Whole genome sequencing of Acanthamoeba isolated from corneal scraping
Whole genome library preparation was performed using Sequencing Ligation kit (SQK-LSK109) and Native barcoding kit (EXP-NBD104) from Oxford Nanopore Technologies. A total of 600 ng of purifiedDNA (as quantified by Qubit) from the sample was end-repaired (NEBnext ultra II end repair kit;NEB#E7546L, New England Biolabs, MA, USA) and cleaned up with 1x AmPure beads (BeckmannCoulter,USA). Native barcode ligation was performed with NEB blunt/ TA ligase (NEB#M0367L, NewEngland Biolabs, MA, USA) and cleaned with 1x AmPure beads. Qubit quantified barcode ligated DNA sample was adapter ligated (AMII) for 10 minutes at 20C usingNEBnext Quick Ligation Module (New England Biolabs, MA, USA) was performed. Library mix wascleaned up using 0.4X AmPure beads (Beckmann Coulter, USA) and library was eluted in 15 l ofelution buffer and used for sequencing on SpotON flowcell (FLO-MIN106). Sequencing was performed on GridION X5 (Oxford Nanopore Technologies, Oxford, UK) using SpotONflow cell (R9.4) in a 48hr sequencing protocol on GridION Release 19.06.9. Nanopore raw reads (fast5format) were basecalled (fastq5 format) using live basecalling algorithm (MinKNOW 18.3.3)