Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol
Open accessAbstract
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.
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Written from the paper’s full text.
TL;DR
This study developed and tested a replicated COI metabarcoding protocol for freshwater macroinvertebrates, using bulk DNA extraction and Illumina MiSeq sequencing. It found a strong biomass–read relationship within a single species, but strong species-specific primer bias across taxa, concluding that PCR-based metabarcoding cannot reliably quantify species abundance or biomass and should be used for presence–absence assessments.
Key findings
- In experiment I, all 31 *Dinocras cephalotes* specimens were recovered across ten replicates, including the smallest specimen at 0.023% (0.145 mg) of total biomass; specimen biomass and sequence abundance showed a highly significant positive linear correlation in all ten replicates (p<0.001, R² = 0.65).
- In experiment II, 83% (43/52) of taxa were reliably recovered; 34 taxa were recovered in all ten replicates. Recovery by group was Ephemeroptera 7/8 (88%), Plecoptera 4/4 (100%), Trichoptera 13/15 (86%), Diptera 7/8 (88%), and Others 12/17 (71%).
- Despite similar biomass, sequence abundance varied by four orders of magnitude among taxa, and there was no correlation between taxon biomass and number of recovered sequences. Primer efficiency was highly species-specific, preventing straightforward abundance or biomass inference.
- LCO and HCO sequencing direction had small effects on final sequence abundance (2.7% and 4.4% differences in experiments I and II), though LCO was 34% more frequent in experiment I (p = 0.006) and no primer bias was detected in experiment II (p = 0.41).
Methods
- Two controlled experiments with ten replicates each were sequenced on two independent MiSeq runs using 300-bp paired-end reads; experiment I used 31 *D. cephalotes* stonefly specimens spanning three orders of magnitude in biomass, and experiment II used 52 freshwater invertebrate taxa with similar tissue biomass.
- DNA was extracted in bulk, amplified in a single PCR step with tagged fusion primers based on standard COI LCO1490/HCO2198, purified, and directly sequenced; experiment I used 1.4% and 20% tissue aliquots, while experiment II pooled roughly equal tissue amounts per taxon.
- Bioinformatic analysis used demultiplexing, cutadapt primer removal, read concatenation, blastn against reference haplotypes for experiment I, and UPARSE OTU clustering at 97% identity with BOLD identification for experiment II; sequence counts were normalised to total sequence abundance and tissue weight.
Limitations
- The paper states that PCR-based metabarcoding cannot accurately estimate species biomass or even abundance in diverse environmental samples because primer efficiency is highly species-specific; it recommends presence–absence metrics and notes that PCR-free approaches are needed for biomass estimates.
- The paper states that only 83% of taxa were recovered with one universal COI primer pair, that barcode reference databases remain incomplete, that DNA-based assessments cannot assign biological traits, and that PCR replication effects were not evaluated due to a small number of replicates.
- Evident from the text: the experiments are limited to freshwater macroinvertebrates from western Germany and a specific MiSeq/COI Folmer protocol; some morphotaxa were identified only to family or order and contained multiple MOTUs, so species-level amplification behaviour may be more variable than the morphotaxon-level summaries suggest.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
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Cites
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Claims the paper makes
0 of 35 cited sources checked · 0 supported · 0 flagged
- Findingown data
Sequence abundance varied by four orders of magnitude between taxa despite the use of similar amounts of biomass.
“However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass.”
Quote in the paperSubstantial claim - Findingown data
There were significant differences between the LCO and HCO primers with respect to the number of reads with no hits in experiment I and final hits in experiment II.
“we found significant differences between the LCO and HCO primers with respect to the number of reads with no hits in experiment I (t-test, p = 0.01, S3A Fig) and final hits in experiment II (t-test, p = 0.01, S3B Fig).”
Quote in the paperNot a substantial claim - Findingown data
Few outliers and low standard deviations were detected for the ratio of LCO- to HCO-based haplotype read abundance for each of the five DNA extractions.
“We detected few outliers and low standard deviations for the ratio of LCO- to HCO-based haplotype read abundance for each of the five DNA extractions (Fig 2B and S8 Fig).”
Quote in the paperNot a substantial claim - Findingown data
The sequencing results for the ten replicates were highly reproducible.
“The sequencing results for the ten replicates were highly reproducible.”
Quote in the paperSubstantial claim - Findingown data
There were no significant differences in sequence abundance among the replicates using different amounts of extracted tissue as a template.
“We did not observe significant differences in sequence abundance among the replicates using different amounts of extracted tissue as a template.”
Quote in the paperSubstantial claim
- Findingown data
From the DNA extractions performed with the TissueLyser LT, six more specimens (2.31%) were recovered than when DNA was extracted with liquid nitrogen.
“From the DNA extractions performed with the TissueLyser LT, six more specimens (2.31%) were recovered than when DNA was extracted with liquid nitrogen.”
Quote in the paperNot a substantial claim - Findingown data
Sequences starting with the LCO primer for the first read were significantly overrepresented in experiment I, being 34% more frequent than sequences beginning with the HCO primer.
“Sequences starting with the LCO primer for the first read were significantly overrepresented in experiment I (34% more frequent than sequences beginning with the HCO primer, t-test, p = 0.006, S4A Fig).”
Quote in the paperNot a substantial claim - Findingown data
A much higher per-site nucleotide diversity was observed using the consensus sequences of the 31 unique Dinocras cephalotes COI haplotypes with both primers than with one primer at a time.
“A much higher per-site nucleotide diversity was observed using the consensus sequences of the 31 unique Dinocras cephalotes COI haplotypes (see experiment I below) (S2 Fig). Fewer peaks of low diversity (only up to 70% identical bases) were detected using both primers than using one primer at a time (100% identical bases).”
Quote in the paperSubstantial claim - Findingown data
In experiment II, no primer bias was detected.
“In experiment II, we did not detect primer bias (t-test, p = 0.41, S4B Fig).”
Quote in the paperNot a substantial claim - Findingown data
Specimens with a low biomass tend to show relatively high variation in sequence abundance among the ten replicates.
“Specimens with a low biomass tend to show relatively high variation in sequence abundance among the ten replicates.”
Quote in the paperSubstantial claim - Findingown data
No substantial differences in recovery rates were observed when different numbers of PCR products were pooled.
“Furthermore, we did not observe substantial differences in recovery rates when different numbers of PCR products were pooled.”
Quote in the paperNot a substantial claim - Findingown data
On average, 99.52% of the hits could be assigned to the specimens used in each extraction.
“On average, 99.52% of the hits could be assigned to the specimens used in each extraction (Fig 3 and S9A Fig).”
Quote in the paperNot a substantial claim - Findingown data
Many of the typical bio-indicator taxa such as Ephemeroptera, Plecoptera, Trichoptera, and Diptera were recovered.
“We recovered many of the typical bio-indicator taxa such Ephemeroptera, Plecoptera, Trichoptera, and Diptera (Table 1).”
Quote in the paperNot a substantial claim - Findingown data
34 taxa were recovered in all ten replicates.
“34 taxa were recovered in all ten replicates (Fig 3).”
Quote in the paperNot a substantial claim - Findingown data
There was slightly more variation in sequence abundance for the replicates in which DNA was extracted from only 1.4% of the total tissue volume than from 20%.
“However, there was slightly more variation in sequence abundance for the replicates in which DNA was extracted from only 1.4% of the total tissue volume than from 20% (S7A Fig).”
Quote in the paperNot a substantial claim - Findingown data
There is a strong negative relationship between specimen weight and variation in sequence abundance.
“We observed a strong negative relationship between specimen weight and variation in sequence abundance, as shown in S5 and S7B Figs.”
Quote in the paperSubstantial claim - Findingown data
There were significant differences in sequence abundance between LCO and HCO for 17 of the 31 stonefly specimens in experiment I.
“there were significant differences in sequence abundance between LCO and HCO for 17 of the 31 stonefly specimens (t-test, p<0.05, S5 Fig).”
Quote in the paperNot a substantial claim - Findingown data
The primers LCO and HCO had small effects on final sequence abundance, with differences of 2.7% and 4.4% for experiment I and II.
“The primers (LCO and HCO) had small effects on final sequence abundance, with differences of 2.7% and 4.4% for experiment I and II.”
Quote in the paperNot a substantial claim - Findingown data
83% of the 52 taxa used to test primer bias were reliably recovered.
“we reliably recovered 83% of the 52 taxa used to test primer bias.”
- Also stated in: § Experiment II · plain
“We were able to reliably recover 83% (43) of the 52 taxa included in experiment II.”
Quote in the paperSubstantial claim - Also stated in: § Experiment II · plain
- Findingown data
There is a distinct positive correlation between species biomass and resulting numbers of MiSeq reads.
“We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads.”
- Also stated in: § Experiment I · boosted
“We found a highly significant positive linear correlation between specimen biomass and sequence abundance in all ten replicates (Fig 2A).”
Quote in the paperSubstantial claim - Also stated in: § Experiment I · boosted
- Findingown data
Even when comparing absolute sequence numbers, the patterns were concordant.
“Even when comparing absolute sequence numbers, the patterns were concordant (Fig 2A).”
Quote in the paperNot a substantial claim - Findingnone given
The results of both experiments were consistent across replicates.
“The results of both experiments were consistent across replicates.”
Quote in the paperNot a substantial claim - Findingreasoning
Some taxa that could only be determined at higher taxonomic level contained different MOTUs with different amplification efficiencies (e.g. the order Arhynchobdellida).
“some taxa that could only be determined at higher taxonomic level, contained different MOTUs with different amplification efficiencies (e.g. the order Arhynchobdellida).”
