scholarly journals StrainSeeker: fast identification of bacterial strains from unassembled sequencing reads using user-provided guide trees.

2016 ◽  
Author(s):  
Mart Roosaare ◽  
Mihkel Vaher ◽  
Lauris Kaplinski ◽  
Mart Mols ◽  
Reidar Andreson ◽  
...  
Keyword(s):  
Rapid Identification ◽  
Clinical Diagnostics ◽  
Bacterial Strains ◽  
Bacterial Genomes ◽  
Newick Format ◽  
Novel Approach ◽  
Custom Made ◽  
High Throughput Detection ◽  
Generation Sequencing ◽  
Guide Trees

Background Fast, accurate and high-throughput detection of bacteria is in great demand. The present work was conducted to investigate the possibility of identifying both known and unknown bacterial strains from unassembled next-generation sequencing reads using custom-made guide trees. Results A program named StrainSeeker was developed that constructs a list of specific k-mers for each node of any given Newick-format tree and enables rapid identification of bacterial genomes within minutes. StrainSeeker has been tested and shown to successfully identify Escherichia coli strains from mixed samples in less than 5 minutes. StrainSeeker can also identify bacterial strains from highly diverse metagenomics samples. StrainSeeker is available at http://bioinfo.ut.ee/strainseeker. Conclusions Our novel approach can be useful for both clinical diagnostics and research laboratories because novel bacterial strains are constantly emerging and their fast and accurate detection is very important.

scholarly journals StrainSeeker: fast identification of bacterial strains from raw sequencing reads using user-provided guide trees

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3353 ◽  
Author(s):  
Märt Roosaare ◽  
Mihkel Vaher ◽  
Lauris Kaplinski ◽  
Märt Möls ◽  
Reidar Andreson ◽  
...  
Keyword(s):  
Bacterial Isolates ◽  
Web Interface ◽  
Bacterial Strains ◽  
Illumina Hiseq ◽  
Guide Tree ◽  
E Coli ◽  
Custom Made ◽  
Tree Branch ◽  
Correct Tree ◽  
Guide Trees

Background Fast, accurate and high-throughput identification of bacterial isolates is in great demand. The present work was conducted to investigate the possibility of identifying isolates from unassembled next-generation sequencing reads using custom-made guide trees. Results A tool named StrainSeeker was developed that constructs a list of specific k-mers for each node of any given Newick-format tree and enables the identification of bacterial isolates in 1–2 min. It uses a novel algorithm, which analyses the observed and expected fractions of node-specific k-mers to test the presence of each node in the sample. This allows StrainSeeker to determine where the isolate branches off the guide tree and assign it to a clade whereas other tools assign each read to a reference genome. Using a dataset of 100 Escherichia coli isolates, we demonstrate that StrainSeeker can predict the clades of E. coli with 92% accuracy and correct tree branch assignment with 98% accuracy. Twenty-five thousand Illumina HiSeq reads are sufficient for identification of the strain. Conclusion StrainSeeker is a software program that identifies bacterial isolates by assigning them to nodes or leaves of a custom-made guide tree. StrainSeeker’s web interface and pre-computed guide trees are available at http://bioinfo.ut.ee/strainseeker. Source code is stored at GitHub: https://github.com/bioinfo-ut/StrainSeeker.

scholarly journals A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marie-Ming Aynaud ◽  
J. Javier Hernandez ◽  
Seda Barutcu ◽  
Ulrich Braunschweig ◽  
Kin Chan ◽  
...  
Keyword(s):  
Large Scale ◽  
Disease Onset ◽  
Clinical Diagnostics ◽  
Viral Pathogens ◽  
Sequencing Platform ◽  
Viral Loads ◽  
Quality Control Metrics ◽  
High Throughput Detection ◽  
Automated Platform ◽  
Generation Sequencing

AbstractPopulation scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe “Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening” (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

scholarly journals SMS: A Novel Approach for Bacterial Strain Genome Reconstruction in Multiple Samples

