Nanopore sequencing of custom mosquito pool
Before sequencing, quality control analysis was done to determine sample concentration using Qubit 4 fluorometer - Qubit 1xdsDNA High Sensitivity (HS) Assay kit (ThermoFisher; Q33231). Sequencing libraries were prepared using the Genomic DNAby Ligation Kit (Oxford Nanopore Technologies; SQK-LSK110). Due to differences in the fragment-length distributions ofsamples, the protocol was altered for some samples to ensure maximum library recovery. This involved using 1X AMPure forthe clean-up of the AMX F adapter ligation reaction and using SFB for the washes during this clean-up reaction. Each librarywas run over one MinION flow cell (Oxford Nanopore Technologies; FLO-MIN106 R9.4.1) on the GridION X5 mk1. Tomaximise sequencing data yields, flow cells were treated with a nuclease flush and reload, using the Flow Cell Wash Kit (OxfordNanopore Technologies; EXP-WSH004). ONT MinKNOW software (version 21.11.7) was used for the sequencing runs thatoriginated fast5 files with read information. Guppy software (version 5.1.13) built in MinKNOW was used for basecallingin the high-accuracy setting, to convert fast5 to fastq files. The Deepseq team (University of Nottingham) performed qualitycontrol of the DNA sent for each pool. Qubit equipment calculated DNA concentrations of 9.28 ng/L, 12.1 ng/L, 32.8ng/L, 29.2 ng/L and 24.2 ng/L for pools 1-5, respectively. The amount used for library preparation was approximately1500 ng for all samples and the final amount loaded in each flow cell was 171.4 ng, 138.6 ng, 97.2 ng, 58.8 ng and 48.9 ng fromeach pool, after library preparation.