scholarly journals Large scale automated phylogenomical analysis of bacterial whole-genome isolates and the Evergreen platform

2019 ◽  
Author(s):  
Judit Szarvas ◽  
Johanne Ahrenfeldt ◽  
Jose Luis Bellod Cisneros ◽  
Martin Christen Frølund Thomsen ◽  
Frank M. Aarestrup ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Pathogenic Bacteria ◽  
Large Scale ◽  
Consensus Sequence ◽  
Daily Basis ◽  
Whole Genome Sequence ◽  
Phylogenomic Analysis ◽  
Whole Genome ◽  
Sequencing Data ◽  
Bioinformatics Pipeline

AbstractPublic health authorities whole-genome sequence thousands of pathogenic isolates each month for microbial diagnostics and surveillance of pathogenic bacteria. The computational methods have not kept up with the deluge of data and need for real-time results.We have therefore created a bioinformatics pipeline for rapid subtyping and continuous phylogenomic analysis of bacterial samples, suited for large-scale surveillance. To decrease the computational burden, a two level clustering strategy is employed. The data is first divided into sets by matching each isolate to a closely related reference genome. The reads then are aligned to the reference to gain a consensus sequence and SNP based genetic distance is calculated between the sequences in each set. Isolates are clustered together with a threshold of 10 SNPs. Finally, phylogenetic trees are inferred from the non-redundant sequences and the clustered isolates are placed on a clade with the cluster representative sequence. The method was benchmarked and found to be accurate in grouping outbreak strains together, while discriminating from non-outbreak strains.The pipeline was applied in Evergreen Online, which processes publicly available sequencing data from foodborne bacterial pathogens on a daily basis, updating the phylogenetic trees as needed. It has so far placed more than 100,000 isolates into phylogenies, and has been able to keep up with the daily release of data. The trees are continuously published on https://cge.cbs.dtu.dk/services/Evergreen

scholarly journals MTBseq: a comprehensive pipeline for whole genome sequence analysis of Mycobacterium tuberculosis complex isolates

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5895 ◽  
Author(s):  
Thomas Andreas Kohl ◽  
Christian Utpatel ◽  
Viola Schleusener ◽  
Maria Rosaria De Filippo ◽  
Patrick Beckert ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Tuberculosis ◽  
Genome Sequence ◽  
Sequence Data ◽  
Whole Genome Sequence ◽  
Whole Genome Sequencing Data ◽  
Phylogenomic Analysis ◽  
Whole Genome ◽  
Sequencing Data ◽  
Desktop Computer

Analyzing whole-genome sequencing data of Mycobacterium tuberculosis complex (MTBC) isolates in a standardized workflow enables both comprehensive antibiotic resistance profiling and outbreak surveillance with highest resolution up to the identification of recent transmission chains. Here, we present MTBseq, a bioinformatics pipeline for next-generation genome sequence data analysis of MTBC isolates. Employing a reference mapping based workflow, MTBseq reports detected variant positions annotated with known association to antibiotic resistance and performs a lineage classification based on phylogenetic single nucleotide polymorphisms (SNPs). When comparing multiple datasets, MTBseq provides a joint list of variants and a FASTA alignment of SNP positions for use in phylogenomic analysis, and identifies groups of related isolates. The pipeline is customizable, expandable and can be used on a desktop computer or laptop without any internet connection, ensuring mobile usage and data security. MTBseq and accompanying documentation is available from https://github.com/ngs-fzb/MTBseq_source.

scholarly journals Population-level genome-wide STR typing in Plasmodium species reveals higher resolution population structure and genetic diversity relative to SNP typing

2021 ◽  
Author(s):  
Jiru Han ◽  
Jacob E Munro ◽  
Anthony Kocoski ◽  
Alyssa E Barry ◽  
Melanie Bahlo
Keyword(s):  
Genetic Diversity ◽  
Large Scale ◽  
Tandem Repeats ◽  
Plasmodium Species ◽  
Whole Genome Sequence ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Sequencing Data ◽  
Genome Wide ◽  
Field Samples