2021 ◽  
Author(s):  
Saidi Wang ◽  
Minerva Fatimae Ventolero ◽  
Haiyan Hu ◽  
Xiaoman Li
Keyword(s):  
Bacterial Strain ◽  
De Novo ◽  
Superior Performance ◽  
Accurate Estimation ◽  
Bacterial Strains ◽  
Bacterial Genomes ◽  
Novel Approach ◽  
Strain Number ◽  
Almost All ◽  
Multiple Samples

Abstract BackgroundThe analysis of the bacterial strains is important for the understanding of drug resistance. Despite the existence of dozens of computational tools for bacterial strain studies, most of them are developed for the limited number of known bacterial strains. Almost all remaining tools are designed to analyze individual samples or local regions instead of the entire bacterial genomes. With multiple shotgun metagenomic samples routinely generated in a project, it is necessary to create methods to reconstruct novel bacterial strain genomes in multiple samples.ResultsWe developed a novel computational approach called SMS to de novo reconstruct bacterial Strain genomes from Multiple shotgun sequencing Samples. Tested on 702 simulated and 195 experimental datasets, SMS reliably identified the strain number, strain abundance, and strain polymorphisms. Compared with two existing approaches, SMS showed superior performance in terms of more accurate estimation of the strain number, abundance and variations.ConclusionsSMS is a useful tool for novel bacterial strain reconstruction in multiple shotgun metagenomic samples.

scholarly journals Diversity of Biodeteriorative Bacterial and Fungal Consortia in Winter and Summer on Historical Sandstone of the Northern Pergola, Museum of King John III’s Palace at Wilanow, Poland

2021 ◽  
Vol 11 (2) ◽  
pp. 620
Author(s):  
Magdalena Dyda ◽  
Agnieszka Laudy ◽  
Przemyslaw Decewicz ◽  
Krzysztof Romaniuk ◽  
Martyna Ciezkowska ◽  
...  
Keyword(s):  
Seasonal Changes ◽  
Microbial Community Composition ◽  
Fungal Communities ◽  
Bacterial Strains ◽  
Microbial Biodiversity ◽  
Acidophilic Bacteria ◽  
Almost All ◽  
Generation Sequencing ◽  
King John

The aim of the presented investigation was to describe seasonal changes of microbial community composition in situ in different biocenoses on historical sandstone of the Northern Pergola in the Museum of King John III’s Palace at Wilanow (Poland). The microbial biodiversity was analyzed by the application of Illumina-based next-generation sequencing methods. The metabarcoding analysis allowed for detecting lichenized fungi taxa with the clear domination of two genera: Lecania and Rhinocladiella. It was also observed that, during winter, the richness of fungal communities increased in the biocenoses dominated by lichens and mosses. The metabarcoding analysis showed 34 bacterial genera, with a clear domination of Sphingomonas spp. across almost all biocenoses. Acidophilic bacteria from Acidobacteriaceae and Acetobacteraceae families were also identified, and the results showed that a significant number of bacterial strains isolated during the summer displayed the ability to acidification in contrast to strains isolated in winter, when a large number of isolates displayed alkalizing activity. Other bacteria capable of nitrogen fixation and hydrocarbon utilization (including aromatic hydrocarbons) as well as halophilic microorganisms were also found. The diversity of organisms in the biofilm ensures its stability throughout the year despite the differences recorded between winter and summer.

scholarly journals 1564. Activity of bacteriophage against multidrug resistant Klebsiella pneumoniae

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S782-S782
Author(s):  
Sailaja Puttagunta ◽  
Maya Kahan-Haanum ◽  
Sharon Kredo-Russo ◽  
Eyal Weinstock ◽  
Efrat Khabra ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Unmet Need ◽  
Multidrug Resistant ◽  
Beta Lactamase ◽  
Bacterial Strains ◽  
Antibiotic Resistant ◽  
Extended Spectrum Beta Lactamase ◽  
Novel Approach ◽  
Infection Types