Quote in the paperSubstantial claim - Findingown data
Identification by COI barcode did not perform worse than identification by morphology.
“Identification by COI barcode did not perform worse than identification by morphology.”
Quote in the paperNot a substantial claim - Findingreasoning
Variation in sequence abundance for each specimen was slightly higher among replicates that had less tissue as starting material.
“Variation in sequence abundance for each specimen was slightly higher among replicates that had less tissue as starting material.”
Quote in the paperNot a substantial claim - Findingreasoning
This trend was stronger for specimens with a smaller biomass.
“This trend was stronger for specimens with a smaller biomass.”
Quote in the paperNot a substantial claim - Findingown data
Irrespective of the tissue volume used for extraction, all 31 specimens were recovered.
“Irrespective of the tissue volume used for extraction, we recovered all 31 specimens.”
Quote in the paperSubstantial claim - Findingown data
11 morphotaxa included multiple species using COI data from the BOLD database.
“and 11 morphotaxa included multiple species using COI data from the BOLD database (S9A Fig).”
Quote in the paperNot a substantial claim - Findingown data
A majority of specimens (83%) were recovered using one universal primer pair.
“Furthermore, we recovered a majority of specimens (83%) using one universal primer pair.”
Quote in the paperNot a substantial claim - Findingown data
Exceptions were Arhynchobdellida, Ceratopogonidae, Scirtidae and Glossosomatidae, where efficiencies differed substantially between the MOTUs.
“Exceptions were Arhynchobdellida, Ceratopogonidae, Scirtidae and Glossosomatidae (S3 Table), where efficiencies differed substantially between the MOTUs.”
Quote in the paperNot a substantial claim - Findingown data
Amplification efficiencies were consistent among replicates of a morphotaxon, in particular when determined to species or genus level.
“While amplification efficiencies were consistent among replicates of a morphotaxon, in particular when determined to species or genus level”
Quote in the paperNot a substantial claim - Findingown data
There was no correlation between taxon biomass and the number of recovered sequences (using the means of ten replicates for each of the 52 taxa).
“There was no correlation between taxon biomass and the number of recovered sequences (using the means of ten replicates for each of the 52 taxa).”
Quote in the paperSubstantial claim - Findingown data
Most morphotaxa represented a single MOTU (i.e. distinct biological species).
“However, most morphotaxa represented a single MOTU (i.e. distinct biological species).”
Quote in the paperNot a substantial claim - Findingown data
Using 31 specimens of a single stonefly species that differed in biomass, the study demonstrated a highly significant correlation between sequence abundance and specimen biomass.
“Using 31 specimens of a single stonefly species (see [43]) that differed in biomass, we demonstrated a highly significant correlation between sequence abundance and specimen biomass.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [43] V Elbrecht, 2014, Genetic diversity and dispersal potential of the stonefly Dinocras cephalotes in a central European low mountain range, Freshw Sci source
Not checked in the free run.
- Findingown data
In 19 of the cases (37%), the morphologically identified taxa were assigned to more than one MOTU (K2P distance of detected haplotypes > 2%).
“In 19 of the cases (37%), the morphologically identified taxa were assigned to more than one MOTU (K2P distance of detected haplotypes > 2%, Fig 3)”
Quote in the paperNot a substantial claim - Findingreasoning
Using the Folmer primers, the number of sequences obtained varied among taxa by several orders of magnitude.
“However, using the Folmer primers, the number of sequences obtained varied among taxa by several orders of magnitude”
Quote in the paperSubstantial claim - Findingown data
OTUs could be reliably assigned to all 52 taxa included in the extraction.
“We were able to reliably assign OTUs to all 52 taxa included in the extraction.”
Quote in the paperSubstantial claim - Findingown data
The number of sequences per replicate was not biased by primer type (LCO or HCO).
“The number of sequences per replicate was not biased by primer type (LCO or HCO)”
Quote in the paperNot a substantial claim - Findingown data
The number of sequences per taxon varied by four orders of magnitude despite the similarity in biomass used for extraction.
“Although many taxa were recovered, the number of sequences per taxon varied by four orders of magnitude despite the similarity in biomass used for extraction (Fig 3).”
Quote in the paperSubstantial claim - Findingown data
In most cases, the sequence abundance obtained for a certain morphotaxon was consistent across replicates after normalising for slight differences in the amount of tissue biomass used.
“In most cases, the sequence abundance obtained for a certain morphotaxon was consistent across replicates after normalising for slight differences in the amount of tissue biomass used.”
Quote in the paperSubstantial claim - Findingown data
The LCO and HCO PCR replicates for DNA extraction yielded highly concordant results.
“Furthermore, the LCO and HCO PCR replicates for DNA extraction yielded highly concordant results”
Quote in the paperNot a substantial claim - Findingown data
In 50% of the cases, barcoding identified specimens at a finer taxonomic level.
“In fact, in 50% of the cases, barcoding identified specimens at a finer taxonomic level”
Quote in the paperNot a substantial claim - Premisecitation
Small organisms may be lost owing to low number of sequence reads.
“small organisms may be lost owing to low number of sequence reads [38].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [38] ME Carew, 2013, Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species, Front Zool source
Not checked in the free run.
- Interpretationown data
The metabarcoding approach yielded reliable results for high-throughput assessments.
“Our metabarcoding approach yielded reliable results for high-throughput assessments.”
Quote in the paperSubstantial claim - Premisecitation
Sampled organisms have vastly different biomasses.
“First, sampled organisms have vastly different biomasses, and thus small organisms may be lost owing to low number of sequence reads [38].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [38] ME Carew, 2013, Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species, Front Zool source
Not checked in the free run.
- Premisecitation
These limitations have direct consequences for inferred ecosystem assessment metrics and thus management decisions.
“these limitations have direct consequences for the inferred ecosystem assessment metrics [10,14] and thus management decisions.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [10] P Haase, 2010, First audit of macroinvertebrate samples from an EU Water Framework Directive monitoring program: human error greatly lowers precision of assessment results, J North Am Benthol Soc source
Not checked in the free run.
- cites [14] ED Stein, 2014, Is DNA Barcoding Actually Cheaper and Faster than Traditional Morphological Methods: Results from a Survey of Freshwater Bioassessment Efforts in the United States?, PLoS ONE source
Not checked in the free run.
- Premisecitation
High sequence similarity in amplicon sequencing can lead to decreased sequence quality on Illumina platforms.
“High sequence similarity in in amplicon sequencing can lead to decreased sequence quality on Illumina platforms [40].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [40] F Krueger, 2011, Large scale loss of data in low-diversity Illumina sequencing libraries can be recovered by deferred cluster calling, PLoS ONE source
Not checked in the free run.
- Interpretationown data
Primer efficiency is highly species-specific.
“the results indicated that primer efficiency is highly species-specific”
Quote in the paperSubstantial claim - Premisecitation
The amplification efficiency of the COI gene varies among species.
“the amplification efficiency of the COI gene varies among species, and this might severely bias results [34,39] particularly in view of the variation in biomass.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [34] M Hajibabaei, 2011, Environmental barcoding: A next-generation sequencing approach for biomonitoring applications using river benthos, PLoS ONE source
Not checked in the free run.
- cites [39] Piñol J, Mir G, Gomez-Polo P, Agustí N. Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods. Mol Ecol Resour. 2014; 1–12. <comment>doi: <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://dx.doi.org/10.1111/1755-0998.12355" xlink:type="simple">10.1111/1755-0998.12355</ext-link></comment> source
Not checked in the free run.
- Premisecitation
This variation in amplification efficiency might severely bias results, particularly in view of the variation in biomass.
“the amplification efficiency of the COI gene varies among species, and this might severely bias results [34,39] particularly in view of the variation in biomass.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [34] M Hajibabaei, 2011, Environmental barcoding: A next-generation sequencing approach for biomonitoring applications using river benthos, PLoS ONE source
Not checked in the free run.
- cites [39] Piñol J, Mir G, Gomez-Polo P, Agustí N. Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods. Mol Ecol Resour. 2014; 1–12. <comment>doi: <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://dx.doi.org/10.1111/1755-0998.12355" xlink:type="simple">10.1111/1755-0998.12355</ext-link></comment> source
Not checked in the free run.
- Interpretationown data
Species-specific primer efficiency prevents straightforward assessments of species abundance and biomass in a sample.
“which would prevent straightforward assessments of species abundance and biomass in a sample.”
Quote in the paperSubstantial claim - Premisecitation
Initial studies on marine benthic taxa show that presence-absence data is sufficient for precise assessment indices.
“Initial studies on marine benthic taxa show that presence-absence data is sufficient for precise assessment indices [51,52]”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [51] JA Ranasinghe, 2012, Performance of Two Southern California Benthic Community Condition Indices Using Species Abundance and Presence-Only Data: Relevance to DNA Barcoding, PLoS ONE source
Not checked in the free run.
- cites [52] E Aylagas, 2014, Environmental status assessment using DNA metabarcoding: towards a genetics based Marine Biotic Index (gAMBI), PLoS ONE source
Not checked in the free run.
- Interpretationreasoning
The result that variation in sequence abundance is higher with less tissue and smaller biomass is expected because stochastic effects increase with reduced specimen biomass.
“This result is expected because stochastic effects increase with reduced specimen biomass.”
Quote in the paperNot a substantial claim- Rests on an assumption: Stochastic effects increase with reduced specimen biomass.
- Interpretationown data
Sequencing direction has a negligible effect when independent replicates are sequenced.
“indicating that sequencing direction has a negligible effect when independent replicates are sequenced.”
Quote in the paperNot a substantial claim - Interpretationown data
The highly concordant LCO and HCO PCR replicates emphasise the overall reliability of the protocol.
“emphasising the overall reliability of our protocol.”
Quote in the paperNot a substantial claim - Premisecitation
The 17% undetected rate is already better than the error rates of several morphology-based studies.
“While this is already better than the error rates of several morphology-based studies (see [10] for a discussion)”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [10] P Haase, 2010, First audit of macroinvertebrate samples from an EU Water Framework Directive monitoring program: human error greatly lowers precision of assessment results, J North Am Benthol Soc source
Not checked in the free run.
- Interpretationnone given
The study established a quick and reliable protocol to assess the macrozoobenthic communities of stream ecosystems.
“In this study, we established a quick and reliable protocol to assess the macrozoobenthic communities of stream ecosystems.”
Quote in the paperSubstantial claim - Interpretationreasoning
The variation in sequence number among taxa using Folmer primers is probably because there were mismatches in the primer binding regions.
“probably because there were mismatches in the primer binding regions.”
Quote in the paperSubstantial claim - Interpretationown data
The ten morphologically identified specimens per morphotaxon included several species.
“indicating that the ten morphologically identified specimens per morphotaxon included several species.”
Quote in the paperNot a substantial claim - Study design and conductown data
The technical accuracy of this method was supported by comparisons among many replicates.
“The technical accuracy of this method was supported by comparisons among many replicates.”
Quote in the paperSubstantial claim - Interpretationown data
Primer efficiencies across different taxa greatly hinder species abundance assessments using PCR-based approaches.
“the results of experiment II show that primer efficiencies across different taxa greatly hinder species abundance assessments using PCR-based approaches”
Quote in the paperSubstantial claim - Premisecitation
Amplification of diverse multitemplate mixtures using universal primers can lead to highly unequal amplification efficiencies among products.
“Amplification of diverse multitemplate mixtures using universal primers can lead to highly unequal amplification efficiencies among products [39,49].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [39] Piñol J, Mir G, Gomez-Polo P, Agustí N. Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods. Mol Ecol Resour. 2014; 1–12. <comment>doi: <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://dx.doi.org/10.1111/1755-0998.12355" xlink:type="simple">10.1111/1755-0998.12355</ext-link></comment> source
Not checked in the free run.
- cites [49] MF Polz, 1998, Bias in template-to-product ratios in multitemplate PCR, Appl Environ Microbiol source
Not checked in the free run.
- Interpretationown data
It is possible to reliably estimate the biomass of a single species using the results of experiment I.
“The results of experiment I show that it is possible to reliably estimate the biomass of a single species”
Quote in the paperSubstantial claim - Interpretationown data
The taxon abundance of diverse environmental samples cannot be reliably assessed.
“However, we also showed that the taxon abundance of diverse environmental samples can not be reliably assessed.”
Quote in the paperSubstantial claim - Interpretationown data
The protocol developed in this study enables the identification of nearly all macrozoobenthic species in an environmental sample.
“The protocol developed in this study enables the identification of nearly all macrozoobenthic species in an environmental sample.”
Quote in the paperSubstantial claim
- Classified as probably not a substantial claim. 23
- May combine two claims. 23
Probably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataThe two independent weight measurements differed in mean by 0.1 mg (SD = 0.03).
“The two independent weight measurements differed in mean by 0.1 mg (SD = 0.03).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataThe MiSeq runs of experiments I and II yielded 9.63 and 14.07 Gb of read data, respectively.
“The MiSeq runs of experiments I and II yielded 9.63 and 14.07 Gb of read data, respectively.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn all 10 replicates, all 31 D. cephalotes specimens were recovered based on their unique haplotypes, including the smallest specimens, which only made up 0.023% (0.145 mg) of the total specimen biomass.
“In all 10 replicates, we recovered all 31 D. cephalotes specimens based on their unique haplotypes, including the smallest specimens, which only made up 0.023% (0.145 mg) of the total specimen biomass (Fig 2A).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe mean normalised sequence abundances had low standard deviations and the linear model fit well (p<0.001, R2 = 0.65).
“The mean normalised sequence abundances had low standard deviations and the linear model fit well (p<0.001, R 2 = 0.65, S8 Fig).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingnone givenFor two specimens, sequence artefacts were recovered; these did not affect further analyses.
“For two specimens, we recovered sequence artefacts (see S6 Fig); these did not affect further analyses.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataBoth MiSeq runs showed good read quality, with sequences having Q30 ≥ 85.3% and 83.3%.
“Both MiSeq runs showed good read quality (sequences with Q30 ≥ 85.3% and 83.3%).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataSequences that did not match the target species were similar to sequences derived from a variety of benthic organisms, but also fungi and plants.
“Sequences that did not match the target species were similar to sequences derived from a variety of benthic organisms, but also fungi and plants.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Findingnone givenUsing the protocol, it is possible to assess community compositions within a week, from sampling to species identification.
“Using our protocol, it is possible to assess community compositions within a week, from sampling to species identification.”
Quote in the paperNot a substantial claimMay combine two claims.
FindingcitationThe study used a universal COI primer with a broad target range, and only 17% of taxa went undetected.
“Furthermore, we used a universal COI primer with a broad target range [41], and only 17% of taxa went undetected.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [41] O Folmer, 1994, DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates, Mol Marine Biol Biotechnol
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataOf all recovered 213 OTUs, 31 could not be identified using the BOLD database.
“Of all recovered 213 OTUs, 31 could not be identified using the BOLD database (S9B Fig).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataA slightly different sequencing efficiency was detected between forward and reverse primers, but this only affected total sequence abundance and did not systematically alter the inferences from the data.
“We detected a slightly different sequencing efficiency between forward and reverse primers, but this only affected total sequence abundance and did not systematically alter the inferences from the data.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
Findingnone givenThe parallel sequencing strategy leads to an increase in per-site sequence diversity and read quality.
“Our parallel sequencing strategy leads to an increase in per-site sequence diversity and read quality.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationDifferent species within a genus or subfamily can have different ecological preferences and stress tolerances and belong to different functional feeding groups.
“different species within a genus or subfamily can have different ecological preferences and stress tolerances and belong to different functional feeding groups [11,12] see [13] for review.”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [11] Merritt RW, Berg MB, Cummins KW. An Introduction to the Aquatic Insects of North America. 2009.
Not checked in the free run.
- cites [12] SU Pauls, 2008, Grazers, shredders and filtering carnivores—The evolution of feeding ecology in Drusinae (Trichoptera: Limnephilidae): Insights from a molecular phylogeny, Mol Phylogenet Evol source
Not checked in the free run.
- cites [13] FC Jones, 2008, Taxonomic sufficiency: The influence of taxonomic resolution on freshwater bioassessments using benthic macroinvertebrates, Environ Rev source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe same PCR master mix was used for all reactions to ensure identical PCR conditions for all replicates.
“The same PCR master mix was used for all reactions to ensure identical PCR conditions for all replicates.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationMany benthic invertebrate larvae are difficult to identify at the species level, so the most practical taxonomic level for identification is often only genus or family.
“many benthic invertebrate larvae are difficult to identify at the species level, and thus the most practical taxonomic level for the identification of these organisms is often only the genus or family [10].”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [10] P Haase, 2010, First audit of macroinvertebrate samples from an EU Water Framework Directive monitoring program: human error greatly lowers precision of assessment results, J North Am Benthol Soc source
Not checked in the free run.
Classified as probably not a substantial claim.
Study design and conductreasoningOnly sequences that had a maximum of five mismatches and gaps were included in the analysis to ensure reliable hits.
“To ensure reliable hits, only sequences that had a maximum of five mismatches and gaps were included in the analysis.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe parallel sequencing approach substantially reduced regions of low per-site diversity, improving read quality.
“The parallel sequencing approach thus substantially reduced regions of low per-site diversity, improving read quality.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationDNA barcoding allows for standardized and accurate species identification.
“DNA barcoding allows for standardized and accurate species identification [15–18].”
Quote in the paperNot a substantial claim0 of 4 sources checked- cites [15] PDN Hebert, 2003, Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species, Proc R Soc Lond B Biol Sci source
Not checked in the free run.
- cites [16] X Zhou, 2009, Towards a comprehensive barcode library for arctic life—Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Canada, Front Zool source
Not checked in the free run.
- cites [17] X Zhou, 2010, Ephemeroptera, Plecoptera, and Trichoptera fauna of Churchill (Manitoba, Canada): insights into biodiversity patterns from DNA barcoding, J North Am Benthol Soc source
Not checked in the free run.
- cites [18] BW Sweeney, 2011, Can DNA barcodes of stream macroinvertebrates improve descriptions of community structure and water quality?, J North Am Benthol Soc source
Not checked in the free run.
May combine two claims.
PremisecitationFrequent identification errors occur and many specimens are not detected in samples.
“frequent identification errors occur and many specimens are not detected in samples [10]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [10] P Haase, 2010, First audit of macroinvertebrate samples from an EU Water Framework Directive monitoring program: human error greatly lowers precision of assessment results, J North Am Benthol Soc source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll analyses were performed with ten replicates to improve statistical robustness.
“All analyses were performed with ten replicates to improve statistical robustness.”
- Also stated in: § Abstract · plain
“Each experiment was replicated ten times to maximise statistical power.”
Quote in the paperNot a substantial claim- Also stated in: § Abstract · plain
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationStein and co-authors showed that ten of 16 assessment metrics had higher statistical power using DNA barcoding than morphological assessment.
“Stein and co-authors showed that ten of 16 assessment metrics had higher statistical power using DNA barcoding than morphological assessment [14].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] ED Stein, 2014, Is DNA Barcoding Actually Cheaper and Faster than Traditional Morphological Methods: Results from a Survey of Freshwater Bioassessment Efforts in the United States?, PLoS ONE source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationFor animals, a 658-bp standardized fragment of the mitochondrial gene COI is typically used.
“For animals, a 658-bp standardized fragment of the mitochondrial gene COI (cytochrome c oxidase subunit 1) is typically used [19].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [19] PDN Hebert, 2003, Biological identifications through DNA barcodes, Proc R Soc Lond B Biol Sci source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationreasoningThese morphotaxa presumably contained several taxonomically distant species, which are unequally well amplified with the Folmer primers.
“Presumably, these morphotaxa contained several taxonomically distant species, which are unequally well amplified with the Folmer primers.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe study used a highly replicated and standardised approach for species detection using DNA metabarcoding.
“We used a highly replicated and standardised approach for species detection using DNA metabarcoding”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Interpretationnone givenSeveral technical and logistic problems have to be overcome before this protocol can be used for large-scale monitoring.
“show that several technical and logistic problems have to be overcome before this protocol can be used for large-scale monitoring.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe amount of tissue used in the DNA extraction was sufficient and did not lead to the systematic exclusion of small specimens as long as tissues are well ground and specimens have similar amplification efficiencies.
“However, our results indicate that the amount of tissue used in the DNA extraction was sufficient and did not lead to the systematic exclusion of small specimens as long as tissues are well ground and specimens have similar amplification efficiencies.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based).
Interpretationnone givenDegraded DNA and contamination as in eDNA studies with amplicons is not a concern for organisms collected directly from streams.
“However degraded DNA and contamination as in eDNA studies with amplicons is not a concern for organisms collected directly from streams.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSeveral studies show that DNA-based assessments are superior to morphological assessments in freshwater ecosystems.
“Several studies show that DNA-based assessments are superior to morphological assessments in freshwater ecosystems (e.g., [14,25,33])”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [14] ED Stein, 2014, Is DNA Barcoding Actually Cheaper and Faster than Traditional Morphological Methods: Results from a Survey of Freshwater Bioassessment Efforts in the United States?, PLoS ONE source
Not checked in the free run.
- cites [25] JK Jackson, 2014, Cryptic biodiversity in streams: a comparison of macroinvertebrate communities based on morphological and DNA barcode identifications, Freshw Sci source
Not checked in the free run.
- cites [33] Zimmermann J, Glöckner G, Jahn R, Enke N, Gemeinholzer B. Metabarcoding vs. morphological identification to assess diatom diversity in environmental studies. Mol Ecol Resour. 2014; 1–17. <comment>doi: <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://dx.doi.org/10.1111/1755-0998.12336" xlink:type="simple">10.1111/1755-0998.12336</ext-link></comment> source
Not checked in the free run.
May combine two claims.
Interpretationown dataIt is not possible to reliably estimate the abundance of a single species because many small organisms generate the same number of sequence reads as a few large organisms.
“but not its abundance because many small organisms generate the same number of sequence reads as a few large organisms.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningIt is not possible to accurately estimate species biomass or even abundance in diverse environmental samples using amplification-based sequencing protocols.
“Thus, it is not possible to accurately estimate species biomass or even abundance in diverse environmental samples using amplification-based sequencing protocols.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe efforts of large national and international barcoding consortia (BOLD, iBOL, and GBOL) have contributed to a substantial increase in both the size and quality of reference databases.
“The efforts of large national and international barcoding consortia (BOLD, iBOL, and GBOL) have contributed to a substantial increase in both the size and quality of reference databases [56]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [56] P Taberlet, 2012, Towards next-generation biodiversity assessment using DNA metabarcoding, Mol Ecol source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.
InterpretationcitationMetabarcoding will widely replace present biomonitoring methods over time because it has higher taxonomic resolution, is cost efficient and fast, and reduces human bias enabling comparisons among studies.
“we are confident that metabarcoding will widely replace present biomonitoring methods over time because 1) it has a higher taxonomic resolution [14], 2) it is cost efficient and fast and, most importantly, 3) it reduces human bias enabling comparisons among studies [57].”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [14] ED Stein, 2014, Is DNA Barcoding Actually Cheaper and Faster than Traditional Morphological Methods: Results from a Survey of Freshwater Bioassessment Efforts in the United States?, PLoS ONE source
Not checked in the free run.
- cites [57] Y Ji, 2013, Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding, Ecol Lett source
Not checked in the free run.
May combine two claims.
Interpretationown dataThe study provides a highly reproducible laboratory protocol for processing macroinvertebrate samples in bulk and identified species using metabarcoding with the standard COI region.
“We provide a highly reproducible laboratory protocol for processing macroinvertebrate samples in bulk and identified species using metabarcoding with the standard COI region.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationPiñol et al. [39] proposed a relationship between biomass and sequence reads, but did not systematically examine this hypothesis.
“Piñol et al. [39] proposed a relationship between biomass and sequence reads, but did not systematically examine this hypothesis.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [39] Piñol J, Mir G, Gomez-Polo P, Agustí N. Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods. Mol Ecol Resour. 2014; 1–12. <comment>doi: <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://dx.doi.org/10.1111/1755-0998.12355" xlink:type="simple">10.1111/1755-0998.12355</ext-link></comment> source
Not checked in the free run.
May combine two claims.
InterpretationreasoningThe observed pattern is consistent with the primer bias already observed between morphotaxa and supports the overall findings of strong taxon-specific amplification bias.
“This is consistent with the primer bias which we already observed between morphotaxa and support the overall findings of strong taxon-specific amplification bias.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).
InterpretationreasoningThe methods developed in this study can easily be adapted to assess the communities of other ecosystems.
“The methods developed in this study can easily be adapted to assess the communities of other ecosystems.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationWhile the conserved ribosomal mitochondrial gene approach could have comparable taxonomic resolution as the COI barcode, it is currently limited by the lack of reference databases.
“However, while this approach could have comparable taxonomic resolution as the COI barcode, it is currently limited by the lack of reference databases [55].”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [55] LJ Clarke, 2014, Environmental metabarcodes for insects: in silicoPCR reveals potential for taxonomic bias, Mol Ecol Resour source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningA combination of DNA species barcodes and ecological traits will maximise the power of metabarcoding for the reliable assessment of ecosystem responses under stress and for biomonitoring.
“A combination of both data types, i.e., DNA species barcodes and ecological traits, will maximise the power of metabarcoding for the reliable assessment of ecosystem responses under stress and for biomonitoring.”
Quote in the paperNot a substantial claim- Rests on an assumption: DNA-based assessments cannot assign biological traits to species.
The paper relies on this without support.
Precise estimates of biomass with respect to specimen recovery and primer bias have not been performed.
“Precise estimates of biomass with respect to specimen recovery and primer bias have not been performed.”
The paper relies on this without support.
COI amplicons are typically similar in base composition.
“COI amplicons are typically similar in base composition; therefore, three strategies were used to increase sequence diversity.”
The paper relies on this without support.
Sanger sequencing requires that each specimen is processed individually in the laboratory, which is costly and extremely time-consuming for routine community assessments involving hundreds or thousands of specimens per sample.
“However, Sanger sequencing requires that each specimen is processed individually in the laboratory, which is costly and extremely time-consuming for routine community assessments involving hundreds or thousands of specimens per sample.”
The paper relies on this without support.
Benthic indicator organisms vary in biomass, and this variation depends on taxonomic group and life stage.
“Benthic indicator organisms vary in biomass, and this variation depends on taxonomic group and life stage.”
The paper relies on this without support.
Morphotaxa could often only be identified at family or order level, and several species per morphotaxon may occur in the sampled aquatic habitats even at the same habitat patch.
“This was expected, as morphotaxa could often only be identified at family or order level, and several species per morphotaxon may occur in the sampled aquatic habitats even at the same habitat patch.”
The paper relies on this without support.
Stochastic effects increase with reduced specimen biomass.
“This result is expected because stochastic effects increase with reduced specimen biomass.”
The paper relies on this without support.
Currently, the monitoring of freshwater ecosystems is based on abundance metrics, which cannot be generated using the metabarcoding solutions currently available.
“Currently, the monitoring of freshwater ecosystems is based on abundance metrics, which cannot be generated using the metabarcoding solutions currently available.”
The paper relies on this without support.
All assessment protocols rely on reference catalogues against which inventory data of a species from an ecosystem are compared.
“All assessment protocols rely on reference catalogues against which inventory data of a species from an ecosystem are compared.”
The paper relies on this without support.
Changes in species traits in a community (e.g. functional feeding groups, tolerance against pollution) are used as indicator values to evaluate the biological significance of inferred community changes.
“In particular, changes in species traits in a community (e.g. functional feeding groups, tolerance against pollution) are used as indicator values to evaluate the biological significance of inferred community changes.”
The paper relies on this without support.
DNA-based assessments cannot assign biological traits to species.
“However, DNA-based assessments cannot assign biological traits to species.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Size filtering increases the likelihood of detecting small-bodied and rare species in samples, as individuals with a larger biomass provide disproportionate amounts of DNA and can therefore be over-represented when metabarcoding bulk samples.
“This was done to increase the likelihood of detecting small-bodied and rare species in samples, as individuals with a larger biomass provide disproportionate amounts of DNA and can therefore be over-represented when metabarcoding bulk samples65.”
From Soundscapes and deep learning enable tracking biodiversity recovery in tropical forests (2023) · cites this paper as [65]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
All 52 taxa assigned OTUs despite taxa going undetected
MinorThe paper says OTUs were reliably assigned to all 52 taxa, but later says 17% of the taxa went undetected. Undetected taxa cannot also have been assigned OTUs.
“We were able to reliably assign OTUs to all 52 taxa included in the extraction.”
“Furthermore, we used a universal COI primer with a broad target range [41], and only 17% of taxa went undetected.”
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Pfrender, M. E., Hawkins, C. P., Bagley, M. J., Courtney, G. W., Creutzburg, B. R., Epler, J. H., Fend, S., Ferrington, L. C., Hartzell, P., Jackson, S. A., et al. (2010) · The Quarterly Review of Biology · doi:10.1086/655118 · Cited by 89 · see citing works
Elbrecht, V., Feld, C. K., Gies, M., Hering, D., Sondermann, M., Tollrian, R., & Leese, F. (2014) · Freshwater Science · doi:10.1086/674536 · Cited by 49 · see citing works
Self-citationHigh‐Throughput Sequencing: A Roadmap Toward Community Ecology
Poisot, T., Péquin, B., & Gravel, D. (2013) · Ecology and Evolution · doi:10.1002/ece3.508 · Cited by 46 · see citing works
The complete mitochondrial genome of the stoneflyDinocras cephalotes(Plecoptera, Perlidae)
Elbrecht, V., Poettker, L., John, U., & Leese, F. (2013) · Mitochondrial DNA · doi:10.3109/19401736.2013.830301 · Cited by 30 · see citing works
Self-citationRanasinghe, J. A., Stein, E. D., Miller, P. E., & Weisberg, S. B. (2012) · PLoS ONE · doi:10.1371/journal.pone.0040875 · Cited by 24 · see citing works
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Environmental DNA metabarcoding: Transforming how we survey animal and plant communities
Deiner, K., Bik, H. M., Mächler, E., Seymour, M., Lacoursière‐Roussel, A., Altermatt, F., Creer, S., Bista, I., Lodge, D. M., de Vere, N., et al. (2017) · Molecular Ecology · doi:10.1111/mec.14350 · Cited by 1,964 · see citing works
“Selected strategies include filtering based on a lower Phred score cut-off, usually set at least above 20 or 30 (Bista (Continues) et al., 2017; Elbrecht & Leese, 2015; H€anfling et al., 2016).”
BackgroundMethodTaberlet, P., Bonin, A., Zinger, L., & Coissac, É. (2018) · doi:10.1093/oso/9780198767220.001.0001 · Cited by 1,061 · see citing works
Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?
Deagle, B. E., Thomas, A. C., McInnes, J. C., Clarke, L. J., Vesterinen, E. J., Clare, E. L., Kartzinel, T. R., & Eveson, J. P. (2018) · Molecular Ecology · doi:10.1111/mec.14734 · Cited by 834 · see citing works
“Many studies have sequenced DNA from insect mock communities; however, rather than considering if read counts are proxies for input biomass, the focus of these studies has generally been to test if taxa can be detected (Alberdi et al. 2017; Clarke et al. 2014; Elbrecht & Leese 2015; Yu et al. 2012).”
BackgroundBeng, K. C., & Corlett, R. T. (2020) · Biodiversity and Conservation · doi:10.1007/s10531-020-01980-0 · Cited by 721 · see citing works
Environmental DNA reveals that rivers are conveyer belts of biodiversity information
Deiner, K., Fronhofer, E. A., Mächler, E., Walser, J., & Altermatt, F. (2016) · Nature Communications · doi:10.1038/ncomms12544 · Cited by 664 · see citing works
Cryptic species as a window into the paradigm shift of the species concept
Fišer, C., Robinson, C. T., & Malard, F. (2018) · Molecular Ecology · doi:10.1111/mec.14486 · Cited by 568 · see citing works
“However, applying this approach to conservation biology (Asmyhr, Linke, Hose, & Nipperess, 2014) and local community ecology (Elbrecht & Leese, 2015; Jones, Ghoorah, & Blaxter, 2011) can be risky.”
MethodPawlowski, J. W., Kelly‐Quinn, M., Altermatt, F., Apothéloz‐Perret‐Gentil, L., Beja, P. R., Boggero, A., Borja, Á., Bouchez, A., Cordier, T., Domaizon, I., et al. (2018) · The Science of The Total Environment · doi:10.1016/j.scitotenv.2018.05.002 · Cited by 562 · see citing works
Self-citationStat, M., Huggett, M. J., Bernasconi, R., DiBattista, J. D., Berry, T. E., Newman, S. J., Harvey, E. S., & Bunce, M. (2017) · Scientific Reports · doi:10.1038/s41598-017-12501-5 · Cited by 546 · see citing works
Deep learning and computer vision will transform entomology
Høye, T. T., Ärje, J., Bjerge, K., Hansen, O. L. P., Iosifidis, A., Leese, F., Mann, H. M. R., Meissner, K. K., Melvad, C., & Raitoharju, J. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2002545117 · Cited by 473 · see citing works
Self-citationElbrecht, V., & Leese, F. (2017) · Frontiers in Environmental Science · doi:10.3389/fenvs.2017.00011 · Cited by 465 · see citing works
“Amplification success of the BF/BR primers was evaluated using 10 mock communities, each containing a set of 52 different freshwater invertebrates also used in previous studies (Elbrecht and Leese, 2015; Elbrecht et al., 2016).”
InfluentialSelf-citationHering, D., Borja, Á., Jones, J. I., Pont, D., Boets, P., Bouchez, A., Bruce, K., Drakare, S., Hänfling, B., Kahlert, M., et al. (2018) · Water Research · doi:10.1016/j.watres.2018.03.003 · Cited by 453 · see citing works
Self-citationHow quantitative is metabarcoding: A meta‐analytical approach
Lamb, P. D., Hunter, E., Pinnegar, J. K., Creer, S., Davies, R. G., & Taylor, M. I. (2018) · Molecular Ecology · doi:10.1111/mec.14920 · Cited by 449 · see citing works
“…& Faecal Fish 16s Diaz‐Real, Serrano, Piriz, and Jovani (2015) 3 454 Individuals Feather mites COI Egge et al. (2013) 11 454 DNA/RNA Haptophytes 18s Elbrecht and Leese (2015) 52 Illumina Biomass Macroinvertebrates (freshwater) COI Elbrecht et al. (2016) 52 Illumina Individuals…”
Elbrecht, V., Vamos, E. E., Meissner, K., Aroviita, J., & Leese, F. (2017) · Methods in Ecology and Evolution · doi:10.1111/2041-210x.12789 · Cited by 385 · see citing works
“DNA metabarcoding can provide much more accurate taxonomic identification than morphology-based methods, and can even be used to detect cryptic species (Elbrecht & Leese 2015).”
BackgroundMethodInfluentialSelf-citationValidation of COI metabarcoding primers for terrestrial arthropods
Elbrecht, V., Braukmann, T., Ivanova, N. V., Prosser, S. W. J., Hajibabaei, M., Wright, M., Zakharov, E., Hebert, P. D. N., & Steinke, D. (2019) · PeerJ · doi:10.7717/peerj.7745 · Cited by 323 · see citing works
“…in-line tags of different length to shift PeerJ reviewing PDF | (2019:07:39441:1:0:NEW 19 Aug 2019) 157 amplicons against each other and sequenced half of the samples in reverse orientation by swapping 158 the Illumina P5 and P7 sequencing adapters on the fusion primers (Elbrecht & Leese 2015).”
BackgroundMethodSelf-citationEstimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding
Krehenwinkel, H., Wolf, M., Lim, J. Y., Rominger, A. J., Simison, W. B., & Gillespie, R. G. (2017) · Scientific Reports · doi:10.1038/s41598-017-17333-x · Cited by 310 · see citing works
Scaling up: A guide to high‐throughput genomic approaches for biodiversity analysis
Porter, T. M., & Hajibabaei, M. (2018) · Molecular Ecology · doi:10.1111/mec.14478 · Cited by 305 · see citing works
“…in copy number, as well as variation in biomass and density among organisms, it has been suggested that a conservative approach is to treat DNA metabarcoding data as presence–absence data only (Elbrecht & Leese, 2015; Hajibabaei, Spall, Shokralla, & van Konynenburg, 2012; Hajibabaei et al., 2011).”
BackgroundUnderstanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies
Kelly, R. P., Shelton, A. O., & Gallego, R. (2019) · Scientific Reports · doi:10.1038/s41598-019-48546-x · Cited by 303 · see citing works
“Taken together, along with other recent work such as [23] and [21], a common 411 understanding of the processes underlying PCR-based studies appears to be coalescing.”
BackgroundResultAncient plant DNA in lake sediments
Parducci, L., Bennett, K. D., Ficetola, G. F., Alsos, I. G., Suyama, Y., Wood, J. R., & Pedersen, M. W. (2017) · New Phytologist · doi:10.1111/nph.14470 · Cited by 276 · see citing works
Cordier, T., Alonso‐Sáez, L., Apothéloz‐Perret‐Gentil, L., Aylagas, E., Bohan, D. A., Bouchez, A., Chariton, A. A., Creer, S., Frühe, L., Keck, F., et al. (2020) · Molecular Ecology · doi:10.1111/mec.15472 · Cited by 282 · see citing works
“…and the variable number of copies of the targeted marker in the genome (Bik et al., 2013; Větrovský & Baldrian 2013), in addition to variations in DNA extractability and primer-specific amplification bias (Elbrecht & Leese, 2015; Krehenwinkel et al., 2017; Piñol, Mir, Gomez-Polo, & Agustí, 2015).”
BackgroundSelf-citationPiñol, J., Senar, M. À., & Symondson, W. O. C. (2018) · Molecular Ecology · doi:10.1111/mec.14776 · Cited by 262 · see citing works
“Consequently, some species are better amplified than others and the proportions in the final mixture do not reflect the original proportion of each species (Elbrecht and Leese, 2015; Leray et al., 2013; Bista et al., 2018).”
BackgroundSrivathsan, A., Lee, L., Katoh, K., Hartop, E., Kutty, S. N., Wong, J., Yeo, D. C. J., & Meier, R. (2021) · BMC Biology · doi:10.1186/s12915-021-01141-x · Cited by 265 · see citing works
“ing samples consisting of hundreds or thousands of specimens [74, 75], large-scale barcoding of individual specimens remains essential for discovering and describing species.”
BackgroundProspects and challenges of environmental DNA (eDNA) monitoring in freshwater ponds
Harper, L. R., Buxton, A. S., Rees, H. C., Bruce, K., Brys, R., Halfmaerten, D., Read, D. S., Watson, H. V., Sayer, C. D., Jones, E. P., et al. (2018) · Hydrobiologia · doi:10.1007/s10750-018-3750-5 · Cited by 268 · see citing works
Benchmarking DNA Metabarcoding for Biodiversity-Based Monitoring and Assessment
Aylagas, E., Borja, Á., Irigoien, X., & Rodríguez‐Ezpeleta, N. (2016) · Frontiers in Marine Science · doi:10.3389/fmars.2016.00096 · Cited by 262 · see citing works
“The abovementioned biases inherent to PCR-based analyses can lead to greater recovery of sequences of some species and the exclusion of others (Elbrecht and Leese, 2015; Piñol et al., 2015).”
BackgroundInfluentialDeep-Sea, Deep-Sequencing: Metabarcoding Extracellular DNA from Sediments of Marine Canyons
Guardiola, M., Uriz, M. J., Taberlet, P., Coissac, É., Wangensteen, O. S., & Turón, X. (2015) · PLoS ONE · doi:10.1371/journal.pone.0139633 · Cited by 255 · see citing works
“Quantitative inference using number of reads of a given MOTU is controversial [5, 65, 66] and it has been suggested that relative abundances, rather than absolute values, are more reliably obtained from metabarcoding data [6, 13].”
BackgroundThomas, A. C., Deagle, B. E., Eveson, J. P., Harsch, C. H., & Trites, A. W. (2015) · Molecular Ecology Resources · doi:10.1111/1755-0998.12490 · Cited by 248 · see citing works
“In those circumstances, it is likely unrealistic to expect accurate estimates of species proportion based on DNA sequence read abundances (e.g. Clarke et al. 2014; Elbrecht & Leese 2015), and correction factors are likely not worth pursuing in that stage of methodological development.”
BackgroundMinimizing polymerase biases in metabarcoding
Nichols, R. V., Vollmers, C., Newsom, L. A., Wang, Y., Heintzman, P. D., Leighton, M., Green, R. E., & Shapiro, B. (2018) · Molecular Ecology Resources · doi:10.1111/1755-0998.12895 · Cited by 245 · see citing works
Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing
Ushio, M., Murakami, H., Masuda, R., Sado, T., Miya, M. U., Sakurai, S., Yamanaka, H., Minamoto, T., & Kondoh, M. (2018) · Metabarcoding and Metagenomics · doi:10.3897/mbmg.2.23297 · Cited by 240 · see citing works
Advances in DNA metabarcoding for food and wildlife forensic species identification
Staats, M., Arulandhu, A. J., Gravendeel, B., Holst‐Jensen, A., Scholtens, I. M. J., Peelen, T., Prins, T. W., & Kok, E. J. (2016) · Analytical and Bioanalytical Chemistry · doi:10.1007/s00216-016-9595-8 · Cited by 244 · see citing works
“PCR bias caused by variable primer– template mismatches across species may limit the quantitative potential of DNA metabarcoding and may cause species to be missed [28, 29].”
BackgroundInfluentialProspects and challenges of implementing DNA metabarcoding for high-throughput insect surveillance
Piper, A. M., Batovska, J., Cogan, N. O. I., Weiss, J., Cunningham, J. P., Rodoni, B., & Blacket, M. J. (2019) · GigaScience · doi:10.1093/gigascience/giz092 · Cited by 239 · see citing works
“Regardless of whether an enrichment or metagenomics approach was used, platform-specific sequencing adapters need to be attached to the molecules (via ligation [151] or 1-step [152] or 2-step PCR [40, 106]) to form “libraries” that can then bind to the flow cell for sequencing (Fig.”
Beyond Biodiversity: Can Environmental DNA (eDNA) Cut It as a Population Genetics Tool?
Adams, C. I. M., Knapp, M., Gemmell, N. J., Jeunen, G., Bunce, M., Lamare, M. D., & Taylor, H. R. (2019) · Genes · doi:10.3390/genes10030192 · Cited by 241 · see citing works
“Additionally, read depth or eDNA copy number may not to correlate with known biomass [98,104,105].”
BackgroundHow to limit false positives in environmental DNA and metabarcoding?
Ficetola, G. F., Taberlet, P., & Coissac, É. (2016) · Molecular Ecology Resources · doi:10.1111/1755-0998.12508 · Cited by 235 · see citing works
“We know that species with just one (singletons) or two reads in one sample are probably artefacts, but if sequencing depth is high in some cases we can find tens of reads, assigned to absent species (De Barba et al. 2014; Elbrecht & Leese 2015).”
BackgroundImplementing and Innovating Marine Monitoring Approaches for Assessing Marine Environmental Status
Danovaro, R., Carugati, L., Berzano, M., Cahill, A., Carvalho, S., Chenuil, A., Corinaldesi, C., Cristina, S., David, R., Dell’Anno, A., et al. (2016) · Frontiers in Marine Science · doi:10.3389/fmars.2016.00213 · Cited by 242 · see citing works
“Conversely, stronger relationships have been reported between biomass and read proportions (Elbrecht and Leese, 2015).”
BackgroundeDNA metabarcoding as a new surveillance approach for coastal Arctic biodiversity
Lacoursière‐Roussel, A., Howland, K. E., Normandeau, É., Grey, E. K., Archambault, P., Deiner, K., Lodge, D. M., Hernandez, C., Leduc, N., & Bernatchez, L. (2018) · Ecology and Evolution · doi:10.1002/ece3.4213 · Cited by 222 · see citing works
“On the other hand, targeting genes with lower taxonomic specificity (e.g., 18S) could improve the detection of biodiversity shifts at higher levels (e.g., phyla level; see Bik et al., 2012; Deagle et al., 2014; Elbrecht & Leese, 2015).”
BackgroundShort COI markers for freshwater macroinvertebrate metabarcoding
Vamos, E. E., Elbrecht, V., & Leese, F. (2017) · Metabarcoding and Metagenomics · doi:10.3897/mbmg.1.14625 · Cited by 219 · see citing works
Self-citationSorting things out: Assessing effects of unequal specimen biomass on DNA metabarcoding
Elbrecht, V., Peinert, B., & Leese, F. (2017) · Ecology and Evolution · doi:10.1002/ece3.3192 · Cited by 209 · see citing works
“We hypothesize that also in more species rich samples, taxa biomass translates directly into read abundance (assuming no primer bias among species, Elbrecht & Leese, 2015).”
InfluentialSelf-citationVasselon, V., Rimet, F., Tapolczai, K., & Bouchez, A. (2017) · Ecological Indicators · doi:10.1016/j.ecolind.2017.06.024 · Cited by 212 · see citing works
Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies
Clarke, L. J., Beard, J. M., Swadling, K. M., & Deagle, B. E. (2017) · Ecology and Evolution · doi:10.1002/ece3.2667 · Cited by 211 · see citing works
“Biases introduced during DNA extraction, PCR amplification, and sequencing are likely to skew the number of reads per taxon, with a disproportionate effect of primertemplate mismatches on PCR- amplification efficiency (Elbrecht & Leese, 2015; Piñol et al., 2015).”
BackgroundOmics Advances in Ecotoxicology
Zhang, X., Xia, P., Wang, P., Yang, J., & Baird, D. J. (2018) · Environmental Science & Technology · doi:10.1021/acs.est.7b06494 · Cited by 204 · see citing works
Censusing marine eukaryotic diversity in the twenty-first century
Leray, M., & Knowlton, N. (2016) · Philosophical Transactions of the Royal Society B Biological Sciences · doi:10.1098/rstb.2015.0331 · Cited by 196 · see citing works
Population‐level inferences from environmental DNA—Current status and future perspectives
Sigsgaard, E. E., Jensen, M. R., Winkelmann, I. E., Møller, P. R., Hansen, M. M., & Thomsen, P. F. (2019) · Evolutionary Applications · doi:10.1111/eva.12882 · Cited by 194 · see citing works
“…eDNA shedding rates between individuals, and biased PCR amplification or capture efficiency (e.g., Alberdi, Aizpurua, Gilbert, & Bohmann, 2018; Elbrecht & Leese, 2015); (d) co-amplification/co-capture of DNA from closely related species (Burbano et al., 2010; Wilcox et al., 2013); (e) risk of…”
BackgroundMetabarcoding a diverse arthropod mock community
Braukmann, T., Ivanova, N. V., Prosser, S. W. J., Elbrecht, V., Steinke, D., Ratnasingham, S., de Waard, J. R., Sones, J. E., Zakharov, E., & Hebert, P. D. N. (2019) · Molecular Ecology Resources · doi:10.1111/1755-0998.13008 · Cited by 189 · see citing works
“Primer bias arises due to either varying levels of primer mismatch or template degradation (Clarke et al., 2014; Elbrecht & Leese, 2015).”
BackgroundInfluentialSelf-citationMethods for the extraction, storage, amplification and sequencing of DNA from environmental samples
Lear, G., Dickie, I. A., Banks, J. C., Boyer, S., Buckley, H. L., Buckley, T. R., Cruickshank, R., Dopheide, A., Handley, K. M., Hermans, S. M., et al. (2018) · New Zealand Journal of Ecology · doi:10.20417/nzjecol.42.9 · Cited by 181 · see citing works
“Virome: The genetic material from all the viruses associated with a host or environment. specific taxa (Elbrecht & Leese 2015; Aylagas et al. 2016) or sample media (Drummond et al. 2015; Klymus et al. 2107).”
BackgroundTaxonomy‐free molecular diatom index for high‐throughput eDNA biomonitoring
Apothéloz‐Perret‐Gentil, L., Cordonier, A., Straub, F., Iseli, J., Esling, P., & Pawlowski, J. W. (2017) · Molecular Ecology Resources · doi:10.1111/1755-0998.12668 · Cited by 176 · see citing works
“It is widely accepted that different technical and biological biases impact the relative abundance of specimens and sequences, making impossible the use of quantitative data in HTS surveys (Elbrecht & Leese 2015).”
BackgroundEstimating intraspecific genetic diversity from community DNA metabarcoding data
Elbrecht, V., Vamos, E. E., Steinke, D., & Leese, F. (2018) · PeerJ · doi:10.7717/peerj.4644 · Cited by 163 · see citing works
“…quality filtering of reads with the 75 recently developed unoise3 denoising strategy (Edgar, 2016) and calibrated this approach using a previously 76 characterized single-species mock sample composed of specimens with known haplotypes (Elbrecht & Leese, 2015; 77 Vamos, Elbrecht & Leese, 2017).”
BackgroundMethodResultInfluentialSelf-citationEnvironmental DNA allows upscaling spatial patterns of biodiversity in freshwater ecosystems
Carraro, L., Mächler, E., Wüthrich, R., & Altermatt, F. (2020) · Nature Communications · doi:10.1038/s41467-020-17337-8 · Cited by 170 · see citing works
Environmental DNA reveals tropical shark diversity in contrasting levels of anthropogenic impact
Bakker, J., Wangensteen, O. S., Chapman, D. D., Boussarie, G., Buddo, D., Guttridge, T. L., Hertler, H., Mouillot, D., Vigliola, L., & Mariani, S. (2017) · Scientific Reports · doi:10.1038/s41598-017-17150-2 · Cited by 171 · see citing works
Random sampling causes the low reproducibility of rare eukaryotic OTUs in Illumina COI metabarcoding
Leray, M., & Knowlton, N. (2017) · PeerJ · doi:10.7717/peerj.3006 · Cited by 164 · see citing works
“Differences in primer 73 affinity among taxonomic groups prevent the detection of some OTUs and result in inaccurate 74 estimates of relative abundances, particularly within complex samples (Pinto & Raskin 2012; 75 Deagle et al. 2013; Elbrecht & Leese 2015; Piñol et al. 2015).”
BackgroundCordier, T., Forster, D., Dufresne, Y., Martins, C. I. M., Stoeck, T., & Pawlowski, J. W. (2018) · Molecular Ecology Resources · doi:10.1111/1755-0998.12926 · Cited by 164 · see citing works
“…their formulas, which appears as an insurmountable problem due to the lack of direct relationship between specimen abundance (or biomass) and sequence reads amount in metabarcoding data (Dowle, Pochon, Banks, Shearer, & Wood, 2016; Elbrecht & Leese, 2015; Vivien, Wyler, Lafont, & Pawlowski, 2015).”
BackgroundTesting the potential of a ribosomal 16S marker for DNA metabarcoding of insects
Elbrecht, V., Taberlet, P., Déjean, T., Valentini, A., Usseglio‐Polatera, P., Beisel, J., Coissac, É., Boyer, F., & Leese, F. (2016) · PeerJ · doi:10.7717/peerj.1966 · Cited by 163 · see citing works
“Laboratory conditions and bioinformatic analyses were kept as similar as possible to the study by 69 (Elbrecht & Leese, 2015).”
BackgroundMethodInfluentialSelf-citationTemporal and spatial variation in distribution of fish environmental DNA in England’s largest lake
Handley, L. L., Read, D. S., Winfield, I. J., Kimbell, H. S., Johnson, H. F., Li, J., Hahn, C., Blackman, R. C., Wilcox, R., Donnelly, R., et al. (2019) · Environmental DNA · doi:10.1002/edn3.5 · Cited by 164 · see citing works
Maximizing fish detection with eDNA metabarcoding
Bessey, C., Jarman, S., Berry, O., Olsen, Y. S., Bunce, M., Simpson, T., Power, M., McLaughlin, J., Edgar, G. J., & Keesing, J. K. (2020) · Environmental DNA · doi:10.1002/edn3.74 · Cited by 164 · see citing works
Aylagas, E., Borja, Á., Muxika, I., & Rodríguez‐Ezpeleta, N. (2018) · Ecological Indicators · doi:10.1016/j.ecolind.2018.07.044 · Cited by 162 · see citing works
Drake, L. E., Cuff, J. P., Young, R., Marchbank, A. M., Chadwick, E. A., & Symondson, W. O. C. (2021) · Methods in Ecology and Evolution · doi:10.1111/2041-210x.13780 · Cited by 166 · see citing works
“This will, however, vary with sequencing depth, with relatively greater depths increasing the likelihood of artefacts having more than 10 occurrences (De Barba et al., 2014; Elbrecht & Leese, 2015).”
BackgroundMethodSchenekar, T., Schletterer, M., Lecaudey, L. A., & Weiss, S. J. (2020) · River Research and Applications · doi:10.1002/rra.3610 · Cited by 157 · see citing works
“…sequence variation in the primer annealing sites among individual species, leading to higher efficiency of the PCR reaction for certain species in a mixed DNA template, while other species might get amplified less or not at all (Bylemans, Gleeson, Hardy, & Furlan, 2018; Elbrecht & Leese, 2015).”
BackgroundLeese, F., Sander, M., Buchner, D., Elbrecht, V., Haase, P., & Zizka, V. M. A. (2020) · Environmental DNA · doi:10.1002/edn3.177 · Cited by 157 · see citing works
Self-citationLeese, F., Bouchez, A., Abarenkov, K., Altermatt, F., Borja, Á., Bruce, K., Ekrem, T., ČIAMPOR, F. J., Čiamporová‐Zaťovičová, Z., Costa, F. O., et al. (2018) · Advances in ecological research/Advances in Ecological Research · doi:10.1016/bs.aecr.2018.01.001 · Cited by 151 · see citing works
“Community metabarcoding relies on PCR amplification using “universal”, i.e., nonspecific primers and thus induces amplification bias when the primers inevitably match some taxa better than others (Elbrecht and Leese, 2015; Krehenwinkel et al., 2017).”
MethodSelf-citationVasselon, V., Bouchez, A., Rimet, F., Jacquet, S., Trobajo, R., Corniquel, M., Tapolczai, K., & Domaizon, I. (2017) · Methods in Ecology and Evolution · doi:10.1111/2041-210x.12960 · Cited by 150 · see citing works
“…density and species biomass are major biases likely to affect DNA metabarcoding quantification of multicellular organisms like macroinvertebrates (Elbrecht & Leese, 2015) or fish (Evans et al., 2016), diatoms are unicellular organisms for which gene copy number is mainly affected by the number…”
BackgroundBista, I., Carvalho, G. R., Tang, M., Walsh, K. A., Zhou, X. Y., Hajibabaei, M., Shokralla, S., Seymour, M., Bradley, D. T. C., Liu, S., et al. (2018) · Molecular Ecology Resources · doi:10.1111/1755-0998.12888 · Cited by 147 · see citing works
From metabarcoding to metaphylogeography: separating the wheat from the chaff
Turón, X., Antich, A., Palacín, C., Præbel, K., & Wangensteen, O. S. (2019) · Ecological Applications · doi:10.1002/eap.2036 · Cited by 147 · see citing works
“Further, the quantitative value of metabarcoding data is debatable (Elbrecht and Leese 2015, Wares and Pap-palardo 2016, Pi ~ nol et al. 2019).”
Studying Ecosystems With DNA Metabarcoding: Lessons From Biomonitoring of Aquatic Macroinvertebrates
Bush, A., Compson, Z. G., Monk, W. A., Porter, T. M., Steeves, R., Emilson, E. J. S., Gagné, N., Hajibabaei, M., Roy, M., & Baird, D. J. (2019) · Frontiers in Ecology and Evolution · doi:10.3389/fevo.2019.00434 · Cited by 139 · see citing works
“As stated above, DNA metabarcoding results do not currently produce a reliable signal of abundance or biomass (Elbrecht and Leese, 2015), although at the same time a bias in organism biomass can reduce the detectability of rare taxa (Elbrecht et al.”
BackgroundZaiko, A., Pochon, X., García‐Vázquez, E., Olenin, S., & Wood, S. A. (2018) · Frontiers in Marine Science · doi:10.3389/fmars.2018.00322 · Cited by 145 · see citing works
“Despite recent attempts to develop and optimize standardized protocols for eDNA metabarcoding (Taberlet et al., 2012; Deiner et al., 2015; Elbrecht and Leese, 2015; Aylagas et al., 2016; Porter and Hajibabaei, 2018), they are often restricted to a portion of the biotic community (e.”
