Long read DNA sequencing of Oscheius tipulae: whole organism
To generate fragments of a suitable size range for Oxford Nanopore Promethion sequencing, high molecular weight DNA was diluted to a concentration of 25 ng/uL and fragmented to an average peak size of 25 kb using a Megaruptor-2 instrument (Diagenode). Small fragments <1 kb were removed and the DNA concentrated using bead purification (0.4 x volumes of Ampure-XP beads. Two aliquots of 1 g of sheared O. tipulae DNA and control DNA (lambda 3,2 kb fragment) were subjected to DNA damage repair (NEBNext FFPE DNA Repair Mix; New England Biolabs) followed by DNA End Repair (NEBNext Ultra II End Repair/ dA-tailing Module; New England BioLabs). A second 0.4 x volume Ampure-XP bead clean up was carried out and DNA eluted in sterile distilled H2O. Oxford Nanopore sequencing adapters were ligated to 750 ng of the recovered, end repaired DNA using the Ligation Sequencing kit (SQK-LSK-109; Oxford Nanopore) and NEBNext Ligation Module (New England BioLabs). Following a further 0.4 x volume Ampure-XP purification, the recovered DNA (16.25 fmol) was loaded onto a R9.4.1 PromethION flow cell following the manufacturers instructions and a 60 hr sequencing run was initiated. Raw reads were basecalled using Guppy v2.3.35.