Whole exome sequencing of human normal esophagus tissue adjacent to the tumor
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.