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Asif, Kinza, Denise O’Rourke, Alistair R. Legione, Pollob Shil, Marc S. Marenda, and Amir H. Noormohammadi. "Whole-genome based strain identification of fowlpox virus directly from cutaneous tissue and propagated virus." PLOS ONE 16, no. 12 (December 16, 2021): e0261122. http://dx.doi.org/10.1371/journal.pone.0261122.
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Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn’t compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.
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Therrien, Dustin A., Kranti Konganti, Jason J. Gill, Brian W. Davis, Andrew E. Hillhouse, Jordyn Michalik, H. Russell Cross, Gary C. Smith, Thomas M. Taylor, and Penny K. Riggs. "Complete Whole Genome Sequences of Escherichia coli Surrogate Strains and Comparison of Sequence Methods with Application to the Food Industry." Microorganisms 9, no. 3 (March 16, 2021): 608. http://dx.doi.org/10.3390/microorganisms9030608.
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In 2013, the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) began transitioning to whole genome sequencing (WGS) for foodborne disease outbreak- and recall-associated isolate identification of select bacterial species. While WGS offers greater precision, certain hurdles must be overcome before widespread application within the food industry is plausible. Challenges include diversity of sequencing platform outputs and lack of standardized bioinformatics workflows for data analyses. We sequenced DNA from USDA-FSIS approved, non-pathogenic E. coli surrogates and a derivative group of rifampicin-resistant mutants (rifR) via both Oxford Nanopore MinION and Illumina MiSeq platforms to generate and annotate complete genomes. Genome sequences from each clone were assembled separately so long-read, short-read, and combined sequence assemblies could be directly compared. The combined sequence data approach provides more accurate completed genomes. The genomes from these isolates were verified to lack functional key E. coli elements commonly associated with pathogenesis. Genetic alterations known to confer rifR were also identified. As the food industry adopts WGS within its food safety programs, these data provide completed genomes for commonly used surrogate strains, with a direct comparison of sequence platforms and assembly strategies relevant to research/testing workflows applicable for both processors and regulators.
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Vestal, Grant, Steven Bruzek, Amanda Lasher, Amorce Lima, and Suzane Silbert. "Whole-Genome Sequencing for Bacterial Strain Typing Using the iSeq100 Platform." Infection Control & Hospital Epidemiology 41, S1 (October 2020): s434. http://dx.doi.org/10.1017/ice.2020.1098.
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Background: Hospital-acquired infections pose a significant threat to patient health. Laboratories are starting to consider whole-genome sequencing (WGS) as a molecular method for outbreak detection and epidemiological surveillance. The objective of this study was to assess the use of the iSeq100 platform (Illumina, San Diego, CA) for accurate sequencing and WGS-based outbreak detection using the bioMérieux EPISEQ CS, a novel cloud-based software for sequence assembly and data analysis. Methods: In total, 25 isolates, including 19 MRSA isolates and 6 ATCC strains were evaluated in this study: A. baumannii ATCC 19606, B. cepacia ATCC 25416, E. faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. DNA extraction of all isolates was performed on the QIAcube (Qiagen, Hilden, Germany) using the DNEasy Ultra Clean Microbial kit extraction protocol. DNA libraries were prepared for WGS using the Nextera DNA Flex Library Prep Kit (Illumina) and sequenced at 2×150-bp on the iSeq100 according to the manufacturer’s instructions. The 19 MRSA isolates were previously characterized by the DiversiLab system (bioMérieux, France). Upon validation of the iSeq100 platform, a new outbreak analysis was performed using WGS analysis using EPISEQ CS. ATCC sequences were compared to assembled reference genomes from the NCBI GenBank to assess the accuracy of the iSeq100 platform. The FASTQ files were aligned via BowTie2 version 2.2.6 software, using default parameters, and FreeBayes version 1.1.0.46-0 was used to call homozygous single-nucleotide polymorphisms (SNPs) with a minimum coverage of 5 and an allele frequency of 0.87 using default parameters. ATCC sequences were analyzed using ResFinder version 3.2 and were compared in silico to the reference genome. Results: EPISEQ CS classified 8 MRSA isolates as unrelated and grouped 11 isolates into 2 separate clusters: cluster A (5 isolates) and cluster B (6 isolates) with similarity scores of ≥99.63% and ≥99.50%, respectively. This finding contrasted with the previous characterization by DiversiLab, which identified 3 clusters of 2, 8, and 11 isolates, respectively. The EPISEQ CS resistome data detected the mecA gene in 18 of 19 MRSA isolates. Comparative analysis of the ATCCsequences to the reference genomes showed 99.9986% concordance of SNPs and 100.00% concordance between the resistance genes present. Conclusions: The iSeq100 platform accurately sequenced the bacterial isolates and could be an affordable alternative in conjunction with EPISEQ CS for epidemiological surveillance analysis and infection prevention.Funding: NoneDisclosures: None
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Chattaway, Marie A., Ulf Schaefer, Rediat Tewolde, Timothy J. Dallman, and Claire Jenkins. "Identification of Escherichia coli and Shigella Species from Whole-Genome Sequences." Journal of Clinical Microbiology 55, no. 2 (December 14, 2016): 616–23. http://dx.doi.org/10.1128/jcm.01790-16.
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ABSTRACTEscherichia coliandShigellaspecies are closely related and genetically constitute the same species. Differentiating between these two pathogens and accurately identifying the four species ofShigellaare therefore challenging. The organism-specific bioinformatics whole-genome sequencing (WGS) typing pipelines at Public Health England are dependent on the initial identification of the bacterial species by use of a kmer-based approach. Of the 1,982Escherichia coliandShigellasp. isolates analyzed in this study, 1,957 (98.4%) had concordant results by both traditional biochemistry and serology (TB&S) and the kmer identification (ID) derived from the WGS data. Of the 25 mismatches identified, 10 were enteroinvasiveE. coliisolates that were misidentified asShigella flexneriorS. boydiiby the kmer ID, and 8 wereS. flexneriisolates misidentified by TB&S asS. boydiidue to nonfunctionalS. flexneriO antigen biosynthesis genes. Analysis of the population structure based on multilocus sequence typing (MLST) data derived from the WGS data showed that the remaining discrepant results belonged to clonal complex 288 (CC288), comprising bothS. boydiiandS. dysenteriaestrains. Mismatches between the TB&S and kmer ID results were explained by the close phylogenetic relationship between the two species and were resolved with reference to the MLST data.Shigellacan be differentiated fromE. coliand accurately identified to the species level by use of kmer comparisons and MLST. Analysis of the WGS data provided explanations for the discordant results between TB&S and WGS data, revealed the true phylogenetic relationships between different species ofShigella, and identified emerging pathoadapted lineages.
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Rabinowitz, Peter, Bar Zilberman, Yair Motro, Marilyn C. Roberts, Alex Greninger, Lior Nesher, Shalom Ben-Shimol, et al. "Whole Genome Sequence Analysis of Brucella melitensis Phylogeny and Virulence Factors." Microbiology Research 12, no. 3 (August 24, 2021): 698–710. http://dx.doi.org/10.3390/microbiolres12030050.
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Brucellosis has a wide range of clinical severity in humans that remains poorly understood. Whole genome sequencing (WGS) analysis may be able to detect variation in virulence genes. We used Brucella melitensis sequences in the NCBI Sequence Read Archive (SRA) database to assemble 248 whole genomes, and additionally, assembled 27 B. melitensis genomes from samples of human patients in Southern Israel. We searched the 275 assembled genomes for the 43 B. melitensis virulence genes in the Virulence Factors of Pathogenic Bacteria Database (VFDB) and 10 other published putative virulence genes. We explored pan-genome variation across the genomes and in a pilot analysis, explored single nucleotide polymorphism (SNP) variation among the ten putative virulence genes. More than 99% of the genomes had sequences for all Brucella melitensis virulence genes included in the VFDB. The 10 other virulence genes of interest were present across all the genomes, but three of these genes had SNP variation associated with particular Brucella melitensis genotypes. SNP variation was also seen within the Israeli genomes obtained from a small geographic region. While the Brucella genome is highly conserved, this novel and large whole genome study of Brucella demonstrates the ability of whole genome and pan-genome analysis to screen multiple genomes and identify SNP variation in both known and novel virulence genes that could be associated with differential disease virulence. Further development of whole genome techniques and linkage with clinical metadata on disease outcomes could shed light on whether such variation in the Brucella genome plays a role in pathogenesis.
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Gunasekara, A. W. A. C. W. R., L. G. T. G. Rajapaksha, and T. L. Tung. "Whole-genome sequence analysis through online web interfaces: a review." Genomics & Informatics 20, no. 1 (March 31, 2022): e3. http://dx.doi.org/10.5808/gi.20038.
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The recent development of whole-genome sequencing technologies paved the way for understanding the genomes of microorganisms. Every whole-genome sequencing (WGS) project requires a considerable cost and a massive effort to address the questions at hand. The final step of WGS is data analysis. The analysis of whole-genome sequence is dependent on highly sophisticated bioinformatics tools that the research personal have to buy. However, many laboratories and research institutions do not have the bioinformatics capabilities to analyze the genomic data and therefore, are unable to take maximum advantage of whole-genome sequencing. In this aspect, this study provides a guide for research personals on a set of bioinformatics tools available online that can be used to analyze whole-genome sequence data of bacterial genomes. The web interfaces described here have many advantages and, in most cases exempting the need for costly analysis tools and intensive computing resources.
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Atxaerandio-Landa, Aitor, Ainhoa Arrieta-Gisasola, Lorena Laorden, Joseba Bikandi, Javier Garaizar, Irati Martinez-Malaxetxebarria, and Ilargi Martinez-Ballesteros. "A Practical Bioinformatics Workflow for Routine Analysis of Bacterial WGS Data." Microorganisms 10, no. 12 (November 29, 2022): 2364. http://dx.doi.org/10.3390/microorganisms10122364.
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The use of whole-genome sequencing (WGS) for bacterial characterisation has increased substantially in the last decade. Its high throughput and decreasing cost have led to significant changes in outbreak investigations and surveillance of a wide variety of microbial pathogens. Despite the innumerable advantages of WGS, several drawbacks concerning data analysis and management, as well as a general lack of standardisation, hinder its integration in routine use. In this work, a bioinformatics workflow for (Illumina) WGS data is presented for bacterial characterisation including genome annotation, species identification, serotype prediction, antimicrobial resistance prediction, virulence-related genes and plasmid replicon detection, core-genome-based or single nucleotide polymorphism (SNP)-based phylogenetic clustering and sequence typing. Workflow was tested using a collection of 22 in-house sequences of Salmonella enterica isolates belonging to a local outbreak, coupled with a collection of 182 Salmonella genomes publicly available. No errors were reported during the execution period, and all genomes were analysed. The bioinformatics workflow can be tailored to other pathogens of interest and is freely available for academic and non-profit use as an uploadable file to the Galaxy platform.
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Ruppitsch, Werner, Ariane Pietzka, Karola Prior, Stefan Bletz, Haizpea Lasa Fernandez, Franz Allerberger, Dag Harmsen, and Alexander Mellmann. "Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Listeria monocytogenes." Journal of Clinical Microbiology 53, no. 9 (July 1, 2015): 2869–76. http://dx.doi.org/10.1128/jcm.01193-15.
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Whole-genome sequencing (WGS) has emerged today as an ultimate typing tool to characterizeListeria monocytogenesoutbreaks. However, data analysis and interlaboratory comparability of WGS data are still challenging for most public health laboratories. Therefore, we have developed and evaluated a newL. monocytogenestyping scheme based on genome-wide gene-by-gene comparisons (core genome multilocus the sequence typing [cgMLST]) to allow for a unique typing nomenclature. Initially, we determined the breadth of theL. monocytogenespopulation based on MLST data with a Bayesian approach. Based on the genome sequence data of representative isolates for the whole population, cgMLST target genes were defined and reappraised with 67L. monocytogenesisolates from two outbreaks and serotype reference strains. The Bayesian population analysis generated fiveL. monocytogenesgroups. Using all available NCBI RefSeq genomes (n= 36) and six additionally sequenced strains, all genetic groups were covered. Pairwise comparisons of these 42 genome sequences resulted in 1,701 cgMLST targets present in all 42 genomes with 100% overlap and ≥90% sequence similarity. Overall, ≥99.1% of the cgMLST targets were present in 67 outbreak and serotype reference strains, underlining the representativeness of the cgMLST scheme. Moreover, cgMLST enabled clustering of outbreak isolates with ≤10 alleles difference and unambiguous separation from unrelated outgroup isolates. In conclusion, the novel cgMLST scheme not only improves outbreak investigations but also enables, due to the availability of the automatically curated cgMLST nomenclature, interlaboratory exchange of data that are crucial, especially for rapid responses during transsectorial outbreaks.
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Chiaverini, Alexandra, Mostafa Y. Abdel-Glil, Jörg Linde, Domenico Galante, Valeria Rondinone, Antonio Fasanella, Cesare Cammà, Nicola D’Alterio, Giuliano Garofolo, and Herbert Tomaso. "Whole Genome Sequencing for Studying Bacillus anthracis from an Outbreak in the Abruzzo Region of Italy." Microorganisms 8, no. 1 (January 8, 2020): 87. http://dx.doi.org/10.3390/microorganisms8010087.
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Anthrax is a serious infectious disease caused by the gram-positive and spore-forming bacterium Bacillus anthracis. In Italy, anthrax is an endemic disease with sporadic cases each year and few outbreaks, especially in Southern Italy. However, new foci have been discovered in zones without previous history of anthrax. During summer 2016, an outbreak of anthrax caused the death of four goats in the Abruzzo region, where the disease had not been reported before. In order to investigate the outbreak, we sequenced one strain and compared it to 19 Italian B. anthracis genomes. Furthermore, we downloaded 71 whole genome sequences representing the global distribution of canonical SNP lineages and used them to verify the phylogenetic positioning. To this end, we analyzed and compared the genome sequences using canonical SNPs and the whole genome SNP-based analysis. Our results demonstrate that the outbreak strain belonged to the Trans-Eurasian (TEA) group A.Br.011/009, which is the predominant clade in Central-Southern Italy. In conclusion, the high genomic relatedness of the Italian TEA strains suggests their evolution from a common ancestor, while the spread is supposedly driven by trade as well as human and transhumance activities. Here, we demonstrated the capabilities of whole genome sequencing (WGS), which can be used as a tool for outbreak analyses and surveillance activities.
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Petronella, Nicholas, Palni Kundra, Olivia Auclair, Karine Hébert, Mary Rao, Kyle Kingsley, Katrien De Bruyne, et al. "Changes detected in the genome sequences of Escherichia coli, Listeria monocytogenes, Vibrio parahaemolyticus, and Salmonella enterica after serial subculturing." Canadian Journal of Microbiology 65, no. 11 (November 2019): 842–50. http://dx.doi.org/10.1139/cjm-2019-0235.
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Whole genome sequencing (WGS) is rapidly replacing other molecular techniques for identifying and subtyping bacterial isolates. The resolution or discrimination offered by WGS is significantly higher than that offered by other molecular techniques, and WGS readily allows infrequent differences that occur between 2 closely related strains to be found. In this investigation, WGS was used to identify the changes that occurred in the genomes of 13 strains of bacterial foodborne pathogens after 100 serial subcultures. Pure cultures of Shiga-toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Vibrio parahaemolyticus were subcultured daily for 100 successive days. The 1st and 100th subcultures were whole-genome sequenced using short-read sequencing. Single nucleotide polymorphisms (SNPs) were identified between the 1st and final culture using 2 different approaches, and multilocus sequence typing of the whole genome was also performed to detect any changes at the allelic level. The number of observed genomic changes varied by strain, species, and the SNP caller used. This study provides insight into the genomic variation that can be detected using next-generation sequencing and analysis methods after repeated subculturing of 4 important bacterial pathogens.
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Joensen, Katrine G., Anna M. M. Tetzschner, Atsushi Iguchi, Frank M. Aarestrup, and Flemming Scheutz. "Rapid and EasyIn SilicoSerotyping of Escherichia coli Isolates by Use of Whole-Genome Sequencing Data." Journal of Clinical Microbiology 53, no. 8 (May 13, 2015): 2410–26. http://dx.doi.org/10.1128/jcm.00008-15.
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Accurate and rapid typing of pathogens is essential for effective surveillance and outbreak detection. Conventional serotyping ofEscherichia coliis a delicate, laborious, time-consuming, and expensive procedure. With whole-genome sequencing (WGS) becoming cheaper, it has vast potential in routine typing and surveillance. The aim of this study was to establish a valid and publicly available tool for WGS-basedin silicoserotyping ofE. coliapplicable for routine typing and surveillance. A FASTA database of specific O-antigen processing system genes for O typing and flagellin genes for H typing was created as a component of the publicly available Web tools hosted by the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org). AllE. coliisolates available with WGS data and conventional serotype information were subjected to WGS-based serotyping employing this specific SerotypeFinder CGE tool. SerotypeFinder was evaluated on 682E. coligenomes, 108 of which were sequenced for this study, where both the whole genome and the serotype were available. In total, 601 and 509 isolates were included for O and H typing, respectively. The O-antigen geneswzx,wzy,wzm, andwztand the flagellin genesfliC,flkA,fllA,flmA, andflnAwere detected in 569 and 508 genome sequences, respectively. SerotypeFinder for WGS-based O and H typing predicted 560 of 569 O types and 504 of 508 H types, consistent with conventional serotyping. In combination with other available WGS typing tools,E. coliserotyping can be performed solely from WGS data, providing faster and cheaper typing than current routine procedures and making WGS typing a superior alternative to conventional typing strategies.
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Yu, Jun, Jian Wang, and Huanming Yang. "An International Campaign for Agricultural and Livestock Genomics (CALG)." Asia-Pacific Biotech News 06, no. 24 (November 25, 2002): 958–65. http://dx.doi.org/10.1142/s0219030302001970.
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A coordinated international effort to sequence agricultural and livestock genomes has come to its time. While human genome and genomes of many model organisms (related to human health and basic biological interests) have been sequenced or plugged in the sequencing pipelines, agronomically important crop and livestock genomes have not been given high enough priority. Although we are facing many challenges in policy-making, grant funding, regional task emphasis, research community consensus and technology innovations, many initiatives are being announced and formulated based on the cost-effective and large-scale sequencing procedure, known as whole genome shotgun (WGS) sequencing that produces draft sequences covering a genome from 95 percent to 99 percent. Identified genes from such draft sequences, coupled with other resources, such as molecular markers, large-insert clones and cDNA sequences, provide ample information and tools to further our knowledge in agricultural and environmental biology in the genome era that just comes to its accelerated period. If the campaign succeeds, molecular biologists, geneticists and field biologists from all countries, rich or poor, would be brought to the same starting point and expect another astronomical increase of basic genomic information, ready to convert effectively into knowledge that will ultimately change our lives and environment into a greater and better future. We call upon national and international governmental agencies and organizations as well as research foundations to support this unprecedented movement.
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Roosaare, Märt, Mikk Puustusmaa, Märt Möls, Mihkel Vaher, and Maido Remm. "PlasmidSeeker: identification of known plasmids from bacterial whole genome sequencing reads." PeerJ 6 (April 2, 2018): e4588. http://dx.doi.org/10.7717/peerj.4588.
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BackgroundPlasmids play an important role in the dissemination of antibiotic resistance, making their detection an important task. Using whole genome sequencing (WGS), it is possible to capture both bacterial and plasmid sequence data, but short read lengths make plasmid detection a complex problem.ResultsWe developed a tool named PlasmidSeeker that enables the detection of plasmids from bacterial WGS data without read assembly. The PlasmidSeeker algorithm is based onk-mers and usesk-mer abundance to distinguish between plasmid and bacterial sequences. We tested the performance of PlasmidSeeker on a set of simulated and real bacterial WGS samples, resulting in 100% sensitivity and 99.98% specificity.ConclusionPlasmidSeeker enables quick detection of known plasmids and complements existing tools that assemble plasmids de novo. The PlasmidSeeker source code is stored on GitHub:https://github.com/bioinfo-ut/PlasmidSeeker.
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Zhao, S., G. H. Tyson, Y. Chen, C. Li, S. Mukherjee, S. Young, C. Lam, J. P. Folster, J. M. Whichard, and P. F. McDermott. "Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp." Applied and Environmental Microbiology 82, no. 2 (October 30, 2015): 459–66. http://dx.doi.org/10.1128/aem.02873-15.
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ABSTRACTThe objectives of this study were to identify antimicrobial resistance genotypes forCampylobacterand to evaluate the correlation between resistance phenotypes and genotypes usingin vitroantimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114Campylobacterspecies isolates (82C. coliand 32C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, includingtet(O),blaOXA-61,catA,lnu(C),aph(2″)-Ib,aph(2″)-Ic,aph(2′)-If,aph(2″)-Ig,aph(2″)-Ih,aac(6′)-Ie-aph(2″)-Ia,aac(6′)-Ie-aph(2″)-If,aac(6′)-Im,aadE,sat4,ant(6′),aad9,aph(3′)-Ic, andaph(3′)-IIIa, and mutations in two housekeeping genes (gyrAand 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs.
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Mage, Rose G. "Genes encoding proteins of immunological interest in the Oryctolagus cuniculus (rabbit) whole genome sequences and ENCODE (81.19)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 81.19. http://dx.doi.org/10.4049/jimmunol.182.supp.81.19.
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Abstract Rabbits are a valuable resource for diagnostic and therapeutic antibodies, are becoming increasingly important for vaccine development and as animal models of human diseases. They remain a major source of polyclonal and monoclonal antibodies because unique characteristics of their immune system result in antibodies of high affinity and specificity. The whole genome shotgun (2x WGS) "unfinished Oryctolagus cuniculus database" had serious gaps, but assemblies of the 2x WGS sequence were useful for designing primers for PCR by predicting mRNA sequences and exon boundaries. Deeper 7x WGS coverage was completed in 2008 and assembly of a high quality draft rabbit genome sequence is nearing completion. The "Thorbecke Inbred Rabbit" chosen for sequencing at Broad Institute was less heterozygous than outbred NZW but the Trace Archive of Oryctolagus cuniculus (WGS) reveals heterozygosity at the immunoglobulin heavy chain locus. The ENCODE Project with ~ 1% of rabbit genomic sequence from a different animal has rabbit genes of immunological interest including a gene cluster syntenic with human and mouse containing IL13, IL3, IL4, IL5 and IRF1. Rabbit genome sequences can contribute comparative genomic data, reveal variants of genes of immunological interest, and genetic contributions to animal and human disease susceptibilities. This research was supported by the Intramural Research Program of the NIH, NIAID.
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Carattoli, Alessandra, Ea Zankari, Aurora García-Fernández, Mette Voldby Larsen, Ole Lund, Laura Villa, Frank Møller Aarestrup, and Henrik Hasman. "In SilicoDetection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing." Antimicrobial Agents and Chemotherapy 58, no. 7 (April 28, 2014): 3895–903. http://dx.doi.org/10.1128/aac.02412-14.
