Whole-Genome Sequencing of Two Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from India Syed Beenish Rufai, a Sarman Singh a,b a Division of Clinical Microbiology and Molecular Medicine, All India Institute of Medical Sciences, New Delhi, India b Clinical Microbiology and Molecular Medicine, All India Institute of Medical Sciences, Bhopal, India ABSTRACT The emergence of extensively drug-resistant tuberculosis (XDR-TB) pres- ents a considerable challenge and a public health concern due to the high mortality rate of this disease. Whole-genome sequencing (WGS) of XDR-TB isolates is thus es- sential for understanding the mechanism of drug resistance. Here we report whole- genome sequences of two XDR-TB strains of two lineages from India. N ext-generation sequencing (NGS) of whole genomes has paved the way for understanding the mechanisms of drug resistance at the genomic level (1). This information can lead to timely and appropriate treatment strategies to reduce further transmission of the resistant strains. Two indigenous strains of Mycobacterium tuber- culosis (both from sputum samples from pulmonary TB patients) were isolated from the North-Western Provinces of India and given the laboratory identification (ID) numbers L-182 and L-31, respectively. Both sputum samples were processed using the N-acetyl- L-cysteine-sodium citrate-NaOH (NALC-NaOH) method. For each isolate, 500 l of the decontaminated sample was inoculated in Bactec-MGIT960 per the manufacturer’s instructions (Becton Dickinson, Sparks, MD, USA) (2). Identification and characterization of the M. tuberculosis isolates was done by using multiplex PCR as published earlier (3). The first- and second-line drug susceptibility testing of the above M. tuberculosis cultures, which identified the two isolates as XDR-MTB, was performed by using a Bactec MGIT-960 system (BD, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions (4). The DNA of these isolates was extracted by using the chloroform- isoamyl-alcohol method (5, 6) followed by molecular characterization by application of a spoligotyping technique using a commercial kit (Ocimum Biosolutions, India). The spoligotyping method, which was done per the manufacturer’s instructions (7), iden- tified the genetic lineages of the M. tuberculosis isolates. The spoligotyping analysis confirmed that one isolate belonged to the Beijing (L-182) strain lineage and the other one (L-31) to the Central Asian strain lineage. Whole-genome sequencing of both isolates was performed using the Ion Torrent PGM platform on an Ion 316v2 chip. Briefly, the genomic DNA library was prepared using an Ion Xpress Plus fragment library kit and an Ion Xpress barcode adapter kit. Size selection of a library for targeted read length was carried out by using a size fraction- ation method with 2% EGel SizeSelect gel according to the manufacturer’s instructions (Ion Torrent, Life Technologies). A barcoded target library of 200 bp was prepared for L-182 and L-31, which resulted in totals of 856,225 and 854,481 reads with mean read lengths of 181 bp and 167 bp, respectively. Template preparation from the diluted library was performed using an Ion PGM template OT2 200 kit (Ion Torrent, Life Technologies). Template amplification and enrichment were performed using the Ion OneTouch 2 system, which consists of two Ion OneTouch 2 instruments and the Ion OneTouch ES enrichment system (Ion Torrent). The whole-genome sequencing was performed using an Ion PGM machine (Ion Torrent). The Torrent Suite software plugin Citation Rufai SB, Singh S. 2019. Whole- genome sequencing of two extensively drug- resistant Mycobacterium tuberculosis isolates from India. Microbiol Resour Announc 8:e00007-19. https://doi.org/10.1128/MRA .00007-19. Editor Frank J. Stewart, Georgia Institute of Technology Copyright © 2019 Rufai and Singh. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Sarman Singh, sarman.singh@gmail.com. Received 4 January 2019 Accepted 17 January 2019 Published 14 February 2019 GENOME SEQUENCES crossm Volume 8 Issue 7 e00007-19 mra.asm.org 1