M220 focused

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens of the primary tumor from each patient were collected for analysis. The black PREP FFPE DNA Kit (Analytik Jena, Germany) was used to isolate DNA from the FFPE tissue specimens. Whole blood samples were centrifuged for 10 minutes (1,600 g) at room temperature to isolate lymphocytes, and a Tiangen Whole Blood DNA Kit (Tiangen, Beijing, China) was used to extract DNA from peripheral blood lymphocytes according to the manufacturer's instructions. Genomic DNA was sheared into 150-200 bp fragments with a Covaris M220 focused ultrasonicator (Covaris, Massachusetts, USA), and a DNA fragment library was constructed using a KAPA HTP Library Preparation Kit for the Illumina platform (KAPA Biosystems, Massachusetts, USA) according to the manufacturer's instructions. The DNA library was captured with a 543-gene plate designed based on the NimbleGen SeqCap EZ library (Roche, Wisconsin, USA), which includes key tumor-related genes. Captured samples were subjected to paired-end sequencing on the Illumina HiSeq X-Ten (cohort 1) or NovaSeq 6000 (cohort 2) platform.

For whole-genome sequencing with next-generation sequencing (NGS) technology, a Covaris M220 focused ultrasonicator (Covaris, Woburn, MA, USA) was used to fragment the extracted DNA, targeting peak fragment lengths of 400 bp. The fragmented DNA was purified and concentrated with magnetic Agencourt AMPure XP Beads (Beckman Coulter, Beverly, MA, USA) and used for barcoded NGS library preparation with the GeneRead™ DNA Library L Prep Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Agencourt AMPure XP Beads (Beckman Coulter, Beverly, MA, USA) were used for the purification and double size selection of the NGS library fragments. The NGS library concentration was determined using a QIAseq Library Quant Assay Kit (Qiagen, Hilden, Germany) and a Qubit v.3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The emulsion PCR and enrichment were carried out using the Ion PGM™ Hi-Q™ View OT2 Kit reagents (ThermoFisher Scientific–Ion Torrent, Carlsbad, CA, USA) according to the manufacturer’s instructions. The sequencing of the NGS library was performed on the Ion PGM platform using the Ion PGM™ Hi-Q™ View Sequencing Kit reagents (ThermoFisher Scientific–Ion Torrent, Carlsbad, CA, USA).

Good quality FFPE DNA was sonicated using the Covaris M220 focused ultrasonicator to an average fragment size of 300 bp; poor and very poor quality samples did not require further fragmentation (Supplementary Figure S1). DNA was then repaired using the NEBNext^® FFPE DNA Repair Mix, according the manufacturer’s protocol (New England Biolabs, Hitchin, UK). Library preparation was performed using the NEBNext^® Ultra II™ DNA Library Prep Kit for Illumina^® according to the manufacturer’s protocol for FFPE samples (New England Biolabs; half volumes of all reagents were used in the pilot experiment) and 10 cycles of library amplification, during which the library was indexed using custom PE1.0 (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) and indexed reverse primer (5′-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′; index sequence underlined). For the pilot experiment, the library was dual indexed using NEBNext^® Multiplex Oligos for Illumina^® (NEB). The library was purified and quantified using the same methods as described for DDAT. We chose the NEBNext Ultra II DNA Library Prep Kit for comparison as although many WGS library preparation reagents are now commercially available, this kit is very widely used in the community, and as such is a relevant comparison for our DDAT methodology.