Assembling the genome of an extremely drug-resistant pathogen

Globally, with over 10 million new cases and 1.6 million deaths each year, and a growing number of multi- and extremely-drug resistant strains, tuberculosis (TB) is a major threat to human health1. Gaining a comprehensive understanding of genetic mechanisms contributing to its transmission and resistance is therefore paramount for combatting drug-resistant TB in endemic settings.

Short-read sequencing has been used for Mycobacterium tuberculosis (MTB) assembly previously yet it has limited ability to resolve long, repetitive regions and structural variants, and is hindered by the relatively high GC content (65%) of the MTB genome.

Using the Oxford Nanopore MinION, Bainomugisa et al. performed whole-genome sequencing of the modern Beijing lineage strain of TB responsible for drug resistance outbreaks in the Western Province of Papua New Guinea2. The team sequenced and assembled a high-quality, complete genome, including all PE/PPE (proline-glutamate/proline-proline-glutamate) gene families, which are thought to contribute to virulence but are often excluded from short-read sequencing analysis due to their repetitive and GC-rich nature.

With a sequencing depth of 238x obtained from a single MinION Flow Cell, a final assembly of 4,404,064 bp was obtained in a single contig with >99.9% accuracy. Furthermore, 166/168 PE/PPE genes had 100% breadth of coverage at 299.87x depth, compared to a short-read dataset of this strain where only 92/168 of these genes obtained 100% coverage at an average depth of 46.3x. Finally, genome-wide variant calling identified three deleted genic regions and a 4,490 bp insertion that were both absent from the H37RV reference strain genome (Figure 1).

Figure 1: Integrative Genomic Viewer (IGV) alignments of short-read data from different M. tuberculosis lineages (A-G) mapped against the nanopore draft genome. Left image: a 4,490 bp insertion spanning 7 annotated genes. Right image: a 390 bp insertion. Image courtesy of Dr. Lachlan Coin, The University of Queensland, Australia.
  1. WHO (2018) Tuberculosis [Online]. Available at: en/news-room/fact-sheets/detail/ tuberculosis [Accessed: 17 September 2019]
  2. Bainomugisa, A. et al. 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. Microb. Genom. doi: 10.1099/ mgen.0.000188 (2018).