scholarly journals Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Elton J. R. Vasconcelos ◽  
Chayan Roy ◽  
Joseph A. Geiger ◽  
Kristina M. Oney ◽  
Melody Koo ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Next Generation Sequencing ◽  
Complete Analysis ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
Spotted Fever Group ◽  
Next Generation ◽  
Vector Borne ◽  
16 S Rrna ◽  
Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

scholarly journals Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

2020 ◽  
Vol 9 (1) ◽  
pp. 21
Author(s):  
Abdul Ghafar ◽  
Anson V. Koehler ◽  
Ross S. Hall ◽  
Charles G. Gauci ◽  
Robin B. Gasser ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Water Buffalo ◽  
Hypervariable Region ◽  
Small Subunit ◽  
Rrna Gene ◽  
Next Generation ◽  
Small Subunit Rrna ◽  
Tropical Theileriosis ◽  
Ecological Zones ◽  
Generation Sequencing

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

scholarly journals Next-Generation Sequencing for Pathogen Identification in Infected Foot Ulcers

2020 ◽  
Vol 5 (4) ◽  
pp. 2473011420S0002
Author(s):  
Yoonjung Choi ◽  
Irvin Oh
Keyword(s):  
Next Generation Sequencing ◽  
False Positive ◽  
Bacterial Genome ◽  
Rrna Gene ◽  
Next Generation ◽  
Antibiotic Resistant ◽  
Streptococcus Species ◽  
Resistant Genes ◽  
Standard Culture ◽  
Generation Sequencing

Category: Other Introduction/Purpose: Foot infections are often polymicrobial with diverse microbiomes. Accurate identification of the main pathogen in diabetic foot ulcer (DFU) remain challenging due to contamination or negative cultures often leading to ineffective post-surgical antibiotic treatment. Application of molecular diagnostics, such as next generation sequencing (NGS) has been explored as an alternative to standard culture in orthopaedic infections. NGS is highly sensitive and detects an entire bacterial genome along with pharmacologic resistant genes in a given sample. We sought to investigate the potential use of NGS for accurate diagnosis and quantification of various species in infected DFU. We hypothesize that NGS will provide a more accurate means of diagnosing and profiling microorganisms in infected DFU compared to the standard culture method. Methods: We investigated 30 infected DFU patients who underwent surgical treatment by a single academic orthopaedic surgeon from October 2018 to September 2019. The average age of the patient was 60.4 (range 33-82) years-old. Surgical procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Quantitative PCR was performed to determine the bacterial burden present in the sample. DNA was amplified by PCR from a highly conserved region of the rRNA gene in the bacteria (16S rRNA). Once a high level of DNA was generated and determined, it was compared against NIH GenBank database. Concordance between the standard culture and NGS was assessed. Results: In 28 of 29 patients, pathogens were identified by both NGS and culture, with complete consistency of organisms in 13 cases (concordance rate: 43.3%). NGS provided relative quantitative measures and the presence of antibiotic resistant genes for each pathogen. In NGS, Anaerococcus species (79.3%) was the most common organism, followed by Streptococcus species (44.8%), Prevotella species (44.8%), Finegoldia magna (44.8%). In culture, S. aureus (58.6%) was the most common, followed by Streptococcus species (34.5%), coagulase-negative Staphylococci (24.1%), Corynebacterium species (20.7%). On average, NGS revealed 5.1 (1-11) number of pathogens, whereas standard culture revealed 2.6 (1-6) pathogens in a given sample. NGS identified 2 cases with false positive standard culture and detected antibiotic resistant organisms in 15 specimens. Conclusion: NGS is an emerging method of microbial identification in orthopedic infection. It is particularly helpful in profiling diverse microbes in polymicrobial infected DFU. It can identify major pathogens and may correct false positive or false negative culture. NGS may allow a faster invitation of postoperative targeted antibiotic therapy. [Table: see text]

scholarly journals Clinical Application of Next-Generation Sequencing of Plasma Cell-Free DNA for Genotyping Untreated Advanced Non-Small Cell Lung Cancer

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2707
Author(s):  
Maria Gabriela O. Fernandes ◽  
Natália Cruz-Martins ◽  
Conceição Souto Moura ◽  
Susana Guimarães ◽  
Joana Pereira Reis ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Clinical Outcomes ◽  
Test Performance ◽  
Molecular Diagnostic ◽  
Test Accuracy ◽  
Newly Diagnosed ◽  
Next Generation ◽  
Clinical Value ◽  
Generation Sequencing