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ABSTRACTIn the work presented here, we designed and developed two easy-to-use Web tools forin silicodetection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the familyEnterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multidrug-resistantEnterobacteriaceaespecies by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the familyEnterobacteriaceaein the NCBI nucleotide database were collected to build a consensus database for integration into a Web tool called PlasmidFinder that can be used for replicon sequence analysis of raw, contig group, or completely assembled and closed plasmid sequencing data. The PlasmidFinder database currently consists of 116 replicon sequences that match with at least at 80% nucleotide identity all replicon sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated fromwww.pubmlst.organd integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection ofSalmonella entericaserovar Typhimurium isolates. PlasmidFinder identified a total of 103 replicons and between zero and five different plasmid replicons within each of 49S. Typhimurium draft genomes tested. The pMLST Web tool was able to subtype genomic sequencing data of plasmids, revealing both known plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in clinically relevant bacterial pathogens.
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Belkadi, Aziz, Alexandre Bolze, Yuval Itan, Aurélie Cobat, Quentin B. Vincent, Alexander Antipenko, Lei Shang, Bertrand Boisson, Jean-Laurent Casanova, and Laurent Abel. "Whole-genome sequencing is more powerful than whole-exome sequencing for detecting exome variants." Proceedings of the National Academy of Sciences 112, no. 17 (March 31, 2015): 5473–78. http://dx.doi.org/10.1073/pnas.1418631112.
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We compared whole-exome sequencing (WES) and whole-genome sequencing (WGS) in six unrelated individuals. In the regions targeted by WES capture (81.5% of the consensus coding genome), the mean numbers of single-nucleotide variants (SNVs) and small insertions/deletions (indels) detected per sample were 84,192 and 13,325, respectively, for WES, and 84,968 and 12,702, respectively, for WGS. For both SNVs and indels, the distributions of coverage depth, genotype quality, and minor read ratio were more uniform for WGS than for WES. After filtering, a mean of 74,398 (95.3%) high-quality (HQ) SNVs and 9,033 (70.6%) HQ indels were called by both platforms. A mean of 105 coding HQ SNVs and 32 indels was identified exclusively by WES whereas 692 HQ SNVs and 105 indels were identified exclusively by WGS. We Sanger-sequenced a random selection of these exclusive variants. For SNVs, the proportion of false-positive variants was higher for WES (78%) than for WGS (17%). The estimated mean number of real coding SNVs (656 variants, ∼3% of all coding HQ SNVs) identified by WGS and missed by WES was greater than the number of SNVs identified by WES and missed by WGS (26 variants). For indels, the proportions of false-positive variants were similar for WES (44%) and WGS (46%). Finally, WES was not reliable for the detection of copy-number variations, almost all of which extended beyond the targeted regions. Although currently more expensive, WGS is more powerful than WES for detecting potential disease-causing mutations within WES regions, particularly those due to SNVs.
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Hyun, Jungwon, Sujin Oh, Yun Ji Hong, and Kyoung Un Park. "Prediction of various blood group systems using Korean whole-genome sequencing data." PLOS ONE 17, no. 6 (June 3, 2022): e0269481. http://dx.doi.org/10.1371/journal.pone.0269481.
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Aims This study established blood group analysis methods using whole-genome sequencing (WGS) data and conducted blood group analyses to determine the domestic allele frequency using public data from the Korean whole sequence analysis of the Korean Reference Genome Project conducted by the Korea Disease Control and Prevention Agency (KDCA). Materials and methods We analyzed the differences between the human reference sequences (hg19) and the conventional reference cDNA sequences of blood group genes using the Clustal Omega website, and established blood group analysis methods using WGS data for 41 genes, including 39 blood group genes involved in 36 blood group antigens, as well as the GATA1 and KLF1 genes, which are erythrocyte-specific transcription factor genes. Using CLC genomics Workbench 11.0 (Qiagen, Aarhus, Denmark), variant analysis was performed on these 41 genes in 250 Korean WGS data sets, and each blood group’s genotype was predicted. The frequencies for major alleles were also investigated and compared with data from the Korean rare blood program (KRBP) and the Erythrogene database (East Asian and all races). Results Among the 41 blood group-related genes, hg19 showed variants in the following genes compared to the conventional reference cDNA: GYPA, RHD, RHCE, FUT3, ACKR1, SLC14A1, ART4, CR1, and GCNT2. Among 250 WGS data sets from the Korean Reference Genome Project, 70.6 variants were analyzed in 205 samples; 45 data samples were excluded due to having no variants. In particular, the FUT3, GNCT2, B3GALNT1, CR1, and ACHE genes contained numerous variants, with averages of 21.1, 13.9, 13.4, 9.6, and 7.0, respectively. Except for some blood groups, such as ABO and Lewis, for which it was difficult to predict the alleles using only WGS data, most alleles were successfully predicted in most blood groups. A comparison of allele frequencies showed no significant differences compared to the KRBP data, but there were differences compared to the Erythrogene data for the Lutheran, Kell, Duffy, Yt, Scianna, Landsteiner-Wiener, and Cromer blood group systems. Numerous minor blood group systems that were not available in the KRBP data were also included in this study. Conclusions We successfully established and performed blood group analysis using Korean public WGS data. It is expected that blood group analysis using WGS data will be performed more frequently in the future and will contribute to domestic data on blood group allele frequency and eventually the supply of safe blood products.
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Roberts, Scott C., Egon A. Ozer, Teresa Zembower, and Chao Qi. "864. Whole Genome Sequencing is Unable to Track Candida auris Transmission." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S470—S471. http://dx.doi.org/10.1093/ofid/ofaa439.1053.
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Abstract Background Candida auris (C. auris), an emerging yeast species, is often drug-resistant and has caused outbreaks in healthcare settings. Surging C. auris cases at our institution prompted whole genome sequencing (WGS) of patient and environmental specimens and comparison to local and international isolates. Methods WGS was performed on clinical and environmental isolates obtained from Northwestern Memorial Hospital (NMH) from June 2018 to December 2019. Genome sequences were compared against isolates from other institutions in the Chicagoland area obtained from a reference lab (ACL) and from the CDC. Two isolates underwent long-read sequencing on the Oxford Nanopore GridION platform to obtain closed genomes. WGS was performed on the remaining isolates with the Illumina MiSeq platform. Results Twenty isolates from NMH, five from ACL, and two from the CDC underwent WGS to yield 12.6 Mb genomes. Any two NMH isolates differed from each other by a maximum of 36 single nucleotide variants (SNV) (Figure 1). Two patients thought to be part of a transmission cluster (isolates CA06 and CA07), differed by 7 SNVs. No phylogenetic grouping between hospital systems across Chicagoland was observed. Isolates from room surfaces from a C. auris patient differed by 1-6 SNVs from each other and from 7-8 SNVs from the patient isolate. Samples taken from different body sites of another patient differed by 4-9 SNVs. Average SNV counts were lower among nosocomially acquired cases when compared to C. auris isolates present on admission (Figure 2). All NMH isolates were fluconazole sensitive, but a fluconazole resistant ACL isolate differed from a sensitive NMH isolate by only 4 SNVs. Figure 1: Phylogenetic tree of all NMH and ACL isolates with fluconazole sensitivities Figure 2: Observed pairwise SNP differences between nosocomial and POA strains Conclusion WGS of C. auris did not reveal identical isolates in any instance, even from the same patient or the patients and their environment. Generally, lower numbers of SNVs were observed for intra- versus inter-institutional isolates. More work is needed to determine the use, if any, of WGS in outbreak investigations. Disclosures All Authors: No reported disclosures
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Chen, Xun, and Dawei Li. "ERVcaller: identifying polymorphic endogenous retrovirus and other transposable element insertions using whole-genome sequencing data." Bioinformatics 35, no. 20 (March 21, 2019): 3913–22. http://dx.doi.org/10.1093/bioinformatics/btz205.
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Abstract Motivation Approximately 8% of the human genome is derived from endogenous retroviruses (ERVs). In recent years, an increasing number of human diseases have been found to be associated with ERVs. However, it remains challenging to accurately detect the full spectrum of polymorphic (unfixed) ERVs using whole-genome sequencing (WGS) data. Results We designed a new tool, ERVcaller, to detect and genotype transposable element (TE) insertions, including ERVs, in the human genome. We evaluated ERVcaller using both simulated and real benchmark WGS datasets. Compared to existing tools, ERVcaller consistently obtained both the highest sensitivity and precision for detecting simulated ERV and other TE insertions derived from real polymorphic TE sequences. For the WGS data from the 1000 Genomes Project, ERVcaller detected the largest number of TE insertions per sample based on consensus TE loci. By analyzing the experimentally verified TE insertions, ERVcaller had 94.0% TE detection sensitivity and 96.6% genotyping accuracy. Polymerase chain reaction and Sanger sequencing in a small sample set verified 86.7% of examined insertion statuses and 100% of examined genotypes. In conclusion, ERVcaller is capable of detecting and genotyping TE insertions using WGS data with both high sensitivity and precision. This tool can be applied broadly to other species. Availability and implementation http://www.uvm.edu/genomics/software/ERVcaller.html. Supplementary information Supplementary data are available at Bioinformatics online.
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Rueca, Martina, Emanuela Giombini, Giulia Gramigna, Cesare Ernesto Maria Gruber, Lavinia Fabeni, Angela Corpolongo, Valentina Mazzotta, et al. "Molecular Characterization of Whole-Genome SARS-CoV-2 from the First Suspected Cases of the XE Variant in the Lazio Region, Italy." Diagnostics 12, no. 9 (September 14, 2022): 2219. http://dx.doi.org/10.3390/diagnostics12092219.
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We report two cases of SARS-CoV-2 recombinant variant XE detected in nasopharyngeal swabs (NPS) of hospitalized patients with no evident epidemiological link in Lazio, Central Italy. Whole-Genome Sequencing (WGS) performed on an Ion Torrent GSS5 platform according to Italian flash surveys showed genomes corresponding to the PANGOLIN unclassified lineage and the Nextclade XE clade. Further analyses were then carried out to investigate more deeply the genetic characteristics of these XE-like sequences. When phylogenetic trees, by using IQ-TREE, were built splitting the genome into two regions according to the putative XE recombination site, the upstream and downstream regions were seen to be clustered near BA.1 and BA.2 sequences, respectively. However, our XE-like sequences clustered separately, with a significant bootstrap, from the classified European and Italian XE strains, although the recombination site between BA.1 and BA.2 was identified at the nucleotide site 11556 by RDP4 software, consistent with the putative XE breakpoint. These findings show the risk of the introduction of novel recombinant variants of SARS-CoV-2 and the existence of XE-like strains, phylogenetically separated, that could make their exact taxonomy difficult. It follows the need for continued SARS-CoV-2 surveillance by WGS.
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Muttineni, Radhakrishna, Nagamani Kammili, Thrilok Chander Bingi, Raja Rao M., Kalyani Putty, Pankaj Singh Dholaniya, Ravi Kumar Puli, et al. "Clinical and whole genome characterization of SARS-CoV-2 in India." PLOS ONE 16, no. 2 (February 2, 2021): e0246173. http://dx.doi.org/10.1371/journal.pone.0246173.
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We report clinical profile of hundred and nine patients with SARS CoV-2 infection, and whole genome sequences (WGS) of seven virus isolates from the first reported cases in India, with various international travel histories. Comorbidities such as diabetes, hypertension, and cardiovascular disease were frequently associated with severity of the disease. WBC and neutrophil counts showed an increase, while lymphocyte counts decreased in patients with severe infection suggesting a possible neutrophil mediated organ damage, while immune activity may be diminished with decrease in lymphocytes leading to disease severity. Increase in SGOT, SGPT and blood urea suggests the functional deficiencies of liver, heart, and kidney in patients who succumbed to the disease when compared to the group of recovered patients. The WGS analysis showed that these isolates were classified into two clades: I/A3i, and A2a (four according to GISAID: O, L, GR, and GH). Further, WGS phylogeny and travel history together indicate possible transmission from Middle East and Europe. Three S protein variants: Wuhan reference, D614G, and Y28H were identified predicted to possess different binding affinities to host ACE2.
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Olivieri, David N., and Francisco Gambón-Deza. "Iterative Variable Gene Discovery from Whole Genome Sequencing with a Bootstrapped Multiresolution Algorithm." Computational and Mathematical Methods in Medicine 2019 (February 11, 2019): 1–13. http://dx.doi.org/10.1155/2019/3780245.
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In jawed vertebrates, variable (V) genes code for antigen-binding regions of B and T lymphocyte receptors, which generate a specific response to foreign pathogens. Obtaining the detailed repertoire of these genes across the jawed vertebrate kingdom would help to understand their evolution and function. However, annotations of V-genes are known for only a few model species since their extraction is not amenable to standard gene finding algorithms. Also, the more distant evolution of a taxon is from such model species, and there is less homology between their V-gene sequences. Here, we present an iterative supervised machine learning algorithm that begins by training a small set of known and verified V-gene sequences. The algorithm successively discovers homologous unaligned V-exons from a larger set of whole genome shotgun (WGS) datasets from many taxa. Upon each iteration, newly uncovered V-genes are added to the training set for the next predictions. This iterative learning/discovery process terminates when the number of new sequences discovered is negligible. This process is akin to “online” or reinforcement learning and is proven to be useful for discovering homologous V-genes from successively more distant taxa from the original set. Results are demonstrated for 14 primate WGS datasets and validated against Ensembl annotations. This algorithm is implemented in the Python programming language and is freely available at http://vgenerepertoire.org.
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GRAHAM, R. M. A., C. J. DOYLE, and A. V. JENNISON. "Real-time investigation of a Legionella pneumophila outbreak using whole genome sequencing." Epidemiology and Infection 142, no. 11 (February 27, 2014): 2347–51. http://dx.doi.org/10.1017/s0950268814000375.
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SUMMARYLegionella pneumophila is the main pathogen responsible for outbreaks of Legionnaires' disease, which can be related to contaminated water supplies such as cooling towers or water pipes. We combined conventional molecular methods and whole genome sequence (WGS) analysis to investigate an outbreak of L. pneumophila in a large Australian hospital. Typing of these isolates using sequence-based typing and virulence gene profiling, was unable to discriminate between outbreak and non-outbreak isolates. WGS analysis was performed on isolates during the outbreak, as well as on unlinked isolates from the Public Health Microbiology reference collection. The more powerful resolution provided by analysis of whole genome sequences allowed outbreak isolates to be distinguished from isolates that were temporally and spatially unassociated with the outbreak, demonstrating that this technology can be used in real-time to investigate L. pneumophila outbreaks.
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Khoder, May, Marwan Osman, Issmat I. Kassem, Rayane Rafei, Ahmad Shahin, Pierre Edouard Fournier, Jean-Marc Rolain, and Monzer Hamze. "Whole Genome Analyses Accurately Identify Neisseria spp. and Limit Taxonomic Ambiguity." International Journal of Molecular Sciences 23, no. 21 (November 3, 2022): 13456. http://dx.doi.org/10.3390/ijms232113456.
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Genome sequencing facilitates the study of bacterial taxonomy and allows the re-evaluation of the taxonomic relationships between species. Here, we aimed to analyze the draft genomes of four commensal Neisseria clinical isolates from the semen of infertile Lebanese men. To determine the phylogenetic relationships among these strains and other Neisseria spp. and to confirm their identity at the genomic level, we compared the genomes of these four isolates with the complete genome sequences of Neisseria gonorrhoeae and Neisseria meningitidis and the draft genomes of Neisseria flavescens, Neisseria perflava, Neisseria mucosa, and Neisseria macacae that are available in the NCBI Genbank database. Our findings revealed that the WGS analysis accurately identified and corroborated the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) species identities of the Neisseria isolates. The combination of three well-established genome-based taxonomic tools (in silico DNA-DNA Hybridization, Ortho Average Nucleotide identity, and pangenomic studies) proved to be relatively the best identification approach. Notably, we also discovered that some Neisseria strains that are deposited in databases contain many taxonomical errors. The latter is very important and must be addressed to prevent misdiagnosis and missing emerging etiologies. We also highlight the need for robust cut-offs to delineate the species using genomic tools.
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Lei, Haiyan, Tianwei Li, Shien Tsai, Robert J. Biggar, Francis Nkrumah, Janet Neequayee, Marina Gutierrez, et al. "Whole Genome Sequences of Epstein-Barr Viruses Associated with Burkitt Lymphoma Tissues." Blood 124, no. 21 (December 6, 2014): 2987. http://dx.doi.org/10.1182/blood.v124.21.2987.2987.
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Abstract Epstein-Barr virus (EBV), first discovered from Burkitt's lymphoma (BL), is a class 1 carcinogen that is now associated with a wide range of hematologic and epithelial cancers, including lymphoma nasopharyngeal carcinoma (NPC), gastric cancer, Hodgkin lymphoma and some AIDS-associated B cell lymphoma. Although almost all BL cases from Africa and NPC in China are EBV-positive, consistent with a direct role of EBV in tumor causation, the precise nature of the mechanisms of causation remains to be elucidated. Of interest, EBV is ubiquitous and causes asymptomatic lifelong infection. Up to 95% of developing world population is infected at an early age. In contrast, the geographical patterns of EBV-associated cancers and their peaks age-incidences vary. For example, BL incidence is highest in equatorial Africa, where peak risk occurs in children aged 5-9 years. By contrast, NPC incidence is highest in Southern China and also parts of northern Africa; with peak risk in the elderly. These variations have led to speculation about presence of EBV variants with different penetration and expression. Previous studies attempting to examine this question have focused on genetic variation in one or only a few EBV genes at a time, precluding firm conclusions about genetic variation. Whole EBV genome analysis in tumor and non-diseased tissue from the same individual as well from healthy individuals in at risk populations may facilitate discovery of sequence heterogeneity that might be associated with cancer risk. Since the first genome of EBV was published, 23 whole EBV genomes have been sequenced, including from 3 BL cell lines, 5 immortalized B cells of normal blood donors (B95-8 plus WT-EBV), and 13 NPC biopsies. No EBV genomes have however been sequenced to date directly from BL biopsies and from healthy individuals from the same region. The goal of this study is to obtain a comprehensive assessment of EBV genome in BL tissue, and to determine how these sequences differ from EBV genomes in matched non-tumor reservoir of same individuals and from EBV genomes in healthy individuals from same regions. We have available 50 BL biopsies, 37 representing endemic BL from Africa, 13 from South America, and normal tissue from healthy individuals from the same region. We are reporting preliminary data obtained from whole genome sequences of EBV genomes from six BL biopsies from West Africa and South America obtained using high-throughput sequencing (HTS) Illumina MiSeq platform. Using the WT-EBV as a reference, EBV genomes in the BL biopsies showed considerable variation ranging from 550-1200 variations per genome (Fig 1). Most were single nucleotide variations. Insertions and deletions ranged between 15 and 51 per genome. As much as one third of variations resulted in amino acid changes. Surprisingly some of the BL biopsies contained EBV genomes with heterozygous reads, suggesting that ongoing mutations in the EBV genome occurred after clonal expansion of BL cells. Novel variations were observed in BZLF1 suggesting a possible influence of variation on regulation of EBV lytic cycle. Using an in-house EBV genome database prepared for comparative analysis that contained genomic DNA sequences of the 23 published EBV genomes, sequence comparison and phylogenetic analysis showed a much greater sequence diversity among EBV sequences from BL biopsies on a whole-genome level. Based upon these results, we are proposing to expand EBV genome wide sequencing from the remaining BL biopsies as well as from paired normal subjects to determine variations commonly associated with BL and to understand how these EBV genomic variations contribute to BL pathogenesis in different geographic areas. Fig 1 Distribution of variations across the EBV genome in select BL biopsies Fig 1. Distribution of variations across the EBV genome in select BL biopsies Disclosures No relevant conflicts of interest to declare.
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Goyal, Manisha, Lysiane Hauben, Hannes Pouseele, Magali Jaillard, Katrien De Bruyne, Alex van Belkum, and Richard Goering. "Retrospective Definition of Clostridioides difficile PCR Ribotypes on the Basis of Whole Genome Polymorphisms: A Proof of Principle Study." Diagnostics 10, no. 12 (December 12, 2020): 1078. http://dx.doi.org/10.3390/diagnostics10121078.
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Clostridioides difficile is a cause of health care-associated infections. The epidemiological study of C. difficile infection (CDI) traditionally involves PCR ribotyping. However, ribotyping will be increasingly replaced by whole genome sequencing (WGS). This implies that WGS types need correlation with classical ribotypes (RTs) in order to perform retrospective clinical studies. Here, we selected genomes of hyper-virulent C. difficile strains of RT001, RT017, RT027, RT078, and RT106 to try and identify new discriminatory markers using in silico ribotyping PCR and De Bruijn graph-based Genome Wide Association Studies (DBGWAS). First, in silico ribotyping PCR was performed using reference primer sequences and 30 C. difficile genomes of the five different RTs identified above. Second, discriminatory genomic markers were sought with DBGWAS using a set of 160 independent C. difficile genomes (14 ribotypes). RT-specific genetic polymorphisms were annotated and validated for their specificity and sensitivity against a larger dataset of 2425 C. difficile genomes covering 132 different RTs. In silico PCR ribotyping was unsuccessful due to non-specific or missing theoretical RT PCR fragments. More successfully, DBGWAS discovered a total of 47 new markers (13 in RT017, 12 in RT078, 9 in RT106, 7 in RT027, and 6 in RT001) with minimum q-values of 0 to 7.40 × 10−5, indicating excellent marker selectivity. The specificity and sensitivity of individual markers ranged between 0.92 and 1.0 but increased to 1 by combining two markers, hence providing undisputed RT identification based on a single genome sequence. Markers were scattered throughout the C. difficile genome in intra- and intergenic regions. We propose here a set of new genomic polymorphisms that efficiently identify five hyper-virulent RTs utilizing WGS data only. Further studies need to show whether this initial proof-of-principle observation can be extended to all 600 existing RTs.
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Zhang, Chao, Kyle Cleveland, Felice Schnoll-Sussman, Bridget McClure, Michelle Bigg, Prashant Thakkar, Nikolaus Schultz, Eric G. Pamer, Doron Betel, and Manish A. Shah. "Identification of the gastric microbiome from endoscopic biopsy samples using whole genome sequencing." Journal of Clinical Oncology 33, no. 3_suppl (January 20, 2015): 8. http://dx.doi.org/10.1200/jco.2015.33.3_suppl.8.
