TWIST CVRP of Cimex sp.
The sequencing libraries were generated from homogenized, pooled (n = 4) bat bugs using the combination of Twist Library Preparation Enzymatic Fragmentation (EF) kit 2.0 (#104211) and hybridization with Twist Comprehensive Viral Research Panel (#1035550), according to the manufacturers protocol (Twist Total Nucleic Acids Library Preparation EF Kit 2.0 for Viral Pathogen Detection and Characterization Protocol and Twist Target Enrichment Standard Hybridization v1 protocol). Briefly, RNA was extracted using Direct-zol RNA Miniprep Kits (R2053) and the extracted nucleic acid was converted to cDNA using ProtoScript II First Strand cDNA Synthesis kit (E6560S) and Random Primer 6 (S1230S) from New England Biolab (NEB). The NEB Next Ultra II Non-Directional RNA second Strand Synthesis kit (E6111S) was subsequently used to convert the single-stranded cDNA to double stranded DNA (dsDNA). The Illumina TruSeq-compatible libraries were generated using the Twist Library Preparation EF kit and the generated libraries were pooled with 6 libraries per pool. The pooled libraries went to hybridization capture using the Twist Comprehensive Viral Research Panel. Following the enrichment, the enriched libraries were pooled again in equimolar ratios and sequencing with loading concentration of 6pM and spiked with 1% Phix control V3 using Illumina Miseq. The pooled library was diluted and denatured according to the standard Miseq System Denature and Dilute Libraries Guide (Document # 15039740v10), and sequenced to generate paired-end 75bp reads using a 150 cycle Miseq V3 reagent kit (illumina, MS-102-3001).