scholarly journals Environmental DNA Barcode Sequence Capture: Targeted, PCR-free Sequence Capture for Biodiversity Analysis from Bulk Environmental Samples

2016 ◽  
Author(s):  
Shadi Shokralla ◽  
Joel F. Gibson ◽  
Ian King ◽  
Donald J. Baird ◽  
Daniel H. Janzen ◽  
...  
Keyword(s):  
Environmental Samples ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Dna Barcode ◽  
Environmental Dna ◽  
Amplicon Sequencing ◽  
Marker Genes ◽  
Hybridization Capture ◽  
Sequence Capture ◽  
Wide Range

ABSTRACTEnvironmental DNA analysis using PCR amplified marker genes has been a key application of high-throughput sequencing (HTS). However, PCR bias is a major drawback to gain accurate qualitative and quantitative biodiversity data. We developed a PCR-free approach using enrichment baits for species-specific mitochondrial cytochrome c oxidase 1(COI) DNA barcodes. The sequence capture was tested on species-rich bulk terrestrial and aquatic benthic samples. Hybridization capture recovered an average of 6 and 4.7 more arthropod orders than amplicon sequencing for terrestrial and benthic samples, respectively. For the terrestrial sample, the four most abundant arthropod orders comprised 94.0% of the sample biomass. These same four orders comprised 95.5% and 97.5% of the COI sequences recovered by amplification and capture, respectively. Hybridization capture recovered three arthropod orders that were detected by biomass analysis, but not by amplicon sequencing and two other insect orders that were not detected by either biomass or amplicon methods. These results indicate the advantage of using sequence capture for a more accurate analysis of biodiversity in bulk environmental samples. The protocol can be easily customized to other DNA barcode markers or gene regions of interest for a wide range of taxa or for a specific target group.

scholarly journals Natrix: a Snakemake-based workflow for processing, clustering, and taxonomically assigning amplicon sequencing reads

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Marius Welzel ◽  
Anja Lange ◽  
Dominik Heider ◽  
Michael Schwarz ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Workflow Management ◽  
Amplicon Sequencing ◽  
Version Control ◽  
Marker Genes ◽  
Sequencing Data ◽  
Taxonomic Assignment ◽  
Ecological Processes ◽  
Link Type ◽  
User Friendly

Abstract Background Sequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires efficient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an efficient workflow management system. Results We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub (https://github.com/MW55/Natrix) or as a Docker container on DockerHub (https://hub.docker.com/r/mw55/natrix). Conclusion Natrix is a user-friendly and highly extensible workflow for processing Illumina amplicon data.

scholarly journals Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays

2009 ◽  
Vol 75 (22) ◽  
pp. 7253-7260 ◽  
Author(s):  
R. van Doorn ◽  
M. M. Klerks ◽  
M. P. E. van Gent-Pelzer ◽  
A. G. C. L. Speksnijder ◽  
G. A. Kowalchuk ◽  
...  
Keyword(s):  
High Throughput ◽  
Environmental Samples ◽  
False Negative ◽  
Environmental Dna ◽  
Target Range ◽  
Gene Target ◽  
Soil Dna ◽  
Control Approach ◽  
Wide Range ◽  
Accurate Quantification

ABSTRACT PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.

scholarly journals Species detections in environmental DNA using metabarcoding: a useful tool in estuaries monitoring

2015 ◽  
Author(s):  
Adrián Mártinez-Marqués ◽  
Carlos Enrique Carleos ◽  
Eva García-Vazquez ◽  
Yaisel J. Borrell Pichs
Keyword(s):  
Dna Analysis ◽  
Anthropogenic Activities ◽  
Dna Barcode ◽  
Environmental Dna ◽  
Coi Gene ◽  
Universal Primers ◽  
Mitochondrial Coi ◽  
Mitochondrial Coi Gene ◽  
Cantabrian Coast ◽  
Taxonomic Groups

Estuaries are amongst the most productive habitats in Earth, producing more organic materia than forests, meadows or agricultural lands. In addition, estuaries exhibit high, and precious, biodiversity levels. In this study an environmental DNA analysis of the two most important estuaries in Asturias (Cantabrian Coast, north Iberia) in terms of food production (Ría del Eo and Ría de Villaviciosa) was carried out. The objective was to monitor aquatic biodiversity and also to detect alien species that can be associated with anthropogenic activities (e.g.: aquaculture). To achieve these objectives, a metabarcoding methodology based in NGS (next generation sequencing) and the mitochondrial COI gene as a DNA Barcode was used. Results showed that this methodology was useful to detect the presence of three different non-native genera (Crepidula, Lymnaea, Macrobrachium) that are probably parasitating species cultured in these estuaries. It is true that Metabarcoding has still unsolved problems such as the lack of 100% universal primers and paucity of referenced sequences for some taxonomic groups in the databases. However, it represents already a powerful tool to manage the resources of these important ecosystems and to guarantee their long-term sustainailibity.

scholarly journals Non-invasive surveys of mammalian viruses using environmental DNA

