Dataset Information

IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes.

ABSTRACT: The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment.We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed.This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.

SUBMITTER: Reyes A

PROVIDER: S-EPMC3443423 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes.

Reyes Alejandro A Sandoval Andrea A Cubillos-Ruiz Andrés A Varley Katherine E KE Hernández-Neuta Ivan I Samper Sofía S Martín Carlos C García María Jesús MJ Ritacco Viviana V López Lucelly L Robledo Jaime J Zambrano María Mercedes MM Mitra Robi D RD Del Portillo Patricia P

BMC genomics 20120615

<h4>Background</h4>The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multip ...[more]

PMID: 22703188

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Project description:BackgroundSeveral technical hurdles and limitations have restricted the use of IS6110 restriction fragment length polymorphism (IS6110 RFLP), the most effective typing method for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS6110-5'3'FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5' and 3' IS6110 polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS6110-5'3'FP to be used as an alternative to IS6110 RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility.ObjectivesHere we introduced critical amendments to the original IS6110-5'3'FP protocol and compared its performance to that of 24-loci multiple interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR), the current standard method for TB transmission analyses.MethodsIS6110-5'3'FP protocol modifications involved: (i) the generation of smaller-sized polymorphic fragments for efficient cloning and PCR amplification, (ii) omission of the plasmid amplification step in E. coli for shorter turnaround times, (iii) the use of more stable fluorophores for increased sensitivity, (iv) automated subtraction of background fluorescent signals, and (v) the automated conversion of fluorescent peaks into binary data.ResultsIn doing so, the overall turnaround time of IS6110-5'3'FP was reduced to 4 hours. The new protocol allowed detecting almost all 5' and 3' IS6110 polymorphic fragments of any given strain, including IS6110 high-copy number Beijing strains. IS6110-5'3'FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the M. tuberculosis lineage 4.ConclusionsThe IS6110-5'3'FP protocol described herein reached the optimal discriminatory potential of IS6110 fingerprinting and proved more accurate than 24-loci MIRU-VNTR in estimating recent TB transmission. The method, which is highly cost-effective, was rendered versatile enough to prompt its evaluation as an automatized solution for a TB integrated molecular surveillance.

Dataset Information

IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes.

Publications

IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes.

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