Phylogenomics Sheds Light on the population structure of Mycobacterium bovis from a Multi-Host Tuberculosis System

AbstractMolecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. The genetic relatedness of M. bovis isolates from cattle and wildlife have also suggested sustained transmission within this multi-host system. However, while previous surveillance highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric and provided valuable information on the prevalence and spatial occurrence of TB, links at the wildlife-livestock interfaces were established mainly via genotype associations. Therefore, evidence at a local fine scale of transmission events linking wildlife hosts and cattle remains lacking. Here, we explore the advantages of whole genome sequencing (WGS) applied to cattle, red deer and wild boar isolates to reconstruct the evolutionary dynamics of M. bovis and to identify putative pathogen transmission events. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with geographic specificities, as well as the establishment of a SNPs catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to reconstruct transmission networks, which together with inferred secondary cases, further structured this M. bovis population by host species and geographic location.WGS of M. bovis isolates from Portugal is reported for the first time, refining the spatiotemporal context of transmission events and providing further support to the key role of red deer and wild boar on the persistence of animal TB in this Iberian multi-host system.

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Whole Genome Sequencing Refines Knowledge on the Population Structure of Mycobacterium bovis from a Multi-Host Tuberculosis System

Microorganisms ◽

10.3390/microorganisms9081585 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1585

Author(s):

Ana C. Reis ◽

Liliana C. M. Salvador ◽

Suelee Robbe-Austerman ◽

Rogério Tenreiro ◽

Ana Botelho ◽

...

Keyword(s):

Population Structure ◽

Whole Genome Sequencing ◽

Wild Boar ◽

Genome Sequencing ◽

Mycobacterium Bovis ◽

Red Deer ◽

Variable Number Tandem Repeat ◽

Variant Calling ◽

Whole Genome ◽

Network Analyses

Classical molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought the first insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. Previous surveillance provided valuable information on the prevalence and spatial occurrence of TB and highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric. However, links at the wildlife–livestock interfaces were established mainly via classical genotype associations. Here, we apply whole genome sequencing (WGS) to cattle, red deer and wild boar isolates to reconstruct the M. bovis population structure in a multi-host, multi-region disease system and to explore links at a fine genomic scale between M. bovis from wildlife hosts and cattle. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with apparent geographic specificities, as well as the establishment of an SNP catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to further structure this M. bovis population by host species and TB hotspots, providing a baseline for network analyses in different epidemiological and disease control contexts. WGS of M. bovis isolates from Portugal is reported for the first time in this pilot study, refining the spatiotemporal context of TB at the wildlife–livestock interface and providing further support to the key role of red deer and wild boar on disease maintenance. The SNP diversity observed within this dataset supports the natural circulation of M. bovis for a long time period, as well as multiple introduction events of the pathogen in this Iberian multi-host system.

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Population Structure of Mycobacterium bovis in Germany: a Long-Term Study Using Whole-Genome Sequencing Combined with Conventional Molecular Typing Methods

Journal of Clinical Microbiology ◽

10.1128/jcm.01573-20 ◽

2020 ◽

Vol 58 (11) ◽

Author(s):

Thomas A. Kohl ◽

Katharina Kranzer ◽

Sönke Andres ◽

Thierry Wirth ◽

Stefan Niemann ◽

...

Keyword(s):

Population Structure ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Mycobacterium Bovis ◽

Variable Number Tandem Repeat ◽

Epidemiological Surveillance ◽

Whole Genome ◽

Content Type ◽

Long Term Study ◽

Wide Range

ABSTRACT Mycobacterium bovis is the primary cause of bovine tuberculosis (bTB) and infects a wide range of domestic animal and wildlife species and humans. In Germany, bTB still emerges sporadically in cattle herds, free-ranging wildlife, diverse captive animal species, and humans. In order to understand the underlying population structure and estimate the population size fluctuation through time, we analyzed 131 M. bovis strains from animals (n = 38) and humans (n = 93) in Germany from 1999 to 2017 by whole-genome sequencing (WGS), mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, and spoligotyping. Based on WGS data analysis, 122 out of the 131 M. bovis strains were classified into 13 major clades, of which 6 contained strains from both human and animal cases and 7 only strains from human cases. Bayesian analyses suggest that the M. bovis population went through two sharp anticlimaxes, one in the middle of the 18th century and another one in the 1950s. WGS-based cluster analysis grouped 46 strains into 13 clusters ranging in size from 2 to 11 members and involving strains from distinct host types, e.g., only cattle and also mixed hosts. Animal strains of four clusters were obtained over a 9-year span, pointing toward autochthonous persistent bTB infection cycles. As expected, WGS had a higher discriminatory power than spoligotyping and MIRU-VNTR typing. In conclusion, our data confirm that WGS and suitable bioinformatics constitute the method of choice to implement prospective molecular epidemiological surveillance of M. bovis. The population of M. bovis in Germany is diverse, with subtle, but existing, interactions between different host groups.

