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Graph construction method impacts variation representation and analyses in a bovine super-pangenome.Genome Biology May 2023Several models and algorithms have been proposed to build pangenomes from multiple input assemblies, but their impact on variant representation, and consequently...
BACKGROUND
Several models and algorithms have been proposed to build pangenomes from multiple input assemblies, but their impact on variant representation, and consequently downstream analyses, is largely unknown.
RESULTS
We create multi-species super-pangenomes using pggb, cactus, and minigraph with the Bos taurus taurus reference sequence and eleven haplotype-resolved assemblies from taurine and indicine cattle, bison, yak, and gaur. We recover 221 k nonredundant structural variations (SVs) from the pangenomes, of which 135 k (61%) are common to all three. SVs derived from assembly-based calling show high agreement with the consensus calls from the pangenomes (96%), but validate only a small proportion of variations private to each graph. Pggb and cactus, which also incorporate base-level variation, have approximately 95% exact matches with assembly-derived small variant calls, which significantly improves the edit rate when realigning assemblies compared to minigraph. We use the three pangenomes to investigate 9566 variable number tandem repeats (VNTRs), finding 63% have identical predicted repeat counts in the three graphs, while minigraph can over or underestimate the count given its approximate coordinate system. We examine a highly variable VNTR locus and show that repeat unit copy number impacts the expression of proximal genes and non-coding RNA.
CONCLUSIONS
Our findings indicate good consensus between the three pangenome methods but also show their individual strengths and weaknesses that need to be considered when analysing different types of variants from multiple input assemblies.
Topics: Animals; Cattle; Genome; Minisatellite Repeats; Sequence Analysis, DNA
PubMed: 37217946
DOI: 10.1186/s13059-023-02969-y -
Journal of Microbiological Methods Aug 2023Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission...
Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission dynamics of brucellosis worldwide. We aimed to compare the population structure of the genomes of 53 Brazilian Brucella abortus isolates using eight different genotyping methods: multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphism (SNP) detection (parSNP and NASP) from whole genomes. The strains were isolated from six different Brazilian states between 1977 and 2008 and had previously been analyzed using MLVA8, MLVA11, and MLVA16. Their whole genomes were sequenced, assembled, and subjected to MLST9 MLST21, cgMLST, and SNP analyses. All the genotypes were compared by hierarchical grouping method based on the average distances between the correlation matrices of each technique. MLST9 and MLST21 had the lowest level of resolution, both revealing only four genotypes. MLVA8, MLVA11, and MLVA16 had progressively increasing levels of resolution as more loci were analyzed, identifying 6, 16, and 44 genotypes, respectively. cgMLST showed the highest level of resolution, identifying 45 genotypes, followed by the SNP-based methods, both of which had 44 genotypes. In the assessed population, MLVA was more discriminatory than MLST and was easier and cheaper to perform. SNP techniques and cgMLST provided the highest levels of resolution and the results from the two methods were in close agreement. In conclusion, the choice of genotyping technique can strongly affect one's ability to make meaningful epidemiological conclusions but is dependent on available resources: while the VNTR based techniques are more indicated to high prevalence scenarios, the WGS methods are the ones with the best discriminative power and therefore recommended for outbreaks investigation.
Topics: Humans; Brucella abortus; Genotyping Techniques; Genotype; Multilocus Sequence Typing; Brucellosis; Minisatellite Repeats; Phylogeny
PubMed: 37343840
DOI: 10.1016/j.mimet.2023.106772 -
Transactions of the Royal Society of... Aug 2021Multidrug-resistant TB (MDR-TB) outbreaks have occurred in the Thamaka district, Kanchanaburi province in Thailand.
BACKGROUND
Multidrug-resistant TB (MDR-TB) outbreaks have occurred in the Thamaka district, Kanchanaburi province in Thailand.
METHODS
Seventy-two isolates, which included 7% mono-, 30.6% MDR and extensively drug-resistant TB (XDR-TB), were genotyped by spoligotyping, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) and single nucleotide polymorphism genotyping, and their drug resistance was analysed.
