Human cot 1 dna

An aliquot of each metagenomic library was subjected to viral sequence enrichment using the Comprehensive Viral Research Panel (CVRP; Twist Biosciences, South San Francisco, CA, USA) (Berg et al., 2020 (link)). Briefly, libraries were pooled on an equimolar basis in sets of 16–24 (each set containing a positive and negative control) such that each pool contained ∼3 μg total DNA, as assessed by a Qubit Flex fluorimeter. The pools were dried down using a vacuum centrifuge, then resuspended in a solution of Human Cot-1 DNA and xGen Universal Nextera Blockers (Integrated DNA Technologies, Coralville, IA). Hybridization of the CVRP probes to the NGS libraries was performed per manufacturer’s instructions for 16 hr. Hybridized sequences were separated from non-hybridized sequences by affinity interaction on Streptavidin beads (Twist Biosciences), amplified using a KAPA library amplification kit (Roche, Basal, Switzerland), and re-purified using magnetic PCR beads (Twist Biosciences). The resulting libraries were analyzed for size and concentration as above.

The FFPE and Non-FFPE samples were pooled separately using 500 ng of library for each sample. These two pools of libraries were then hybridized in solution to the HGSC VCRome 2.1 design (Bainbridge et al, 2011 (link)) (42 Mb [mega base]; NimbleGen) according to the manufacturer’s protocol NimbleGen SeqCap EZ Exome Library SR User’s Guide (Version 2.2) with minor revisions. For ∼3,500 clinically relevant genes that had low coverage (<20× coverage at ∼2.72 Mb sequencing data) probes were supplemented with PKv1 and PKv2 reagent spiked into the VCRome 2.1. Human COT1 DNA and xGen Universal Blocking oligonucleotides (Integrated DNA Technologies) were added into the hybridization to block repetitive genomic sequences and the adaptor sequences and hybridization was carried out at 42°C for 72 h. Post-capture LM-PCR amplification was performed using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Cat no. KK2612; Kapa Biosystems, Inc.) with 12 cycles of amplification. After the final AMPure XP bead purification, quantity and size of the capture library was analyzed using the Agilent Bioanalyzer 2100 DNA Chip 7500.