Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing.
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.
Hodges
Emily
E
Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
Rooks
Michelle
M
Xuan
Zhenyu
Z
Bhattacharjee
Arindam
A
Benjamin Gordon
D
D
Brizuela
Leonardo
L
Richard McCombie
W
W
Hannon
Gregory J
GJ
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D015723:Gene Library; D023281:Genomics / Q000295:instrumentation / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data
D017931:DNA Primers