Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing

Nat Protoc. 2009;4(6):960-74. doi: 10.1038/nprot.2009.68. Epub 2009 May 28.

Abstract

Complementary techniques that deepen information content and minimize reagent costs are required to realize the full potential of massively parallel sequencing. Here, we describe a resequencing approach that directs focus to genomic regions of high interest by combining hybridization-based purification of multi-megabase regions with sequencing on the Illumina Genome Analyzer (GA). The capture matrix is created by a microarray on which probes can be programmed as desired to target any non-repeat portion of the genome, while the method requires only a basic familiarity with microarray hybridization. We present a detailed protocol suitable for 1-2 microg of input genomic DNA and highlight key design tips in which high specificity (>65% of reads stem from enriched exons) and high sensitivity (98% targeted base pair coverage) can be achieved. We have successfully applied this to the enrichment of coding regions, in both human and mouse, ranging from 0.5 to 4 Mb in length. From genomic DNA library production to base-called sequences, this procedure takes approximately 9-10 d inclusive of array captures and one Illumina flow cell run.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • DNA Primers
  • Gene Library
  • Genomics / instrumentation
  • Genomics / methods
  • Genomics / statistics & numerical data
  • Humans
  • Mice
  • Nucleic Acid Hybridization / methods
  • Oligonucleotide Array Sequence Analysis / instrumentation
  • Oligonucleotide Array Sequence Analysis / methods*
  • Oligonucleotide Array Sequence Analysis / statistics & numerical data
  • Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / instrumentation
  • Sequence Analysis, DNA / methods*
  • Sequence Analysis, DNA / statistics & numerical data

Substances

  • DNA Primers