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Mycobacterium bovisPersistence in Two Different Captive Wild Animal Populations in Germany: a Longitudinal Molecular Epidemiological Study Revealing Pathogen Transmission by Whole-Genome Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.00302-18 ◽

2018 ◽

Vol 56 (9) ◽

Cited By ~ 6

Author(s):

Thomas A. Kohl ◽

Christian Utpatel ◽

Stefan Niemann ◽

Irmgard Moser

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bovine Tuberculosis ◽

Animal Health ◽

Variable Number Tandem Repeat ◽

Pathogen Transmission ◽

Whole Genome ◽

The European Union ◽

Content Type ◽

Animal Populations

ABSTRACTBovine tuberculosis (bTB) caused byMycobacterium bovisis a transmissible disease notifiable to the World Organization for Animal Health and to the European Union, with ongoing efforts of surveillance and eradication in every EU member state. In Germany, a country which has been declared officially free from bovine tuberculosis since 1997 by the EU,M. bovisinfections still occur sporadically in cattle and other mammals, including humans. Here, the transmission routes of a bTB outbreak in a wildlife park in Germany affecting different cervid species, bison, lynx, and pot-bellied pigs were followed by employing whole-genome sequencing (WGS) combined with spoligotyping and mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing. One singleM. bovisstrain persisted from 2002 to 2015, and transmission between the park and a distantly located captive cervid farm was verified. The spoligotyping patterns remained identical, while MIRU-VNTR typing of 24 loci of the standardized panel and locus 2163a as an additional locus revealed one change at locus 2165 in a strain from a fallow deer and one at locus 2461 in isolates from red deer over the whole time period. WGS analysis confirmed the close relatedness of the isolates, with a maximum of 12 single nucleotide polymorphisms (SNPs) detected between any two sequenced isolates. In conclusion, our data confirm a longitudinal outbreak ofM. bovisin a German wildlife park and provide the first insights into the dynamics of different genotyping markers inM. bovis.

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Factors that Influence Mycobacterium bovis Infection in Red Deer and Wild Boar in an Epidemiological Risk Area for Tuberculosis of Game Species in Portugal

Transboundary and Emerging Diseases ◽

10.1111/tbed.12439 ◽

2015 ◽

Vol 64 (3) ◽

pp. 793-804 ◽

Cited By ~ 7

Author(s):

S. Madeira ◽

A. Manteigas ◽

R. Ribeiro ◽

J. Otte ◽

A. Pina Fonseca ◽

...

Keyword(s):

Wild Boar ◽

Mycobacterium Bovis ◽

Red Deer ◽

Risk Area ◽

Game Species ◽

Epidemiological Risk

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Whole-Genome Sequence of Mycobacterium bovis W-1171, Isolated from the Laryngopharyngeal Lymph Node of a Wild Boar in South Korea

Genome Announcements ◽

10.1128/genomea.01464-15 ◽

2015 ◽

Vol 3 (6) ◽

Author(s):

Narae Kim ◽

Yunho Jang ◽

So-young Park ◽

Woong-seog Song ◽

Jong-Taek Kim ◽

...

Keyword(s):

Lymph Node ◽

South Korea ◽

Wild Boar ◽

Mycobacterium Bovis ◽

Genome Sequence ◽

Whole Genome Sequence ◽

Whole Genome ◽

Protein Coding ◽

Protein Coding Genes ◽

Free Ranging

Mycobacterium bovis W-1171 was isolated from a wild boar living in a free-ranging field in Gyeonggido, South Korea. The whole-genome sequence of this strain was determined in 50 contigs, which was 4,304,865 bp with a 65.57% G+C content. In total 3,945 protein-coding genes were predicted from this assembly.

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Long-term molecular surveillance provides clues on a cattle origin for Mycobacterium bovis in Portugal

Scientific Reports ◽

10.1038/s41598-020-77713-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ana C. Reis ◽

Rogério Tenreiro ◽

Teresa Albuquerque ◽

Ana Botelho ◽

Mónica V. Cunha

Keyword(s):