RESULTS
The spoligotyping results showed that Beijing spoligo-international type (SIT)1 was predominant (n=38; 52.8%) while the remaining were non-Beijing sublineages (n=34). The MIRU-VNTR analysis showed that Beijing isolates, most of which belonged to the modern type (n=37), formed 5 clusters and 13 individual patterns. In katG, only mutation Ser315Thr was identified. In rpoB, Ser531Leu was predominant, except for His526Arg and Leu533Pro, which were found in two isolates. A cluster of 14 Beijing strains contained these common mutations and shared the MIRU-VNTR genotype with isolates in the Thamaka district that had spread previously. Two U SIT523 isolates contained the mutations A1400G in rrs and Asp94Gly in gyrA genes, indicating a spread of XDR-TB.
CONCLUSIONS
Most mutations were associated with drug resistance and the specific MDR Beijing and XDR-TB in U SIT523 isolates remain. This genotyping is a key tool for tracking TB transmission in the Thamaka district of Thailand.
Topics: Antitubercular Agents; Disease Outbreaks; Drug Resistance, Multiple, Bacterial; Genotype; Humans; Minisatellite Repeats; Mycobacterium tuberculosis; Thailand; Tuberculosis, Multidrug-Resistant
PubMed: 33320938
DOI: 10.1093/trstmh/traa148 -
International Journal of... 2022Tuberculosis (TB) remains a serious public health burden in Korea. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) is preferred for...
Validation and comparative analysis of kogene mycobacterial interspersed repetitive unit-variable number of tandem repeat typing kit and its application on clinically isolated samples from national tuberculosis hospital, Republic of Korea.
BACKGROUND
Tuberculosis (TB) remains a serious public health burden in Korea. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) is preferred for epidemiological TB investigation. Until recently, the difficulty lies in epidemiological TB investigation due to the absence of commercialized MIRU-VNTR in Korea. Here, we have evaluated the newly designed MIRU-VNTR kit by Kogenebiotech, Korea.
MATERIALS AND METHODS
A total of 200 samples, where 100 are Mycobacrerium tuberculosis (M. tuberculosis), and the other 100 are non-M. tuberculosis, were used. Initially, the Kogenebiotech MIRU-VNTR typing kit (KoMIRU) was compared with Multilocus Variable Number Tandem Repeat Genotyping of M. tuberculosis typing kit (MVNTR) by Philip Supply for validation purpose. Then, Limit of Detection for DNA copies was optimized. Finally, KoMIRU and Genoscreen MIRU-VNTR typing kit (GeMIRU) were tested and comparatively analyzed for its specificity and sensitivity.
RESULTS
The study showed that the KoMIRU has slightly higher discriminatory power over MVNTR, 100% versus 97.5%. In comparative analysis, the KoMIRU has shown comparable capability as GeMIRU, showing 100% for sensitivity and specificity with a 95% CI value of 96.38 to 100.00%. Also, no discrepancies were observed on discriminated lineage strains between KoMIRU and GeMIRU. Out of 100, 84 were identified as Beijing strains, and remains were identified as NEW-1 (n = 8), Uganda (n = 6), East African Indian (EAI) (n = 6), Turkey (n = 2), and Haarlem (n = 1).
CONCLUSION
In this study, KoMIRU has shown a comparable capability to GeMIRU. Furthermore, previous researches had suggested an association between lineage strains and drug resistance; hence, the implementation of KoMIRU can help in TB control and prevention.
Topics: Bacterial Typing Techniques; DNA, Bacterial; Genotype; Hospitals, Chronic Disease; Humans; Interspersed Repetitive Sequences; Minisatellite Repeats; Mycobacterium tuberculosis; Tuberculosis, Lymph Node
PubMed: 35295020
DOI: 10.4103/ijmy.ijmy_8_22 -
BMC Infectious Diseases Jun 2021Cholera has been present and recurring in Zambia since 1977. However, there is a paucity of data on genetic relatedness and diversity of the Vibrio cholerae isolates...
BACKGROUND
Cholera has been present and recurring in Zambia since 1977. However, there is a paucity of data on genetic relatedness and diversity of the Vibrio cholerae isolates responsible for these outbreaks. Understanding whether the outbreaks are seeded from existing local isolates or if the outbreaks represent separate transmission events can inform public health decisions.
RESULTS
Seventy-two V. cholerae isolates from outbreaks in 2009/2010, 2016, and 2017/2018 in Zambia were characterized using multilocus variable number tandem repeat analysis (MLVA) and whole genome sequencing (WGS). The isolates had eight distinct MLVA genotypes that clustered into three MLVA clonal complexes (CCs). Each CC contained isolates from only one outbreak. The results from WGS revealed both clustered and dispersed single nucleotide variants. The genetic relatedness of isolates based on WGS was consistent with the MLVA, each CC was a distinct genetic lineage and had nearest neighbors from other East African countries. In Lusaka, isolates from the same outbreak were more closely related to themselves and isolates from other countries than to isolates from other outbreaks in other years.
