Comparison of an Automated Repetitive Sequence-based PCR Microbial Typing System with IS6110-Restriction Fragment Length Polymorphism for Epidemiologic Investigation of Clinical Mycobacterium tuberculosis Isolates in Korea

Tuberculosis remains a severe public health problem worldwide. Presently, genotyping is used for conducting epidemiologic and clinical studies on tuberculosis cases. We evaluated the efficacy of the repetitive sequence-based PCR (rep-PCR)-based DiversiLab™ system (bioMérieux, France) over the IS6110-restriction fragment length polymorphism analysis for detecting Mycobacterium tuberculosis. In all, 89 clinical M. tuberculosis isolates collected nationwide from Korea were used. The DiversiLab system allocated the 89 isolates to 8 groups with 1 unique isolate when a similarity level of 95% was applied. Seventy-six isolates of the Beijing family and 13 isolates of non-Beijing family strains were irregularly distributed regardless of rep-PCR groups. The DiversiLab system generated a rapid, sensitive, and standardized result. It can be used to conduct molecular epidemiologic studies to identify clinical M. tuberculosis isolates in Korea

Human evolution in Siberia: from frozen bodies to ancient DNA

<p>Abstract</p> <p>Background</p> <p>The Yakuts contrast strikingly with other populations from Siberia due to their cattle- and horse-breeding economy as well as their Turkic language. On the basis of ethnological and linguistic criteria as well as population genetic studies, it has been assumed that they originated from South Siberian populations. However, many questions regarding the origins of this intriguing population still need to be clarified (e.g. the precise origin of paternal lineages and the admixture rate with indigenous populations). This study attempts to better understand the origins of the Yakuts by performing genetic analyses on 58 mummified frozen bodies dated from the 15^thto the 19^thcentury, excavated from Yakutia (Eastern Siberia).</p> <p>Results</p> <p>High quality data were obtained for the autosomal STRs, Y-chromosomal STRs and SNPs and mtDNA due to exceptional sample preservation. A comparison with the same markers on seven museum specimens excavated 3 to 15 years ago showed significant differences in DNA quantity and quality. Direct access to ancient genetic data from these molecular markers combined with the archaeological evidence, demographical studies and comparisons with 166 contemporary individuals from the same location as the frozen bodies helped us to clarify the microevolution of this intriguing population.</p> <p>Conclusion</p> <p>We were able to trace the origins of the male lineages to a small group of horse-riders from the Cis-Baïkal area. Furthermore, mtDNA data showed that intermarriages between the first settlers with Evenks women led to the establishment of genetic characteristics during the 15^thcentury that are still observed today.</p

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.)

Predicting haplogroups using a versatile machine learning program (PredYMaLe) on a new mutationally balanced 32 Y-STR multiplex (CombYplex): Unlocking the full potential of the human STR mutation rate spectrum to estimate forensic parameters

We developed a new mutationally well-balanced 32 Y-STR multiplex (CombYplex) together with a machine learning (ML) program PredYMaLe to assess the impact of STR mutability on haplogourp prediction, while respecting forensic community criteria (high DC/HD). We designed CombYplex around two sub-panels M1 and M2 characterized by average and high-mutation STR panels. Using these two sub-panels, we tested how our program PredYmale reacts to mutability when considering basal branches and, moving down, terminal branches. We tested first the discrimination capacity of CombYplex on 996 human samples using various forensic and statistical parameters and showed that its resolution is sufficient to separate haplogroup classes. In parallel, PredYMaLe was designed and used to test whether a ML approach can predict haplogroup classes from Y-STR profiles. Applied to our kit, SVM and Random Forest classifiers perform very well (average 97 %), better than Neural Network (average 91 %) and Bayesian methods (< 90 %)

Genotyping of single nucleotide polymorphisms in DNA extracted from ancient human tissues : application to the study of ancient Siberian populations

Dans ce travail de thèse, une approche méthodologique originale basée sur l'analyse de différents polymorphismes ponctuels (ou SNP) a été utilisée sur des échantillons anciens dans le but d'étudier l'histoire de populations sibériennes du passé. Des SNP du génome humain ont été analysés à partir d'échantillons anciens provenant du Sud de la Sibérie afin d'apporter de nouveaux éléments quant au peuplement de cette région. Les données génétiques obtenues indiquent qu aux Ages du Bronze et du Fer, cette région était peuplée de sujets au teint pâle, aux cheveux clairs et aux yeux bleus qui étaient originaires d'Europe de l'Est. Ces résultats ont permis de mettre en évidence l'existence de mouvements de populations d'Europe de l'Est jusqu au Sud de la Sibérie avant 1800 avant J.-C. De plus, au vu de données archéologiques et linguistiques, il semblerait que ces mouvements puissent être liés à l'expansion vers l'Est de peuples ayant parlé le proto indo-européen. L'analyse de SNP du génome d'agents pathogènes responsables de la tuberculose menée à partir de vertèbres yakoutes présentant des lésions osseuses évocatrices d'une atteinte tuberculeuse a permis d'appréhender l'état sanitaire de la population yakoute (Sibérie Orientale). En effet, le fait d'avoir pu diagnostiquer la tuberculose chez 3 de ces sujets et d'identifier M. tuberculosis comme agent responsable de l'infection a apporté, pour la première fois, la preuve de la présence de la tuberculose humaine en Yakoutie aux XVIIème et XVIIIème siècles. La qualité des résultats obtenus ainsi que les hypothèses originales qui ont pu être proposées attestent de l'intérêt que présente l'analyse de SNP pour les études d'ADN ancien.In the present work, an original methodological approach based on the study of various single nucleotide polymorphisms (SNPs) was used on ancient DNA samples to study the history of ancient Siberian populations. On the one hand, SNPs of the human genome were analyzed on ancient South Siberian samples in order to shed light on the peopling of this region. The resulting data indicate that, at the Bronze and Iron Ages, this region was inhabited by fair-skinned, light-haired and blue- or green-eyed people that originated from Eastern Europe. Our results thus show that population movements from Eastern Europe to South Siberia occurred before 1800 Before Christ. Moreover, according to archeological and linguistic data, it seems that these movements were linked to the eastward expansion of people who spoke the ancestral Indo-European or Proto-Indo-European language. On the other hand, SNPs enabling the distinction between common tuberculosis pathogens were analyzed on ancient Yakut vertebrae with lytic lesions evocative of the diagnosis of this disease in order to investigate the sanitary state of the Yakut population (Oriental Siberia). The fact that tuberculosis was diagnosed in 3 cases and that M. tuberculosis was found to be responsible for these infections provide for the first time the proof that human tuberculosis was present in Yakutia in the 17th and 18th centuries. The quality of the ancient data obtained during this work as well as the possibility to propose new hypotheses give evidence that SNPs are valuable markers for ancient DNA studies. It also demonstrates that the SNaPshotTM -based minisequencing is a method of choice for SNP typing on degraded DNA templates