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

Nilgiriwala, Kayzad; Rabodoarivelo, Marie Sylvianne; Hall, Michael B.; Patel, Grishma; Mandal, Ayan; Mishra, Shefali; Andrianomanana, Fanantenana Randria; Dingle, Kate E.; Rodger, Gillian; George, Sophie; Crook, Derrick W.; Hoosdally, Sarah; Mistry, Nerges; Rakotosamimanana, Niaina; Iqbal, Zamin; Lapierre, Simon; Walker, Timothy M.

doi:10.1128/jcm.01578-22

Cited by 13 publications

(18 citation statements)

References 19 publications

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“…Since we have not explored the potential of culture-free WGS as a diagnostic tool for TB, we performed as many runs as necessary to achieve sufficient coverage and depth to reach our goal, regardless of the sequencing cost per sample. With this effort we were able to perform culture-free sequencing, with high quality, in 76% (61/80) of diagnostic samples, suggesting that further improvements of the technique will be needed as shown by others 15 . In fact, while previous studies showed that smear-negative and scanty sputa could be enriched and sequenced occasionally [10][11][12][13] , a formal testing of the limit of detection is still needed.…”

Section: Discussionmentioning

confidence: 72%

Genetic diversity within diagnostic sputum samples is mirrored in the culture ofMycobacterium tuberculosis

Mariner-Llicer,

Goig,

Torres-Puente

et al. 2024

Preprint

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Culturing Mycobacterium tuberculosis (MTB) from tuberculosis cases is the basis for many research and clinical applications. Paradoxically, it is assumed to impose a diversity bottleneck, which, if true, would entail unexplored consequences. The alternative, culture-free sequencing from diagnostic samples, is a promising but challenging approach both to obtain and analyse the MTB genome from the complex sample. This study obtains high-quality genomes of sputum-culture pairs from two different settings after developing a workflow for sequencing from sputum and a tailored bioinformatics pipeline. Our approach reveals that 88% of variants called in culture-free sequencing analysis are false positives due to supplementary alignments, mostly in enriched-sputa samples. Overall, contrary to the bottleneck dogma, we identify a 97% variant agreement within sputum-culture pairs, with a high correlation also in the variants frequency (0.98). Our findings extrapolate to all publicly available data, thus demonstrating that in most cases culture accurately mirrors clinical samples.

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Section: Discussionmentioning

confidence: 72%

Genetic diversity within diagnostic sputum samples is mirrored in the culture ofMycobacterium tuberculosis

Mariner-Llicer,

Goig,

Torres-Puente

et al. 2024

Preprint

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“…We generated an in silico metagenomic readset from a variety of organisms (human, bacteria, virus) at ratios commonly seen in M. tuberculosis metagenomic samples (see Generating in silico metagenomic reads) [8]. After removing Nanopore reads shorter than 500bp or with an ambiguous base, we were left with 234984 reads with a total of 2.48 gigabases [17].…”

Section: Resultsmentioning

confidence: 99%

“…We simulated metagenomic Nanopore and Illumina sequencing reads to a mixture ratio that approximates that found in patient sputa [8], albeit with a slightly higher mycobacterial component. In total, 4.5 and 0.9 gigabases were generated for Nanopore and Illumina, respectively, at proportions: 46% each for bacteria and human, 6% M. tuberculosis complex (MTBC), and 1% each for virus and non-tuberculous mycobacteria (NTM).…”

Section: Generating In Silico Metagenomic Readsmentioning

confidence: 99%

“…While M. tuberculosis contamination removal and read classification have been assessed previously [9,15], these studies have focused on Illumina sequencing data. However, Nanopore data is becoming a popular choice for M. tuberculosis metagenomic studies [8,16]. As Nanopore sequencing can be performed on a laptop, analysis pipelines should use computational resources that make them executable on such machines.…”

Section: Introductionmentioning

confidence: 99%

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Pangenome databases provide superior host removal and mycobacteria classification from clinical metagenomic data

