WGS of E. faecalis
Genomic DNA was extracted from overnight freshly grown cultures of these isolates using the HiPurA Bacterial Genomic DNA Isolation Kit (HiMedia, India). The genomic DNA was quality checked using agarose gel electrophoresis; NanoDrop spectrophotometer (ThermoFisher, USA) and Qubit assay (ThermoFisher, USA). DNA samples showing the intact bands on agarose gel with a 50 ng/l concentration and A260/280 ratio of 1.80 were selected for further processing. Sequencing libraries were prepared using the KAPA HyperPlus kit as per the manufacturer`s protocol (Roche, Switzerland). Briefly, genomic DNA was subjected to random enzymatic fragmentation followed by end repair and adapter ligation. The libraries were purified using the DNA beads and subjected to a few cycles of PCR amplification. The final libraries were quantified using the Qubit assay and size distribution was checked with TapeStation system (Agilent, USA). The libraries were subjected to 2 x 150 base pair sequencing on NovaSeq6000 platform (Illumina, USA). Raw sequencing data was quality-filtered using Trimmomatic (v0.36) (Bolger et al., 2014). During the quality filtering, the sequencing reads were scanned with a 5-base wide sliding window and a cut was made when the average quality score per base dropped below Q30. Bases at 5` and 3` ends of reads falling below Q20 quality scores were also removed. After quality filtering, sequencing reads shorter than 75 bases were also removed. The quality-filtered sequencing reads were aligned with the reference genome (E. faecalis EnGen0336), followed by consensus generation using samtools (v1.17) (Li et al., 2009). The genome annotation of the assembled genomes was done using Prokka (v1.14.6) to identify and annotate genes, features, and functional elements (Seemann, 2014). Additionally, the coverage and depth of the sequences were determined using BEDtools (v2.30.0) (Quinlan and Hall, 2010).
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