scholarly journals Assessment of metagenomic sequencing and qPCR for detection of influenza D virus in bovine respiratory tract samples

2020 ◽  
Author(s):  
Maodong Zhang ◽  
Yanyun Huang ◽  
Dale L. Godson ◽  
Champika Fernando ◽  
Trevor W. Alexander ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Sequence Detection ◽  
Nasal Swabs ◽  
Sequencing Platforms ◽  
Species Specific

AbstractHigh throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What’s In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

scholarly journals Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 814 ◽  
Author(s):  
Maodong Zhang ◽  
Yanyun Huang ◽  
Dale L. Godson ◽  
Champika Fernando ◽  
Trevor W. Alexander ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
Sequence Detection ◽  
Nasal Swabs ◽  
Sequencing Platforms ◽  
Species Specific

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What’s In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

scholarly journals Sequencing and Computational Approaches to Identification and Characterization of Microbial Organisms

2013 ◽  
Vol 5 ◽  
pp. BECB.S10886 ◽  
Author(s):  
Brijesh Singh Yadav ◽  
Venkateswarlu Ronda ◽  
Dinesh P. Vashista ◽  
Bhaskar Sharma
Keyword(s):  
Sequence Data ◽  
Microbial Interactions ◽  
Microbial Pathogens ◽  
Nucleotide Sequence Data ◽  
Computational Approaches ◽  
Microbial Detection ◽  
Sequencing Technologies ◽  
Sequencing Platforms ◽  
Identification And Characterization

The recent advances in sequencing technologies and computational approaches are propelling scientists ever closer towards complete understanding of human-microbial interactions. The powerful sequencing platforms are rapidly producing huge amounts of nucleotide sequence data which are compiled into huge databases. This sequence data can be retrieved, assembled, and analyzed for identification of microbial pathogens and diagnosis of diseases. In this article, we present a commentary on how the metagenomics incorporated with microarray and new sequencing techniques are helping microbial detection and characterization.

scholarly journals The Power of Using Novel Nanopore Sequencing Technology for Diagnosis, Genomic and Pathological Studies of Covid-19

2021 ◽  
Vol 2 (3) ◽  
pp. 4014-4028
Author(s):  
Chenghao Du
Keyword(s):  
Analytical Methods ◽  
Diagnostic Methods ◽  
Public Access ◽  
Genomic Research ◽  
Metagenomic Sequencing ◽  
Nanopore Sequencing ◽  
The Novel ◽  
Sequencing Technology ◽  
Different Types ◽  
Sequencing Platforms

The novel coronavirus disease 2019 (COVID‐19), originally identified in December 2019 Wuhan, China, has propagated to worldwide pandemic, causing many cases of death and morbidity. Since the development of COVID-19 vaccines is still under experimental stages without public access, different types of testing and detection ensuring rapid and accurate results are urgently required to prevent delaying isolation of infected patients. The traditional diagnostic and analytical methods of COVID-19 relied heavily on nucleic acid and antibody-antigen methods but are subject to assembly bias, restricted by reading length, showed some false positive/negative results and had a long turnaround time. Hence, three styles of nanopore sequencing techniques as complementary tools for COVID-19 diagnosis and analysis are introduced. The long-read nanopore sequencing technology has been adopted in metagenomic and pathological studies of virosphere including SARS-CoV-2 recently by either metagenomically, directly or indirectly sequencing the viral genomic RNA of SARS-CoV-2 in real-time to detect infected specimens for early isolation and treatment, to investigate the transmission and evolutionary routes of SARS-CoV-2 as well as its pathogenicity and epidemiology. In this article, the Nanopore-Based Metagenomic Sequencing, Direct RNA Nanopore Sequencing (DRS), and Nanopore Targeted Sequencing (NTS) become the main focus of the novel COVID-19 detecting analytical methods in sequencing platforms, which are discussed in comparison with other traditional and popular diagnostic methods. Finally, different types of nanopore sequencing platforms that are developed by Oxford Nanopore Technologies (ONT) due to various purposes and demands in viral genomic research are briefly discussed.

scholarly journals DNA Metabarcoding for the Characterization of Terrestrial Microbiota—Pitfalls and Solutions

2021 ◽  
Vol 9 (2) ◽  
pp. 361
Author(s):  
Davide Francioli ◽  
Guillaume Lentendu ◽  
Simon Lewin ◽  
Steffen Kolb
Keyword(s):  
High Throughput Sequencing ◽  
Methodological Approach ◽  
Soil Microbial Communities ◽  
Cost Effective ◽  
Trophic Levels ◽  
Gene Markers ◽  
Dna Metabarcoding ◽  
Terrestrial Environments ◽  
Sequencing Platforms

