Nimblegen seqcap hybridization and wash kit

NimbleGen SeqCap Hybridization and Wash kit (Roche NimbleGen, Switzerland) were used according to the technical note for targeted region selection. Hybridization probe can capture bulk library DNA from 8–12 indexed Illumina libraries. The probe library was designed through the NimbleDesign portal (Version 2) using genome build hg19 NCBI Build 37.1/GRCh37. All hybridization and wash operations were conducted with the NimbleGen SeqCap Hybridization and Wash kit (Roche NimbleGen, Basel, Switzerland) following the manufacturer’s manual. After hybridization, two 50 µL library amplification PCR were performed for 14 amplification cycles respectively, and then pooled and purified with Agencourt AMPure XP beads (Beckman-Coulter). Finally, quantification and length determination of the prepared library were performed before sequenced on an Illumina HiSeq X Ten using 150-bp paired-end runs

Targeted region selection was performed with the NimbleGen SeqCap Hybridization and Wash Kit (Roche, Swiss). A 1-µg mixed DNA library from 8-12 indexed Illumina libraries was captured with a hybridization probe. The probe library was designed through the NimbleDesign portal (Version 02) using genome build hg19 NCBI Build 37.1/GRCh37. The panel was 40 kb, targeting the regions of genes KRAS, NRAS, PIK3CA, BRAF, EGFR, EML4, ALK, SLC34A2, and ROS1. The hybridization and washing were conducted according to the manufacturer's protocol, and the captured DNA fragments were divided into two 50 µL reactions and amplified with 14 PCR cycles. The two reactions were pooled and purified with Agencourt AMPure XP beads. The captured product was sequenced using 150 bp paired-end runs on the Illumina NextSeq CN500 after quantification by the Qubit dsDNA Assay Kit and determination of fragment length by the Agilent 2100 Bioanalyzer with the DNA 1000 Kit. The median yield of each library was ~11.0 M reads, and the mean depth was ~10,000 X.