2.4. Targeted NGS and Analysis of bTMB and Other Biomarkers
cancer
CANCER
Next-generation sequencing (NGS) was performed using CT-ULTRA, a targeted NGS system designed for ctDNA analysis, on patients’ samples with more than 10 ng of cfDNA. The NGS targeted 118 genes, including the entire exons of 88 genes, 30 hotspots and the partial introns of 4 genes often rearranged in cancer. The library preparation for each cfDNA was performed by sequential reactions of end repair, A-base tailing and ligation with a TruSeq adaptor using a SureSelectXT Reagent kit (Agilent Technologies, Santa Clara, CA, USA). After quantification using a Qubit™ (Thermo Fisher Scientific), ten libraries with sample-specific six base pair (bp) barcodes were pooled for a total of 1500 ng to be used for the hybrid capture using an Agilent SureSelectXT custom kit (OP_AMC_ctV1.3 RNA bait; Agilent Technologies). The DNA libraries that passed quality checks were sequenced using the HiSeq X Ten platform (Illumina, San Diego, CA, USA) for paired-end sequencing. Sequenced reads were aligned to the human reference genome (NCBI build 37) with Burrows–Wheeler Aligner (version 0.5.9, broadinstitute, Boston, MA, USA) using the default options. A polymerase chain reaction (PCR) de-duplication was performed using Picard’s MarkDuplicates package. After the initial alignment process, the reads were realigned at common indel positions with the Genome Analysis Toolkit (GATK) IndelRealigner. Then the recalibration of the base quality was done using the GATK Table-Recalibration and used as the final Binary Alignment Map (BAM).