Whole exome sequencing of human gastric cancer surgical section from esophagus
DNA was extracted from the FFPE tissue using the AnaPrep FFPE DNA extraction kit following the manufactural instruction. Whole-exome DNA was captured from total genomic DNA using the SeqCap EZ System from NimbleGen according to the manufacturers instructions. Briefly, genomic DNA was sheared, size selected to roughly 200-250 base pairs, and the ends were repaired and ligated to specific adapters and multiplexing indexes. Fragments were then incubated with SeqCap biotinylated DNA baits followed by the LM-PCR, and the RNA-DNA hybrids were purified using streptavidin-coated magnetic beads. The RNA baits were then digested to release the targeted DNA fragments, followed by a brief amplification of 15 or less PCR cycles. Sequencing was performed on Illumina Hiseq 3000 for a pair read 150 run. Data quality check was done on Illumina SAV. De-multiplexing was performed with Illumina Bcl2fastq2 v 2.17 program.