Highly Parallel and Short-Acting Amplification with Locus-Specific Primers to Detect Single Nucleotide Polymorphisms by the DigiTag2 Assay

The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance) were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM) and specific (concentration of unexpected amplicons <2 nM) amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites) in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays

Tuberculosis in the Caribbean: using spacer oligonucleotide typing to understand strain origin and transmission.

We used direct repeat (DR)-based spacer oligonucleotide typing (spoligotyping) (in association with double-repetitive element polymerase chain reaction, IS6110-restriction fragment length polymorphism [RFLP], and sometimes DR-RFLP and polymorphic GC-rich sequence-RFLP) to detect epidemiologic links and transmission patterns of Mycobacterium tuberculosis on Martinique, Guadeloupe, and French Guiana. In more than a third of the 218 strains we typed from this region, clusters and isolates shared genetic identity, which suggests epidemiologic links. However, because of limited epidemiologic information, only 14.2% of the strains could be directly linked. When spoligotyping patterns shared by two or more isolates were pooled with 392 spoligotypes from other parts of the world, new matches were detected, which suggests imported transmission. Persisting foci of endemic disease and increased active transmission due to high population flux and HIV-coinfection may be linked to the recent reemergence of tuberculosis in the Caribbean. We also found that several distinct families of spoligotypes are overrepresented in this region

The Forest behind the Tree: Phylogenetic Exploration of a Dominant Mycobacterium tuberculosis Strain Lineage from a High Tuberculosis Burden Country

BACKGROUND: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. CONCLUSIONS/SIGNIFICANCE: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications

High-Throughput Method for Detecting Genomic-Deletion Polymorphisms

DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms

Congruence analysis between MIRU-VNTR typing, deligotyping and spoligotyping.

<p>A selection of 137 isolates was used, representing the diversity of the different lineages and subgroups predicted based on MIRU-VNTR typing, spoligotyping and the MIRU-VNTR<i>Plus</i> database (see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-g001" target="_blank">Fig. 1</a>). A. A MIRU-VNTR-based dendrogram was generated using the neighbor-joining algorithm and rooted using a <i>M. prototuberculosis</i> C/D genotype (alias <i>M. canettii</i>) as outgroup. Solid coloured circles on tree nodes indicate MIRU-VNTR groupings that are monophyletic when compared to LSP-based (deligotyping) and/or (when deligotyping was not informative) spoligotyping-based groupings. Partially monophyletic groupings are indicated by coloured rings. B. MIRU-VNTR-based minimum spanning tree. The same isolates were used as in the neighbor-joining tree. Colours and grouping names correspond to those of panel A. Distances between circles are proportional to the number of allele differences between MIRU-VNTR genotypes; circle sizes are proportional to the numbers of isolates sharing an identical genotype. Del, deligotyping probe; RD, region of difference (reference LSP, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-t001" target="_blank">Table 1</a>); spol, spoligotyping spacer; LAMu, unclassified LAM isolate. Color codes of phylogenetic groups (see text for further description): yellow, Indo-Oceanic (LSP)/EAI (spoligotyping); grey, S; light purple, X; red, Haarlem; blue, Brazil 1; dark purple; Brazil 2; pink, LAM II; khaki, LAM I; green, LAM III.</p

Discriminatory powers of different genotyping methods used.

<p>Discriminatory powers of different genotyping methods used.</p

Distribution of <i>M. tuberculosis</i> lineages in the study.

a<p>Nomenclatures corresponding to Gagneux et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone.0018256-Gagneux2" target="_blank">[30]</a>/Brudey et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone.0018256-Brudey1" target="_blank">[48]</a>, except for Brazil-1 and -2 named according to this study.</p

Genotypic diversity of <i>M. tuberculosis</i> isolates from 3 Brazilian regions.

<p>Colour-coded 24-locus MIRU-VNTR alleles and spoligotypes from 361 isolates are represented. A MIRU-VNTR-based dendrogram was generated using the neighbor-joining algorithm and rooted using a <i>M. prototuberculosis</i> C/D genotype (alias <i>M. canettii</i>) as outgroup. <i>M. tuberculosis</i> strain lineages and branches shown at the right were identified by analyzing the congruence of MIRU-VNTR typing and spoligotyping results within this collection and submitting the isolate genotypes to the MIRU-VNTR<i>Plus</i> identification database (see text). X gr1 and gr2 (groups 1 and 2) correspond to X del26/RD183 and Xdel29/RD193 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-g003" target="_blank">Fig. 3</a>, respectively.</p

<i>M. tuberculosis</i> strain cluster distribution among three Brazilian regions.

<p>Molecular clusters are defined by isolates sharing identical 24-locus MIRU-VNTR genotypes and spoligotypes.</p