MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes of<i>Mycobacterium tuberculosis</i>

Rajwani, Rahim; Shehzad, Sheeba; Siu, G. G.

doi:10.7717/peerj.5090

Cited by 13 publications

(4 citation statements)

References 28 publications

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“…Slightly adapted script from what was set-up for runs were written to annotate these genomes in term of spoligotype profiles and lineage/sublineage assignation. MIRU-Profiler was used to infer MIRU types from assembled genomes [24]. Resulting patterns were entered in TBminer to identify most likely MTC sublineage assignation according to MIRU-VNTR or spoligo profile or their combination [25].…”

Section: Methodsmentioning

confidence: 99%

Exhaustive reconstruction of the CRISPR locus inMycobacterium tuberculosiscomplex using short reads

Guyeux

Sola

Refrégier

2019

Preprint

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Spoligotyping, a graphical partial display of the CRISPR locus that can be produced in vitro or in silico, is an important tool for analyzing the diversity of given Mycobacterium tuberculosis complex (MTC) isolates. As other CRISPR loci, this locus is made up of an alternation between direct repeats and spacers, and flanked by cas genes. Unveiling the genetic mechanisms of its evolution requires to have a fairly large amount of fully reconstructed loci among all MTC lineages.In this article, we point out and resolve the problem of CRISPR reconstruction based on short read sequences. We first show that more than 1/3 of the currently assembled genomes available for this complex contain a CRISPR locus erroneously reconstructed, and errors can be very significant. Second, we present a new computational method allowing this locus to be reconstructed extensively and reliably in silico using short read sequencing runs. Third, using this method, we describe new structural characteristics of CRISPR locus by lineages. We show how both the classical experimental in vitro approach and the basic in silico spoligotyping provided by existing analytic tools miss a whole diversity of this locus in MTC, by not capturing duplications, spacer and direct repeats variants, and IS6110 insertion locations. This description is extended in a second article that presents general rules for the evolution of the CRISPR locus in MTC.This work opens new perspectives for a larger exploration of CRISPR loci diversity and of mechanisms involved in its evolution and its functionality.

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Section: Methodsmentioning

confidence: 99%

Exhaustive reconstruction of the CRISPR locus inMycobacterium tuberculosiscomplex using short reads

Guyeux

Sola

Refrégier

2019

Preprint

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“…Scripts adapted from those set-up for SRR were written to extract information from complete genomes: 43-spacers spoligotype profiles, lineage/sublineage assignation according to SNPs. MIRU-Profiler was used to infer MIRU types from complete genomes [ 36 ]. Resulting patterns were entered in the on-line tool TBminer ( http://info-demo.lirmm.fr/tbminer/index.php ) to identify most likely MTC lineage and sublineage assignation according to MIRU-VNTR, spoligo profile, or their combination [ 26 ].…”

Section: Design and Implementationmentioning

confidence: 99%

CRISPRbuilder-TB: “CRISPR-builder for tuberculosis”. Exhaustive reconstruction of the CRISPR locus in mycobacterium tuberculosis complex using SRA

et al. 2021

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Mycobacterium tuberculosis complex (MTC) CRISPR locus diversity has long been studied solely investigating the presence/absence of a known set of spacers. Unveiling the genetic mechanisms of its evolution requires a more exhaustive reconstruction in a large amount of representative strains. In this article, we point out and resolve, with a new pipeline, the problem of CRISPR reconstruction based directly on short read sequences in M. tuberculosis. We first show that the process we set up, that we coin as “CRISPRbuilder-TB” (https://github.com/cguyeux/CRISPRbuilder-TB), allows an efficient reconstruction of simulated or real CRISPRs, even when including complex evolutionary steps like the insertions of mobile elements. Compared to more generalist tools, the whole process is much more precise and robust, and requires only minimal manual investigation. Second, we show that more than 1/3 of the currently complete genomes available for this complex in the public databases contain largely erroneous CRISPR loci. Third, we highlight how both the classical experimental in vitro approach and the basic in silico spoligotyping provided by existing analytic tools miss a whole diversity of this locus in MTC, by not capturing duplications, spacer and direct repeats variants, and IS6110 insertion locations. This description is extended in a second article that describes MTC-CRISPR diversity and suggests general rules for its evolution. This work opens perspectives for an in-depth exploration of M. tuberculosis CRISPR loci diversity and of mechanisms involved in its evolution and its functionality, as well as its adaptation to other CRISPR locus-harboring bacterial species.

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“…Notwithstanding, the relatively low performance of spoligotyping compared to SNP barcoding and cgMLST, highlights the urgent need for a paradigm shift by migrating towards WGS-based approaches for pathogen typing, and start building 'new' historical datasets comprising full genomic information (19,79). Although WGS-based in silico MIRU-VNTR typing was not included in our workflow, similar to spoligotyping, it has been reported to suffer on May 30, 2021 by guest http://jcm.asm.org/ Downloaded from from lower accuracy when only short-read data is available (80), and would therefore equally benefit from being phased out in favor of the more performant WGS-based typing methods. Neither cgMLST nor SNP barcoding offer enough resolution to detect closely related outbreaks characterized by only a few SNPs difference (81).…”

Section: Discussionmentioning

confidence: 99%

A Bioinformatics Whole-Genome Sequencing Workflow for Clinical Mycobacterium tuberculosis Complex Isolate Analysis, Validated Using a Reference Collection Extensively Characterized with Conventional Methods and In Silico Approaches

et al. 2021

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The use of whole genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. For Mycobacterium tuberculosis (MTB), in particular, WGS has the benefit of drastically reducing the time to generate results compared to most conventional phenotypic methods. Consequently, a multitude of solutions for analyzing WGS MTB data have been developed, but their successful integration in clinical and national reference laboratories is hindered by the requirement for their validation, for which a consensus framework is still largely absent. We developed a bioinformatics workflow for (Illumina) WGS-based routine typing of MTB Complex (MTBC) member isolates allowing complete characterization including (sub)species confirmation and identification (16S, csb/RD, hsp65), Single Nucleotide Polymorphism (SNP)-based antimicrobial resistance (AMR) prediction, and pathogen typing (spoligotyping, SNP barcoding, and core genome MultiLocus Sequence Typing). Workflow performance was validated on a per-assay basis using a collection of 238 in-house sequenced MTBC isolates, extensively characterized with conventional molecular biology-based approaches supplemented with public data. For SNP-based AMR prediction, results from molecular genotyping methods were supplemented with in silico modified datasets allowing to greatly increase the set of evaluated mutations. The workflow demonstrated very high performance with performance metrics >99% for all assays, except for spoligotyping where sensitivity dropped to ∼90%. The validation framework for our WGS-based bioinformatics workflow can aid standardization of bioinformatics tools by the MTB community and other SNP-based applications regardless of the targeted pathogen(s). The bioinformatics workflow is available for academic and non-profit usage through the Galaxy instance of our institute at https://galaxy.sciensano.be.

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MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes ofMycobacterium tuberculosis

Cited by 13 publications

References 28 publications

Exhaustive reconstruction of the CRISPR locus inMycobacterium tuberculosiscomplex using short reads

Exhaustive reconstruction of the CRISPR locus inMycobacterium tuberculosiscomplex using short reads

CRISPRbuilder-TB: “CRISPR-builder for tuberculosis”. Exhaustive reconstruction of the CRISPR locus in mycobacterium tuberculosis complex using SRA

A Bioinformatics Whole-Genome Sequencing Workflow for Clinical Mycobacterium tuberculosis Complex Isolate Analysis, Validated Using a Reference Collection Extensively Characterized with Conventional Methods and In Silico Approaches

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