Enabling large-scale feather mite studies: an Illumina DNA metabarcoding pipeline

AbstractIn the era of emergence and re-emergence of vector-borne diseases, a high throughput trap-based insect monitoring is essential for the identification of invasive species, study of mosquito populations and risk assessment of disease outbreaks. Insect DNA metabarcoding technology has emerged as a highly promising methodology for unbiased and large-scale surveillance. Despite significant attempts to introduce DNA metabarcoding in mosquito or other insect surveillance qualitative and quantitative metabarcoding remains a challenge. In the present study, we have developed a methodology of in-tandem identification and quantification using cytochrome oxidase subunit I (COI) combined with a secondary multilocus identification and quantification involving three loci of 28S ribosomal DNA. The presented methodology was able to identify individual species in pools of mosquitoes with 95.94% accuracy and resolve with high accuracy (p = 1, χ2 = 2.55) mosquito population composition providing a technology capable of revolutionizing mosquito surveillance through metabarcoding. The methodology, given the respective dataset, has the potential to be applied to various small animal populations.

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Message in a Bottle, Metabarcoding Enables Biodiversity Comparisons Across Ecoregions

10.1101/2021.07.05.451165 ◽

2021 ◽

Author(s):

Dirk Steinke ◽

Stephanie L deWaard ◽

Jayme E Sones ◽

Natalia V. Ivanova ◽

Sean WJ Prosser ◽

...

Keyword(s):

Species Composition ◽

Large Scale ◽

Spatial And Temporal Variation ◽

Geographical Distance ◽

Transient Species ◽

Arthropod Communities ◽

Biogeographic Patterns ◽

Arthropod Species ◽

Dna Metabarcoding ◽

Basic Understanding

Background Traditional biomonitoring approaches have delivered a basic understanding of biodiversity, but they cannot support the large scale assessments required to manage and protect entire ecosystems. This study employed DNA metabarcoding to assess spatial and temporal variation in species richness and diversity in arthropod communities from 52 protected areas spanning three Canadian ecoregions. Results This study revealed the presence of 26,263 arthropod species in the three ecoregions and indicated that at least another 3,000 to 5,000 await detection. Results further demonstrate that communities are more similar within than between ecoregions, even after controlling for geographical distance. Overall alphadiversity declined from east to west, reflecting a gradient in habitat disturbance. Shifts in species composition were high at every site with turnover greater than nestedness, suggesting the presence of many transient species. Conclusions Differences in species composition among their arthropod communities confirm that ecoregions are a useful synoptic for biogeographic patterns and for structuring conservation efforts. The present results also demonstrate that metabarcoding enables large scale monitoring of shifts in species composition, making it possible to move beyond the biomass measurements that have been the key metric employed in prior efforts to track change in arthropod communities.

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Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

10.7287/peerj.preprints.3456v3 ◽

2018 ◽

Author(s):

Vasco Elbrecht ◽

Dirk Steinke

Keyword(s):

High Throughput ◽

Illumina Sequencing ◽

Large Scale ◽

High Throughput Sequencing ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

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Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

10.7287/peerj.preprints.3456v2 ◽

2018 ◽

Author(s):

Vasco Elbrecht ◽

Dirk Steinke

Keyword(s):

High Throughput ◽

Illumina Sequencing ◽

Large Scale ◽

High Throughput Sequencing ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

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Large-Scale Monitoring of Plants through Environmental DNA Metabarcoding of Soil: Recovery, Resolution, and Annotation of Four DNA Markers

PLoS ONE ◽

10.1371/journal.pone.0157505 ◽

2016 ◽

Vol 11 (6) ◽

pp. e0157505 ◽

Cited By ~ 50

Author(s):

Nicole A. Fahner ◽

Shadi Shokralla ◽

Donald J. Baird ◽

Mehrdad Hajibabaei

Keyword(s):

Dna Markers ◽

Large Scale ◽

Environmental Dna ◽

Dna Metabarcoding ◽

Soil Recovery

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Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

10.7287/peerj.preprints.3456 ◽

2018 ◽

Author(s):

Vasco Elbrecht ◽

Dirk Steinke

Keyword(s):

High Throughput ◽

Illumina Sequencing ◽

Large Scale ◽

High Throughput Sequencing ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

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Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

10.7287/peerj.preprints.3456v4 ◽

2018 ◽

Cited By ~ 1

Author(s):

Vasco Elbrecht ◽

Dirk Steinke

Keyword(s):

High Throughput ◽

Illumina Sequencing ◽

Large Scale ◽

High Throughput Sequencing ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

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Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring

10.7287/peerj.preprints.3456v5 ◽

2018 ◽

Author(s):

Vasco Elbrecht ◽

Dirk Steinke

Keyword(s):

High Throughput ◽

Illumina Sequencing ◽

Large Scale ◽

High Throughput Sequencing ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

Positive Controls

The viability of DNA metabarcoding for assessment of freshwater macrozoobenthos has been demonstrated over the past years. It matured to a stage where it can be applied to monitoring at a large scale, keeping pace with increased high throughput sequencing (HTS) capacity. However, workflows and sample tagging need to be optimized to accommodate for hundreds of samples within a single sequencing run. We here conceptualize a streamlined metabarcoding workflow, in which samples are processed in 96-well plates. Each sample is replicated starting with tissue extraction. Negative and positive controls are included to ensure data reliability. With our newly developed fusion primer sets for the BF2+BR2 primer pair up to three 96-well plates (288 wells) can be uniquely tagged for a single Illumina sequencing run. By including Illumina indices, tagging can be extended to thousands of samples. We hope that our metabarcoding workflow will be used as a practical guide for future large-scale biodiversity assessments involving freshwater invertebrates. However, we also want to point out that this is just one possible metabarcoding approach, and that we hope this article will stimulate discussion and publication of alternatives and extensions.

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Assessing strengths and weaknesses of DNA metabarcoding based macroinvertebrate identification for routine stream monitoring

10.7287/peerj.preprints.2759 ◽

2017 ◽

Author(s):

Vasco Elbrecht ◽

Edith Vamos ◽

Kristian Meissner ◽

Jukka Aroviita ◽

Florian Leese

Keyword(s):

Large Scale ◽

Monitoring Program ◽

Ecological Status ◽

Taxonomic Resolution ◽

Great Promise ◽

Morphological Identification ◽

Full Potential ◽

Stream Monitoring ◽

Dna Metabarcoding ◽

Primer Sets

1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 2) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. 3) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R2 were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as expensive as morphology-based identification. 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

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Marine environments

10.1093/oso/9780198767220.003.0013 ◽

2018 ◽

Author(s):

Pierre Taberlet ◽

Aurélie Bonin ◽

Lucie Zinger ◽

Eric Coissac

Keyword(s):

Large Scale ◽

Marine Invertebrates ◽

Current Knowledge ◽

Basic Research ◽

Marine Biodiversity ◽

Marine Environments ◽

Marine Microbiology ◽

Environmental Genomics ◽

Dna Metabarcoding ◽

Novel Taxa

Chapter 13 “Marine environments” focuses on different applications of eDNA to study marine biodiversity. After a brief description of the current knowledge on DNA cycle in pelagic and benthic environments, this chapter revisits how DNA metabarcoding, and more generally environmental genomics have revolutionized the field of marine microbiology through the discovery of novel taxa and by unveiling large-scale patterns of diversity for marine bacteria, protists, and viruses. This chapter then presents recent applications of DNA metabarcoding for both basic research or biomonitoring purposes to study marine invertebrates and fish populations and diversity, as well as the detection of invasive species. Current gaps and methodological challenges are also discussed.

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