Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.
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.
Gnirke
Andreas
A
Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA. gnirke@broad.mit.edu
Melnikov
Alexandre
A
Maguire
Jared
J
Rogov
Peter
P
LeProust
Emily M
EM
Brockman
William
W
Fennell
Timothy
T
Giannoukos
Georgia
G
Fisher
Sheila
S
Russ
Carsten
C
Gabriel
Stacey
S
Jaffe
David B
DB
Lander
Eric S
ES
Nusbaum
Chad
C
D001482:Base Composition / Q000235:genetics; D001499:Bayes Theorem; D019921:Biotinylation; D005091:Exons / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D009841:Oligonucleotides