Resuspension buffer rsb

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.