Qubit dsdna double stranded dna br assay kit

Genomic DNA was extracted from mid-logarithmic phase bacteria growing in 7H9 media using Qiagen UCP Pathogen kit (Qiagen). Illumina libraries were prepared from 830–931 ng of mechanically fragmented DNA (300 bp, Covaris M instrument per manufacturer protocol) using the Kapa Hyper prep kit and quantified using the Qubit double-stranded DNA (dsDNA) BR assay kit (Thermo Fisher Scientific). Fragment size was assessed on a fragment analyzer (Advanced Analytical Technologies). Libraries were multiplexed and sequenced as 75-base-long single-end reads on an Illumina NextSeq 500 instrument. Reads were adapted and quality trimmed with Trimmomatic v0.33 with the following threshold: quality threshold of 15 and minimum length before dropping reads of 40. Trimmed reads were then mapped onto the Msmeg mc²155 reference genome (RefSeq NC_008596.1) using Bowtie2 v2.2.5. Variant calling was performed using Varscan (min coverage: 5; min reads: 5; average quality: 15; min variant allele frequency: 0.01; min for homoplasy: 0.9). Vcf were merged with bcftools option –merge.