TY - JOUR
T1 - Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing
AU - Hodges, Emily
AU - Rooks, Michelle
AU - Xuan, Zhenyu
AU - Bhattacharjee, Arindam
AU - Gordon, D. Benjamin
AU - Brizuela, Leonardo
AU - McCombie, W. Richard
AU - Hannon, Gregory J.
PY - 2009
Y1 - 2009
N2 - 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 lg 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.
AB - 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 lg 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.
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U2 - 10.1038/nprot.2009.68
DO - 10.1038/nprot.2009.68
M3 - Article
C2 - 19478811
AN - SCOPUS:66749159688
SN - 1754-2189
VL - 4
SP - 960
EP - 978
JO - Nature Protocols
JF - Nature Protocols
IS - 6
ER -