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8 Background: H. pylori is the primary risk factor for gastric cancer (GC). However, virtually all infected patients do not develop GC, and H. pylori eradication does not reduce GC risk. We established a whole genome sequencing (WGS) pipeline to detect the gastric microbiome from small endoscopic samples directly. With it, we are not only be able to accurately detect H. pylori infection in GC, but also examine the microbiome in samples from other malignancies. Methods: Patients (pts) undergoing upper endoscopy without chronic inflammatory disease or chronic NSAID use were eligible for participation, categorized as follows: (1) active infection (H. pylori identified on biopsy, or positive CLOtest), (2) prior infection (previous H. pylori treatment, or ELISA IgG positive), and (3) gastric cancer. Endoscopic biopsies from 3 locations in the antrum/body were freshly frozen for WGS on a HiSeq2500 platform at ~10X coverage. Our customized computational pipeline successively removes human sequences, with remaining un-mapped reads aligned to 1,400 unique bacteria genomes. Results: 16 biopsy samples taken from 10 pts were sequenced. All H. pylori positive samples detected by our pipeline have been successfully validated by qPCR. Surprisingly, H. pylori was identified in previously treated pts. We also applied our pipeline to control samples to evaluate the performance of bacterial identification. Negative sample are blood samples from HapMap projects with human DNA only. In contrast, positive samples are bacteria enriched samples from Human Microbiome Project. Our identifications matched the previously reported results. Finally, we profiled 37 gastric cancer tumors and matching normal WGS samples from TCGA and found that 40% of them are H. pylori positive. This result is a novel discovery that was not reported in the TCGA GC study. Conclusions: This is the first demonstration of detailed unbiased microbiome detection performed from gastric endoscopic biopsy samples using WGS. We found that standard treatment does not always eradicate H. pylori possibly explaining why H. pylori treatment fails to reduce cancer risk. We also found that ~40% of gastric cancers have evidence of persistent H. pylori bacterial content.
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Zainulabid, Ummu Afeera, Aini Syahida Mat Yassim, Mushtaq Hussain, Ayesha Aslam, Sharmeen Nellisa Soffian, Mohamad Shafiq Mohd Ibrahim, Norhidayah Kamarudin, Mohd Nazli Kamarulzaman, How Soon Hin, and Hajar Fauzan Ahmad. "Whole genome sequence analysis showing unique SARS-CoV-2 lineages of B.1.524 and AU.2 in Malaysia." PLOS ONE 17, no. 2 (February 25, 2022): e0263678. http://dx.doi.org/10.1371/journal.pone.0263678.
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SARS-CoV-2 has spread throughout the world since its discovery in China, and Malaysia is no exception. WGS has been a crucial approach in studying the evolution and genetic diversity of SARS-CoV-2 in the ongoing pandemic. Despite considerable number of SARS-CoV-2 genome sequences have been submitted to GISAID and NCBI databases, there is still scarcity of data from Malaysia. This study aims to report new Malaysian lineages of the virus, responsible for the sustained spikes in COVID-19 cases during the third wave of the pandemic. Patients with nasopharyngeal and/or oropharyngeal swabs confirmed COVID-19 positive by real-time RT-PCR with CT value < 25 were chosen for WGS. The selected SARS-CoV-2 isolates were then sequenced, characterized and analyzed along with 986 sequences of the dominant lineages of D614G variants currently circulating throughout Malaysia. The prevalence of clade GH and G formed strong ground for the presence of two Malaysian lineages of AU.2 and B.1.524 that has caused sustained spikes of cases in the country. Statistical analysis on the association of gender and age group with Malaysian lineages revealed a significant association (p <0.05). Phylogenetic analysis revealed dispersion of 41 lineages, of these, 22 lineages are still active. Mutational analysis showed presence of unique G1223C missense mutation in transmembrane domain of the spike protein. For better understanding of the SARS-CoV-2 evolution in Malaysia especially with reference to the reported lineages, large scale studies based on WGS are warranted.
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Castro, Vinicius Silva, Rodrigo Ortega Polo, Eduardo Eustáquio de Souza Figueiredo, Emmanuel Wihkochombom Bumunange, Tim McAllister, Robin King, Carlos Adam Conte-Junior, and Kim Stanford. "Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses." PLOS ONE 16, no. 9 (September 3, 2021): e0257168. http://dx.doi.org/10.1371/journal.pone.0257168.
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Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.
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Ritter, Deborah, Kimberly Walker, Myoung Kwon, Premal Lulla, Catherine M. Bollard, Andrea Sheehan, Helen E. Heslop, Richard A. Gibbs, David A. Wheeler, and Martha P. Mims. "Whole genome sequencing of sporadic Burkitt lymphoma in HIV-infected and uninfected patients." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 8577. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.8577.
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8577 Background: Burkitt Lymphoma is defined by canonical translocations between MYC and immunoglobulin IgH, IgK or IgL (8:14, 8:2, 8:22, respectively), and is commonly associated with HIV. The identification of HIV from sequenced samples is critical to understanding HIV-associated Burkitt Lymphoma. While recent novel gene mutations (ID3 and TCF3) have been implicated in functional roles, concomitant genomic structural variants and the interaction of HIV with structural variation is less well defined. Methods: We sequenced the whole genomes of 15 patients with 100bp paired-end reads on Illumina Hi-Seq platform, resulting in an average insert size of 278 (+/- 63) and coverage of 60X tumor and 30X normal. We included 7 HIV-negative, and 8 HIV-positive subjects. Sequencing reads were mapped to the reference genome using BWA. Large-scale structural variation was detected by the BreakDancer and Crest programs. Functional annotation was used to prioritize structural variants for validation. Single nucleotide variants and small insertions and deletions were detected by CARNAC, a somatic variation discovery pipeline. The subset of WGS reads that failed to align to the human reference genome were tested for the presence of HIV sequences by comparing the unmapped reads to a database of viral DNA sequences which included the common subtypes of HIV defined by Los Alamos. Reads matching HIV or EBV with an expectation value of <10-4 were analyzed to determine virus coverage and viral integration sites. Results: Canonical MYC-IgH translocations were identified in 9/15 (60%) tumor samples, with 2 additional subjects harboring either a deletion or an inversion near exon1 of MYC; 4 had no MYC rearrangement. MYC translocations occurred equally in both groups. TP53 and SMARC4 point mutations were observed recurrently in the HIV uninfected group but not in the HIV infected patients. Variable levels of HIV DNA sequence were observed in normal tissue of all HIV infected patients. Conclusions: Whole genome sequencing has identified known somatic variants in HIV infected and uninfected patients. Two genes, TP53 and SMARC4, appear to be differentially mutated, but additional samples are needed to achieve statistical significance.
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Low, Andrew J., Adam G. Koziol, Paul A. Manninger, Burton Blais, and Catherine D. Carrillo. "ConFindr: rapid detection of intraspecies and cross-species contamination in bacterial whole-genome sequence data." PeerJ 7 (May 31, 2019): e6995. http://dx.doi.org/10.7717/peerj.6995.
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Whole-genome sequencing (WGS) of bacterial pathogens is currently widely used to support public-health investigations. The ability to assess WGS data quality is critical to underpin the reliability of downstream analyses. Sequence contamination is a quality issue that could potentially impact WGS-based findings; however, existing tools do not readily identify contamination from closely-related organisms. To address this gap, we have developed a computational pipeline, ConFindr, for detection of intraspecies contamination. ConFindr determines the presence of contaminating sequences based on the identification of multiple alleles of core, single-copy, ribosomal-protein genes in raw sequencing reads. The performance of this tool was assessed using simulated and lab-generated Illumina short-read WGS data with varying levels of contamination (0–20% of reads) and varying genetic distance between the designated target and contaminant strains. Intraspecies and cross-species contamination was reliably detected in datasets containing 5% or more reads from a second, unrelated strain. ConFindr detected intraspecies contamination with higher sensitivity than existing tools, while also being able to automatically detect cross-species contamination with similar sensitivity. The implementation of ConFindr in quality-control pipelines will help to improve the reliability of WGS databases as well as the accuracy of downstream analyses. ConFindr is written in Python, and is freely available under the MIT License at github.com/OLC-Bioinformatics/ConFindr.
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Hunter, Zachary, Lian Xu, Yangsheng Zhou, Guang Yang, Xia Liu, Yang Cao, Christina Hanzis, et al. "Whole-Genome Sequencing Results From 30 Patients with Waldenstrom's Macroglobulinemia." Blood 118, no. 21 (November 18, 2011): 434. http://dx.doi.org/10.1182/blood.v118.21.434.434.
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Abstract Abstract 434 Introduction: The primary oncogenetic event resulting in malignant transformation in Waldenstrom's Macroglobulinemia (WM) remains to be delineated. We therefore employed whole genome sequencing (WGS) to help identify potential somatic variants in WM. Patients and Methods: Thirty patients meeting consensus criteria for the diagnosis of WM were included for these studies, whose characteristics are depicted in Table 1. CD19-magnetic bead sorting was used for isolation of bone marrow LPC. CD19-depleted PB mononuclear cells were collected as matched normal tissue. For 10 patients, WGS of tumor and matched normal samples was performed, and for 20 additional patients tumor samples alone were completely sequenced. Library construction and WGS was performed by Complete Genomics Inc. Read sequences were aligned to the NCBI Build 37. High confidence somatic variants were identified using cgatools version 1.3. Novel non-synonymous exonic variants for familial and sporadic LPL/WM patients were identified using ANNOVAR using to filter against several large databases including dbSNP version 132, the November 2010 release version of the 1,000 genomes project, and a 46 healthy donor dataset from Complete Genomics, Inc. based on KnownGene annotations. Variants filtered out in this process were checked against the dbSNP132 flagged SNP database for potential clinical significance. Data was further annotated against the Database of Genomic Variants and the Segmental Duplication Database, TargetScan, and transcription factor binding site data from the ENCODE project. When applicable, variants were scored using SIFT, PolyPhen2 and Mutation Taster. Copy number neutral loss of heterozygosity (CNLOH) was identified from the rate of heterozygous variants per 500,000 base pairs, CG content adjusted coverage data, and allele imbalance calculated from the percentage of total reads supporting the less covered allele. Results: Tumor and normal genomes were both sequenced to an average of 66X (range 60–91X) coverage of mapped individual reads. The average gross mapped yield for these genomes was 186.89 (range 171.56–262.03 Gb). Acquired copy number changes were common, and included losses in chromosome 6q (13/30; 43%), gains in chromosome 4 (7/30; 23%), and gains in 6p (3/30; 10%). Large regions of CNLOH were observed in 9/30 (30%) of patients occurring in chromosomes 1, 2, 3, 5, 9, 11, 17, 21, and X. The most frequent somatic variant occurred at position 38182641 in chromosome 3p22.2 in the myeloid differentiation primary response (MYD88) gene, resulting in a non-synonymous change at amino acid position 265 from leucine to proline (L265P) in 26/30 (86.7%) patients. Of these, 4/26 (15%) had a CNLOH covering this position making the variant effectively homozygous. Additional somatic variants occurred in transporter 2, ATP-binding cassette, sub-family B (TAP2) gene in 7/30 (23%) patients; chemokine (C-X-C motif) receptor 4 (CXCR4) gene in 6/30 (20%) patients. Somatic variants were also identified in the coding regions of low density lipoprotein receptor-related protein 1B (LRP1B) gene in 5/30 (17%) patients; mesothelin (MSLN) gene in 4/30 (13%) patients; AT rich interactive domain 1A (ARID1A) gene in 3/30 (10%) patients; histone cluster 1, H1e (HIST1H1E) in 3/30 (10%) patients, and Rap guanine nucleotide exchange factor 3 (RAPGEF3) in 3/30 (10%) patients. Conclusions: The results of this study provide the first reporting of comprehensive WGS efforts in patients with WM, and reveal recurring somatic variants in genes with important regulatory functions including MYD88, TAP2, and CXCR4. Structural and functional validation studies are ongoing and will be updated at the meeting. The results of these studies provide important new insights into the pathogenesis of WM. Disclosures: No relevant conflicts of interest to declare.
