries. Nucleic Acids Research, 38, e116-. ISSN 0305-1048.

Full text not available from this repository.

Official URL: http://dx.doi.org/10.1093/nar/gkq072

Abstract

Microarray-based enrichment of selected genomic loci is a powerful method for genome complexity reduction for next-generation sequencing. Since the vast majority of exons in vertebrate genomes are smaller than 150 nt, we explored the use of short fragment libraries (85-110 bp) to achieve higher enrichment specificity by reducing carryover and adverse effects of flanking intronic sequences. High enrichment specificity (60-75%) was obtained with a relative even base coverage. Up to 98% of the target-sequence was covered more than 20x at an average coverage depth of about 200x. To verify the accuracy of SNP/mutation detection, we evaluated 384 known non-reference SNPs in the targeted regions. At approximately 200x average sequence coverage, we were able to survey 96.4% of 1.69 Mb of genomic sequence with only 4.2% false negative calls, mostly due to low coverage. Using the same settings, a total of 1197 novel candidate variants were detected. Verification experiments revealed only eight false positive calls, indicating an overall false positive rate of less than 1 per approximately 200,000 bp. Taken together, short fragment libraries provide highly efficient and flexible enrichment of exonic targets and yield relatively even base coverage, which facilitates accurate SNP and mutation detection. Raw sequencing data, alignment files and called SNPs have been submitted into GEO database http://www.ncbi.nlm.nih.gov/geo/ with accession number GSE18542.

Item Type:Article
Institutes:Hubrecht Instituut
ID Code:9532
Deposited On:11 Feb 2011 01:00
Last Modified:07 Sep 2011 17:01

Repository Staff Only: item control page