scholarly journals M. BOVIS BCG-1 (RUSSIA) SUB-STRAIN GENOME STABILITY INVESTIGATION WITHIN THE ENTIRE PRODUCTION PROCESS

2018 ◽  
pp. 58-67
Author(s):  
E. V. Otrashevskaya ◽  
V. N. Vinokurova ◽  
E. A. Shilikov ◽  
E. A. Sotnikova ◽  
T. A. Perevyshina ◽  
...  
Keyword(s):  
Production Process ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Stability ◽  
Genome Structure ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Vaccine Production ◽  
The Stability ◽  
Strain Genome

Aim. The aim of the current study was to analyze the genome structure of the M. bovis BCG-1 (Russia) sub-strain, used for the vaccine production, as well as its genome stability within the entire production process. Materials and methods. Whole genome sequencing and M. bovis BCG-1 (Russia) working seed lot and for the last production passage of the sub-strain cultivation from a number of the vaccine batches. Additionally, VNTR sequences of 24 locus analyses, RD patterns comparison, as well as spoligotyping were performed. Results. The whole genome sequence of the M. bovis BCG-1 (Russia) working seed lot was assembled, annotated and deposited to GenBank. On the basis of DU2- and RD-regions analyzes M. bovis BCG-1 (Russia) sub-strain was confirmed to be belonged to BCG Russia strains of DU2-I group. Whole genome sequencing followed by comparative analysis of RD patterns and SNPs confirmed the stability of the vaccine sub-strain genome from the working seed lot to a number of the vaccine batches obtained within the two-years period. VNTR profile and spoligopattern exactly matched the M. bovis BCG-1 (Russia). Conclusion. Thus the M. bovis BCG-1 (Russia) sub-strain genome identity and stability have been studied and demonstrated. The obtained result confirmed the vaccine production process consistency.

scholarly journals SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weili Cai ◽  
Schyler Nunziata ◽  
John Rascoe ◽  
Michael J. Stulberg
Keyword(s):  
Whole Genome Sequencing ◽  
Molecular Characterization ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genome Coverage ◽  
Metagenomic Sample ◽  
Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

Abstract 343: Bayesian Selection of Modifier Genes in Hypertrophic Cardiomyopathy Through Whole Genome Sequencing

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Matthew Wheeler ◽  
Daryl Waggott ◽  
Megan Grove ◽  
Frederick Dewey ◽  
Cuiping Pan ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
A Priori ◽  
Copy Number Variants ◽  
Whole Genome Sequence ◽  
Monogenic Disease ◽  
Whole Genome ◽  
Genetic Modifiers ◽  
Structural Variants

Background: Technological advances have greatly reduced the cost of whole genome sequencing. For single individuals clinical application is apparent, while exome sequencing in tens of thousands of people has allowed a more global view of genetic variation that can inform interpretation of specific variants in individuals. We hypothesized that genome sequencing of patients with monogenic cardiomyopathy would facilitate discovery of genetic modifiers of phenotype. Methods and Results: We identified 48 individuals diagnosed with cardiomyopathy and with putative mutations in MYH7, the gene encoding beta myosin heavy chain. We carried out whole genome sequencing and applied a newly developed analytical pipeline optimized for discovery of genes modifying severity of clinical presentation and outcomes. Using a combination of external priors and rare variant burden tests we scored genes as potential modifiers. There were 96 genes that reached a modifier score of 6 out of 12 or better (9=2, 8=8, 7=17, 6=69). We identified NCKAP1, a gene that regulates actin filament dynamics, and CAMSAP1, a calmodulin regulate gene that regulates microtubule dynamics, as top scoring modifiers of hypertrophic cardiomyopathy phenotypes (score=9) while LDB2, RYR2, FBN1 and ATP1A2 had modifier scores of 8. Of the top scoring genes, 21 out of 96 were identified as candidates a priori. Our candidate prioritization scheme identified the previously described modifiers of cardiomyopathy phenotype, FHOD3 and MYBPC3, as top scoring genes. We identified structural variants in 21 clinically sequenced cardiomyopathy associated genes, 13 of which were at less than 10% frequency. Copy number variants in ILK and CSRP3 were nominally associated with ejection fraction (p=0.03), while 8 genes showed copy gains (GLA, FKTN, SGCD, TTN, SOS1, ANKRD1, VCL and NEBL). Structural variants were found in CSRP3, MYL3 and TNNC1, all of which have been implicated as causative for HCM. Conclusion: Evaluation of the whole genome sequence, even in the case of putatively monogenic disease, leads to important diagnostic and scientific insights not revealed by panel-based sequencing.