Short tandem repeats (STRs) are highly informative genetic markers that have been used extensively in population genetics analysis. They are an important source of genetic diversity and can also have functional impact. Despite the availability of bioinformatic methods that permit large-scale genome-wide genotyping of STRs from whole genome sequencing data, they have not previously been applied to sequencing data from large collections of malaria parasite field samples. Here, we have genotyped STRs using HipSTR in more than 3,000 Plasmodium falciparum and 174 Plasmodium vivax published whole-genome sequence data from samples collected across the globe. High levels of noise and variability in the resultant callset necessitated the development of a novel method for quality control of STR genotype calls. A set of high-quality STR loci (6,768 from P. falciparum and 3,496 from P. vivax) were used to study Plasmodium genetic diversity, population structures and genomic signatures of selection and these were compared to genome-wide single nucleotide polymorphism (SNP) genotyping data. In addition, the genome-wide information about genetic variation and other characteristics of STRs in P. falciparum and P. vivax have been made available in an interactive web-based R Shiny application PlasmoSTR (https://github.com/bahlolab/PlasmoSTR).

scholarly journals Epidemic Clostridioides difficile Ribotype 027 Lineages: Comparisons of Texas Versus Worldwide Strains

2019 ◽  
Vol 6 (2) ◽  
Author(s):  
Bradley T Endres ◽  
Khurshida Begum ◽  
Hua Sun ◽  
Seth T Walk ◽  
Ali Memariani ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Phylogenetic Trees ◽  
Large Scale ◽  
The United States ◽  
Whole Genome Sequence ◽  
Snp Analysis ◽  
Whole Genome ◽  
Ribotype 027 ◽  
Clostridioides Difficile

Abstract Background The epidemic Clostridioides difficile ribotype 027 strain resulted from the dissemination of 2 separate fluoroquinolone-resistant lineages: FQR1 and FQR2. Both lineages were reported to originate in North America; however, confirmatory large-scale investigations of C difficile ribotype 027 epidemiology using whole genome sequencing has not been undertaken in the United States. Methods Whole genome sequencing and single-nucleotide polymorphism (SNP) analysis was performed on 76 clinical ribotype 027 isolates obtained from hospitalized patients in Texas with C difficile infection and compared with 32 previously sequenced worldwide strains. Maximum-likelihood phylogeny based on a set of core genome SNPs was used to construct phylogenetic trees investigating strain macro- and microevolution. Bayesian phylogenetic and phylogeographic analyses were used to incorporate temporal and geographic variables with the SNP strain analysis. Results Whole genome sequence analysis identified 2841 SNPs including 900 nonsynonymous mutations, 1404 synonymous substitutions, and 537 intergenic changes. Phylogenetic analysis separated the strains into 2 prominent groups, which grossly differed by 28 SNPs: the FQR1 and FQR2 lineages. Five isolates were identified as pre-epidemic strains. Phylogeny demonstrated unique clustering and resistance genes in Texas strains indicating that spatiotemporal bias has defined the microevolution of ribotype 027 genetics. Conclusions Clostridioides difficile ribotype 027 lineages emerged earlier than previously reported, coinciding with increased use of fluoroquinolones. Both FQR1 and FQR2 ribotype 027 epidemic lineages are present in Texas, but they have evolved geographically to represent region-specific public health threats.

scholarly journals Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation

2020 ◽  
Author(s):  
Caroline Charre ◽  
Christophe Ginevra ◽  
Marina Sabatier ◽  
Hadrien Regue ◽  
Grégory Destras ◽  
...  
Keyword(s):  
Large Scale ◽  
Consensus Sequence ◽  
Amplicon Sequencing ◽  
Reference Sequence ◽  
High Threshold ◽  
Whole Genome ◽  
Bioinformatics Pipeline ◽  
Viral Loads ◽  
Two Samples ◽  
Complete Genomes

AbstractSince the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialized kits including a hybridization capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP) and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples, with mNGS and RVOP producing the most complete genomes. For mid viral loads, 2/8 and 1/8 genomes were incomplete (<99%) with mNGS and both CleanPlex and RVOP, respectively. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques yielding complete genomes for 7/8 samples. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in two samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34-nt deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a default of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer annealing or probe-hybridizing regions and would require regular updates of the technique according to SARS-CoV-2 evolution.

scholarly journals Ethnically diverse urban transmission networks of Neisseria gonorrhoeae without evidence of HIV serosorting

2019 ◽  
Vol 96 (2) ◽  
pp. 106-109
Author(s):  
Jayshree Dave ◽  
John Paul ◽  
Thomas Joshua Pasvol ◽  
Andy Williams ◽  
Fiona Warburton ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Ethnic Groups ◽  
Antimicrobial Susceptibility ◽  
Sequence Data ◽  
Small Sample ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Sequencing Data ◽  
Transmission Networks ◽  
Hiv Serosorting

