The pre-capture libraries were pooled as a 4-plex (approximately 500 ng/sample, 1 ug per pool) and hybridized in solution to the HGSC CORE design42 (link) (52Mb, NimbleGen) according to the manufacturer’s protocol (NimbleGen SeqCap EZ Exome Library SR User’s Guide) with minor revisions. Human COT1 DNA and full-length Illumina adaptor-specific blocking oligonucleotides were added into the hybridization to block repetitive genomic sequences and the adaptor sequences, followed by post-capture LM-PCR amplification using the 2X SOLiD Library High Fidelity Amplification Mix with 14 cycles of amplification. After the final SPRI bead purification, quantity and size of the capture library was analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500. The efficiency of the capture was evaluated by performing a qPCR-based quality assay on the four standard NimbleGen internal controls. Successful enrichment of the capture libraries was estimated to range from a 6 to 9 of ΔCt value over the non-enriched samples.
Protocol detail
Exome Capture and Sequencing Protocol
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Assay
Block
Dna chip
Exome
Genomic
Human
Hybridization
Library
Oligonucleotides
Repetitive sequences
Corresponding Organization :
Other organizations : Baylor College of Medicine, University of California, San Francisco, University of Toronto, The University of Texas Health Science Center at Houston
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Variable analysis
independent variables
- Hybridization of the pre-capture libraries to the HGSC CORE design
- Addition of human COT1 DNA and full-length Illumina adaptor-specific blocking oligonucleotides into the hybridization
- Post-capture LM-PCR amplification with 14 cycles of amplification
- Efficiency of the capture, estimated by performing a qPCR-based quality assay on the four standard NimbleGen internal controls
- Quantity and size of the capture library, analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500
- Pre-capture libraries were pooled as a 4-plex (approximately 500 ng/sample, 1 ug per pool)
- Hybridization was performed according to the manufacturer's protocol (NimbleGen SeqCap EZ Exome Library SR User's Guide) with minor revisions
- Four standard NimbleGen internal controls used for the qPCR-based quality assay
- Non-enriched samples used as a comparison for the efficiency of the capture, estimated by the ΔCt value
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