WGS of H37Rv: delta BioB-Rv1591
Methods: Between 150 and 200ng of genomic DNA was sheared acoustically and HiSeq sequencing libraries were prepared using the KAPA Hyper Prep Kit (Roche). PCR amplification of the libraries was carried out for 10 cycles. 5-10x106 50-bp paired-end reads were obtained for each sample on an Illumina HiSeq 2500 using the TruSeq SBS Kit v3 (Illumina). Post-run demultiplexing and adapter removal were performed and fastq files were inspected using fastqc (Andrews S. (2010). FastQC: a quality control tool for high throughput sequence data. Available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Trimmed fastq files were then aligned to the reference genome (M. tuberculosis str. H37RvCO; NCBI Reference Sequence: NZ_CM001515.1) using bwa mem (Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25:1754-60). Bam files were sorted and merged using samtools (Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Genome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics, 25, 2078-9.). Read groups were added and bam files de-duplicated using Picard tools and GATK best-practices were followed for SNP and indel detection (A framework for variation discovery and genotyping using next-generation DNA sequencing dataDePristo M, Banks E, Poplin R, Garimella K, Maguire J, Hartl C, Philippakis A, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell T, Kernytsky A, Sivachenko A, Cibulskis K, Gabriel S, Altshuler D, Daly M, 2011 NATURE GENETICS 43:491-498). Acknowledgements: We acknowledge the use of the Integrated Genomics Operation Core at MSKCC, funded by the NCI Cancer Center Support Grant (CCSG, P30 CA08748), Cycle for Survival and the Marie-Jose and Henry R. Kravis Center for Molecular Oncology.