Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

Detection of the A189G mtDNA heteroplasmic mutation in relation to age in modern and ancient bones.

International audienceThe aim of this study was to demonstrate the presence of the A189G age-related point mutation on DNA extracted from bone. For this, a peptide nucleic acid (PNA)/DNA sequencing method which can determine an age threshold for the appearance of the mutation was used. Initially, work was done in muscle tissue in order to evaluate the sensitivity of the technique and afterwards in bone samples from the same individuals. This method was also applied to ancient bones from six well-preserved skeletal remains. The mutation was invariably found in muscle, and at a rate of up to 20% in individuals over 60 years old. In modern bones, the mutation was detected in individuals aged 38 years old or more, at a rate of up to 1%, but its occurrence was not systematic (only four out of ten of the individuals over 50 years old carried the heteroplasmy). For ancient bones, the mutation was also found in the oldest individuals according to osteologic markers. The study of this type of age-related mutation and a more complete understanding of its manifestation has potentially useful applications. Combined with traditional age markers, it could improve identification accuracy in forensic cases or in anthropological studies of ancient populations

Revising mtDNA haplotypes of the ancient Hungarian conquerors with next generation sequencing

As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.We thank S. Lecher, S. Li and J. Zallet for technical support. Calculations were performed at the sciCORE scientific computing core facility at the University of Basel. This work was supported by the Swiss National Science Foundation (grants 310030_166687 (S.G.) and 320030_153442 (M.E.) and Swiss HIV Cohort Study grant 740 to L.F.), the European Research Council (309540-EVODRTB to S.G.), TB-PAN-NET (FP7-223681 to S.N.), PathoNgenTrace projects (FP7-278864-2 to S.N.), SystemsX.ch (S.G.), the German Center for Infection Research (DZIF; S.N.), the Novartis Foundation (S.G.), the Natural Science Foundation of China (91631301 to Q.G.), and the National Institute of Allergy and Infectious Diseases (5U01-AI069924-05) of the US National Institutes of Health (M.E.)

Identification and Genotyping of Mycobacterium tuberculosis Complex Species by Use of a SNaPshot Minisequencing-Based Assay▿

The aim of the present study was to investigate the use of the SNaPshot minisequencing method for the identification of Mycobacterium tuberculosis complex (MTBC) isolates to the species level and for further genotyping of M. tuberculosis isolates. We developed an innovative strategy based on two multiplex allele-specific minisequencing assays that allowed detection of eight species-specific and eight lineage-specific single nucleotide polymorphisms (SNPs). Each assay consisted of an eightplex PCR amplification, followed by an eightplex minisequencing reaction with the SNaPshot multiplex kit (Applied Biosystems) and, finally, analysis of the extension products by capillary electrophoresis. The whole strategy was developed with a panel of 56 MTBC strains and 15 negative controls. All MTBC strains tested except one M. africanum clinical isolate were accurately identified to the species level, and all M. tuberculosis isolates were successfully further genotyped. This two-step strategy based on SNaPshot minisequencing allows the simultaneous differentiation of closely related members of the MTBC, the distinction between principal genetic groups, and the characterization of M. tuberculosis isolates into one of the seven prominent SNP cluster groups (SCGs) and could be a useful tool for diagnostic and epidemiological purposes

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Single Nucleotide Polymorphism Genotyping Assay Using iPLEX Gold Technology for Identification of Mycobacterium tuberculosis Complex Species and Lineages▿

The major goal of the present study was to investigate the potential use of a novel single nucleotide polymorphism (SNP) genotyping technology, called iPLEX Gold (Sequenom), for the simultaneous analysis of 16 SNPs that have been previously validated as useful for identification of Mycobacterium tuberculosis complex (MTBC) species and classification of MTBC isolates into distinct genetic lineages, known as principal genetic groups (PGGs) and SNP cluster groups (SCGs). In this context, we developed a 16-plex iPLEX assay based on an allele-specific-primer single-base-extension reaction using the iPLEX Gold kit (Sequenom), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis on the commercially available Sequenom MassARRAY platform. This assay was tested on a panel of 55 well-characterized MTBC strains that were also genotyped for the same loci using the previously reported SNaPshot assay, as well as 10 non-MTBC mycobacteria and 4 bacteria not belonging to the genus Mycobacterium. All MTBC samples were successfully analyzed with the iPLEX assay, which yielded clear allelic data for 99.9% of the SNPs (879 out of 880). No false-positive results were obtained with the negative controls. Compared to the SNaPshot assay, the newly developed 16-plex iPLEX assay produced fully concordant results that allowed reliable differentiation of MTBC species and recognition of lineages, thus demonstrating its potential value in diagnostic, epidemiological, and evolutionary applications. Compared to the SNaPshot approach, the implementation of the iPLEX technology could offer a higher throughput and could be a more flexible and cost-effective option for microbiology laboratories

"OpenLAB": A 2-hour PCR-based practical for high school students.

The Strasbourg University PhD school in Life and Health Sciences launched an initiative called "OpenLAB." This project was developed in an effort to help high school teenagers understand theoretical and abstract concepts in genetics. A second objective of this program is to help students in defining their future orientation and to attract them to biology. The general idea is a 2-hour PCR-based practical that is developed around a fictitious criminal investigation. The practical is taught by PhD graduate students who bring all the required reagents and modern equipment into the classroom. Running the PCR provides free time dedicated to discussions with students about their future plans after the high school diploma. A specific website and a powerpoint presentation were developed to provide appropriate scientific information. Starting on a modest scale in Strasbourg in December 2008, "OpenLAB" was rapidly and well received all around, visiting 53 classes spread over a 200 km area in Alsace until May 2009. It permitted interactions with almost one thousand students in their last year of high school, with the prospect to visit 20% more classes this school year. Our experience, along with feedback from students and their teachers, suggests that it is possible to reach out to many students and have a strong impact with a rather limited budget.journal article2010 Sepimporte

mRNA profiling for the identification of blood—Results of a collaborative EDNAP exercise

A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests