Can DNA-based ecosystem assessments quantify species abundance? Testing primer bias and biomass - sequence relationships with an innovative metabarcoding protocol

Species Abundance ◽

Reference Database ◽

Dna Metabarcoding ◽

Coi Barcoding ◽

Species Abundances ◽

Species Specific ◽

Sequence Relationships

Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity should rely on presence-absence metrics.

High-throughput DNA barcoding of oligochaetes for abundance-based indices to assess the biological quality of sediments in streams and lakes

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65394 ◽

2021 ◽

Vol 4 ◽

Author(s):

Régis Vivien ◽

Laure Apothéloz-Perret-Gentil ◽

Jan Pawlowski ◽

Inge Werner ◽

Michel Lafont ◽

...

Keyword(s):

Dna Barcoding ◽

High Throughput ◽

Species Abundance ◽

Genetic Identification ◽

Dna Barcodes ◽

Biological Quality ◽

Dna Metabarcoding ◽

Morphological Approach

Aquatic oligochaete communities are valuable indicators of the biological quality of sediments in streams and lakes, but identification of specimens to the species level based on morphological features requires solid expertise in taxonomy and is possible only for a fraction of specimens present in a sample. The identification of aquatic oligochaetes using DNA barcodes would facilitate their use in biomonitoring and allow a wider use of this taxonomic group for ecological diagnoses. Previous approaches based on DNA metabarcoding of samples composed of total sediments or pools of specimens have been proposed for assessing the biological quality of ecosystems, but such methods do not provide precise information on species abundance, which limits the value of resulting ecological diagnoses. Here, we tested how a DNA barcoding approach based on high-throughput sequencing of sorted and genetically tagged specimens performed to assess oligochaete species diversity and abundance and the biological quality of sediments in streams and lakes. We applied both molecular and morphological approaches at 13 sites in Swiss streams and at 7 sites in Lake Geneva. We genetically identified 33 or 66 specimens per site. For both approaches, we used the same index calculations. We found that the ecological diagnoses derived from the genetic approach matched well with those of the morphological approach and that the genetic identification of only 33 specimens per site provided enough ecological information for correctly estimating the biological quality of sediments in streams and lakes.

DNA-Based Herbal Teas’ Authentication: An ITS2 and psbA-trnH Multi-Marker DNA Metabarcoding Approach

Plants ◽

10.3390/plants10102120 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2120

Author(s):

Jessica Frigerio ◽

Giulia Agostinetto ◽

Valerio Mezzasalma ◽

Fabrizio De De Mattia ◽

Massimo Labra ◽

...

Keyword(s):

Quality Control ◽

Quantitative Analysis ◽

Medicinal Plants ◽

High Throughput ◽

The Other ◽

Plant Component ◽

Identification Rate ◽

Dna Metabarcoding ◽

Therapeutic Properties

Medicinal plants have been widely used in traditional medicine due to their therapeutic properties. Although they are mostly used as herbal infusion and tincture, employment as ingredients of food supplements is increasing. However, fraud and adulteration are widespread issues. In our study, we aimed at evaluating DNA metabarcoding as a tool to identify product composition. In order to accomplish this, we analyzed fifteen commercial products with DNA metabarcoding, using two barcode regions: psbA-trnH and ITS2. Results showed that on average, 70% (44–100) of the declared ingredients have been identified. The ITS2 marker appears to identify more species (n = 60) than psbA-trnH (n = 35), with an ingredients’ identification rate of 52% versus 45%, respectively. Some species are identified only by one marker rather than the other. Additionally, in order to evaluate the quantitative ability of high-throughput sequencing (HTS) to compare the plant component to the corresponding assigned sequences, in the laboratory, we created six mock mixtures of plants starting both from biomass and gDNA. Our analysis also supports the application of DNA metabarcoding for a relative quantitative analysis. These results move towards the application of HTS analysis for studying the composition of herbal teas for medicinal plants’ traceability and quality control.

PLANiTS: a curated sequence reference dataset for plant ITS DNA metabarcoding

Database ◽

10.1093/database/baz155 ◽

2020 ◽

Vol 2020 ◽

Cited By ~ 6

Author(s):

Elisa Banchi ◽

Claudio G Ametrano ◽

Samuele Greco ◽

David Stanković ◽

Lucia Muggia ◽

...

Keyword(s):

High Throughput ◽

Its Region ◽

Computational Effort ◽

Its Sequences ◽

Reference Dataset ◽

Bioinformatic Pipeline ◽

Taxonomic Level ◽

Dna Metabarcoding ◽

Reference Databases

Abstract DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called ‘better clustering for QIIME’ (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100 224 sequences), PLANiTS2 (96 771 sequences) and PLANiTS (97 550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering. Database URL: https://github.com/apallavicini/bc4q; https://github.com/apallavicini/PLANiTS.

Sorting things out - assessing effects of unequal specimen biomass on DNA metabarcoding

10.7287/peerj.preprints.2561v1 ◽

2016 ◽

Cited By ~ 1

Author(s):

Vasco Elbrecht ◽

Bianca Peinert ◽

Florian Leese

Keyword(s):

High Throughput ◽

Sequencing Depth ◽

West Germany ◽

Coi Gene ◽

Mountain Stream ◽

Detection Rates ◽

Size Classes ◽

Dna Metabarcoding ◽

Size Sorting

1) Environmental bulk samples often contain many taxa with biomass differences of several orders of magnitude. This can be problematic in DNA metabarcoding and metagenomic high throughput sequencing approaches, as large specimens contribute over proportionally much DNA template. Thus a few specimens of high biomass will dominate the dataset, potentially leading to smaller specimens remaining undetected. Sorting of samples and balancing the amounts of tissue used per size fraction should improve detection rates, but has not been systematically tested. 2) Here we tested the effects of size sorting on taxa detection using freshwater macroinvertebrates. Kick sampling was performed at two locations of a low-mountain stream in West Germany, specimens were morphologically identified and sorted into small, medium and large size classes (< 2.5x5, 5x10 and up to 10x20 mm). Tissue from the 3 size categories was extracted individually, and pooled to simulate bulk samples that were not sorted and samples which were sorted and then pooled proportionately by specimen size. DNA from all 5 extractions of both samples was amplified using 4 different freshwater primer sets for the COI gene and sequenced on a HiSeq Illumina sequencer. 3) Sorting taxa by size and pooling them proportionately according to their abundance lead to a more equal amplification compared to the processing of complete samples without sorting. The sorted samples recovered 30% more taxa than the unsorted samples, at the same sequencing depth. Our results imply that sequencing depth can be decreased ~ 5 fold when sorting the samples into three size classes. 4) Our results demonstrate that even a coarse size sorting can substantially improve detection rates. While high throughput sequencing will become more accessible and cheaper within the next years, sorting bulk samples by specimen biomass is a simple yet efficient method to reduce current sequencing costs.

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 ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

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.

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 ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

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.

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 ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

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.

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 ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

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.

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 ◽

Scaling Up ◽

The Past ◽

Dna Metabarcoding ◽

Primer Sets ◽

Fusion Primer ◽

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.