scholarly journals Whole-Genome Sequencing Allows for Improved Identification of Persistent Listeria monocytogenes in Food-Associated Environments

2015 ◽  
Vol 81 (17) ◽  
pp. 6024-6037 ◽  
Author(s):  
Matthew J. Stasiewicz ◽  
Haley F. Oliver ◽  
Martin Wiedmann ◽  
Henk C. den Bakker
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genetic Determinants ◽  
Single Nucleotide ◽  
Content Type ◽  
Food Borne ◽  
Clonal Spread

ABSTRACTWhile the food-borne pathogenListeria monocytogenescan persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study ofL. monocytogenesin retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping ofL. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced strains, and (iii) identify genetic determinants ofL. monocytogenespersistence. WGS analysis revealed three prophage regions that explained differences between three pairs of phylogenetically similar populations with pulsed-field gel electrophoresis types that differed by ≤3 bands. WGS-SNP-based phylogenetics found that putatively persistentL. monocytogenesrepresent SNP patterns (i) unique to a single retail deli, supporting persistence within the deli (11 clades), (ii) unique to a single state, supporting clonal spread within a state (7 clades), or (iii) spanning multiple states (5 clades). Isolates that formed one of 11 deli-specific clades differed by a median of 10 SNPs or fewer. Isolates from 12 putative persistence events had significantly fewer SNPs (median, 2 to 22 SNPs) than between isolates of the same subtype from other delis (median up to 77 SNPs), supporting persistence of the strain. In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis. No individual genes were enriched among persistent isolates compared to sporadic isolates. Our data show that WGS analysis improves food-borne pathogen subtyping and identification of persistent bacterial pathogens in food associated environments.

scholarly journals Allele-Specific Quantification of Structural Variations in Cancer Genomes

2016 ◽  
Author(s):  
Yang Li ◽  
Shiguo Zhou ◽  
David C. Schwartz ◽  
Jian Ma
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Graphical Model ◽  
Genome Structure ◽  
Cancer Genome ◽  
Whole Genome ◽  
Structural Variations ◽  
Cancer Genomes ◽  
Allele Specific

AbstractOne of the hallmarks of cancer genome is aneuploidy, resulting in abnormal copy numbers of alleles. Structural variations (SVs) can further modify the aneuploid cancer genomes into a mixture of rearranged genomic segments with extensive range of somatic copy number alterations (CNAs). Indeed, aneuploid cancer genomes have significantly higher rate of CNAs and SVs. However, although methods have been developed to identify SVs and allele-specific copy number of genome (ASCNG) separately, no existing algorithm can simultaneously analyze SVs and ASCNG. Such integrated approach is particularly important to fully understand the complexity of cancer genomes. Here we introduce a new algorithm called Weaver to provide allele-specific quantification of SVs and CNAs in aneuploid cancer genomes. Weaver uses a probabilistic graphical model by utilizing cancer whole genome sequencing data to simultaneously estimate the digital copy number and inter-connectivity of SVs. Our simulation evaluation, comparison with single-molecule Optical Mapping analysis, and real data applications (including MCF-7, HeLa, and TCGA whole genome sequencing samples) demonstrated that Weaver is highly accurate and can greatly refine the analysis of complex cancer genome structure.

scholarly journals Five-year Microevolution of a Multidrug-Resistant Mycobacterium Tuberculosis Strain within a Patient with Inadequate Compliance to Treatment.

