Kapa hyperprep library preparation kit

To obtain ~500-bp DNA fragments, 10–20 ng/μL of DNA extract in 100 μL chilled TLE buffer (10mM Tris, pH8.0, 0.1mM EDTA) was sheared using a Bioruptor (Diagenode) on high power for up to 15 min. Fragment sizes were assessed on 1.5% agarose gels. Sheared DNA was bead-purified using 1.2X volume of Agencourt AMPure XP Beads (Beckman Coulter Cat. # A63880) and eluted in 50 μL water. Library preparation (end repair, A-tailing and adapter-ligation) was performed using the KAPA HyperPrep Library Preparation Kit (Cat. # KR0961/KK8503). Double-stranded DNA adapters contained a random 12-bp dUMI sequence and a defined 5-bp spacer sequence added to Illumina TruSeq adaptor sequences [61 (link)] (S1 Fig). Subsequently, DNA was bead-purified with 1X volume of beads and eluted in 50 μL water.
DNA libraries were subjected to pre-enrichment amplification with primers mws13 & mws20 (S1 Table and S1 Fig). PCR conditions were: 95°C 4 mins; 5–8 cycles of 98°C 20 sec, 60°C 45 sec, 72°C 45 sec; 72°C 3 mins, 4°C hold. If the bead-purified elution from the end repair and adapter step had more than 240 ng total, it was divided into 50 μL PCR reactions of ≤240 ng and pooled after amplification. PCR products were then bead-purified as above with 1.2X volume beads and elution in 100 μL water, quantified with Nanodrop, and their sizes assessed using a Bio-analyzer (Agilent DNA 7500) or Qiaxcel (QIAGEN AM420).

A focus of tumor with higher than 70% tumor cellularity was marked on an H&E-stained section. Matching areas of the tumor were macrodissected for DNA sequencing. Normal squamous epithelium from the excised skin was used as a matched control. Briefly, we used a KAPA HyperPrep library preparation kit (Kapa Biosystems, Wilmington, MA, USA) to prepare indexed libraries from 200 ng genomic DNA that had been sheared using a Biorupter Ultrasonicator (Diagenode, Denville, NJ, USA). The multiplexed library pool was hybridized to the MD Anderson T200.1 probe pool (Roche NimbleGen, Madison, WI, USA). Following hybridization and reaction cleanup, the enriched libraries were amplified with seven cycles of postcapture PCR, then assessed for target enrichment by quantitative PCR. Sequencing was performed in one lane of a HiSeq4000 Sequencer (Illumina, San Diego, CA, USA) using a 150 bp paired-end configuration.

250 ng of DNA were used for mechanical DNA shearing that was performed in a total volume of 52 µL with the Covaris S220 through microTube-50 AFA Fiber Screw-Cap (Covaris®) using a setting of 30% duty factor, 100 W peak incidence power, and 1000 cycles per burst for 150 s. Note that the concentrations of genomic DNA from 5WBF-treated blood samples were often very low or even below the Qubit® detection threshold (Additional file 1: Table S3). Then, 52 µL of undiluted genomic DNA were used for mechanical DNA shearing. Genomic DNA libraries were constructed for high throughput sequencing using the KAPA HyperPrep Library Preparation Kit (Kapa Biosystems, Woburn, MA). DNA libraries were checked for quality and quantity using Qubit® for concentration and BioAnalyser 2100 Agilent for fragment size. Libraries were sequenced at 150 bp paired-end using an Illumina NextSeq 500 instrument at the GENOM’IC platform from Institut Cochin (Paris, France).