targeted DNA sequencing of human cfDNA
Cell free DNA was extracted from 4 mL plasma using the QiAmp Circulating Nucleic acid kit (QIAGEN GmbH, Germany) and was quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) with the dsDNA High Sensitivity kit (Thermo Fisher Scientific).Libraries were generated with a customized QIAseq Targeted DNA Panel Kit (QIAGEN GmbH, Germany),according to the manufacturers protocol. Libraries were quantified by qRT-PCR using KAPA Library Quantification Kit Illumina Platforms (Roche Diagnostics). Pooled libraries (10 per run) were loaded at a combined concentration of 10 pM and analyzed by paired-end sequencing on a Illumina MiSeq instrument using the MiSeq Reagent Micro kit v2 (2 150 bp) for up to 8 million paired-end reads using a custom sequencing primer (QIAseq A Read 1 Primer).