Whole-Genome Sequencing of Anaerobic Bacteria

Bacteria were grown in thioglycolate broth (Becton Dickinson, USA) for 48 h in anaerobic conditions (85% N₂, 5% H₂, and 10% CO₂) to obtain a cell pellet from 1 mL of culture. DNA was extracted using DNeasy Mini Kit (Qiagen, Hilden Germany) as previously described [15 ,26 (link),27 ]. The quality and yield of genomic DNA was verified by agarose gel electrophoresis and a Nanodrop 2000 (ThermoFischer Scientific, USA). Whole-genome sequencing (WGS) was done using Illumina HiSeq 2000 with PE 150 plus index read (Illumina, San Diego, CA). Adapters and phiX Illumina standards were informatically removed before quality control of the reads was done using FastQC. The coverage was calculated with the formula C = LN/G, where L is the read length, N is the number of reads and G is the genome length [28 ]. The genome sequences were assembled and annotated as previously described [15 ,16 (link),26 (link)]. Briefly, the reads were assembled using Shovill (https://github.com/tseemann/shovill) with the default settings. Automated gene annotation was done with Prokka v.1.14.5 using default parameters [29 ].