Qsep100 system

Following vendor manual instructions, genomic DNA and mRNA from FFPE samples were extracted using an AllPrep DNA/RNA FFPE tissue kit. This extraction kit is manufactured by Qiagen in Germany. Concentrations of DNA and cDNA were estimated using Qubit 3.0 from Thermo Fisher Scientific in the USA. In addition, DNA fragment distribution was assessed by a Qsep100 system from Bioptic, Taiwan, China.

Genomic DNA (gDNA) was extracted from frozen tumor tissues using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), or from formalin-fixed paraffin-embedded (FFPE) tumor tissues using a GeneRead DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), based on the manufacturer’s guidelines. The concentration of gDNA was measured using Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and the gDNA was quantified by Qsep100 System (BIOptic, China).
Total RNA was extracted from frozen tumor tissues using Trizol and RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quantity was assessed on Nanodrop, and the integrity of total RNA was estimated by RNA 6000 Nano Kit on Aligent 2100 Bioanalyzer.

Sequencing libraries were prepared using 20–50 ng ctDNA as sample inputs following the protocol of the AVENIO cfDNA Expanded kit (77 genes, Roche, Switzerland) or the AVENIO cfDNA Surveillance kit (197 genes, Roche, Switzerland). Quality control measures were carried out to assess the concentration and ideal peak size of the enriched library. The ideal peak size was assessed using the Qsep100 system (BIOptic, New Taipei, Taiwan). The expected sequencing library size was more than 300 bp. Sequencing was conducted using the 300-cycle NextSeq 500/550 Mid Output kit v2.5 on the Illumina NextSeq 550 System (Illumina, San Diego, CA, USA).