CONCLUSIONS
Our observations are consistent with i) the presence of random mutation and alternative mechanisms of nucleotide variation, and ii) three separate transmission events of V. cholerae into Lusaka, Zambia. We suggest that locally, case-area targeted invention strategies and regionally, well-coordinated plans be in place to effectively control future cholera outbreaks.
Topics: Cholera; Cluster Analysis; Disease Outbreaks; Genetic Variation; Genotype; Humans; Minisatellite Repeats; Vibrio cholerae O1; Whole Genome Sequencing; Zambia
PubMed: 34126945
DOI: 10.1186/s12879-021-06259-5 -
Journal of Visualized Experiments : JoVE Jul 2021In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and...
In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences. This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard. Following this guide, students should be able to: • Harvest and extract DNA from buccal mucosa epithelial cells • Perform a PCR experiment and understand the function of various reaction components • Analyze the amplicons by agarose gel electrophoresis and interpret the results • Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences.
Topics: Alleles; DNA Fingerprinting; Humans; Laboratories; Minisatellite Repeats; Paternity; Phylogeny
PubMed: 34338668
DOI: 10.3791/62305 -
Infection, Genetics and Evolution :... Aug 2019Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries and 6th among the countries with the highest prevalence of drug-resistant TB. However,...
Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries and 6th among the countries with the highest prevalence of drug-resistant TB. However, insufficient data are available on the genetic structure of M. tuberculosis strains circulating in this country. The objective of this study was to explore the genetic diversity of multidrug-resistant M. tuberculosis isolates from Punjab, Pakistan with a combination of spoligotyping and 24-loci MIRU-VNTR typing. Among a total of 127 MDR isolates studies, 53 spoligotypes were obtained, split into 14 clusters (n = 88, 69.3%, 2-29 isolates per cluster) and 39 (30.7%) unique patterns. At the phylogenetic level, the most prevalent sublineage was CAS1_DELHI (n = 53, 41.7%), mostly represented by ST 1942 (n = 29, 22.8%), followed by T1 (n = 14, 11%) and Beijing (n = 10, 7.8%). The remaining nine sublineages (CAS, MANU2, EAI5, T2, LAM10_CAM, H1, X1, H4 and CAS2) involved altogether 24 (18.9%) isolates. Twenty-six (20.5%) isolates could not be assigned to any specific clade. MIRU-VNTR typing identified 123 (96.8%), 97 (76.4%) and 65 (51.2%) unique types with a tolerance of 0, 1, and 2 locus differences between the patterns. Upon combined spoligotyping and MIRU-VNTR typing analysis, 123 (96.8%), 108 (85%), and 91 (71.7%) unique types were identified if a tolerance of 0, 1, and 2 locus differences in the MIRU-VNTR patterns was assumed, respectively. Based on the clustering results, the transmission rate for MDR-TB cases under the study was calculated at 3.2%, 15%, and 28.3%. Overall, three clades, namely CAS1_DELHI, T1, and Beijing accounted for the majority of MDR-TB cases in Pakistan. Up to a third of the cases were clustered upon combined spoligotyping and MIRU-VNTR typing, suggesting a moderate level of active transmission.
Topics: Bacterial Typing Techniques; Genetic Variation; Humans; Minisatellite Repeats; Molecular Epidemiology; Multilocus Sequence Typing; Mycobacterium tuberculosis; Pakistan; Phylogeny; Tuberculosis, Multidrug-Resistant
PubMed: 30822549
DOI: 10.1016/j.meegid.2019.02.029 -
Scientific Reports Oct 2019Plasmodiophora brassicae is a soil-borne pathogen that attacks roots of cruciferous plants causing clubroot disease. The pathogen belongs to the Plasmodiophorida order...
