Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.

Nat Biotechnol
Authors
Keywords
Abstract

Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that approximately 60% of target bases in the exonic 'catch', and approximately 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

Year of Publication
2009
Journal
Nat Biotechnol
Volume
27
Issue
2
Pages
182-9
Date Published
2009 Feb
ISSN
1546-1696
URL
DOI
10.1038/nbt.1523
PubMed ID
19182786
PubMed Central ID
PMC2663421
Links
Grant list
U54 HG003067 / HG / NHGRI NIH HHS / United States
U54 HG003067-05 / HG / NHGRI NIH HHS / United States
HG03067-05 / HG / NHGRI NIH HHS / United States