Abstract Background The prevalence of extended-spectrum beta-lactamase (ESBL) producing and carbapenem resistant (CR) Klebsiella pneumoniae (KP) has significantly risen in all geographic regions. Infections due to these bacteria are associated with high mortality across different infection types. Even with newer options, there remains an unmet need for safe and effective therapeutic options to treat infections caused by ESBL and CR KP. Phage therapy offers a novel approach with an unprecedented and orthogonal mechanism of action for treatment of diseases caused by pathogenic bacterial strains that are insufficiently addressed by available antibiotics. Phage-based therapies confer a high strain-level specificity and have a strong intrinsic safety profile. Here we describe the identification of novel phages that can effectively target antibiotic resistant KP strains. Host range of the 21 phages on 33 strain KP panel via solid culture infectivity assays. Red marks resistance to infection while sensitivity to phage is marked in green Methods KP clinical strains were isolated from human stool specimens preserved in glycerol. Selective culturing was carried, followed by testing of individual colonies for motility, indole and urease production, sequenced and analyzed by Kleborate tool to determine antibiotic resistant genes. Natural phages were isolated from plaques that developed on susceptible bacterial targets, sequenced and characterized. Results Antibiotic-resistant KP strains encoding beta lactamase genes or a carbapenemase (n=33) were isolated from healthy individuals (n=3), and patients with inflammatory bowel disease (n=26) or primary sclerosing cholangitis (n=3). Isolates sequencing revealed bla CTX-M15 and/or bla SHV encoding strains and carbapenamase KPC-2. A panel of 21 phages targeting the beta-lactamase- and carbapenemase-producing KP strains were identified. Phage sequencing revealed that all phages belong to the Caudovirales order and include 6 Siphoviridae, 14 Myoviridae, and 1 Podoviridae. In vitro lytic activity of the phages was tested on the isolated bacteria and revealed a coverage of 70% of the 33 isolated antibiotic resistant strains, >50% of which were targeted by multiple phages. Conclusion Collectively, these results demonstrate the feasibility of identifying phage with potent activity against antibiotic resistant KP strains, and may provide a novel therapeutic approach for treatment of ESBL and CR KP infections. Disclosures All Authors: No reported disclosures

scholarly journals Next-Generation Sequencing: From Basic Research to Diagnostics

2009 ◽  
Vol 55 (4) ◽  
pp. 641-658 ◽  
Author(s):  
Karl V Voelkerding ◽  
Shale A Dames ◽  
Jacob D Durtschi
Keyword(s):  
Next Generation Sequencing ◽  
Dna Sequencing ◽  
Single Molecule ◽  
Basic Research ◽  
Clinical Diagnostics ◽  
Massively Parallel ◽  
Next Generation ◽  
New Paradigm ◽  
The Impact ◽  
Generation Sequencing

Abstract Background: For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). Content: This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. Summary: In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

scholarly journals Erratum to: Evaluating next-generation sequencing for direct clinical diagnostics in diarrhoeal disease

2017 ◽  
Vol 36 (7) ◽  
pp. 1339-1342
Author(s):  
K. G. Joensen ◽  
A. L. Ø. Engsbro ◽  
O. Lukjancenko ◽  
R. S. Kaas ◽  
O. Lund ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Diarrhoeal Disease ◽  
Clinical Diagnostics ◽  
Next Generation ◽  
Generation Sequencing

Concealed cardiomyopathy as a frequent cause of idiopathic ventricular fibrillation in a representative Czech cohort of survivors of sudden cardiac arrest (SCA)

EP Europace ◽  
2021 ◽  
Vol 23 (Supplement_3) ◽  
Author(s):  
A Krebsova ◽  
P Votypka ◽  
P Peldova ◽  
J Haskova ◽  
T Tavacova ◽  
...  
Keyword(s):  
Cardiac Arrest ◽  
Genetic Testing ◽  
Ventricular Fibrillation ◽  
Dna Sequencing ◽  
Clinical Diagnostics ◽  
Idiopathic Ventricular Fibrillation ◽  
Custom Made ◽  
Diagnostic Work ◽  
Work Up ◽  
Diagnostic Work Up

