Portable sequencing of Mycobacterium tuberculosis for clinical and epidemiological applications

Abstract

With >1 million associated deaths in 2020, human tuberculosis (TB) caused by the bacteria Mycobacterium tuberculosis remains one of the deadliest infectious diseases. A plethora of genomic tools and bioinformatics pipelines have become available in recent years to assist the whole genome sequencing of M. tuberculosis. The Oxford Nanopore Technologies (ONT) portable sequencer is a promising platform for cost-effective application in clinics, including personalizing treatment through detection of drug resistance-associated mutations, or in the field, to assist epidemiological and transmission investigations. In this study, we performed a comparison of 10 clinical isolates with DNA sequenced on both long-read ONT and (gold standard) short-read Illumina HiSeq platforms. Our analysis demonstrates the robustness of the ONT variant calling for single nucleotide polymorphisms, despite the high error rate. Moreover, because of improved coverage in repetitive regions where short sequencing reads fail to align accurately, ONT data analysis can incorporate additional regions of the genome usually excluded (e.g. pe/ppe genes). The resulting extra resolution can improve the characterization of transmission clusters and dynamics based on inferring closely related isolates. High concordance in variants in loci associated with drug resistance supports its use for the rapid detection of resistant mutations. Overall, ONT sequencing is a promising tool for TB genomic investigations, particularly to inform clinical and surveillance decision-making to reduce the disease burden.

Functional genetic variation in pe/ppe genes contributes to diversity in Mycobacterium tuberculosis lineages and potential interactions with the human host

“…In a recent study, Marin et al (2022) found several pe / ppe genes with good mappability and variant detection with short-read sequencing platforms that could be included in WGS analysis with confidence. The majority of these genes belonged to class C in our analysis, congruent with a better coverage of the conserved pe / ppe genes found also in previous studies ( Gómez-González et al, 2022 ), while class K genes were found with lower scores for Illumina mappability. Long read sequencing technologies can be of use to overcome this problem ( Gómez-González et al, 2022 ).…”

“…The majority of these genes belonged to class C in our analysis, congruent with a better coverage of the conserved pe / ppe genes found also in previous studies ( Gómez-González et al, 2022 ), while class K genes were found with lower scores for Illumina mappability. Long read sequencing technologies can be of use to overcome this problem ( Gómez-González et al, 2022 ). We analyzed PacBio assemblies to provide the most comprehensive picture to date of genetic diversity in all 169 pe / ppe genes.…”

Advantages of long- and short-reads sequencing for the hybrid investigation of the Mycobacterium tuberculosis genome

“…The introduction of the LRS technology represents a valid alternative to the SRS approaches, because it allowed a better characterization of the MTB genome, e.g., InDel and repetitive regions. Comparing the results between SRS and LRS, different studies highlight genome regions where the SRS lacks accuracy due to limit of the technology ( Modlin et al, 2021 ; Peker et al, 2021 ; Gómez-González et al, 2022 ; Marin et al, 2022 ). Although LRS approaches still present a high-error rate (~5–15%), their random nature allows to improve accuracy with higher coverage ( Rhoads and Au, 2015 ; Athanasopoulou et al, 2021 ; Amoutzias et al, 2022 ).…”

Pangenome databases improve host removal and mycobacteria classification from clinical metagenomic data

“…Each human readset was randomly downsampled to 1 Gbp using rasusa (v0.7.1) [ 51 ]. For the M. tuberculosis component, we used Illumina HiSeq 4000 (accession: ERR245682) and Nanopore R10.3 (accession: ERR8170871) [ 52 ] (we used R10.3 as there were no R10.4 M. tuberculosis WGS datasets publicly available). For the bacterial component, we used Illumina MiSeq (accession: ERR7255689) and Nanopore R10.4 (accession: ERR7287988) reads from the ZymoBIOMICS HMW DNA Standard D6322 (Zymo Research), which contains 7 bacterial and 1 fungal strain(s) [ 53 ]—none of which are Mycobacterium .…”