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McDermott, Patrick F., Gregory H. Tyson, Claudine Kabera, Yuansha Chen, Cong Li, Jason P. Folster, Sherry L. Ayers, Claudia Lam, Heather P. Tate, and Shaohua Zhao. "Whole-Genome Sequencing for Detecting Antimicrobial Resistance in Nontyphoidal Salmonella." Antimicrobial Agents and Chemotherapy 60, no. 9 (July 5, 2016): 5515–20. http://dx.doi.org/10.1128/aac.01030-16.
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ABSTRACTLaboratory-basedin vitroantimicrobial susceptibility testing is the foundation for guiding anti-infective therapy and monitoring antimicrobial resistance trends. We used whole-genome sequencing (WGS) technology to identify known antimicrobial resistance determinants among strains of nontyphoidalSalmonellaand correlated these with susceptibility phenotypes to evaluate the utility of WGS for antimicrobial resistance surveillance. Six hundred fortySalmonellaof 43 different serotypes were selected from among retail meat and human clinical isolates that were tested for susceptibility to 14 antimicrobials using broth microdilution. The MIC for each drug was used to categorize isolates as susceptible or resistant based on Clinical and Laboratory Standards Institute clinical breakpoints or National Antimicrobial Resistance Monitoring System (NARMS) consensus interpretive criteria. Each isolate was subjected to whole-genome shotgun sequencing, and resistance genes were identified from assembled sequences. A total of 65 unique resistance genes, plus mutations in two structural resistance loci, were identified. There were more unique resistance genes (n =59) in the 104 human isolates than in the 536 retail meat isolates (n =36). Overall, resistance genotypes and phenotypes correlated in 99.0% of cases. Correlations approached 100% for most classes of antibiotics but were lower for aminoglycosides and beta-lactams. We report the first finding of extended-spectrum β-lactamases (ESBLs) (blaCTX-M1andblaSHV2a) in retail meat isolates ofSalmonellain the United States. Whole-genome sequencing is an effective tool for predicting antibiotic resistance in nontyphoidalSalmonella, although the use of more appropriate surveillance breakpoints and increased knowledge of new resistance alleles will further improve correlations.
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Zhou, Chenxi, Tania Duarte, Rocio Silvestre, Genoveva Rossel, Robert O. M. Mwanga, Awais Khan, Andrew W. George, et al. "Insights into population structure of East African sweetpotato cultivars from hybrid assembly of chloroplast genomes." Gates Open Research 2 (September 5, 2018): 41. http://dx.doi.org/10.12688/gatesopenres.12856.1.
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Background: The chloroplast (cp) genome is an important resource for studying plant diversity and phylogeny. Assembly of the cp genomes from next-generation sequencing data is complicated by the presence of two large inverted repeats contained in the cp DNA. Methods: We constructed a complete circular cp genome assembly for the hexaploid sweetpotato using extremely low coverage (<1×) Oxford Nanopore whole-genome sequencing (WGS) data coupled with Illumina sequencing data for polishing. Results: The sweetpotato cp genome of 161,274 bp contains 152 genes, of which there are 96 protein coding genes, 8 rRNA genes and 48 tRNA genes. Using the cp genome assembly as a reference, we constructed complete cp genome assemblies for a further 17 sweetpotato cultivars from East Africa and an I. triloba line using Illumina WGS data. Analysis of the sweetpotato cp genomes demonstrated the presence of two distinct subpopulations in East Africa. Phylogenetic analysis of the cp genomes of the species from the Convolvulaceae Ipomoea section Batatas revealed that the most closely related diploid wild species of the hexaploid sweetpotato is I. trifida. Conclusions: Nanopore long reads are helpful in construction of cp genome assemblies, especially in solving the two long inverted repeats. We are generally able to extract cp sequences from WGS data of sufficiently high coverage for assembly of cp genomes. The cp genomes can be used to investigate the population structure and the phylogenetic relationship for the sweetpotato.
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Zhou, Chenxi, Tania Duarte, Rocio Silvestre, Genoveva Rossel, Robert O. M. Mwanga, Awais Khan, Andrew W. George, et al. "Insights into population structure of East African sweetpotato cultivars from hybrid assembly of chloroplast genomes." Gates Open Research 2 (July 21, 2020): 41. http://dx.doi.org/10.12688/gatesopenres.12856.2.
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Background: The chloroplast (cp) genome is an important resource for studying plant diversity and phylogeny. Assembly of the cp genomes from next-generation sequencing data is complicated by the presence of two large inverted repeats contained in the cp DNA. Methods: We constructed a complete circular cp genome assembly for the hexaploid sweetpotato using extremely low coverage (<1×) Oxford Nanopore whole-genome sequencing (WGS) data coupled with Illumina sequencing data for polishing. Results: The sweetpotato cp genome of 161,274 bp contains 152 genes, of which there are 96 protein coding genes, 8 rRNA genes and 48 tRNA genes. Using the cp genome assembly as a reference, we constructed complete cp genome assemblies for a further 17 sweetpotato cultivars from East Africa and an I. triloba line using Illumina WGS data. Analysis of the sweetpotato cp genomes demonstrated the presence of two distinct subpopulations in East Africa. Phylogenetic analysis of the cp genomes of the species from the Convolvulaceae Ipomoea section Batatas revealed that the most closely related diploid wild species of the hexaploid sweetpotato is I. trifida. Conclusions: Nanopore long reads are helpful in construction of cp genome assemblies, especially in solving the two long inverted repeats. We are generally able to extract cp sequences from WGS data of sufficiently high coverage for assembly of cp genomes. The cp genomes can be used to investigate the population structure and the phylogenetic relationship for the sweetpotato.
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Merker, Jason D., Krishna Roskin, Dana Ng, Cuiping Pan, Dianna G. Fisk, Carol D. Jones, Linda Gojenola, et al. "Whole Genome Sequence Analysis of Primary Myelofibrosis." Blood 120, no. 21 (November 16, 2012): 2863. http://dx.doi.org/10.1182/blood.v120.21.2863.2863.
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Abstract Abstract 2863 Background: JAK2 V617F is a recurrent, activating mutation in patients (pts) with BCR-ABL1-negative MPNs. Mutations in codon 515 of MPL occur in 1–5% and 5–10% of ET and PMF pts, respectively, and similar to JAK2 V617F, lead to constitutive JAK-STAT signaling. The acquisition of multiple mutational events affecting the JAK-STAT axis or components of the epigenetic machinery is common in MPNs and likely contributes to phenotypic diversity, including progression to acute myeloid leukemia. Mutated genes thus far implicated in MPN initiation and/or progression include TET2, CBL, SH2B3, ASXL1, DNMT3A, IDH1/2, IKZF1, EZH2, SRSF2, and TP53. In order to identify novel somatic mutations associated with classic BCR-ABL1-negative MPNs, we performed whole genome sequencing of DNA extracted from peripheral blood granulocytes and cultured skin fibroblasts from a patient with PMF and a known MPL W515K mutation. Methods: Whole genome sequencing (WGS) was undertaken in a 55 year-old man with untreated PMF four years after initial diagnosis. His DIPSS Plus risk group was low (score 0). His karyotype was normal, and molecular testing revealed wild-type JAK2 in addition to the MPL W515K mutation. WGS of purified granulocytes and paired cultured skin fibroblasts was performed using both Illumina HiSeq and Complete Genomics (CGI) platforms. The resulting data were analyzed using multiple independent aligners and variant callers. Amplicon-based targeted resequencing with the Illumina MiSeq platform was used to evaluate additional patient samples for recurrent mutations. Stanford institutional review board approval and informed pt consent was obtained for these analyses. Results: The PMF genome was sequenced to 88X (Illumina) and 128X (CGI) average fold coverage, and the cultured skin fibroblast genome was sequenced to 47X (Illumina) and 126X (CGI). The PMF genome had a low somatic mutation rate, consistent with that observed for other sequenced hematopoietic tumor genomes, with a low number of true somatic mutation calls. To definitively identify true mutations among various sequencing artefacts and germline variants, we use cultured skin fibroblasts which can be prepared with no contamination by neoplastic cells. In addition to re-identification of the MPL W515K mutation, this approach identified six additional somatic mutations that alter gene coding regions, splice sites, or known regulatory regions: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a splice-site mutation in CAP2; three nonsynonymous point mutations in KIAA0355, SOX30, MFRP; and a 19-base pair (bp) deletion involving a regulatory region in the 5'-untranslated region (5'-UTR) of BRD2, a bromodomain-containing protein implicated in transcriptional regulation (Table). CARD6, BRD2, and KIAA0355, an uncharacterized protein, are expressed by the granulocytes derived from this patient, supporting a potential role in the development of PMF in this pt. Using massively parallel sequencing, we are currently examining the transcribed region of BRD2 and the coding region of the other five genes in additional pt samples. Analysis of the first 87 samples (MF=47, PV=20, ET=20) out of a cohort of 180 MPN pts has thus far not identified recurrent somatic mutations in these six genes. Conclusion: High-coverage genome sequencing of neoplastic and germline cells from a patient with MPL-mutated PMF identified six additional somatic mutations of potential functional significance. Work is ongoing to determine if somatic mutations in these genes are found in other patients with BCR-ABL1-negative MPNs, and their pathogenetic relevance to MPN biology. Disclosures: Snyder: Illumina: Consultancy; GenapSys: Membership on an entity's Board of Directors or advisory committees; Personalis: Consultancy, Founder Other.
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Letko, Anna, Fabienne Leuthard, Vidhya Jagannathan, Daniele Corlazzoli, Kaspar Matiasek, Daniela Schweizer, Marjo K. Hytönen, Hannes Lohi, Tosso Leeb, and Cord Drögemüller. "Whole Genome Sequencing Indicates Heterogeneity of Hyperostotic Disorders in Dogs." Genes 11, no. 2 (February 4, 2020): 163. http://dx.doi.org/10.3390/genes11020163.
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Craniomandibular osteopathy (CMO) and calvarial hyperostotic syndrome (CHS) are proliferative, non-neoplastic disorders affecting the skull bones in young dogs. Different forms of these hyperostotic disorders have been described in many dog breeds. However, an incompletely dominant causative variant for CMO affecting splicing of SLC37A2 has been reported so far only in three Terrier breeds. The purpose of this study was to identify further possible causative genetic variants associated with CHS in an American Staffordshire Terrier, as well as CMO in seven affected dogs of different breeds. We investigated their whole-genome sequences (WGS) and filtered variants using 584 unrelated genomes, which revealed no variants shared across all affected dogs. However, filtering for private variants of each case separately yielded plausible dominantly inherited candidate variants in three of the eight cases. In an Australian Terrier, a heterozygous missense variant in the COL1A1 gene (c.1786G>A; p.(Val596Ile)) was discovered. A pathogenic missense variant in COL1A1 was previously reported in humans with infantile cortical hyperostosis, or Caffey disease, resembling canine CMO. Furthermore, in a Basset Hound, a heterozygous most likely pathogenic splice site variant was found in SLC37A2 (c.1446+1G>A), predicted to lead to exon skipping as shown before in SLC37A2-associated canine CMO of Terriers. Lastly, in a Weimaraner, a heterozygous frameshift variant in SLC35D1 (c.1021_1024delTCAG; p.(Ser341ArgfsTer22)) might cause CMO due to the critical role of SLC35D1 in chondrogenesis and skeletal development. Our study indicates allelic and locus heterogeneity for canine CMO and illustrates the current possibilities and limitations of WGS-based precision medicine in dogs.