Plasmodiophora brassicae is a soil-borne pathogen that attacks roots of cruciferous plants causing clubroot disease. The pathogen belongs to the Plasmodiophorida order in Phytomyxea. Here we used long-read SMRT technology to clarify the P. brassicae e3 genomic constituents along with comparative and phylogenetic analyses. Twenty contigs representing the nuclear genome and one mitochondrial (mt) contig were generated, together comprising 25.1 Mbp. Thirteen of the 20 nuclear contigs represented chromosomes from telomere to telomere characterized by [TTTTAGGG] sequences. Seven active gene candidates encoding synaptonemal complex-associated and meiotic-related protein homologs were identified, a finding that argues for possible genetic recombination events. The circular mt genome is large (114,663 bp), gene dense and intron rich. It shares high synteny with the mt genome of Spongospora subterranea, except in a unique 12 kb region delimited by shifts in GC content and containing tandem minisatellite- and microsatellite repeats with partially palindromic sequences. De novo annotation identified 32 protein-coding genes, 28 structural RNA genes and 19 ORFs. ORFs predicted in the repeat-rich region showed similarities to diverse organisms suggesting possible evolutionary connections. The data generated here form a refined platform for the next step involving functional analysis, all to clarify the complex biology of P. brassicae.
Topics: Cell Nucleus; Genome, Mitochondrial; Genome, Protozoan; Mitochondria; Molecular Sequence Annotation; Phylogeny; Plant Diseases; Plasmodiophorida
PubMed: 31673019
DOI: 10.1038/s41598-019-52274-7 -
PLoS Genetics Aug 2020Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that...
Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that human TRIB3 promoter contains a 33-bp variable number tandem repeat (VNTR) and characterize the heterogeneity and function of this genetic element. Analysis of human populations around the world uncovered the existence of alleles ranging from 1 to 5 copies of the repeat, with 2-, 3- and 5-copy alleles being the most common but displaying considerable geographical differences in frequency. The repeated sequence overlaps a C/EBP-ATF transcriptional regulatory element and is highly conserved, but not repeated, in various mammalian species, including great apes. The repeat is however evident in Neanderthal and Denisovan genomes. Reporter plasmid experiments in human cell culture reveal that an increased copy number of the TRIB3 promoter 33-bp repeat results in increased transcriptional activity. In line with this, analysis of whole genome sequencing and RNA-Seq data from human cohorts demonstrates that the copy number of TRIB3 promoter 33-bp repeats is positively correlated with TRIB3 mRNA expression level in many tissues throughout the body. Moreover, the copy number of the TRIB3 33-bp repeat appears to be linked to known TRIB3 eQTL SNPs as well as TRIB3 SNPs reported in genetic association studies. Taken together, the results indicate that the promoter 33-bp VNTR constitutes a causal variant for TRIB3 expression variation between individuals and could underlie the results of SNP-based genetic studies.
Topics: Cell Cycle Proteins; Estonia; Female; Gene Expression Regulation; Genetic Heterogeneity; Genetics, Population; Genotype; Humans; Male; Minisatellite Repeats; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; RNA-Seq; Repressor Proteins; Whole Genome Sequencing
PubMed: 32745133
DOI: 10.1371/journal.pgen.1008981 -
Tuberculosis (Edinburgh, Scotland) May 2022To describe the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) and factors associated with its transmission in Singapore.
OBJECTIVE
To describe the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) and factors associated with its transmission in Singapore.
METHODS
Spoligotyping, 24-loci mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR) typing and demographic data from the national TB notification registry of MTBC culture-positive cases notified from January 2011 to December 2017 were analysed.
RESULTS
Of the 12,046 culture-positive cases notified, complete spoligotyping and MIRU-VNTR typing results were available for 8690 (72.1%) belonging to 4950 (57.0%) local-born and 3740 (43.0%) foreign-born persons. From these, 4810 (55.3%) were identified in 883 clusters. The proportion of recent transmission was 45.2%. The East-Asian Lineage 2 accounted for 4045 (47.1%) of isolates, and disproportionately accounted for large clusters. Clustered cases were more likely to be older than 50 years, male, Malay, local-born, Singapore citizens or Permanent Residents, of lower socioeconomic status, imprisoned; to harbour East-Asian Lineage 2 strain; to have cavitary pulmonary TB, positive sputum smear or be recalcitrant treatment defaulters. They were less likely to have multidrug-resistant, or isoniazid or rifampicin mono-resistant TB.
CONCLUSION
We demonstrated the diversity of MTBC strains and, notwithstanding the likely over-estimation of clustering using these genotyping methods, elucidated factors associated with TB transmission in Singapore.
Topics: Genotype; Humans; Male; Minisatellite Repeats; Molecular Epidemiology; Mycobacterium tuberculosis; Singapore; Tuberculosis, Pulmonary
PubMed: 35395568
DOI: 10.1016/j.tube.2022.102204