title: Targeted capture sequencing with UMI analysis
design: All genomic laboratory work was conducted by the NCI Cancer Genomics Research Laboratory. A total of 200 ng of DNA was extracted for each sample using Agencourt AMPure XP reagent (Beckman Coulter Life Sciences, Indianapolis, IN) following all manufacturer's provided protocols. The KAPA HyperPlus kit was used to prepare an adapter ligated library using xGen Dual Index UMI adapters (IDT, Coralville, IA) according to manufacturer's protocol. The fragmentation time was optimized to generate sequencing libraries with sequence insert sizes ranging from 250-350 base pairs (bp). For pre-hybridization, genomic DNA sample libraries were amplified by ligation-mediated PCR consisting of one reaction of 20 μL library DNA, 25 μL 2x KAPA HiFi HotStart ReadyMix, and 5μL 10x Library Amplification Primer Mix. The Library Amplification Primer Mix includes two primers with sequences: 5'-AATGATACGGCGACCACCGA-3' and 5'-CAAGCAGAAGACGGCATACGA-3'. Polymerase chain reaction (PCR) cycling conditions were 98˚C for 45 seconds, followed by 5 cycles of 98˚C for 15 seconds, 60˚C for 30 seconds and 72˚C for 30 seconds. The final step was an extension at 72˚C for a duration of 1 minute. The finished reaction was held at 4˚C until further downstream processing. Amplified material was cleaned using Agencourt AMPure XP Reagent (Beckman Coulter Life Sciences, Indianapolis, IN) according to provided protocols.  The resulting amplified sample libraries were quantified using Quant-iT™ PicoGreen dsDNA Reagent (ThermoFisher Scientific, Waltham, MA). For liquid phase sequence capture, amplified sample libraries with unique barcoded adapters were combined in equal amounts into 1.1 μg pools prior to hybridization for multiplex sequence capture. Targeted sequence capture was performed with Roche NimbleGen's SeqCap EZ Choice Library, targeting 650 genes implicated in CHIP, DNA repair pathways and radiation sensitivity (Supplemental Table 1) and covering approximately 3.8 Mb of genomic content (Roche NimbleGen, Inc., Madison, WI). Prior to hybridization the following components were added to the 1.1 μg pooled sample library: 2ul xGEN Universal Blocker (IDT, Coralville, IA) and 5 μL of 1 mg/mL COT-1 DNA (ThermoFisher Scientific, Waltham, MA). Pooled libraries were dried down by puncturing a hole in the plate seal and processing in an Eppendorf 5301 Vacuum Concentrator (Eppendorf, Hauppauge, NY) set to 60˚C for approximately 1 hour. A total of 7.5 μL of NimbleGen Hybridization Buffer and 3.0 μL of NimbleGen Hybridization Component A were added to each dried pool and placed in a heating block for 10 minutes at 95˚C. The mixture was then transferred to 4.5 μL of EZ Exome Probe Library and hybridized at 47˚C for 64 to 72 hours.  The final washing and recovery of captured DNA were performed as described in NimbleGen SeqCap EZ Library SR Protocol. Pools of captured DNA were amplified by ligation-mediated PCR consisting of one reaction for each pool containing 20 μL captured library DNA, 25 μL 2x KAPA HiFi HotStart ReadyMix, and 5 μL 10x Library Amplification primer mix. The Library Amplification primer mix includes two primers with sequences: 5'-AATGATACGGCGACCACCGA-3' and 5'-CAAGCAGAAGACGGCATACGA-3'. PCR cycling conditions were 98˚C for 45 seconds, followed by 10 cycles of 98˚C for 15 seconds, 60˚C for 30 seconds and 72˚C for 30 seconds. The last step was an extension at 72˚C for 60 seconds. The final reaction was kept at 4˚C until additional processing.  The amplified material was cleaned with Agencourt AMPure XP Reagent (Beckman Coulter Life Sciences, Indianapolis, IN) according to NimbleGen SeqCap EZ Library SR protocol.  Pools of amplified captured DNA were then quantified using Kapa's Library Quantification Kit for Illumina (Kapa Biosystems) on the LightCycler 480 (Roche, Indianapolis, IN). The resulting post-capture enriched multiplexed sequencing libraries were loaded on a NovaSeq 6000 (Illumina, San Diego, CA) and paired-end sequencing was performed using read lengths of 2×150 bp.
library_name: SC109464_targetseq