Wild Boar ◽

Mycobacterium Bovis ◽

Red Deer ◽

Robust Statistics ◽

Probability Model ◽

Ecological Processes ◽

Spatiotemporal Clustering ◽

History Of ◽

Bayesian Inference Framework

AbstractAnimal tuberculosis (TB), caused by Mycobacterium bovis, is maintained in Portugal in a multi-host system, with cattle, red deer and wild boar, playing a central role. However, the ecological processes driving transmission are not understood. The main aim of this study was thus to contribute to the reconstruction of the spatiotemporal history of animal TB and to refine knowledge on M. bovis population structure in order to inform novel intervention strategies. A collection of 948 M. bovis isolates obtained during long-term surveillance (2002–2016, 15 years) of cattle (n = 384), red deer (n = 303) and wild boar (n = 261), from the main TB hotspot areas, was characterized by spoligotyping and 8 to 12-loci MIRU-VNTR. Spoligotyping identified 64 profiles and MIRU-VNTR distinguished 2 to 36 subtypes within each spoligotype, enabling differentiation of mixed or clonal populations. Common genotypic profiles within and among livestock and wildlife in the same spatiotemporal context highlighted epidemiological links across hosts and regions, as for example the SB0119-M205 genotype shared by cattle in Beja district or SB0121-M34 shared by the three hosts in Castelo Branco and Beja districts. These genomic data, together with metadata, were integrated in a Bayesian inference framework, identifying five ancestral M. bovis populations. The phylogeographic segregation of M. bovis in specific areas of Portugal where the disease persists locally is postulated. Concurrently, robust statistics indicates an association of the most probable ancient population with cattle and Beja, providing a clue on the origin of animal TB epidemics. This relationship was further confirmed through a multinomial probability model that assessed the influence of host species on spatiotemporal clustering. Two significant clusters were identified, one that persisted between 2004 and 2010, in Beja district, with Barrancos county at the centre, overlapping the central TB core area of the Iberian Peninsula, and highlighting a significant higher risk associated to cattle. The second cluster was predominant in the 2012–2016 period, holding the county Rosmaninhal at the centre, in Castelo Branco district, for which wild boar contributed the most in relative risk. These results provide novel quantitative insights beyond empirical perceptions, that may inform adaptive TB control choices in different regions.

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From whole genome sequencing data toward a simple genotyping tool: application to the animal pathogen Mycobacterium bovis

10.26226/morressier.56d5ba2ad462b80296c965c0 ◽

2016 ◽

Author(s):

Lorraine Michelet

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Mycobacterium Bovis ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data

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Clinical-grade whole-genome sequencing and 3′ transcriptome analysis of colorectal cancer patients

Genome Medicine ◽

10.1186/s13073-021-00852-8 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Agata Stodolna ◽

Miao He ◽

Mahesh Vasipalli ◽

Zoya Kingsbury ◽

Jennifer Becq ◽

...

Keyword(s):

Colorectal Cancer ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Transcriptome Analysis ◽

Variant Calling ◽

Standard Of Care ◽

Genomic Variation ◽

Whole Genome ◽

Clinical Grade ◽

Pathway Gene

Abstract Background Clinical-grade whole-genome sequencing (cWGS) has the potential to become the standard of care within the clinic because of its breadth of coverage and lack of bias towards certain regions of the genome. Colorectal cancer presents a difficult treatment paradigm, with over 40% of patients presenting at diagnosis with metastatic disease. We hypothesised that cWGS coupled with 3′ transcriptome analysis would give new insights into colorectal cancer. Methods Patients underwent PCR-free whole-genome sequencing and alignment and variant calling using a standardised pipeline to output SNVs, indels, SVs and CNAs. Additional insights into the mutational signatures and tumour biology were gained by the use of 3′ RNA-seq. Results Fifty-four patients were studied in total. Driver analysis identified the Wnt pathway gene APC as the only consistently mutated driver in colorectal cancer. Alterations in the PI3K/mTOR pathways were seen as previously observed in CRC. Multiple private CNAs, SVs and gene fusions were unique to individual tumours. Approximately 30% of patients had a tumour mutational burden of > 10 mutations/Mb of DNA, suggesting suitability for immunotherapy. Conclusions Clinical whole-genome sequencing offers a potential avenue for the identification of private genomic variation that may confer sensitivity to targeted agents and offer patients new options for targeted therapies.

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Whole genome resequencing and custom genotyping unveil clonal lineages in ‘Malbec’ grapevines (Vitis vinifera L.)

Scientific Reports ◽

10.1038/s41598-021-87445-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Luciano Calderón ◽

Nuria Mauri ◽

Claudio Muñoz ◽

Pablo Carbonell-Bejerano ◽

Laura Bree ◽

...

Keyword(s):

Genetic Diversity ◽

Somatic Mutations ◽

Clonal Propagation ◽

Variant Calling ◽

Vitis Vinifera L ◽

Whole Genome ◽

Single Nucleotide Variants ◽

Genome Resequencing ◽

Diversity Pattern ◽

Whole Genome Resequencing

AbstractGrapevine cultivars are clonally propagated to preserve their varietal attributes. However, genetic variations accumulate due to the occurrence of somatic mutations. This process is anthropically influenced through plant transportation, clonal propagation and selection. Malbec is a cultivar that is well-appreciated for the elaboration of red wine. It originated in Southwestern France and was introduced in Argentina during the 1850s. In order to study the clonal genetic diversity of Malbec grapevines, we generated whole-genome resequencing data for four accessions with different clonal propagation records. A stringent variant calling procedure was established to identify reliable polymorphisms among the analyzed accessions. The latter procedure retrieved 941 single nucleotide variants (SNVs). A reduced set of the detected SNVs was corroborated through Sanger sequencing, and employed to custom-design a genotyping experiment. We successfully genotyped 214 Malbec accessions using 41 SNVs, and identified 14 genotypes that clustered in two genetically divergent clonal lineages. These lineages were associated with the time span of clonal propagation of the analyzed accessions in Argentina and Europe. Our results show the usefulness of this approach for the study of the scarce intra-cultivar genetic diversity in grapevines. We also provide evidence on how human actions might have driven the accumulation of different somatic mutations, ultimately shaping the Malbec genetic diversity pattern.

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