ObjectiveWe aimed to characterise gonorrhoea transmission patterns in a diverse urban population by linking genomic, epidemiological and antimicrobial susceptibility data.MethodsNeisseria gonorrhoeae isolates from patients attending sexual health clinics at Barts Health NHS Trust, London, UK, during an 11-month period underwent whole-genome sequencing and antimicrobial susceptibility testing. We combined laboratory and patient data to investigate the transmission network structure.ResultsOne hundred and fifty-eight isolates from 158 patients were available with associated descriptive data. One hundred and twenty-nine (82%) patients identified as male and 25 (16%) as female; four (3%) records lacked gender information. Self-described ethnicities were: 51 (32%) English/Welsh/Scottish; 33 (21%) white, other; 23 (15%) black British/black African/black, other; 12 (8%) Caribbean; 9 (6%) South Asian; 6 (4%) mixed ethnicity; and 10 (6%) other; data were missing for 14 (9%). Self-reported sexual orientations were 82 (52%) men who have sex with men (MSM); 49 (31%) heterosexual; 2 (1%) bisexual; data were missing for 25 individuals. Twenty-two (14%) patients were HIV positive. Whole-genome sequence data were generated for 151 isolates, which linked 75 (50%) patients to at least one other case. Using sequencing data, we found no evidence of transmission networks related to specific ethnic groups (p=0.64) or of HIV serosorting (p=0.35). Of 82 MSM/bisexual patients with sequencing data, 45 (55%) belonged to clusters of ≥2 cases, compared with 16/44 (36%) heterosexuals with sequencing data (p=0.06).ConclusionWe demonstrate links between 50% of patients in transmission networks using a relatively small sample in a large cosmopolitan city. We found no evidence of HIV serosorting. Our results do not support assortative selectivity as an explanation for differences in gonorrhoea incidence between ethnic groups.

scholarly journals Whole Genome Sequencing of the Mutamouse Model Reveals Strain- and Colony-Level Variation, and Genomic Features of the Transgene Integration Site

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Matthew J. Meier ◽  
Marc A. Beal ◽  
Andrew Schoenrock ◽  
Carole L. Yauk ◽  
Francesco Marchetti
Keyword(s):  
Integration Site ◽  
Whole Genome Sequence ◽  
Comparative Genomic ◽  
Whole Genome ◽  
Missense Mutations ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
High Coverage ◽  
Deletion Event ◽  
Transgene Integration

Abstract The MutaMouse transgenic rodent model is widely used for assessing in vivo mutagenicity. Here, we report the characterization of MutaMouse’s whole genome sequence and its genetic variants compared to the C57BL/6 reference genome. High coverage (>50X) next-generation sequencing (NGS) of whole genomes from multiple MutaMouse animals from the Health Canada (HC) colony showed ~5 million SNVs per genome, ~20% of which are putatively novel. Sequencing of two animals from a geographically separated colony at Covance indicated that, over the course of 23 years, each colony accumulated 47,847 (HC) and 17,677 (Covance) non-parental homozygous single nucleotide variants. We found no novel nonsense or missense mutations that impair the MutaMouse response to genotoxic agents. Pairing sequencing data with array comparative genomic hybridization (aCGH) improved the accuracy and resolution of copy number variants (CNVs) calls and identified 300 genomic regions with CNVs. We also used long-read sequence technology (PacBio) to show that the transgene integration site involved a large deletion event with multiple inversions and rearrangements near a retrotransposon. The MutaMouse genome gives important genetic context to studies using this model, offers insight on the mechanisms of structural variant formation, and contributes a framework to analyze aCGH results alongside NGS data.

scholarly journals Large-Scale Whole-Genome Sequencing Reveals the Genetic Architecture of Primary Membranoproliferative GN and C3 Glomerulopathy

2020 ◽  
Vol 31 (2) ◽  
pp. 365-373 ◽  
Author(s):  
Adam P. Levine ◽  
Melanie M.Y. Chan ◽  
Omid Sadeghi-Alavijeh ◽  
Edwin K.S. Wong ◽  
H. Terence Cook ◽  
...  
Keyword(s):  
Large Scale ◽  
Rare Variants ◽  
Alternative Pathway ◽  
Atypical Hemolytic Uremic Syndrome ◽  
Gene Mutations ◽  
Whole Genome Sequence ◽  
European Ancestry ◽  
Whole Genome ◽  
C3 Glomerulopathy ◽  
Complement Gene

