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

Nat Biotechnol. 2009 Feb;27(2):182-9. doi: 10.1038/nbt.1523. Epub 2009 Feb 1.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition / genetics
  • Bayes Theorem
  • Biotinylation
  • Exons / genetics
  • Genomics / methods*
  • Humans
  • Nucleic Acid Hybridization
  • Oligonucleotide Array Sequence Analysis
  • Oligonucleotides / metabolism*
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide / genetics
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods*

Substances

  • Oligonucleotides