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Health of the Czech Republic Introduction The complex diagnostic work up in SCA survivors often does not yield a concrete cardiological diagnosis. Moreover, there is conflicting evidence whether genetic testing could support or guide clinical diagnostics. Purpose To assess the molecular architecture of idiopathic ventricular fibrillation in cases without apparent evidence of specific structural or arrhythmic cardiac disease at initial diagnostic work up in a representative Czech cohort. Patients and Methods Between 2013 - 2020 we have ascertained 100 SCA survivors (54 M / 46 F; age range at cardiac arrest 5-69 years). Genetic counselling was followed by massively parallel DNA sequencing using custom-made panels comprising 100 cardiac conditions-related genes. Subsequently, thorough cardiological screening examinations in first degree relatives were carried out. Presence of pathogenic variants was validated by Sanger DNA sequencing and through family segregation analyses. Results Highly likely or certain molecular aetiology (i.e. based on the presence of Class 4 or 5 variants) was disclosed in 20/100 (20%) in PKP2 (3x), SCN5A (4x), RYR2 (3x), TTN (2x), PLN, FLNC, PRKAG2, KCNH2, KCNQ1, SLC4A, TNNT2, and DSP. Interestingly, the KCNE1 p.Asp85Asn (LQT 5 lite) variant, was detected in further 3/100 cases, representing a recognized risk factor for ventricular arrhythmias. Conclusions Genetic testing facilitates stratification of the cause of arrhythmia in a substantial portion of SCA survivors. The utility of positive outcomes of genetic testing was substantiated in 10/20 gene positive patients, where the genetic stratification led to diagnosis of concealed arrhythmogenic cardiomyopathy, whose extent of morphological changes was under the diagnostic sensibility of imaging modalities or ECG. Our results enable individualized care in SCA survivors and assure targeted preventive approaches in their relatives.

scholarly journals Novel High-Throughput Detection Method To Assess Bacterial Surfactant Production

2010 ◽  
Vol 76 (16) ◽  
pp. 5363-5372 ◽  
Author(s):  
Adrien Y. Burch ◽  
Briana K. Shimada ◽  
Patrick J. Browne ◽  
Steven E. Lindow
Keyword(s):  
Pseudomonas Syringae ◽  
High Throughput ◽  
Water Drop ◽  
Bacterial Strains ◽  
Swarming Motility ◽  
Detection Assay ◽  
Wide Range ◽  
Surfactant Production ◽  
Transposon Mutants ◽  
High Throughput Detection

ABSTRACT A novel biosurfactant detection assay was developed for the observation of surfactants on agar plates. By using an airbrush to apply a fine mist of oil droplets, surfactants can be observed instantaneously as halos around biosurfactant-producing colonies. This atomized oil assay can detect a wide range of different synthetic and bacterially produced surfactants. This method could detect much lower concentrations of many surfactants than a commonly used water drop collapse method. It is semiquantitative and therefore has broad applicability for uses such as high-throughput mutagenesis screens of biosurfactant-producing bacterial strains. The atomized oil assay was used to screen for mutants of the plant pathogen Pseudomonas syringae pv. syringae B728a that were altered in the production of biosurfactants. Transposon mutants displaying significantly altered surfactant halos were identified and further analyzed. All mutants identified displayed altered swarming motility, as would be expected of surfactant mutants. Additionally, measurements of the transcription of the syringafactin biosynthetic cluster in the mutants, the principal biosurfactant known to be produced by B728a, revealed novel regulators of this pathway.

scholarly journals Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species

2013 ◽  
Vol 10 (1) ◽  
pp. 45 ◽  
Author(s):  
Melissa E Carew ◽  
Vincent J Pettigrove ◽  
Leon Metzeling ◽  
Ary A Hoffmann
Keyword(s):  
Next Generation Sequencing ◽  
Environmental Monitoring ◽  
Rapid Identification ◽  
Next Generation ◽  
Bioindicator Species ◽  
Generation Sequencing
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