title: GSM6437415: DNA-seq - Patient 65; Homo sapiens; OTHER
library_construction_protocol: Sample DNA was extracted from peripheral blood mononuclear cells isolated by gradient centrifugation and re-suspended in a concentration of 500 ng in 50µl of low TE buffer. Paired-end indexed libraries were prepared using the Sureselect XT Low Input Library prep protocol on the Agilent Bravo liquid handler following the manufacturer's protocol (NewEngland Biolabs, Ipswitch, MA, and Agilent Technologies, Ankeny, IA). Briefly, 200ng of target DNA was fragmented using the Covaris LE220 plus sonicator. The settings of duty factor 30%, peak incident power (PIP) 450, cycles per burst 200, time 180 seconds generated double-stranded DNA fragments with blunt or sticky ends with a fragment size mode of between 150-200bp. The ends were repaired using the Sureselect End-Repair-A-Tailing enzyme mix. Adapter ligated DNA fragments were size selected to enrich for 200 bp inserts (~320 bp total library size) using AMPURE XP bead purification. The size selected adapter-modified fragments were enriched, and specific indexes were added by 12 cycles of PCR using universal index primers. The concentration and size distribution of the libraries was determined on an Agilent Bioanalyzer DNA 1000 chip. The Custom Capture hybrid-target enrichment probes were designed using Agilent SureSelect design software (Agilent Technologies, Santa Clara, CA). The targeted gene panel was comprised of 62,962 single probes with size 1.805Mbp, and covered the coding regions, UTRs, and overlapping intron/exon regions for 205 genes described and / or enriched for CH mutations (see Appendix: Target Gene Panel). The custom capture was carried out using the Agilent Bravo liquid handler following Agilent's SureSelect XT Low. Purified capture products were then amplified using the SureSelect Post-Capture primer mix for 14 cycles. Libraries were validated and quantified on the Agilent Bioanalyzer. Samples were sequenced by 150 paired end reads, 21 samples to a Flow Cell, on an Illumina NovaSeq 6000 SP with an expected depth of ≥1,000X coverage (Illumina, SanDiego, CA).