scholarly journals Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis

2015 ◽  
Vol 54 (3) ◽  
pp. 556-564 ◽  
Author(s):  
Célia Leão ◽  
Robert J. Goldstone ◽  
Josephine Bryant ◽  
Joyce McLuckie ◽  
João Inácio ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Mycobacterium Avium ◽  
Discriminatory Power ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Phylogenetic Groups ◽  
Single Nucleotide ◽  
Content Type

Typing ofMycobacterium aviumsubspeciesparatuberculosisstrains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole-genome sequencing have extended possibilities for the characterization ofMycobacterium aviumsubspeciesparatuberculosis, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133Mycobacterium aviumsubspeciesparatuberculosisisolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPsMycobacterium aviumsubspeciesparatuberculosisisolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit–variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing ofMycobacterium aviumsubspeciesparatuberculosisisolates.

scholarly journals Studying Bordetella pertussis Populations by Use of SNPeX, a Simple High-Throughput Single Nucleotide Polymorphism Typing Method

2015 ◽  
Vol 53 (3) ◽  
pp. 838-846 ◽  
Author(s):  
Anne Zeddeman ◽  
Sandra Witteveen ◽  
Marieke J. Bart ◽  
Marjolein van Gent ◽  
Han G. J. van der Heide ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Bordetella Pertussis ◽  
Primer Extension ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Single Nucleotide Primer Extension

Large outbreaks of pertussis occur despite vaccination. A first step in the analyses of outbreaks is strain typing. However, the typing ofBordetella pertussis, the causative agent of pertussis, is problematic because the available assays are insufficiently discriminatory, not unequivocal, time-consuming, and/or costly. Here, we describe a single nucleotide primer extension assay for the study ofB. pertussispopulations, SNPeX (single nucleotide primer extension), which addresses these problems. The assay is based on the incorporation of fluorescently labeled dideoxynucleotides (ddNTPs) at the 3′ end of allele-specific poly(A)-tailed primers and subsequent analysis with a capillary DNA analyzer. Each single nucleotide polymorphism (SNP) primer has a specific length, and as a result, up to 20 SNPs can be determined in one SNPeX reaction. Importantly, PCR amplification of target DNA is not required. We selected 38 SNPeX targets from the whole-genome sequencing data of 74B. pertussisstrains collected from across the world. The SNPeX-based phylogenetic trees preserved the general tree topology ofB. pertussispopulations based on whole-genome sequencing, with a minor loss of details. We envisage a strategy whereby SNP types (SnpTs) are quickly identified with the SNPeX assay during an outbreak, followed by whole-genome sequencing (WGS) of a limited number of isolates representing predominant SnpTs and the incorporation of novel SNPs in the SNPeX assay. The flexibility of the SNPeX assay allows the method to evolve along with the pathogen, making it a promising method for studying outbreaks ofB. pertussisand other pathogens.

scholarly journals Whole genome sequencing of a single Bos taurus animal for single nucleotide polymorphism discovery

2009 ◽  
Vol 10 (8) ◽  
pp. R82 ◽  
Author(s):  
Sebastian H Eck ◽  
Anna Benet-Pagès ◽  
Krzysztof Flisikowski ◽  
Thomas Meitinger ◽  
Ruedi Fries ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Bos Taurus ◽  
Single Nucleotide Polymorphism Discovery ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Polymorphism Discovery

scholarly journals Single nucleotide polymorphism in the rpoB gene Mycobacterium tuberculosis from Papua-Indonesia and its impact on rifampicin resistance: A whole genome sequencing analysis

2021 ◽  
Vol 15 (2) ◽  
pp. 1
Author(s):  
Yustinus Maladan ◽  
Tri Wahyuni ◽  
Hana Krismawati
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Mycobacterium Tuberculosis ◽  
Single Nucleotide Polymorphisms ◽  
Rna Polymerase ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

In the antibiotic era, Tuberculosis (TB) drugs resistance especially Rifampicin (RIF) is highly reported around the world. Resistance of RIF is caused by the mutation of genes that associated with RIF receptor. The aims of this study are detecting the Single Nucleotide Polymorphism of Rifampicin resistant genes using Whole Genome Sequencing (WGS) and analysing the profile of protein changing caused by SNP. Twenty Mycobacterium tuberculosis culture samples were passed on WGS procedure and 19 samples were adequate to further bioinformatics analysis. Single Nucleotide Polymorphisms Analysis was done using TBprofiler. Based on TBProfiler, seventeen samples were resistant to rifampicin. The mutations that cause the resistance are S450L, D435Y, H445Y, 430P, Q432K. Other Single Nucleotide Polymorphisms H835R, V534M and R224C were also found. The H835R mutants are present together with the S450L, V534M with S450L mutants, and R224C with Q432K mutants. Native protein for RNA Polymerase Subunit β used was the result of separation from the crystal structure of Mycobacterium tuberculosis H37Rv RNA polymerase (PDB: 5UHB). Binding affinity RIF to RNA Polymerase Subunit β calculated using AutoDock vina. Construction of mutant 3D structures using FoldX5. From the analysis, it was found that seventeen samples were resistant to rifampicin and two samples did not contain SNP which could cause resistance to rifampicin.

scholarly journals Screening of triploid with low-coverage whole-genome sequencing by a single-nucleotide polymorphism-based test in miscarriage tissue

2019 ◽  
Vol 36 (12) ◽  
pp. 2525-2531
Author(s):  
Qian Geng ◽  
Xiaoli Cui ◽  
Yaqi Zhang ◽  
Lijuan Zhang ◽  
Cai Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Single Nucleotide Polymorphism ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Uniparental Disomy ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Quadratic Curve ◽  
Low Coverage

Abstract Purpose To establish a single-nucleotide polymorphism-based analysis (SBA) method to identify triploidy in the miscarriage tissue by using low-coverage whole-genome sequencing (LC-WGS). Methods The method was established by fitting a quadratic curve model by counting the distribution of three heterozygous mutation content intervals. The triploid test result was mainly determined by the opening direction and the axis of symmetry of the quadratic curve, and Z test between the same batch samples was also used for auxiliary judgment. Results Two hundred thirteen diploid samples and 8 triploid samples were used for establishment of the analytical method and 203 unknown samples were used for blind testing. In the blind testing, we found 2 cases positive for triploidy. After chromosome microarray analysis (CMA) and mass spectrometry verification, we found that both samples were true positives. We randomly selected 5 samples from the negative samples for mass spectrometry verification, and the results showed that these samples were all true negatives. Conclusions Our method achieved accurate detection of triploidy in the miscarriage tissue and has the potential to detect more chromosomal abnormality types such as uniparental disomy (UPD) using a single LC-WGS approach.

scholarly journals Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection

2015 ◽  
Vol 53 (10) ◽  
pp. 3334-3340 ◽  
Author(s):  
Angela J. Taylor ◽  
Victoria Lappi ◽  
William J. Wolfgang ◽  
Pascal Lapierre ◽  
Michael J. Palumbo ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Molecular Subtyping ◽  
Foodborne Outbreaks

Salmonella entericaserovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis ofS. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmentalS. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of theS. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method forS. Enteritidis.

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