Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.
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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
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Journal | Nat Biotechnol
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Volume | 27
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Issue | 2
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Pages | 182-9
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Date Published | 2009 Feb
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ISSN | 1546-1696
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URL | |
DOI | 10.1038/nbt.1523
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PubMed ID | 19182786
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PubMed Central ID | PMC2663421
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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
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