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Zhang, Gong, Yongjian Zhang, and Jingjie Jin. "The Ultrafast and Accurate Mapping Algorithm FANSe3: Mapping a Human Whole-Genome Sequencing Dataset Within 30 Minutes." Phenomics 1, no. 1 (February 2021): 22–30. http://dx.doi.org/10.1007/s43657-020-00008-5.
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AbstractAligning billions of reads generated by the next-generation sequencing (NGS) to reference sequences, termed “mapping”, is the time-consuming and computationally-intensive process in most NGS applications. A Fast, accurate and robust mapping algorithm is highly needed. Therefore, we developed the FANSe3 mapping algorithm, which can map a 30 × human whole-genome sequencing (WGS) dataset within 30 min, a 50 × human whole exome sequencing (WES) dataset within 30 s, and a typical mRNA-seq dataset within seconds in a single-server node without the need for any hardware acceleration feature. Like its predecessor FANSe2, the error rate of FANSe3 can be kept as low as 10–9 in most cases, this is more robust than the Burrows–Wheeler transform-based algorithms. Error allowance hardly affected the identification of a driver somatic mutation in clinically relevant WGS data and provided robust gene expression profiles regardless of the parameter settings and sequencer used. The novel algorithm, designed for high-performance cloud-computing after infrastructures, will break the bottleneck of speed and accuracy in NGS data analysis and promote NGS applications in various fields. The FANSe3 algorithm can be downloaded from the website: http://www.chi-biotech.com/fanse3/.
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Ledwaba, Maphuti Betty, Barbara Akorfa Glover, Itumeleng Matle, Giuseppe Profiti, Pier Luigi Martelli, Rita Casadio, Katiuscia Zilli, et al. "Whole Genome Sequence Analysis of Brucella abortus Isolates from Various Regions of South Africa." Microorganisms 9, no. 3 (March 11, 2021): 570. http://dx.doi.org/10.3390/microorganisms9030570.
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The availability of whole genome sequences in public databases permits genome-wide comparative studies of various bacterial species. Whole genome sequence-single nucleotide polymorphisms (WGS-SNP) analysis has been used in recent studies and allows the discrimination of various Brucella species and strains. In the present study, 13 Brucella spp. strains from cattle of various locations in provinces of South Africa were typed and discriminated. WGS-SNP analysis indicated a maximum pairwise distance ranging from 4 to 77 single nucleotide polymorphisms (SNPs) between the South African Brucella abortus virulent field strains. Moreover, it was shown that the South African B. abortus strains grouped closely to B. abortus strains from Mozambique and Zimbabwe, as well as other Eurasian countries, such as Portugal and India. WGS-SNP analysis of South African B. abortus strains demonstrated that the same genotype circulated in one farm (Farm 1), whereas another farm (Farm 2) in the same province had two different genotypes. This indicated that brucellosis in South Africa spreads within the herd on some farms, whereas the introduction of infected animals is the mode of transmission on other farms. Three B. abortus vaccine S19 strains isolated from tissue and aborted material were identical, even though they originated from different herds and regions of South Africa. This might be due to the incorrect vaccination of animals older than the recommended age of 4–8 months or might be a problem associated with vaccine production.
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Moghnia, Ola H., and Nourah A. Al-Sweih. "Whole Genome Sequence Analysis of Multidrug Resistant Escherichia coli and Klebsiella pneumoniae Strains in Kuwait." Microorganisms 10, no. 3 (February 25, 2022): 507. http://dx.doi.org/10.3390/microorganisms10030507.
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The spread of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae is a global concern. The management of infections caused by multidrug resistance (MDR) isolates poses substantial clinical challenges in both hospitals and communities. This study aimed to investigate the genetic characteristics and variations of MDR E. coli and K. pneumoniae isolates. Bacterial identification and antibiotic susceptibility testing against 19 antibiotics were performed by standard methods. Whole genome sequencing (WGS) was carried out on eight carbapenem-resistant isolates using an Illumina MiSeq platform. The assembled draft genomes were annotated, then sequences were blasted against antimicrobial resistance (AMR) genes database. WGS detected several resistance genes mediating the production of β-lactamases, including carbapenems and extended-spectrum β-lactamase genes as (blaOXA-1/-48, blaKPC-2/-29, blaCMY-4/-6, blaSHV-11/-12, blaTEM-1, blaCTX-M-15, blaOKP-B, blaACT and blaEC). Furthermore quinolone resistance including oqxA/oqxB, aac(6′)-Ib-cr5, gyrA_D87N, gyrA_S83F, gyrA_S83L, parC_S80I, parE_S458A, parE_I355T, parC_S80I, and qnrB1. In addition to aminoglycoside modifying enzymes genes (aph(6)-Id, aph(3″)-Ib, aac(3)-IIa, aac(6′)-Ib, aadA1, aadA2 and aadA5), trimethoprim-sulfamethoxazole (dfrA12/A14/A17 and sul1/sul2), tetracycline (tetA and tetB), fosfomycin (fosA and uhpT_E350Q) resistance genes, while other genes were detected conferring chloramphenicol (floR, catA2, and efflux pump cmIA5), macrolides resistance (mph(A) and erm(B), and quaternary ammonium efflux pump qacEdelta. Bleomycin and colistin resistance genes were detected as ble and pmrB_R256G, respectively. Comprehensive analysis of MDR strains provided by WGS detected variable antimicrobial resistance genes and their precise resistance mechanism. WGS is essential for control and prevention strategies to combat the growing threat of AMR and the implementation of multifaceted interventions are needed.
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Kozyreva, Varvara K., Chau-Linda Truong, Alexander L. Greninger, John Crandall, Rituparna Mukhopadhyay, and Vishnu Chaturvedi. "Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory." Journal of Clinical Microbiology 55, no. 8 (June 7, 2017): 2502–20. http://dx.doi.org/10.1128/jcm.00361-17.
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ABSTRACT Public health microbiology laboratories (PHLs) are on the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole-genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards. Here, we describe the validation of WGS on the Illumina platform for routine use in PHLs according to Clinical Laboratory Improvements Act (CLIA) guidelines for laboratory-developed tests (LDTs). We developed a validation panel comprising 10 Enterobacteriaceae isolates, 5 Gram-positive cocci, 5 Gram-negative nonfermenting species, 9 Mycobacterium tuberculosis isolates, and 5 miscellaneous bacteria. The genome coverage range was 15.71× to 216.4× (average, 79.72×; median, 71.55×); the limit of detection (LOD) for single nucleotide polymorphisms (SNPs) was 60×. The accuracy, reproducibility, and repeatability of base calling were >99.9%. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from multilocus sequence typing (MLST) and genome-wide SNP-based phylogenetic assays were 100%. The following objectives were accomplished: (i) the establishment of the performance specifications for WGS applications in PHLs according to CLIA guidelines, (ii) the development of quality assurance and quality control measures, (iii) the development of a reporting format for end users with or without WGS expertise, (iv) the availability of a validation set of microorganisms, and (v) the creation of a modular template for the validation of WGS processes in PHLs. The validation panel, sequencing analytics, and raw sequences could facilitate multilaboratory comparisons of WGS data. Additionally, the WGS performance specifications and modular template are adaptable for the validation of other platforms and reagent kits.
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Amaral, Gilda Rose S., Graciela M. Dias, Michiyo Wellington-Oguri, Luciane Chimetto, Mariana E. Campeão, Fabiano L. Thompson, and Cristiane C. Thompson. "Genotype to phenotype: identification of diagnostic vibrio phenotypes using whole genome sequences." International Journal of Systematic and Evolutionary Microbiology 64, Pt_2 (February 1, 2014): 357–65. http://dx.doi.org/10.1099/ijs.0.057927-0.
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Vibrios are ubiquitous in the aquatic environment and can be found in association with animal or plant hosts. The range of ecological relationships includes pathogenic and mutualistic associations. To gain a better understanding of the ecology of these microbes, it is important to determine their phenotypic features. However, the traditional phenotypic characterization of vibrios has been expensive, time-consuming and restricted in scope to a limited number of features. In addition, most of the commercial systems applied for phenotypic characterization cannot characterize the broad spectrum of environmental strains. A reliable and possible alternative is to obtain phenotypic information directly from whole genome sequences. The aim of the present study was to evaluate the usefulness of whole genome sequences as a source of phenotypic information. We performed a comparison of the vibrio phenotypes obtained from the literature with the phenotypes obtained from whole genome sequences. We observed a significant correlation between the previously published phenotypic data and the phenotypic data retrieved from whole genome sequences of vibrios. Analysis of 26 vibrio genomes revealed that all genes coding for the specific proteins involved in the metabolic pathways responsible for positive phenotypes of the 14 diagnostic features (Voges–Proskauer reaction, indole production, arginine dihydrolase, ornithine decarboxylase, utilization of myo-inositol, sucrose and l-leucine, and fermentation of d-mannitol, d-sorbitol, l-arabinose, trehalose, cellobiose, d-mannose and d-galactose) were found in the majority of the vibrios genomes. Vibrio species that were negative for a given phenotype revealed the absence of all or several genes involved in the respective biochemical pathways, indicating the utility of this approach to characterize the phenotypes of vibrios. The absence of the global regulation and regulatory proteins in the Vibrio parahaemolyticus genome indicated a non-vibrio phenotype. Whole genome sequences represent an important source for the phenotypic identification of vibrios.
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Nannya, Yasuito, Yoshida Kenichi, Keisuke Kataoka, Yasunobu Nagata, Tetsuichi Yoshizato, Tomoki Naoe, Hitoshi Kiyoi, et al. "Integrated Molecular Analysis of Myelodysplastic Syndromes Using Whole Genome Sequencing." Blood 128, no. 22 (December 2, 2016): 5512. http://dx.doi.org/10.1182/blood.v128.22.5512.5512.