Background: Analysis of circulating tumor DNA (ctDNA) has remarkable potential as a non-invasive lung cancer molecular diagnostic method. This prospective study addressed the clinical value of a targeted-gene amplicon-based plasma next-generation sequencing (NGS) assay to detect actionable mutations in ctDNA in patients with newly diagnosed advanced lung adenocarcinoma. Methods: ctDNA test performance and concordance with tissue NGS were determined, and the correlation between ctDNA findings, clinical features, and clinical outcomes was evaluated in 115 patients with paired plasma and tissue samples. Results: Targeted-gene NGS-based ctDNA and NGS-based tissue analysis detected 54 and 63 genomic alterations, respectively; 11 patients presented co-mutations, totalizing 66 hotspot mutations detected, 51 on both tissue and plasma, 12 exclusively on tissue, and 3 exclusively on plasma. NGS-based ctDNA revealed a diagnostic performance with 81.0% sensitivity, 95.3% specificity, 94.4% PPV, 83.6% NPV, test accuracy of 88.2%, and Cohen’s Kappa 0.764. PFS and OS assessed by both assays did not significantly differ. Detection of ctDNA alterations was statistically associated with metastatic disease (p = 0.013), extra-thoracic metastasis (p = 0.004) and the number of organs involved (p = 0.010). Conclusions: This study highlights the potential use of ctDNA for mutation detection in newly diagnosed NSCLC patients due to its high accuracy and correlation with clinical outcomes.

scholarly journals Serra da Estrela PDO Cheese Microbiome as Revealed by Next Generation Sequencing

2021 ◽  
Vol 9 (10) ◽  
pp. 2007
Author(s):  
Rui Rocha ◽  
Manuela Vaz Velho ◽  
Joana Santos ◽  
Paulo Fernandes
Keyword(s):  
Next Generation Sequencing ◽  
Relative Abundance ◽  
Raw Materials ◽  
Rrna Gene ◽  
Next Generation ◽  
Candida Spp ◽  
Characterization Study ◽  
Materials Used ◽  
Serra Da Estrela ◽  
Generation Sequencing

Serra da Estrela PDO cheese is the oldest traditional cheese manufactured in Portugal. In this work, its microbiome as well as the main raw materials used in cheese production, raw ewes’ milk and thistle flowers (Cynara cardunculus L.), were characterized using next generation sequencing. Samples were accordingly retrieved from a local producer over two consecutive production campaigns and at different time periods within each campaign. The bacterial and fungi communities associated with each matrix were accessed through sequencing of V3−V4 and Internal Transcribed Spacer 2 regions of rRNA gene amplicons, respectively. A high microbial diversity was found associated to each matrix, differing significantly (p < 0.05) from each other. Over 500 taxa were identified in each analyzed matrix, ranging from dominant (relative abundance > 1%), sub-dominant (0.01−1%) and rare taxa (<0.01%). Specifically, in cheese, 30 taxa were present in all analyzed samples (core taxa), including species of Leuconostoc spp. and Lactococcus spp. for bacteria and Candida spp., Debaryomyces spp. and Yarrowia spp. for fungi, that were cumulatively the most prevalent genera in Serra da Estrela PDO cheese (average relative abundance ≥10%). Ultimately, this characterization study may contribute to a better understanding of the microbial dynamics of this traditional PDO product, namely the influence of raw materials on cheese microbiome, and could assist producers interested in preserving the identity, quality and safety of Serra da Estrela PDO cheese.

scholarly journals Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

2021 ◽  
Vol 15 (10) ◽  
pp. e0009779
Author(s):  
Fakhriddin Sarzhanov ◽  
Funda Dogruman-Al ◽  
Monica Santin ◽  
Jenny G. Maloney ◽  
Ayse Semra Gureser ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Gastrointestinal Symptoms ◽  
Molecular Methods ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Next Generation ◽  
Dientamoeba Fragilis ◽  
Significant Difference ◽  
Generation Sequencing ◽  
Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

scholarly journals Identification of Genetic Hereditary Predisposition to Hematologic Malignancies By Clinical Next-Generation Sequencing

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3854-3854 ◽  
Author(s):  
Amy E Knight Johnson ◽  
Lucia Guidugli ◽  
Kelly Arndt ◽  
Gorka Alkorta-Aranburu ◽  
Viswateja Nelakuditi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Family Members ◽  
Hematologic Malignancies ◽  
Dyskeratosis Congenita ◽  
Molecular Diagnostic ◽  
Next Generation ◽  
Hereditary Predisposition ◽  
Ngs Data ◽  
Generation Sequencing

