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 Genome-Wide Identification of 5-Methylcytosine Sites in Bacterial Genomes By High-Throughput Sequencing of MspJI Restriction Fragments

2021 ◽  
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

ABSTRACTSingle-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

scholarly journals Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0247541
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Victoria Wu ◽  
Richard J. Roberts
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Specific Sequence ◽  
Genome Wide ◽  
Cost Effective Alternative ◽  
Simple Column ◽  
Sequencing Platforms

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.

scholarly journals Technological advancements and their importance for nematode identification

SOIL ◽  
2016 ◽  
Vol 2 (2) ◽  
pp. 257-270 ◽  
Author(s):  
Mohammed Ahmed ◽  
Melanie Sapp ◽  
Thomas Prior ◽  
Gerrit Karssen ◽  
Matthew Alan Back
Keyword(s):  
High Throughput ◽  
Crop Production ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Biological Indicators ◽  
Rapid Identification ◽  
Community Studies ◽  
Terrestrial Environments ◽  
Traditional Taxonomy ◽  
Sequencing Platforms

Abstract. Nematodes represent a species-rich and morphologically diverse group of metazoans known to inhabit both aquatic and terrestrial environments. Their role as biological indicators and as key players in nutrient cycling has been well documented. Some plant-parasitic species are also known to cause significant losses to crop production. In spite of this, there still exists a huge gap in our knowledge of their diversity due to the enormity of time and expertise often involved in characterising species using phenotypic features. Molecular methodology provides useful means of complementing the limited number of reliable diagnostic characters available for morphology-based identification. We discuss herein some of the limitations of traditional taxonomy and how molecular methodologies, especially the use of high-throughput sequencing, have assisted in carrying out large-scale nematode community studies and characterisation of phytonematodes through rapid identification of multiple taxa. We also provide brief descriptions of some the current and almost-outdated high-throughput sequencing platforms and their applications in both plant nematology and soil ecology.

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 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 UAV-GPR Fusion Approach for the Characterization of a Quarry Excavation Area in Falconara Albanese, Southern Italy

Drones ◽  
2021 ◽  
Vol 5 (2) ◽  
pp. 40
Author(s):  
Annamaria Saponaro ◽  
Giovanni Dipierro ◽  
Emanuele Cannella ◽  
Antonio Panarese ◽  
Angelo Maurizio Galiano ◽  
...  
Keyword(s):  
Methodological Approach ◽  
Research Work ◽  
Mining Industry ◽  
Cost Effective ◽  
Railway Ballast ◽  
Mining Operations ◽  
Innovative Methodology ◽  
Data Acquisition And Processing ◽  
Excavation Area

The characterization of a quarry site which is suitable for railway ballast aggregate production represents a big challenge for the mining industry. The knowledge of structural discontinuities within local geological materials is fundamental to guide mining operations, optimize investments, and guarantee quarry security. This research work presents an innovative methodology for the subsurface investigation of a quarry excavation area down to a depth of about 50 m in Falconara Albanese, Calabria, Italy. The proposed methodological approach incorporates photogrammetry, drone technology, and GPR data acquisition and processing. Photogrammetry represents the first step for obtaining a 3D topographical model reconstruction of the whole quarry, helping to detail the acquisition approach and properly plan the subsequent drone survey. In particular, two 120 MHz antennas have been mounted on the drone and two profiles have been acquired above and across the quarry. Results show the presence of fractured material and demonstrate the applicability of the method for identification of areas that are more suitable for railway ballast production. The presented method is therefore capable of detecting subsurficial fractures at a quarry site by means of a relatively fast and cost-effective procedure. Results are achieved within the framework of an industry project.

scholarly journals Application of Multiplex PCR for Characterization of Human Embryonic Stem Cells (hESCs) and Its Differentiated Progenies

