Cbot 2

Seven sections were cut from the FFPE tumor blocks. The middle five were subjected to DNA extraction whilst section one and seven, stained with hematoxylin and eosin, served as references to locate regions with high tumor cell concentrations. After macrodissection, DNA extraction was performed with the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. DNA shearing to 200 bp fragments was executed by Covaris’ Adaptive Focused Acoustics technology using an M220 Focused-ultrasonicator (Covaris, Woburn, Massachusetts). Using 200 ng of starting material, library construction was completed by use of the NEXTflex Rapid DNA-Seq Kit and NEXTflex DNA Barcodes (Bioo Scientific, Austin, TX). After pooling, cluster generation and sequencing were executed by respectively a cBot 2 and HiSeq 3000 system (Illumina, Essex, UK). The minimal number of reads (single-end (SE); 50-cycle mode) per sample was intended to be at least 15 million (mean coverage of 0.25×).

Blood samples were collected in 10‐mL cell‐free DNA BCT tubes (Streck) or PAXgene Blood DNA Tubes (Qiagen). Within 24 hours after collection, plasma isolation was executed by centrifugation (4°C; 10 minutes at 1600 g; 10 minutes at 16 000 g, or 15 minutes at 1900 g, respectively). The supernatant was transferred to a new tube and cfDNA was extracted from 3.5‐mL plasma using the Maxwell RSC ccfDNA Plasma Kit (Promega), following the manufacturer's instructions.
Using 25 μL of cfDNA, library preparation was executed on a Hamilton Star liquid handler using the NEXTflex Cell Free DNA‐Seq Library Prep Kit (Bioo Scientific) and NEXTflex DNA Barcodes (Bioo Scientific). After pooling, cluster generation and sequencing were completed by respectively a cBot 2 and HiSeq 3000 system (Illumina). The minimal number of reads (single‐read; 50‐cycle mode) per sample was set to 15 million.

TruSeq DNA PCR-free Library Preparation Kit (Illumina, San Diego, CA) was performed following manufacturer’s instructions. Briefly, genomic DNA (gDNA) was diluted to 20 ng/μL using Resuspension Buffer (RSB, Illumina) and 55 μL were transferred to Covaris microTubes (Covaris, Woburn, MA). The normalized gDNA was then sheared on an LE220 focused-ultrasonication system (Covaris) to achieve target peak of 450 bp with an Average Power of 81.0 W (SonoLab settings: duty factor, 18.0%; peak incident power, 45.0 watts; 200 cycles per burst; treatment duration, 60 s; water bath temperature, 5–8.5 °C). The quality of the final DNA libraries was assessed (High Sensitivity dsDNA, AATI). Per manufacturer’s protocol, library peak size was in the range of 550 to 620 bp. The DNA libraries were quantified by real- time quantitative PCR, using the KAPA SYBR FAST Library Quantification Kit (KAPA Biosystems, Boston, MA) optimized for the Roche LightCycler 480 instrument (Roche). DNA libraries were then normalized to 2 nM and clustered on the Illumina cBot 2 at 200 pM using a HiSeq X Flowcell v2 and the HiSeq X HD Paired-End Cluster Generation Kit v2. Paired-end sequencing was performed with the HiSeq X HD SBS Kit (300 cycles) on the Illumina HiSeq X. Mean genome coverage was 30X and all patient samples exceeded this expectation.