Abstract Introduction: Myelodysplastic syndrome (MDS) and acute leukemia (AL) are a clinically diverse and genetically heterogeneous group of hematologic malignancies. Familial forms of MDS/AL have been increasingly recognized in recent years, and can occur as a primary event or secondary to genetic syndromes, such as inherited bone marrow failure syndromes (IBMFS). It is critical to confirm a genetic diagnosis in patients with hereditary predisposition to hematologic malignancies in order to provide prognostic information and cancer risk assessment, and to aid in identification of at-risk or affected family members. In addition, a molecular diagnosis can help tailor medical management including informing the selection of family members for allogeneic stem cell transplantation donors. Until recently, clinical testing options for this diverse group of hematologic malignancy predisposition genes were limited to the evaluation of single genes by Sanger sequencing, which is a time consuming and expensive process. To improve the diagnosis of hereditary predisposition to hematologic malignancies, our CLIA-licensed laboratory has recently developed Next-Generation Sequencing (NGS) panel-based testing for these genes. Methods: Thirty six patients with personal and/or family history of aplastic anemia, MDS or AL were referred for clinical diagnostic testing. DNA from the referred patients was obtained from cultured skin fibroblasts or peripheral blood and was utilized for preparing libraries with the SureSelectXT Enrichment System. Libraries were sequenced on an Illumina MiSeq instrument and the NGS data was analyzed with a custom bioinformatic pipeline, targeting a panel of 76 genes associated with IBMFS and/or familial MDS/AL. Results: Pathogenic and highly likely pathogenic variants were identified in 7 out of 36 patients analyzed, providing a positive molecular diagnostic rate of 20%. Overall, 6 out of the 7 pathogenic changes identified were novel. In 2 unrelated patients with MDS, heterozygous pathogenic sequence changes were identified in the GATA2 gene. Heterozygous pathogenic changes in the following autosomal dominant genes were each identified in a single patient: RPS26 (Diamond-Blackfan anemia 10), RUNX1 (familial platelet disorder with propensity to myeloid malignancy), TERT (dyskeratosis congenita 4) and TINF2 (dyskeratosis congenita 3). In addition, one novel heterozygous sequence change (c.826+5_826+9del, p.?) in the Fanconi anemia associated gene FANCA was identified. . The RNA analysis demonstrated this variant causes skipping of exon 9 and results in a premature stop codon in exon 10. Further review of the NGS data provided evidence of an additional large heterozygous multi-exon deletion in FANCA in the same patient. This large deletion was confirmed using array-CGH (comparative genomic hybridization). Conclusions: This study demonstrates the effectiveness of using NGS technology to identify patients with a hereditary predisposition to hematologic malignancies. As many of the genes associated with hereditary predisposition to hematologic malignancies have similar or overlapping clinical presentations, analysis of a diverse panel of genes is an efficient and cost-effective approach to molecular diagnostics for these disorders. Unlike Sanger sequencing, NGS technology also has the potential to identify large exonic deletions and duplications. In addition, RNA splicing assay has proven to be helpful in clarifying the pathogenicity of variants suspected to affect splicing. This approach will also allow for identification of a molecular defect in patients who may have atypical presentation of disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Next-generation sequencing in veterinary medicine - a review: part II

2020 ◽  
Vol 51 (3) ◽  
pp. 305-315
Author(s):  
Ksenija Vlahović ◽  
Maja Popović ◽  
Luka Pajurin ◽  
Daniel Špoljarić ◽  
Marina Pavlak ◽  
...  
Keyword(s):  
Veterinary Medicine ◽  
Next Generation Sequencing ◽  
Next Generation ◽  
Generation Sequencing

Nova generacija sekvenciranja predstavlja znatan tehnološki napredak koji omogućuje velik napredak u poznavanju genoma životinja te sve širu primjenu u različitim područjima veterinarske medicine. Danas se napredne tehnologije primjenjuju u sekvenciranju cijelog genoma životinja, sekvenciranju njihovih egzoma, ciljanom sekvenciranju DNK fragmenata i sekvenciranju RNK. Ovaj pregled usmjeren je na trenutačna dostignuća, primjenu i izazove povezane s uporabom naprednih tehnologija sekvenciranja. Prikazana je i primjena tehnologije nove generacije sekvenciranja u genomici životinja kao i njezin daljnji razvoj i buduća primjena.