Soil-borne microbes are major ecological players in terrestrial environments since they cycle organic matter, channel nutrients across trophic levels and influence plant growth and health. Therefore, the identification, taxonomic characterization and determination of the ecological role of members of soil microbial communities have become major topics of interest. The development and continuous improvement of high-throughput sequencing platforms have further stimulated the study of complex microbiota in soils and plants. The most frequently used approach to study microbiota composition, diversity and dynamics is polymerase chain reaction (PCR), amplifying specific taxonomically informative gene markers with the subsequent sequencing of the amplicons. This methodological approach is called DNA metabarcoding. Over the last decade, DNA metabarcoding has rapidly emerged as a powerful and cost-effective method for the description of microbiota in environmental samples. However, this approach involves several processing steps, each of which might introduce significant biases that can considerably compromise the reliability of the metabarcoding output. The aim of this review is to provide state-of-the-art background knowledge needed to make appropriate decisions at each step of a DNA metabarcoding workflow, highlighting crucial steps that, if considered, ensures an accurate and standardized characterization of microbiota in environmental studies.

scholarly journals Nanopore Metagenomic Sequencing for Detection and Characterization of SARS-CoV-2 in Clinical Samples

2021 ◽  
Author(s):  
Nick P.G. Gauthier ◽  
Cassidy Nelson ◽  
Michael B. Bonsall ◽  
Kerstin Locher ◽  
Marthe Charles ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Clinical Laboratory ◽  
Cost Effective ◽  
Threshold Value ◽  
Clinical Samples ◽  
Diagnostic Strategy ◽  
Metagenomic Sequencing ◽  
Diagnostic Strategies ◽  
Future Work

AbstractThe COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies to detect and characterize pathogens from clinical specimens. The MinION sequencing device allows for rapid, cost-effective, high-throughput sequencing; useful features for translation to clinical laboratory settings. Metagenomic Next-Generation Sequencing (mNGS) approaches provide the opportunity to examine the entire genomic material of a sample; allowing for detection of emerging and clinically relevant pathogens that may be missed in targeted assays. Here we present a pilot study on the performance of Sequence-Independent Single Primer Amplification (SISPA) to amplify RNA randomly for the detection and characterization of SARS-CoV-2. We designed a classifier that corrects for barcode crosstalk between specimens. Our assay yielded 100% specificity overall and 95.2% sensitivity for specimens with a RT-qPCR cycle threshold value less than 30. We assembled 10 complete (>95% coverage at 20x depth), and one near-complete (>80% coverage at 20x depth) genomes from 20 specimens that were classified as positive by mNGS. We characterized these genomes through phylogenetic analysis and found that 10/11 specimens from British Columbia had a closest relative to another British Columbian specimen. Of five samples that we had both assembled genomes, as well as Variant of Concern (VOC) PCR results, we found 100% concordance between these results. Additionally, our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with our VOC PCR technique. This study supports future work examining the broader feasibility of SISPA as a diagnostic strategy for the detection and characterization of viral pathogens.

scholarly journals A workflow for accurate metabarcoding using nanopore MinION sequencing

2020 ◽  
Author(s):  
Bilgenur Baloğlu ◽  
Zhewei Chen ◽  
Vasco Elbrecht ◽  
Thomas Braukmann ◽  
Shanna MacDonald ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Rolling Circle Amplification ◽  
Error Rates ◽  
Read Length ◽  
Taxonomic Assignment ◽  
Major Drawback ◽  
Rolling Circle ◽  
Sequencing Platform ◽  
Sequencing Platforms

AbstractMetabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short-read high-throughput sequencing platforms. The Oxford Nanopore MinION™, a portable sequencing platform, represents a low-cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%.To test if the MinION™ represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full-length consensus DNA barcodes (658bp of cytochrome oxidase I - COI) for a bulk mock sample of 50 aquatic invertebrate species. By applying two different laboratory protocols, we generated two MinION™ runs that were used to build consensus sequences. We also developed a novel Python pipeline, ASHURE, for processing, consensus building, clustering, and taxonomic assignment of the resulting reads.We were able to show that it is possible to reduce error rates to a median accuracy of up to 99.3% for long RCA fragments (>45 barcodes). Our pipeline successfully identified all 50 species in the mock community and exhibited comparable sensitivity and accuracy to MiSeq. The use of RCA was integral for increasing consensus accuracy, but it was also the most time-consuming step during the laboratory workflow and most RCA reads were skewed towards a shorter read length range with a median RCA fragment length of up to 1262bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding but we recommend the exploration of other isothermal amplification procedures to improve consensus length.

