NGS of drug sensitive deleted-CRISPR-Cas MTBC Beijing strain C_0253-12 from patient C
Mycobacterium tuberculosis strain C_0253-12 was isolated and rflp-grade DNA-extracted at the National Public Health Laboratory Tel Aviv, Israel (NPHLTA).  The DNA was sent to the collaborating lab at the Scripps Research Institute in San Diego, California USA (Scripps).  One microgram of each sample was sheared on the Covaris S2. Each sample was then end repaired, a-tailed and adapter ligated.  The samples were cleaned with Agencourt beads and treated with 6 cycles of PCR.  After PCR the samples were cleaned using Zymo 25 columns and loaded on a 2% agarose gel.  The gel was cut from 400-700bp and cleaned again with the Zymo25 column.   The samples were sequenced on an Illumina Miseq system as paired-end reads each with length 300bp. The reads were preprocessed by trimming.  Bioinformatics to examine CRISPR-Cas and other genomic mutations was performed at Scripps by converting bam files to wig files using bamtools, bedtools, and ucsc-tools, and the wig files were imported into the UCSC Microbial Genome Viewer.  Bioinformatics to examine CRISPR-Cas and other genomic mutations was performed at NPHLTA by analyzing cleaned paired-end and single-end reads with BioNumerics version 7.5 (Applied Maths, Belgium).
DOI
10.1186/s12864-017-3560-6.