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Abstract Background Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid malignancies characterized by refractory cytopenias with marrow dysplasia, which frequently progress to acute myeloid leukemia (AML). Although poorly understood in the previous era, the molecular events that underlie the pathogenesis of MDS have been intensively studied using advanced genomics in the past decade and are now fully catalogued into an array of well-defined functional pathways. However, mostly obtained through exome/targeted-capture sequencing, our knowledge about these molecular events is largely confined to those of single nucleotide variations (SNVs) and short indels, as well as arm-level copy number lesions, mostly within the coding sequences. Alterations in the non-coding regions, particularly a diversity of structural variations, in MDS genomes remain to be investigated in most part, even though the relevance of such lesions has recently been unequivocally demonstrated for other cancer types through large-scale whole genome sequencing (WGS) studies. Unfortunately, however, only a small number of MDS samples have been fully analyzes and inspected for genetic alterations using WGS. Patients and Methods In the present study, we performed an integrated, unbiased molecular study of 60 MDS cases, using whole genome sequencing (WGS) in combination with exome and transcriptome sequencing as well as methylome analysis. Paired tumor/germline DNA were obtained from patients' bone marrow and buccal smear samples. Sequencing data were analyzed using novel in-house pipelines, which were tuned to optimize detection of complex structural variations (SVs) and abnormalities in non-coding sequences. For some patients, multiple longitudinal materials were obtained along with their clinical course. Results WGS identified SNVs across the entire genome with a mean of 5.7/Mb/genome with a clear predominance of age-related C to T transitions, followed by other signatures. The spectrum of major targets of somatic mutations successfully recapitulated the previously reported one in MDS, including those involving splicing factors (SRSF2, SF3B1, U2AF1, and ZRSR2), epigenetic regulators (DNMT3A, ASXL1, TET2, BCOR, and EZH2), transcription factors (RUNX1, ETV6, and CUX1), signal transducing molecules (NRAS, KRAS, FLT3, PTPN11, CBL), and other critical molecules (TP53, NPM1, and STAG2). Moreover, other somatic variants within the coding regions were also identified that had already been reported in other human cancers but not in MDS, such as NCOR2X, MUC6, and TIAM2. The analysis of SVs unexpectedly revealed the complexity of MDS genomes. Most of the MDS genomes analyzed had a heavy burden of SVs including tandem duplications, deletions, translocations, and inversions, with a mean of 7.2/genome, which was far more than expected from conventional cytogenetics and array-based karyotyping. Complex rearrangements were common, frequently converging into particular chromosomes, suggesting multiple genetic events at a single genetic insult. Known targets of SNVs and indels were often affected by SVs, which largely escaped from conventional exome and targeted-capture sequencing, including RUNX1, TET2, FHITand other genes, suggesting that conventional platforms may substantially underestimate the frequency of alterations for some genes. Concomitant transcriptome analysis allowed to correlated abnormal splicing with somatic intronic events otherwise undetectable. Furthermore, comprehensive analysis of genomic aberrations in longitudinal samples enabled us to delineate the clonal architecture of the cellular population in MDS and their dynamics during the AML progression or clonal changes caused by AZA treatment. Conclusions Integrated molecular analysis using WGS and other platforms revealed the complexity of MDS genomes previously unexpected and reveal novel genetic alterations. Our results should help to extend our knowledge about the genomic landscape of MDS and provide novel insights into the molecular pathogenesis and clonal dynamics of MDS. Disclosures Kataoka: Kyowa Hakko Kirin: Honoraria; Boehringer Ingelheim: Honoraria; Yakult: Honoraria. Naoe:Pfizer Inc.: Research Funding; CMIC Co., Ltd.: Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Honoraria, Patents & Royalties, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Research Funding; Amgen Astellas BioPharma K.K.: Honoraria; TOYAMA CHEMICAL CO.,LTD.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Honoraria, Patents & Royalties; Celgene K.K.: Honoraria, Research Funding; Sumitomo Dainippon Pharma Co.,Ltd.: Honoraria, Research Funding; Fujifilm Corporation: Honoraria, Patents & Royalties, Research Funding; Bristol-Myers Squibb: Honoraria; Astellas Pharma Inc.: Research Funding. Kiyoi:Celgene Corporation: Consultancy; MSD K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Phizer Japan Inc.: Research Funding; Novartis Pharma K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; AlexionpharmaLLC.: Research Funding. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.
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Heaton, Michael P., Timothy P. L. Smith, Jacky K. Carnahan, Veronica Basnayake, Jiansheng Qiu, Barry Simpson, and Theodore S. Kalbfleisch. "Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with high-altitude pulmonary hypertension." F1000Research 5 (August 16, 2016): 2003. http://dx.doi.org/10.12688/f1000research.9254.1.
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The availability of whole genome sequence (WGS) data has made it possible to discover protein variantsin silico. However, existing bovine WGS databases do not show data in a form conducive to protein variant analysis, and tend to under represent the breadth of genetic diversity in U.S. beef cattle. Thus, our first aim was to use 96 beef sires, sharing minimal pedigree relationships, to create a searchable and publicly viewable set of mapped genomes relevant for 19 popular breeds of U.S. cattle. Our second aim was to identify protein variants encoded by the bovine endothelial PAS domain-containing protein 1 gene (EPAS1), a gene associated with high-altitude pulmonary hypertension in Angus cattle. The identity and quality of genomic sequences were verified by comparing WGS genotypes to those derived from other methods. The average read depth, genotype scoring rate, and genotype accuracy exceeded 14, 99%, and 99%, respectively. The 96 genomes were used to discover four amino acid variants encoded byEPAS1(E270Q, P362L, A671G, and L701F) and confirm two variants previously associated with disease (A606T and G610S). The sixEPAS1missense mutations were verified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assays, and their frequencies were estimated in a separate collection of 1154 U.S. cattle representing 46 breeds. A rooted phylogenetic tree of eight polypeptide sequences provided a framework for evaluating the likely order of mutations and potential impact ofEPAS1alleles on the adaptive response to chronic hypoxia in U.S. cattle. This public, whole genome resource facilitatesin silicoidentification of protein variants in diverse types of U.S. beef cattle, and provides a means of translating WGS data into a practical biological and evolutionary context for generating and testing hypotheses.
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Heaton, Michael P., Timothy P. L. Smith, Jacky K. Carnahan, Veronica Basnayake, Jiansheng Qiu, Barry Simpson, and Theodore S. Kalbfleisch. "Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with pulmonary hypertension." F1000Research 5 (October 5, 2016): 2003. http://dx.doi.org/10.12688/f1000research.9254.2.
Full textAbstract:
The availability of whole genome sequence (WGS) data has made it possible to discover protein variantsin silico. However, existing bovine WGS databases do not show data in a form conducive to protein variant analysis, and tend to under represent the breadth of genetic diversity in global beef cattle. Thus, our first aim was to use 96 beef sires, sharing minimal pedigree relationships, to create a searchable and publicly viewable set of mapped genomes relevant for 19 popular breeds of U.S. cattle. Our second aim was to identify protein variants encoded by the bovine endothelial PAS domain-containing protein 1 gene (EPAS1), a gene associated with pulmonary hypertension in Angus cattle. The identity and quality of genomic sequences were verified by comparing WGS genotypes to those derived from other methods. The average read depth, genotype scoring rate, and genotype accuracy exceeded 14, 99%, and 99%, respectively. The 96 genomes were used to discover four amino acid variants encoded byEPAS1(E270Q, P362L, A671G, and L701F) and confirm two variants previously associated with disease (A606T and G610S). The sixEPAS1missense mutations were verified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assays, and their frequencies were estimated in a separate collection of 1154 U.S. cattle representing 46 breeds. A rooted phylogenetic tree of eight polypeptide sequences provided a framework for evaluating the likely order of mutations and potential impact ofEPAS1alleles on the adaptive response to chronic hypoxia in U.S. cattle. This public, whole genome resource facilitatesin silicoidentification of protein variants in diverse types of U.S. beef cattle, and provides a means of translating WGS data into a practical biological and evolutionary context for generating and testing hypotheses.
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Roy, Sunando, John Hartley, Helen Dunn, Rachel Williams, Charlotte A. Williams, and Judith Breuer. "Whole-genome Sequencing Provides Data for Stratifying Infection Prevention and Control Management of Nosocomial Influenza A." Clinical Infectious Diseases 69, no. 10 (April 17, 2019): 1649–56. http://dx.doi.org/10.1093/cid/ciz020.
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Abstract Background Influenza A virus causes annual epidemics in humans and is associated with significant morbidity and mortality. Haemagglutinin (HA) and neuraminidase (NA) gene sequencing have traditionally been used to identify the virus genotype, although their utility in detecting outbreak clusters is still unclear. The objective of this study was to determine the utility, if any, of whole-genome sequencing over HA/NA sequencing for infection prevention and control (IPC) in hospitals. Methods We obtained all clinical samples from influenza (H1N1)-positive patients at the Great Ormond Street Hospital between January and March 2016. Samples were sequenced using targeted enrichment on an Illumina MiSeq sequencer. Maximum likelihood trees were computed for both whole genomes and concatenated HA/NA sequences. Epidemiological data was taken from routine IPC team activity during the period. Results Complete genomes were obtained for 65/80 samples from 38 patients. Conventional IPC analysis recognized 1 outbreak, involving 3 children, and identified another potential cluster in the haemato-oncology ward. Whole-genome and HA/NA phylogeny both accurately identified the previously known outbreak cluster. However, HA/NA sequencing additionally identified unrelated strains as part of this outbreak cluster. A whole-genome analysis identified a further cluster of 2 infections that had been previously missed and refuted suspicions of transmission in the haemato-oncology wards. Conclusions Whole-genome sequencing is better at identifying outbreak clusters in a hospital setting than HA/NA sequencing. Whole-genome sequencing could provide a faster and more reliable method for outbreak monitoring and supplement routine IPC team work to allow the prevention of transmission.
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Herniou, Elisabeth A., Teresa Luque, Xinwen Chen, Just M. Vlak, Doreen Winstanley, Jennifer S. Cory, and David R. O'Reilly. "Use of Whole Genome Sequence Data To Infer Baculovirus Phylogeny." Journal of Virology 75, no. 17 (September 1, 2001): 8117–26. http://dx.doi.org/10.1128/jvi.75.17.8117-8126.2001.
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ABSTRACT Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) andGranulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses.
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Strauß, Lena, Ulla Ruffing, Salim Abdulla, Abraham Alabi, Ruslan Akulenko, Marcelino Garrine, Anja Germann, et al. "Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology." Journal of Clinical Microbiology 54, no. 4 (January 27, 2016): 1008–16. http://dx.doi.org/10.1128/jcm.03022-15.
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Staphylococcus aureusis a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed anin silicotyping scheme for the software SeqSphere+(Ridom GmbH, Münster, Germany). The implemented target genes (n= 182) correspond to those queried by the IdentibacS. aureusGenotyping DNA microarray (Alere Technologies, Jena, Germany). Thein silicoscheme was evaluated by comparing the typing results of microarray and of WGS for 154 humanS. aureusisolates. A total of 96.8% (n= 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosomemecelement types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.
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Stapleton, Patrick, Alireza Eshaghi, Eddie Chong-King, Mark Cardona, Steve Masney, Aimin Li, Jonathan Gubbay, and Samir Patel. "Molecular Investigation of an Ontario Mumps Outbreak using Whole Genome Sequencing." Open Forum Infectious Diseases 4, suppl_1 (2017): S359. http://dx.doi.org/10.1093/ofid/ofx163.870.
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Abstract Background In early 2017 an outbreak of Mumps virus affected over 100 individuals in the province of Ontario, concurrent with multiple mumps virus outbreaks across North America. Traditional genotyping of mumps outbreaks relies on sequencing a portion of the small hydrophobic (SH) gene, but has limited capability to distinguish between strains of the same genotype. Most mumps cases in Ontario in recent years are of genotype G. We used a novel whole genome sequencing (WGS) protocol to perform a molecular epidemiological investigation of the outbreak. Methods Throat (n = 5) and buccal (n = 15) swabs positive by RT-PCR for SH or Fusion (F) gene targets were cultured in primary Rhesus monkey kidney cells. Cell free viral extract underwent RT-PCR and subsequent PCR amplification using overlapping primer pairs to cover the entire 15 kilobase (kb) genome. The first 8 samples were amplified with 18 pairs of overlapping primers, which was reduced to 9 sets (average fragment size 1.9 kb, range 1.6–2.8 kb) for the final 12 samples. Mumps cDNA libraries were prepared with Nextera XT kit and WGS of the indexed fragments was performed with V2 reagent kits on the Illumina MiSeq instrument. Reference based genome assembly was performed using samtools version 1.4. Phylogenetic analysis was performed by maximum likelihood method in MEGA7. Results We identified two distinct genotype G lineages comprised of 9 patients each and closely related to a 2009–2010 outbreak in Ontario and New York (Figure 1). Inter-lineage single nucleotide polymorphism (SNP) differences ranged from 25 to 31, whereas intra-lineage SNPs ranged from 0 to 8 SNPs. Two outlying sequences, of genotype C and G respectively, may represent sporadic introduction of virus from other areas. Time from virus isolation to SNP based analysis was approximately 4 days. Conclusion WGS of Mumps virus culture isolates using the PCR fragment method identified two distinct genotype G lineages in a large provincial outbreak. This method may aid public health authorities identify separate transmission chains in the case of large outbreaks. Disclosures All authors: No reported disclosures.