2021 ◽  
Author(s):  
Dario Fernández Do Porto ◽  
Johana Monteserin ◽  
Josefina Campos ◽  
Ezequiel J Sosa ◽  
Mario Matteo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Sequence ◽  
Treatment Compliance ◽  
Whole Genome Sequence ◽  
Intermittent Therapy ◽  
Whole Genome ◽  
Short Term ◽  
Depth Analysis

Abstract BackgroundWhole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution —the genetic variability of M. tuberculosis at short time scales— of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported.Case Presentations In this work, we applied whole genome sequencing for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium. tuberculosis isolates obtained from a patient within 57-month of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5' untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patience, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy.ConclusionsThis report highlights the relevance of whole-genome sequencing in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.

scholarly journals Exploring the Diversity of Bacillus whole genome sequencing projects using Peasant, the Prokaryotic Assembly and Annotation Tool

2017 ◽  
Author(s):  
Jonathon Brenner ◽  
Laurynas Kalesinskas ◽  
Catherine Putonti
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Annotation Tool ◽  
Illumina Platform ◽  
Desktop Computer ◽  
High Quality ◽  
Sequencing Project ◽  
Computational Resources

ABSTRACTBackgroundThe persistent decrease in cost and difficulty of whole genome sequencing of microbial organisms has led to a dramatic increase in the number of species and strains characterized from a wide variety of environments. Microbial genome sequencing can now be conducted by small laboratories and as part of undergraduate curriculum. While sequencing is routine in microbiology, assembly, annotation and downstream analyses still require computational resources and expertise, often necessitating familiarity with programming languages. To address this problem, we have created a light-weight, user-friendly tool for the assembly and annotation of microbial sequencing projects.ResultsThe Prokaryotic Assembly and Annotation Tool, Peasant, automates the processes of read quality control, genome assembly, and annotation for microbial sequencing projects. High-quality assemblies and annotations can be generated by Peasant without the need of programming expertise or high-performance computing resources. Furthermore, statistics are calculated so that users can evaluate their sequencing project. To illustrate the computational speed and accuracy of Peasant, the SRA records of 322 Illumina platform whole genome sequencing assays for Bacillus species were retrieved from NCBI, assembled and annotated on a single desktop computer. From the assemblies and annotations produced, a comprehensive analysis of the diversity of over 200 high-quality samples was conducted, looking at both the 16S rRNA phylogenetic marker as well as the Bacillus core genome.ConclusionsPeasant provides an intuitive solution for high-quality whole genome sequence assembly and annotation for users with limited programing experience and/or computational resources. The analysis of the Bacillus whole genome sequencing projects exemplifies the utility of this tool. Furthermore, the study conducted here provides insight into the diversity of the species, the largest such comparison conducted to date.

scholarly journals Evaluation of Pneumococcal Serotyping of Nasopharyngeal-Carriage Isolates by Latex Agglutination, Whole-Genome Sequencing (PneumoCaT), and DNA Microarray in a High-Pneumococcal-Carriage-Prevalence Population in Malawi

2020 ◽  
Vol 59 (1) ◽  
pp. e02103-20
Author(s):  
Todd D. Swarthout ◽  
Andrea Gori ◽  
Naor Bar-Zeev ◽  
Arox W. Kamng’ona ◽  
Thandie S. Mwalukomo ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Dna Microarray ◽  
Latex Agglutination ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Healthy Children ◽  
Vaccine Serotypes ◽  
Relative Abundances ◽  
Expanded Program On Immunization

ABSTRACTAccurate assessment of the serotype distribution associated with pneumococcal colonization and disease is essential for evaluating and formulating pneumococcal vaccines and for informing vaccine policy. For this reason, we evaluated the concordance between pneumococcal serotyping results by latex agglutination, whole-genome sequencing (WGS) with PneumoCaT, and DNA microarray for samples from community carriage surveillance in Blantyre, Malawi. Nasopharyngeal swabs were collected according to WHO recommendations between 2015 and 2017 by using stratified random sampling among study populations. Participants included healthy children 3 to 6 years old (vaccinated with the 13-valent pneumococcal conjugate vaccine [PCV13] as part of the Expanded Program on Immunization [EPI]), healthy children 5 to 10 years old (age-ineligible for PCV13), and HIV-infected adults (18 to 40 years old) on antiretroviral therapy (ART). For phenotypic serotyping, we used a 13-valent latex kit (Statens Serum Institut [SSI], Denmark). For genomic serotyping, we applied the PneumoCaT pipeline to whole-genome sequence libraries. For molecular serotyping by microarray, we used the BUGS Bioscience Senti-SP microarray. A total of 1,347 samples were analyzed. Concordance was 90.7% (95% confidence interval [CI], 89.0 to 92.2%) between latex agglutination and PneumoCaT, 95.2% (95% CI, 93.9 to 96.3%) between latex agglutination and the microarray, and 96.6% (95% CI, 95.5 to 97.5%) between the microarray and PneumoCaT. By detecting additional vaccine serotype (VT) pneumococci carried at low relative abundances (median, 8%), the microarray increased VT detection by 31.5% over that by latex serotyping. To conclude, all three serotyping methods were highly concordant in identifying dominant serotypes. Latex serotyping is accurate in identifying vaccine serotypes and requires the least expertise and resources for field implementation and analysis. However, WGS, which adds population structure, and microarray, which adds multiple-serotype carriage, should be considered at regional reference laboratories for investigating the importance of vaccine serotypes at low relative abundances in transmission and disease.