scholarly journals Tumor BRCA Test for Patients with Epithelial Ovarian Cancer: The Role of Molecular Pathology in the Era of PARP Inhibitor Therapy

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1641 ◽  
Author(s):  
Caterina Fumagalli ◽  
Federica Tomao ◽  
Ilaria Betella ◽  
Alessandra Rappa ◽  
Mariarosaria Calvello ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Next Generation Sequencing ◽  
Epithelial Ovarian Cancer ◽  
Success Rate ◽  
Parp Inhibitor ◽  
Turnaround Time ◽  
Molecular Diagnostic ◽  
Next Generation ◽  
Clinical Implementation ◽  
Generation Sequencing

The PARP inhibitor olaparib has been approved in the maintenance setting of platinum-sensitive epithelial ovarian cancer patients with germline or somatic BRCA1/2 mutation. Therefore, the availability of a tumor BRCA test has become a clinical need. We report the results of the clinical implementation of a tumor BRCA test within the frame of an institutional workflow for the management of patients with nonmucinous and nonborderline epithelial ovarian cancer. In total, 223 patients with epithelial ovarian cancer were prospectively analyzed. BRCA1/2 status was evaluated on formalin-fixed, paraffin-embedded tumor specimens using next-generation sequencing technology. The tumor BRCA test had a success rate of 99.1% (221 of 223 successfully analyzed cases) and a median turnaround time of 17 calendar days. Among the 221 cases, BRCA1 or BRCA2 pathogenic/likely pathogenic mutations were found in 62 (28.1%) cases and variants of uncertain significance in 25 (11.3%) cases. The concordance rate between tumor BRCA test results and germline BRCA1/2 status was 87%, with five cases harboring pathogenic/likely pathogenic somatic-only mutations. The next-generation, sequencing-based tumor BRCA test showed a high success rate and a turnaround time compatible with clinical purposes. The tumor BRCA test could be implemented in a molecular diagnostic setting and it may guide the clinical management of patients with epithelial ovarian cancer.

scholarly journals Next-Generation Sequencing Identifies Gene Mutations That Are Predictive of Malignancy in Residual Needle Rinses Collected From Fine-Needle Aspirations of Thyroid Nodules

2017 ◽  
Vol 142 (2) ◽  
pp. 178-183 ◽  
Author(s):  
Maren Y. Fuller ◽  
Dina Mody ◽  
April Hull ◽  
Kristi Pepper ◽  
Heather Hendrickson ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Thyroid Nodules ◽  
Gene Mutations ◽  
Braf V600e ◽  
Molecular Diagnostic ◽  
Thyroid Malignancy ◽  
Next Generation ◽  
Fine Needle ◽  
Thyroid Fna ◽  
Generation Sequencing

Context.— Thyroid nodules have a prevalence of approximately 70% in adults. Fine-needle aspiration (FNA) is a minimally invasive, cost-effective, standard method to collect tissue from thyroid nodules for cytologic examination. However, approximately 15% of thyroid FNA specimens cannot be unambiguously diagnosed as benign or malignant. Objective.— To investigate whether clinically actionable data can be obtained using next-generation sequencing of residual needle rinse material. Design.— A total of 24 residual needle rinse specimens with malignant (n = 6), indeterminate (n = 9), or benign (n = 9) thyroid FNA diagnoses were analyzed in our clinical molecular diagnostics laboratory using next-generation sequencing assays designed to detect gene mutations and translocations that commonly occur in thyroid cancer. Results were correlated with surgical diagnoses and clinical outcomes. Results.— Interpretable data were generated from 23 of 24 residual needle rinse specimens. Consistent with its well-known role in thyroid malignancy, BRAF V600E mutations were detected in 4 malignant cases. An NRAS mutation was detected in 1 benign case. No mutations were detected from specimens with indeterminate diagnoses. Conclusions.— Our data demonstrate that residual thyroid FNA needle rinses are an adequate source of material for molecular diagnostic testing. Importantly, detection of a mutation implicated in thyroid malignancy was predictive of the final surgical diagnosis and clinical outcome. Our strategy to triage thyroid nodules with indeterminate cytology with molecular testing eliminates the need to perform additional FNA passes into dedicated media or to schedule additional invasive procedures. Further investigation with a larger sample size to confirm the clinical utility of our proposed strategy is underway.

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