2020 ◽  
Author(s):  
Niccolò Alfano ◽  
Anisha Dayaram ◽  
Jan Axtner ◽  
Kyriakos Tsangaras ◽  
Marie-Louise Kampmann ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Environmental Dna ◽  
List Type ◽  
Dna Viruses ◽  
Remote Areas ◽  
Hybridization Capture ◽  
Non Invasive ◽  
Zoonotic Viruses ◽  
Wildlife Pathogens ◽  
Rna And Dna

ABSTRACTEnvironmental DNA (eDNA) and invertebrate-derived DNA (iDNA) have been used to survey biodiversity non-invasively to mitigate difficulties of obtaining wildlife samples, particularly in remote areas or for rare species. Recently, eDNA/iDNA have been applied to monitor known wildlife pathogens, however, most wildlife pathogens are unknown and often evolutionarily divergent.To detect and identify known and novel mammalian viruses from eDNA/iDNA sources, we used a curated set of RNA oligonucleotides as viral baits in a hybridization capture system coupled with high throughput sequencing.We detected multiple known and novel mammalian RNA and DNA viruses from multiple viral families from both waterhole eDNA and leech derived iDNA. Congruence was found between detected hosts and viruses identified in leeches and waterholes.Our results demonstrate that eDNA/iDNA samples represent an effective non-invasive resource for studying wildlife viral diversity and for detecting novel potentially zoonotic viruses prior to their emergence.

scholarly journals Natrix: A Snakemake-based workflow for processing, clustering, and taxonomically assigning amplicon sequencing reads

2020 ◽  
Author(s):  
Marius Welzel ◽  
Anja Lange ◽  
Dominik Heider ◽  
Michael Schwarz ◽  
Bernd Freisleben ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Workflow Management ◽  
Amplicon Sequencing ◽  
Version Control ◽  
Marker Genes ◽  
Sequencing Data ◽  
Taxonomic Assignment ◽  
Ecological Processes ◽  
Sequencing Technologies ◽  
User Friendly

AbstractSequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires effcient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an effcient workflow management system. We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub (https://github.com/MW55/Natrix).

scholarly journals Contribution of DNA barcoding to the study of the bees (Hymenoptera: Apoidea) of Canada: progress to date

2017 ◽  
Vol 149 (6) ◽  
pp. 736-754 ◽  
Author(s):  
Cory S. Sheffield ◽  
Jennifer Heron ◽  
Jason Gibbs ◽  
Thomas M. Onuferko ◽  
Ryan Oram ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Dna Analysis ◽  
Dna Barcode ◽  
Ecological Studies ◽  
Wide Range ◽  
Bee Diversity ◽  
Cryptic Taxa ◽  
Morphological Species ◽  
Taxonomic Studies ◽  
Nesting Preferences

AbstractBees (Hymenoptera: Apoidea, Apiformes) are taxonomically and ecologically diverse, with a wide range of social complexity, nesting preferences, floral associations, and biogeographic restrictions. A Canadian bee checklist, greatly assisted by the gene-assisted approach of DNA barcoding, is nearing completion. Previous evaluation of bee diversity in Canada, assisted by DNA barcoding, was restricted to Nova Scotia, which contains about 25% of the bee species in the country. Here, we summarise efforts to date to build a comprehensive DNA barcode library supporting bee taxonomic studies in Canada, consisting of more than 12 500 barcode-compliant sequences yielding 811 distinct barcode index numbers (BINs). This appears to represent ~95% of the 856 bee species presently recorded from Canada, but comparison with known morphological species in each genus shows that some genera are still under-sampled or may contain cryptic taxa, with much taxonomic work still to be done on bees in Canada. This is particularly true within the taxonomically difficult generaAndrenaFabricius (Andrenidae), HylaeusFabricius (Colletidae),MelissodesLatreille (Apidae),NomadaScopoli (Apidae),OsmiaPanzer (Megachilidae), andSphecodesLatreille (Halictidae). DNA analysis will likely be a key asset in resolving bee taxonomic issues in Canada in the future, and to date has even assisted studies of well-known bee taxa. Here we present summaries of our results, and discuss the use of DNA barcoding to assist future taxonomic work, faunal lists, and ecological studies.

scholarly journals Species detections in environmental DNA using metabarcoding: a useful tool in estuaries monitoring

2015 ◽  
Author(s):  
Adrián Mártinez-Marqués ◽  
Carlos Enrique Carleos ◽  
Eva García-Vazquez ◽  
Yaisel J. Borrell Pichs
Keyword(s):  
Dna Analysis ◽  
Anthropogenic Activities ◽  
Dna Barcode ◽  
Environmental Dna ◽  
Coi Gene ◽  
Universal Primers ◽  
Mitochondrial Coi ◽  
Mitochondrial Coi Gene ◽  
Cantabrian Coast ◽  
Taxonomic Groups

