Nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation

Abstract

Mycobacterium tuberculosis whole-genome sequencing (WGS) using Illumina technology has been widely adopted for genotypic drug susceptibility testing (DST) and outbreak investigation. Oxford Nanopore Technologies is reported to have higher error rates but has not been thoroughly evaluated for these applications. We analyse 151 isolates from Madagascar, South Africa and England with phenotypic DST and matched Illumina and Nanopore data. Using PacBio assemblies, we select Nanopore filters for BCFtools (software) detection of single nucleotide polymorphisms (SNPs). We compare transmission clusters identified by Nanopore and the United Kingdom Health Security Agency Illumina pipeline (COMPASS). We compare Illumina and Nanopore WGS-based DST predictions using Mykrobe (software). Nanopore/BCFtools identifies SNPs with median precision/recall of 99.5/90.2% compared with 99.6/91.9% for Illumina/COMPASS. Using a threshold of 12 SNPs for putative transmission clusters, Illumina identifies 98 isolates as unrelated and 53 as belonging to 19 distinct clusters (size range 2-7). Nanopore reproduces this distribution with addition of 5 singleton isolates to distinct clusters and merging of two cluster pairs. Illumina-based clusters are also replicated using a 5 SNP threshold. Clustering accuracy is maintained using mixed Illumina/Nanopore datasets. Genotyping resistance variants is highly concordant, with 0(4) discordant SNPs (indels) across 151 isolates genotyped at >3000 (60,000) SNPs (indels). We conclude that Illumina and Nanopore sequence data provide comparable cluster-identification and DST results.

Genomic Sequencing from Sputum for Tuberculosis Disease Diagnosis, Lineage Determination and Drug Susceptibility Prediction

“…Illumina data were pre-processed with fastp (v0.23.2) to remove adapter sequences, trim low quality bases from read ends, and remove reads shorter than 30bp (9). Sequencing reads were decontaminated and mapped to a database of common sputum contaminants, and to the M. tuberculosis reference genome (H37Rv (NC_000962.3)), keeping only those reads with a mapping to H37Rv (10, 11). Decontaminated sequence files for each isolate were randomly subsampled with Rasusa (v0.6.0) to a maximum (mean) read depth of 100 (Illumina) and 150 (MinION) (12).…”

“…Mykrobe (v.0.10.0) was used to assign lineage and predict drug susceptibility using parameters described in Hall et. al (10, 11, 13).…”

Evaluation ofMycobacterium Tuberculosisenrichment in metagenomic samples using ONT adaptive sequencing and amplicon sequencing for identification and variant calling

“…While not all parts of the genome were covered, as in WGS, in this case, over 99.6% of genomic positions were not covered by the tested amplicon panel. Taxonomic classification with the amplicon data can be challenging even with longer amplicons 24 , which is especially useful for lineage tracing 25 . In addition, amplification efficiency among different amplicon regions might vary significantly, depending on the sequence complexity length of the amplicon and GC content 26 .…”

Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

“…However, despite its huge potential for pathogen surveillance and global health more broadly [45,46], many endemic countries in Africa have limited capacity for genomic sequencing due to factors including prohibitive costs, barriers to procurement, and a lack of sequencing and computing infrastructure [47]. Oxford Nanopore Technologies (ONT) is being increasingly used for rapid sequencing, diagnostics, antimicrobial susceptibility testing and epidemiological analysis in multiple pathogens, including SARS-CoV-2 [48–50], Zika virus [51,52], Ebola virus [53], Chikungunya virus [54], Mycobacterium tuberculosis [55–57], and bacterial antimicrobial resistance (AMR) and clinical metagenomics [58–68]. ONT devices such as the MinION are portable, relatively cheap and produce sequence data in ‘real-time’, making them well-suited to resource-limited settings including in Low- and Middle-Income Countries (LMIC).…”

“…Oxford Nanopore Technologies (ONT) is being increasingly used for rapid sequencing, diagnostics, antimicrobial susceptibility testing and epidemiological analysis in multiple pathogens, including SARS-CoV-2 [48][49][50], Zika virus [51,52], Ebola virus [53], Chikungunya virus [54], Mycobacterium tuberculosis [55][56][57], and bacterial antimicrobial resistance (AMR) and clinical metagenomics [58][59][60][61][62][63][64][65][66][67][68]. ONT devices such as the MinION are portable, relatively cheap and produce sequence data in 'real-time', making them well-suited to resource-limited settings including in Low-and Middle-Income Countries (LMIC).…”