Sequencing library construction and target enrichment
Before library construction, 1 μg each of genomic DNA extracted from PBL
and tumor specimen was sheared to 300-bp fragments with a Covaris S2
ultrasonicator (Covaris, Woburn, MA, USA). A volume of 20–80 ng DNA
from plasma were used for library construction. Indexed Illumina
next-generation sequencing (NGS) libraries were prepared from PBL DNA
and tumor DNA using the KAPA Library Preparation Kit (Kapa Biosystems,
Wilmington, MA, USA).
Target enrichment was performed with a custom SeqCap EZ Library (Roche
NimbleGen, Madison, WI, USA). To explore the comprehensive genetic
properties of Chinese pan-cancer patients, the capture probe was
designed based on ~1.04 Mb genomic regions of 1021 genes
frequently mutated in common solid tumors. Capture hybridization was
carried out according to the manufacturer’s protocol. Following hybrid
selection, the captured DNA fragments were amplified and then pooled to
generate several multiplex libraries.