scholarly journals SeroBA: rapid high-throughput serotyping of Streptococcus pneumoniae from whole genome sequence data

2017 ◽  
Author(s):  
Lennard Epping ◽  
Andries J. van Tonder ◽  
Rebecca A. Gladstone ◽  
Stephen D. Bentley ◽  
Andrew J. Page ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Validation Dataset ◽  
List Type ◽  
Whole Genome ◽  
Link Type ◽  
Computational Performance ◽  
The Impact

ABSTRACTStreptococcus pneumoniae is responsible for 240,000 - 460,000 deaths in children under 5 years of age each year. Accurate identification of pneumococcal serotypes is important for tracking the distribution and evolution of serotypes following the introduction of effective vaccines. Recent efforts have been made to infer serotypes directly from genomic data but current software approaches are limited and do not scale well. Here, we introduce a novel method, SeroBA, which uses a hybrid assembly and mapping approach. We compared SeroBA against real and simulated data and present results on the concordance and computational performance against a validation dataset, the robustness and scalability when analysing a large dataset, and the impact of varying the depth of coverage in the cps locus region on sequence-based serotyping. SeroBA can predict serotypes, by identifying the cps locus, directly from raw whole genome sequencing read data with 98% concordance using a k-mer based method, can process 10,000 samples in just over 1 day using a standard server and can call serotypes at a coverage as low as 10x. SeroBA is implemented in Python3 and is freely available under an open source GPLv3 license from: https://github.com/sanger-pathogens/seroba.DATA SUMMARYThe reference genome Streptococcus pneumoniae ATCC 700669 is available from National Center for Biotechnology Information (NCBI) with the accession number: FM211187Simulated paired end reads for experiment 2 have been deposited in FigShare: https://doi.org/10.6084/m9.figshare.5086054.v1Accession numbers for all other experiments are listed in Supplementary Table S1 and Supplementary Table S2.I/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. ⊠IMPACT STATEMENTThis article describes SeroBA, a A-mer based method for predicting the serotypes of Streptococcus pneumoniae from Whole Genome Sequencing (WGS) data. SeroBA can identify 92 serotypes and 2 subtypes with constant memory usage and low computational costs. We showed that SeroBA is able to reliably predict serotypes at a depth of coverage as low as 10x and is scalable to large datasets.

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 Whole-genome sequencing reveals progressive versus stable myeloma precursor conditions as two distinct entities

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bénedith Oben ◽  
Guy Froyen ◽  
Kylee H. Maclachlan ◽  
Daniel Leongamornlert ◽  
Federico Abascal ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Sequence ◽  
Monoclonal Gammopathy ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Driver Genes ◽  
Smoldering Myeloma ◽  
Undetermined Significance

AbstractMultiple myeloma (MM) is consistently preceded by precursor conditions recognized clinically as monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM). We interrogate the whole genome sequence (WGS) profile of 18 MGUS and compare them with those from 14 SMMs and 80 MMs. We show that cases with a non-progressing, clinically stable myeloma precursor condition (n = 15) are characterized by later initiation in the patient’s life and by the absence of myeloma defining genomic events including: chromothripsis, templated insertions, mutations in driver genes, aneuploidy, and canonical APOBEC mutational activity. This data provides evidence that WGS can be used to recognize two biologically and clinically distinct myeloma precursor entities that are either progressive or stable.

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