BackgroundPrimary membranoproliferative GN, including complement 3 (C3) glomerulopathy, is a rare, untreatable kidney disease characterized by glomerular complement deposition. Complement gene mutations can cause familial C3 glomerulopathy, and studies have reported rare variants in complement genes in nonfamilial primary membranoproliferative GN.MethodsWe analyzed whole-genome sequence data from 165 primary membranoproliferative GN cases and 10,250 individuals without the condition (controls) as part of the National Institutes of Health Research BioResource–Rare Diseases Study. We examined copy number, rare, and common variants.ResultsOur analysis included 146 primary membranoproliferative GN cases and 6442 controls who were unrelated and of European ancestry. We observed no significant enrichment of rare variants in candidate genes (genes encoding components of the complement alternative pathway and other genes associated with the related disease atypical hemolytic uremic syndrome; 6.8% in cases versus 5.9% in controls) or exome-wide. However, a significant common variant locus was identified at 6p21.32 (rs35406322) (P=3.29×10−8; odds ratio [OR], 1.93; 95% confidence interval [95% CI], 1.53 to 2.44), overlapping the HLA locus. Imputation of HLA types mapped this signal to a haplotype incorporating DQA1*05:01, DQB1*02:01, and DRB1*03:01 (P=1.21×10−8; OR, 2.19; 95% CI, 1.66 to 2.89). This finding was replicated by analysis of HLA serotypes in 338 individuals with membranoproliferative GN and 15,614 individuals with nonimmune renal failure.ConclusionsWe found that HLA type, but not rare complement gene variation, is associated with primary membranoproliferative GN. These findings challenge the paradigm of complement gene mutations typically causing primary membranoproliferative GN and implicate an underlying autoimmune mechanism in most cases.

scholarly journals Whole-Genome Sequence of Leptospira interrogans Serovar Hardjo Subtype Hardjoprajitno Strain Norma, Isolated from Cattle in a Leptospirosis Outbreak in Brazil

2015 ◽  
Vol 3 (6) ◽  
Author(s):  
M. R. V. Cosate ◽  
S. C. Soares ◽  
T. A. Mendes ◽  
R. T. Raittz ◽  
E. C. Moreira ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Pathogenic Bacteria ◽  
Global Distribution ◽  
Whole Genome Sequence ◽  
Leptospira Interrogans ◽  
Whole Genome

Leptospirosis is caused by pathogenic bacteria of the genus Leptospira spp. This neglected re-emergent disease has global distribution and relevance in veterinary production. Here, we report the whole-genome sequence and annotation of Leptospira interrogans serovar Hardjo subtype Hardjoprajitno strain Norma, isolated from cattle in a livestock leptospirosis outbreak in Brazil.

scholarly journals Phylogenomics reveals an extensive history of genome duplication in diatoms (Bacillariophyta)

2017 ◽  
Author(s):  
Matthew Parks ◽  
Teofil Nakov ◽  
Elizabeth Ruck ◽  
Norman J. Wickett ◽  
Andrew J. Alverson
Keyword(s):  
Whole Genome Duplication ◽  
Large Scale ◽  
Genome Duplication ◽  
Gene Tree ◽  
Genomic Diversity ◽  
Phylogenomic Analysis ◽  
Whole Genome ◽  
Gene Trees ◽  
Angiosperm Evolution ◽  
Polyploid Formation

ABSTRACTPremise of the studyDiatoms are one of the most species-rich lineages of microbial eukaryotes. Similarities in clade age, species richness, and contributions to primary production motivate comparisons to flowering plants, whose genomes have been inordinately shaped by whole genome duplication (WGD). These events that have been linked to speciation and increased rates of lineage diversification, identifying WGDs as a principal driver of angiosperm evolution. We synthesized a relatively large but scattered body of evidence that, taken together, suggests that polyploidy may be common in diatoms.MethodsWe used data from gene counts, gene trees, and patterns of synonymous divergence to carry out the first large-scale phylogenomic analysis of genome-scale duplication histories for a phylogenetically diverse set of 37 diatom taxa.Key resultsSeveral methods identified WGD events of varying age across diatoms, though determining the exact number and placement of events and, more broadly, inferences of WGD at all, were greatly impacted by gene-tree uncertainty. Gene-tree reconciliations supported allopolyploidy as the predominant mode of polyploid formation, with particularly strong evidence for ancient allopolyploid events in the thalassiosiroid and pennate diatom clades.ConclusionsWhole genome duplication appears to have been an important driver of genome evolution in diatoms. Denser taxon sampling will better pinpoint the timing of WGDs and likely reveal many more of them. We outline potential challenges in reconstructing paleopolyploid events in diatoms that, together with these results, offer a framework for understanding the evolutionary roles of genome duplication in a group that likely harbors substantial genomic diversity.

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