scholarly journals GrigoraSNPs: Optimized HTS DNA Forensic SNP Analysis

2017 ◽  
Author(s):  
Darrell O. Ricke ◽  
Anna Shcherbina ◽  
Adam Michaleas ◽  
Philip Fremont-Smith
Keyword(s):  
High Throughput ◽  
Tandem Repeats ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Sequence Data ◽  
Snp Analysis ◽  
Analysis Pipeline ◽  
Sequencing Technologies ◽  
High Throughput Dna Sequencing

AbstractHigh throughput DNA sequencing technologies enable improved characterization of forensic DNA samples enabling greater insights into DNA contributor(s). Current DNA forensics techniques rely upon allele sizing of short tandem repeats by capillary electrophoresis. High throughput sequencing enables forensic sample characterizations for large numbers of single nucleotide polymorphism loci. The slowest computational component of the DNA forensics analysis pipeline is the characterization of raw sequence data. This paper optimizes the SNP calling module of the DNA analysis pipeline with runtime results that scale linearly with the number of HTS sequences (patent pending)[1]. GrigoraSNPs can analyze 100 million reads in less than 5 minutes using 3 threads on a 4.0 GHz Intel i7-6700K laptop CPU.

scholarly journals Molecular characterization of Anopheles fluviatilis species complex in the Islamic Republic of Iran

2021 ◽  
Vol 9 (3) ◽  
pp. 257-265
Author(s):  
S. R. Naddaf ◽  
M. A. Oshaghi ◽  
H. Vatandoost ◽  
M. Assmar
Keyword(s):  
Sequence Data ◽  
Islamic Republic ◽  
Anopheles Fluviatilis ◽  
As Species ◽  
Pcr Product ◽  
Islamic Republic Of Iran ◽  
Polymerase Chain ◽  
Second Internal Transcribed Spacer ◽  
Species Specific

A species-specific polymerase chain reaction [PCR] assay was used to identify the species composition of the Anopheles fluviatilis complex in the Islamic Republic of Iran. All the amplified DNA samples from specimens collected from different areas yielded a fragment of 450 bp size, a PCR product corresponding to that of the species denoted as Y. The sequence data from 21 ITS2 [second internal transcribed spacer] regions were compared with those publicly available in the GenBank database and confirmed that the specimens were 100% identical to species Y of India. Species Y is presumably the same as species T that has no role in transmission of malaria in India, whereas An. fluviatilis is known as a secondary vector of malaria in the Islamic Republic of Iran

scholarly journals Characterization of the Viral Microbiome in Patients with Severe Lower Respiratory Tract Infections, Using Metagenomic Sequencing

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e30875 ◽  
Author(s):  
Fredrik Lysholm ◽  
Anna Wetterbom ◽  
Cecilia Lindau ◽  
Hamid Darban ◽  
Annelie Bjerkner ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Lower Respiratory Tract ◽  
Respiratory Tract Infections ◽  
Metagenomic Sequencing ◽  
Lower Respiratory Tract Infections ◽  
Tract Infections

scholarly journals Metagenomic diagnosis of severe psittacosis using multiple sequencing platforms

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Kaiying Wang ◽  
Xiong Liu ◽  
Huiying Liu ◽  
Peihan Li ◽  
Yanfeng Lin ◽  
...  
Keyword(s):  
Antibiotic Susceptibility ◽  
Sequence Data ◽  
Bacterial Load ◽  
Clinical Specimen ◽  
Severe Pneumonia ◽  
Relevant Information ◽  
Diagnostic Techniques ◽  
Chlamydia Psittaci ◽  
Metagenomic Sequencing ◽  
Sequencing Platforms

Abstract Background Chlamydia psittaci is an avian pathogen that can cause lethal human infections. Diagnosis of C. psittaci pneumonia is often delayed due to nonspecific clinical presentations and limited laboratory diagnostic techniques. Results The MinION platform established the diagnosis in the shortest time, while BGISEQ-500 generated additional in-depth sequence data that included the rapid characterization of antibiotic susceptibility. Cytopathy appeared only in cell cultures of BALF. BALF yielded a higher bacterial load than sputum or blood, and may be the most suitable clinical specimen for the genomic diagnosis of severe pneumonia. Conclusions This study indicated that the benefits of metagenomic sequencing include rapid etiologic diagnosis of unknown infections and the provision of additional relevant information regarding antibiotic susceptibility. The continued optimization and standardization of sampling and metagenomic analysis promise to enhance the clinical utility of genomic diagnosis.

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