Library amplification readymix containing kapa hifi dna polymerase

Four pre-capture libraries were pooled together (~750 ng/sample, 3 ug/pool) and then hybridized in solution to the HGSC VCRome 2.1 design1 (Bainbridge et al., 2011 (link)) according to the manufacturer’s protocol NimbleGen SeqCap EZ Exome Library SR User’s Guide (Version 2.2) with minor revisions. Probes for exome coverage across > 3,500 clinically relevant genes that are previously < 20X (~2.72Mb) is supplemented into the VCRome 2.1 probe. Human COT1 DNA was added into the hybridization to block repetitive genomic sequences. Blocking oligonucleotides from Sigma (individually sequence specifically synthesized) or xGen Universal Blocking oligonucleotides (Integrated DNA Technologies) were added into the hybridization to block the adaptor sequences. Hybridization was carried out at 560C for ~16h. Post-capture LM-PCR amplification was performed using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc.) with 12 cycles of amplification. After the final AMPure XP 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 check on the four standard NimbleGen internal controls. Successful enrichment of the capture libraries was estimated to range from a 6 to 9 of ΔC_T value over the non-enriched samples.

For exome capture, FFPE, FF and normal samples were pooled separately as 4 libraries per pool using 250 ng of library for each sample. These pools of libraries were then hybridized separately in solution to the HGSC VCRome 2.1 design (Bainbridge et al., 2011 (link)) (42Mb, NimbleGen) according to the manufacturer’s protocol NimbleGen SeqCap EZ Exome Library SR User’s Guide (Version 2.2) form samples enriched between 2015-2017. Starting in December 2017, probes for exome coverage across >3,500 clinically relevant genes that are previously <20X (~2.72Mb) were supplemented with PKv1 and PKv2 into the VCRome 2.1 probe to enhance capture performance of low coverage regions. Blocking oligonucleotides from Sigma (individually sequence specifically synthesized) or xGen Universal Blocking oligonucleotides (Integrated DNA Technologies) were added into the hybridization to block the adaptor sequences. Hybridization for FF and normal samples was performed at 56°C for ~16h and for FFPE samples hybridization was at 42°C for 72h. Post-capture LM-PCR amplification was performed using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc., Cat # KK2612) with 12 cycles of amplification. After the final AMPure XP bead purification, quantity and size of the capture library was analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500.