Estuaries are amongst the most productive habitats in Earth, producing more organic materia than forests, meadows or agricultural lands. In addition, estuaries exhibit high, and precious, biodiversity levels. In this study an environmental DNA analysis of the two most important estuaries in Asturias (Cantabrian Coast, north Iberia) in terms of food production (Ría del Eo and Ría de Villaviciosa) was carried out. The objective was to monitor aquatic biodiversity and also to detect alien species that can be associated with anthropogenic activities (e.g.: aquaculture). To achieve these objectives, a metabarcoding methodology based in NGS (next generation sequencing) and the mitochondrial COI gene as a DNA Barcode was used. Results showed that this methodology was useful to detect the presence of three different non-native genera (Crepidula, Lymnaea, Macrobrachium) that are probably parasitating species cultured in these estuaries. It is true that Metabarcoding has still unsolved problems such as the lack of 100% universal primers and paucity of referenced sequences for some taxonomic groups in the databases. However, it represents already a powerful tool to manage the resources of these important ecosystems and to guarantee their long-term sustainailibity.

scholarly journals In silico assessment of primers for eDNA studies using PrimerTree and application to characterize the biodiversity surrounding the Cuyahoga River

2015 ◽  
Author(s):  
M.V. Cannon ◽  
J. Hester ◽  
A. Shalkhauser ◽  
E.R. Chan ◽  
K. Logue ◽  
...  
Keyword(s):  
High Throughput ◽  
Dna Sequences ◽  
Water Samples ◽  
High Throughput Sequencing ◽  
Biological Diversity ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Asian Carp ◽  
Broad Perspective ◽  
Wide Range

Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semiaquatic animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity.

scholarly journals Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

2019 ◽  
Author(s):  
Satsuki Tsuji ◽  
Atsushi Maruyama ◽  
Masaki Miya ◽  
Masayuki Ushio ◽  
Hirotoshi Sato ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Water Sample ◽  
Sanger Sequencing ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Environmental Dna ◽  
Intraspecific Diversity ◽  
Survey Method ◽  
Intraspecific Genetic Diversity

AbstractEnvironmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA-based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high-throughput sequencing, denoising was performed using two of the most widely used denoising packages, UNOISE3 and DADA2. Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (UNOISE3) and 41 (DADA2) haplotypes were detected by eDNA analysis. When DADA2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. This study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large-scale capture-based conventional methods, suggesting that it could become a more efficient survey method for investigating intraspecific genetic diversity in the field.

scholarly journals Endogenous Gibbon Ape Leukemia Virus Identified in a Rodent (Melomys burtoni subsp.) from Wallacea (Indonesia)

2016 ◽  
Vol 90 (18) ◽  
pp. 8169-8180 ◽  
Author(s):  
Niccolò Alfano ◽  
Johan Michaux ◽  
Serge Morand ◽  
Ken Aplin ◽  
Kyriakos Tsangaras ◽  
...  
Keyword(s):  
Southeast Asia ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Leukemia Virus ◽  
Southeast Asian ◽  
Content Type ◽  
Hybridization Capture ◽  
Wide Range ◽  
Gibbon Ape Leukemia Virus ◽  
Koala Retrovirus

ABSTRACTGibbon ape leukemia virus (GALV) and koala retrovirus (KoRV) most likely originated from a cross-species transmission of an ancestral retrovirus into koalas and gibbons via one or more intermediate as-yet-unknown hosts. A virus highly similar to GALV has been identified in an Australian native rodent (Melomys burtoni) after extensive screening of Australian wildlife. GALV-like viruses have also been discovered in several Southeast Asian species, although screening has not been extensive and viruses discovered to date are only distantly related to GALV. We therefore screened 26 Southeast Asian rodent species for KoRV- and GALV-like sequences, using hybridization capture and high-throughput sequencing, in the attempt to identify potential GALV and KoRV hosts. Only the individuals belonging to a newly discovered subspecies ofMelomysburtonifrom Indonesia were positive, yielding an endogenous provirus very closely related to a strain of GALV. The sequence of the critical receptor domain for GALV infection in the IndonesianM. burtonisubsp. was consistent with the susceptibility of the species to GALV infection. The second record of a GALV inM. burtoniprovides further evidence thatM. burtoni, and potentially other lineages within the widespread subfamilyMurinae, may play a role in the spread of GALV-like viruses. The discovery of a GALV in the most western part of the Australo-Papuan distribution ofM. burtoni, specifically in a transitional zone between Asia and Australia (Wallacea), may be relevant to the cross-species transmission to gibbons in Southeast Asia and broadens the known distribution of GALVs in wild rodents.IMPORTANCEGibbon ape leukemia virus (GALV) and the koala retrovirus (KoRV) are very closely related, yet their hosts neither are closely related nor overlap geographically. Direct cross-species infection between koalas and gibbons is unlikely. Therefore, GALV and KoRV may have arisen via a cross-species transfer from an intermediate host whose range overlaps those of both gibbons and koalas. Using hybridization capture and high-throughput sequencing, we have screened a wide range of rodent candidate hosts from Southeast Asia for KoRV- and GALV-like sequences. Only aMelomysburtonisubspecies from Wallacea (Indonesia) was positive for GALV. We report the genome sequence of this newly identified GALV, the critical domain for infection of its potential cellular receptor, and its phylogenetic relationships with the other previously characterized GALVs. We hypothesize thatMelomysburtoni, and potentially related lineages with an Australo-Papuan distribution, may have played a key role in cross-species transmission to other taxa.

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