A complete high-quality MinION nanopore assembly of an extensively drug-resistant Mycobacterium tuberculosis Beijing lineage strain identifies novel variation in repetitive PE/PPE gene regions

Abstract

A better understanding of the genomic changes that facilitate the emergence and spread of drug-resistant Mycobacterium tuberculosis strains is currently required. Here, we report the use of the MinION nanopore sequencer (Oxford Nanopore Technologies) to sequence and assemble an extensively drug-resistant (XDR) isolate, which is part of a modern Beijing sub-lineage strain, prevalent in Western Province, Papua New Guinea. Using 238-fold coverage obtained from a single flow-cell, de novo assembly of nanopore reads resulted into one contiguous assembly with 99.92 % assembly accuracy. Incorporation of complementary short read sequences (Illumina) as part of consensus error correction resulted in a 4 404 064 bp genome with 99.98 % assembly accuracy. This assembly had an average nucleotide identity of 99.7 % relative to the reference genome, H37Rv. We assembled nearly all GC-rich repetitive PE/PPE family genes (166/168) and identified variants within these genes. With an estimated genotypic error rate of 5.3 % from MinION data, we demonstrated identification of variants to include the conventional drug resistance mutations, and those that contribute to the resistance phenotype (efflux pumps/transporter) and virulence. Reference-based alignment of the assembly allowed detection of deletions and insertions. MinION sequencing provided a fully annotated assembly of a transmissible XDR strain from an endemic setting and showed its utility to provide further understanding of genomic processes within Mycobacterium tuberculosis.

Applications of sequencing technology in clinical microbial infection

“…73 The Prazsk I team of the Faculty of Medicine at the University of Szeged in Hungary analysed the lytic transcriptome of Varicella-zoster virus (VZV) using the Nanopore sequencing platform, revealing the complex transcriptome structure of VZV. 74 Moon et al 75 77 A team led by Justin O'Grady in the UK used Nanopore sequencing technology to rapidly identify bacterial genera and species and bacterial resistance genes in patients with lower respiratory tract infections. 78 Runtuwene et al 79 used Nanopore sequencing to genotype the malaria parasite-Plasmodium falciparum-and infer its drug resistance status.…”

Characterization of Fecal Microbiota with Clinical Specimen Using Long-Read and Short-Read Sequencing Platform

“…This innovation allows sequencing of the entire 16S rRNA gene, which suggests its potential for more-extensive identification of microorganisms [ 10 ]. Moreover, polymerase chain reaction (PCR)-free long-read sequences lessen the impacts of high-GC regions and amplification-mediated bios to PCR-based short-read sequencing, which leads to more-accurate identification of microorganisms [ 11 ].…”

Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome

“…A MinION flow cell has 512 sensors collecting measurements from 2048 pores. Its minimal initial investment, long reads, and rapid results have enabled many applications for smaller genomes Quick et al 2015;Risse et al 2015;Jansen et al 2017;Bainomugisa et al 2018;Miller et al 2018). Recent runs routinely reach 8 Gb and currently up to 30 Gb, with a big in-field variability and incremental improvements over the years (Schalamun et al 2019).…”