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Nat Biotechnol DOI:10.1038/nbt.1523

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

Publication TypeJournal Article
Year of Publication2009
AuthorsGnirke, A, Melnikov, A, Maguire, J, Rogov, P, LeProust, EM, Brockman, W, Fennell, T, Giannoukos, G, Fisher, S, Russ, C, Gabriel, S, Jaffe, DB, Lander, ES, Nusbaum, C
JournalNat Biotechnol
Date Published2009 Feb
KeywordsBase Composition, Bayes Theorem, Biotinylation, Exons, Genomics, Humans, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis, Oligonucleotides, Polymerase Chain Reaction, Polymorphism, Single Nucleotide, Reproducibility of Results, Sensitivity and Specificity, Sequence Analysis, DNA

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.


Alternate JournalNat. Biotechnol.
PubMed ID19182786
PubMed Central IDPMC2663421
Grant ListU54 HG003067 / HG / NHGRI NIH HHS / United States
U54 HG003067-05 / HG / NHGRI NIH HHS / United States
HG03067-05 / HG / NHGRI NIH HHS / United States