Hall,

Coin

2023

Preprint

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BackgroundCulture-free real-time sequencing of clinical metagenomic samples promises both rapid pathogen detection and antimicrobial resistance profiling. However, this approach introduces the risk of patient DNA leakage. To mitigate this risk, we need near-comprehensive removal of human DNA sequence at the point of sequencing, typically involving use of resource-constrained devices. Existing benchmarks have largely focused on use of standardised databases and largely ignored the computational requirements of depletion pipelines as well as the impact of human genome diversity.ResultsWe benchmarked host removal pipelines on simulated Illumina and Nanopore metagenomic samples. We found that construction of a custom kraken database containing diverse human genomes results in the best balance of accuracy and computational resource usage. In addition, we benchmarked pipelines using kraken and minimap2 for taxonomic classification ofMycobacteriumreads using standard and custom databases. With a database representative of theMycobacteriumgenus, both tools obtained near-perfect precision and recall for classification ofMycobacterium tuberculosis. Computational efficiency of these custom databases was again superior to most standard approaches, allowing them to be executed on a laptop device.ConclusionsNanopore sequencing and a custom kraken human database with a diversity of genomes leads to superior host read removal from simulated metagenomic samples while being executable on a laptop. In addition, constructing a taxon-specific database provides excellent taxonomic read assignment while keeping runtime and memory low. We make all customised databases and pipelines freely available.

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“…Rapid data sharing enables genomic epidemiological analysis, a key part of the global public health response to the COVID‐19 pandemic 25 . Nanopore technology has been used to rapidly sequence the emerging and reemerging pathogens including Zika, Ebola, and other pathogens in multiple outbreaks, where surveillance and genomic investigation capacities are limited 26–30 . Oxford Nanopore has developed workflows for rapid preparation and sequencing of SARS‐CoV‐2 whole genomes, with starter packs that are affordable for LMICs.…”

Section: Discussionmentioning

confidence: 99%

Molecular epidemiology of SARS‐CoV‐2 in Mongolia, first experience with nanopore sequencing in lower‐ and middle‐income countries setting

Erendereg,

Tumurbaatar,

Byambaa

et al. 2023

Immunity Inflam & Disease

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BackgroundCoronavirus disease (COVID‐19) has had a significant impact globally, and extensive genomic research has been conducted on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) lineage patterns and its variants. Mongolia's effective response resulted in low prevalence until vaccinations became available. However, due to the lack of systematically collected data and absence of whole genome sequencing capabilities, we conducted a two‐stepped, nationally representative molecular epidemiologic study of SARS‐CoV‐2 in Mongolia for 2020 and 2021.MethodsWe used retrospective analysis of stored biological samples from November 2020 to October 2021 and a variant‐specific real‐time reverse transcription polymerase chain reaction (RT‐PCR) test to detect SARS‐CoV‐2 variants, followed by whole genome sequencing by Nanopore technology. Samples were retrieved from different sites and stored at −70°C deep freezer, and tests were performed on samples with cycle threshold <30.ResultsOut of 4879 nucleic acid tests, 799 whole genome sequencing had been carried out. Among the stored samples of earlier local transmission, we found the 20B (B.1.1.46) variant predominated in the earlier local transmission period. A slower introduction and circulation of alpha and delta variants were observed compared to global dynamics in 2020 and 2021. Beta or Gamma variants were not detected between November 2020 and September 2021 in Mongolia.ConclusionsSARS‐CoV‐2 variants of concerns including alpha and delta were delayed in circulation potentially due to public health stringencies in Mongolia. We are sharing our initial experience with whole genome sequencing of SARS‐CoV‐2 from Mongolia, where sequencing data is sparse.

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

Abstract: Universal access to drug susceptibility testing for newly diagnosed tuberculosis patients is recommended. Access to culture-based diagnostics remains limited, and targeted molecular assays are vulnerable to emerging resistance mutations.

Cited by 13 publications

References 19 publications

Genetic diversity within diagnostic sputum samples is mirrored in the culture ofMycobacterium tuberculosis

Genetic diversity within diagnostic sputum samples is mirrored in the culture ofMycobacterium tuberculosis

Pangenome databases provide superior host removal and mycobacteria classification from clinical metagenomic data

Molecular epidemiology of SARS‐CoV‐2 in Mongolia, first experience with nanopore sequencing in lower‐ and middle‐income countries setting

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