2010 ◽  
Vol 15 (6) ◽  
pp. 630-643 ◽  
Author(s):  
Murali Krishna Mamidi ◽  
Rajarshi Pal ◽  
Ramesh Bhonde ◽  
Zubaidah Zakaria ◽  
Satish Totey
Keyword(s):  
Gene Expression ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Turnaround Time ◽  
Mouse Embryonic Fibroblast ◽  
Gene Markers ◽  
Real Time Quantitative Pcr ◽  
Hesc Lines

Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan® low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.

scholarly journals Measuring genetic differentiation from Pool-seq data

2018 ◽  
Author(s):  
Valentin Hivert ◽  
Raphël Leblois ◽  
Eric J. Petit ◽  
Mathieu Gautier ◽  
Renaud Vitalis
Keyword(s):  
Genetic Differentiation ◽  
High Throughput Sequencing ◽  
Model Misspecification ◽  
Cost Effective ◽  
Sequencing Data ◽  
Sequencing Errors ◽  
Cost Effective Alternative ◽  
Method Of Moments Estimator ◽  
Dna Pool

AbstractThe recent advent of high throughput sequencing and genotyping technologies enables the comparison of patterns of polymorphisms at a very large number of markers. While the characterization of genetic structure from individual sequencing data remains expensive for many non-model species, it has been shown that sequencing pools of individual DNAs (Pool-seq) represents an attractive and cost-effective alternative. However, analyzing sequence read counts from a DNA pool instead of individual genotypes raises statistical challenges in deriving correct estimates of genetic differentiation. In this article, we provide a method-of-moments estimator of FST for Pool-seq data, based on an analysis-of-variance framework. We show, by means of simulations, that this new estimator is unbiased, and outperforms previously proposed estimators. We evaluate the robustness of our estimator to model misspecification, such as sequencing errors and uneven contributions of individual DNAs to the pools. Last, by reanalyzing published Pool-seq data of different ecotypes of the prickly sculpin Cottus asper, we show how the use of an unbiased FST estimator may question the interpretation of population structure inferred from previous analyses.

scholarly journals Prospects and challenges of implementing DNA metabarcoding for high-throughput insect surveillance

GigaScience ◽  
2019 ◽  
Vol 8 (8) ◽  
Author(s):  
Alexander M Piper ◽  
Jana Batovska ◽  
Noel O I Cogan ◽  
John Weiss ◽  
John Paul Cunningham ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Scale Up ◽  
Pest Species ◽  
Rapid Diagnostics ◽  
Invasive Insects ◽  
Sequencing Technologies ◽  
Dna Metabarcoding ◽  
Diagnostic Applications ◽  
Sequencing Platforms

AbstractTrap-based surveillance strategies are widely used for monitoring of invasive insect species, aiming to detect newly arrived exotic taxa as well as track the population levels of established or endemic pests. Where these surveillance traps have low specificity and capture non-target endemic species in excess of the target pests, the need for extensive specimen sorting and identification creates a major diagnostic bottleneck. While the recent development of standardized molecular diagnostics has partly alleviated this requirement, the single specimen per reaction nature of these methods does not readily scale to the sheer number of insects trapped in surveillance programmes. Consequently, target lists are often restricted to a few high-priority pests, allowing unanticipated species to avoid detection and potentially establish populations.DNA metabarcoding has recently emerged as a method for conducting simultaneous, multi-species identification of complex mixed communities and may lend itself ideally to rapid diagnostics of bulk insect trap samples. Moreover, the high-throughput nature of recent sequencing platforms could enable the multiplexing of hundreds of diverse trap samples on a single flow cell, thereby providing the means to dramatically scale up insect surveillance in terms of both the quantity of traps that can be processed concurrently and number of pest species that can be targeted. In this review of the metabarcoding literature, we explore how DNA metabarcoding could be tailored to the detection of invasive insects in a surveillance context and highlight the unique technical and regulatory challenges that must be considered when implementing high-throughput sequencing technologies into sensitive diagnostic applications.

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