BackgroundStrategies for sample labelling and library preparation in DNA metabarcoding studies
Bohmann, K., Elbrecht, V., Carøe, C., Bista, I., Leese, F., Bunce, M., Yu, D. W., Seymour, M., Dumbrell, A. J., & Creer, S. (2021) · Molecular Ecology Resources · doi:10.1111/1755-0998.13512 · Cited by 143 · see citing works
Self-citationEnvironmental DNA detection of rare and invasive fish species in two Great Lakes tributaries
Balasingham, K. D., Walter, R. P., Mandrak, N. E., & Heath, D. D. (2017) · Molecular Ecology · doi:10.1111/mec.14395 · Cited by 139 · see citing works
“…a semiquantitative measure of signal strength and can, thus, be used as a proxy for detection confidence (stronger signal indicates greater confidence) or, more indirectly, species abundance (Amend, Seifert, & Bruns, 2010; Carew, Pettigrove, Metzeling, & Hoffmann, 2013; Elbrecht & Leese, 2015).”
MethodMorinière, J., Balke, M., Doczkal, D., Geiger, M. F., Hardulak, L. A., Haszprunar, G., Hausmann, A., Hendrich, L., Regalado, L., Rulik, B., et al. (2019) · Molecular Ecology Resources · doi:10.1111/1755-0998.13022 · Cited by 140 · see citing works
“…of invasive and pest species (Ashfaq & Hebert, 2016; L. A. Hardulak et al. in prep), macrozoobenthos sampling for assessing water and stream health (Elbrecht & Leese, 2015; Serrana, Miyake, Gamboa, & Watanabe, 2018), faeces analyses for dietary inference (De Barba et al., 2014; Hawlitschek,…”
BackgroundBylemans, J., Gleeson, D. M., Hardy, C. M., & Furlan, E. M. (2018) · Ecology and Evolution · doi:10.1002/ece3.4387 · Cited by 137 · see citing works
“However, primers used in the initial amplification of barcode sequences can introduce significant biases (Clarke, Beard, Swadling, & Deagle, 2017; Elbrecht & Leese, 2015; Tremblay et al., 2015).”
A performance evaluation of targeted eDNA and eDNA metabarcoding analyses for freshwater fishes
Bylemans, J., Gleeson, D. M., Duncan, R. P., Hardy, C. M., & Furlan, E. M. (2019) · Environmental DNA · doi:10.1002/edn3.41 · Cited by 132 · see citing works
Macher, J., Vivancos, A., Piggott, J. J., Centeno, F. C., Matthaei, C. D., & Leese, F. (2018) · Molecular Ecology Resources · doi:10.1111/1755-0998.12940 · Cited by 136 · see citing works
“This is a suitable alternative to rarefaction (Elbrecht & Leese, 2015).”
MethodSelf-citationGeiger, M. F., Morinière, J., Hausmann, A., Haszprunar, G., Wägele, W. J., Hebert, P. D. N., & Rulik, B. (2016) · Biodiversity Data Journal · doi:10.3897/bdj.4.e10671 · Cited by 130 · see citing works
“For example, because species diversity is relatively low in freshwater ecosystems, metabarcoding has been quite successful in these settings (Elbrecht and Leese 2015, Dowle et al. 2015, Valentini et al. 2016).”
Background“Pitfalls in relative abundance estimation using eDNA metabarcoding”
Fonseca, V. G. (2018) · Molecular Ecology Resources · doi:10.1111/1755-0998.12902 · Cited by 129 · see citing works
Hajibabaei, M., Porter, T. M., Wright, M., & Rudar, J. (2019) · PLoS ONE · doi:10.1371/journal.pone.0220953 · Cited by 129 · see citing works
Zhang, S., Lu, Q., Wang, Y., Wang, X., Zhao, J., & Yao, M. (2019) · Molecular Ecology Resources · doi:10.1111/1755-0998.13105 · Cited by 129 · see citing works
“…can confound the relationship between species abun‐ dance and eDNA concentrations, but the most influential factor is likely to be biased primer binding and amplification of differ‐ ent taxa during PCR (Elbrecht & Leese, 2015; Li, Handley, Read, & Hänfling, 2018; Pinõl, Senar, & Symondson, 2018).”
BackgroundElbrecht, V., Beermann, A. J., Goessler, G., Neumann, J., Tollrian, R., Wagner, R., Wlecklik, A., Piggott, J. J., Matthaei, C. D., & Leese, F. (2016) · Freshwater Biology · doi:10.1111/fwb.12713 · Cited by 123 · see citing works
Self-citationShirazi, S., Meyer, R. S., & Shapiro, B. (2021) · Ecology and Evolution · doi:10.1002/ece3.8239 · Cited by 124 · see citing works
DNA Sequencing as a Tool to Monitor Marine Ecological Status
Goodwin, K. D., Thompson, L., Duarte, B., Kahlke, T., Thompson, A. R., Marques, J. C., & Caçador, I. (2017) · Frontiers in Marine Science · doi:10.3389/fmars.2017.00107 · Cited by 124 · see citing works
“Some studies report adequate estimates of relative abundance (Aylagas et al., 2014, 2016; Elbrecht and Leese, 2015; Tan et al., 2015; Thomsen and Willerslev, 2015) even though read abundance does not necessarily provide a direct correlation to organism abundance because of issues that include…”
BackgroundMethodInfluentialMetabarcoding From Microbes to Mammals: Comprehensive Bioassessment on a Global Scale
Compson, Z. G., McClenaghan, B., Singer, G. A. C., Fahner, N. A., & Hajibabaei, M. (2020) · Frontiers in Ecology and Evolution · doi:10.3389/fevo.2020.581835 · Cited by 118 · see citing works
“For example, a DNA metabarcoding study using a mock community of aquatic macroinvertebrates found four orders of magnitude differences in sequence abundances of 52 taxa despite standardization of biomass prior to PCR amplification and sequencing (Elbrecht and Leese, 2015).”
Scaling up DNA metabarcoding for freshwater macrozoobenthos monitoring
Elbrecht, V., & Steinke, D. (2018) · Freshwater Biology · doi:10.1111/fwb.13220 · Cited by 119 · see citing works
“With the newly designed BF2 + BR2 fusion primers developed in this publication up to three 96- well plates can be multiplexed for a single run n1 1 2 3 4 5 6pos 7 9 10 11 12 13 n28 14 16 n3 17 18 19 2015 21 23 24 25 n4 26 2722 28 29 30 31 32 33 34n5 35 37 38 n6 39 40 4136 1 n7 3 4 5 6 72 8 10 11 12 13 n8 149 15 17 18…”
BackgroundMethodResultInfluentialSelf-citationLaroche, O., Wood, S. A., Tremblay, L. A., Ellis, J. I., Lear, G., & Pochon, X. (2017) · Marine Pollution Bulletin · doi:10.1016/j.marpolbul.2017.11.042 · Cited by 119 · see citing works
Vasselon, V., Domaizon, I., Rimet, F., Kahlert, M., & Bouchez, A. (2017) · Freshwater Science · doi:10.1086/690649 · Cited by 117 · see citing works
“…amplification efficiency that may differ among species; DNA sequencing errors and bioinformatics filtering that may affect DNA sequence reliability (Jeon et al. 2008, Amend et al. 2010, Bragg et al. 2013, Weber and Pawlowski 2013, Deiner et al. 2015, Elbrecht and Leese 2015, Schmidt et al. 2015).”
BackgroundMata, V. A., Rebelo, H., Amorim, F., McCracken, G. F., Jarman, S., & Beja, P. R. (2018) · Molecular Ecology · doi:10.1111/mec.14779 · Cited by 117 · see citing works
“Another problem is the imperfect correlation between the proportions of sequencing reads and biomass, making it hard to establish the contribution of each prey item to the overall diet (Deagle, Thomas, Shaffer, Trites, & Jarman, 2013; Elbrecht & Leese, 2015; Piñol, Mir, Gomez‐Polo, & Agustí, 2015).”
BackgroundEcogenomics of Zooplankton Community Reveals Ecological Threshold of Ammonia Nitrogen
Yang, J., Zhang, X., Xie, Y., Song, C., Sun, J., Zhang, Y., Giesy, J. P., & Yu, H. (2017) · Environmental Science & Technology · doi:10.1021/acs.est.6b05606 · Cited by 114 · see citing works
PrimerMiner : an r package for development and in silico validation of DNA metabarcoding primers
Elbrecht, V., & Leese, F. (2016) · Methods in Ecology and Evolution · doi:10.1111/2041-210x.12687 · Cited by 114 · see citing works
“…regions like the cytochrome c oxidase subunit I (COI) fragment for animals often show highly variable primer binding sites (Deagle et al. 2014; Sharma & Kobayashi 2014) leading sometimes to substantial primer bias and species remaining undetected (Pi ~ nol et al. 2014; Elbrecht & Leese 2015).”
BackgroundSelf-citationCoble, A. A., Flinders, C., Homyack, J. A., Penaluna, B. E., Cronn, R., & Weitemier, K. (2018) · The Science of The Total Environment · doi:10.1016/j.scitotenv.2018.08.370 · Cited by 110 · see citing works
Impacts of DNA extraction and PCR on DNA metabarcoding estimates of soil biodiversity
Dopheide, A., Xie, D., Buckley, T. R., Drummond, A. J., & Newcomb, R. D. (2018) · Methods in Ecology and Evolution · doi:10.1111/2041-210x.13086 · Cited by 112 · see citing works
“A common strategy to limit these effects is the exclusion of low- abundance sequences or OTUs (Elbrecht & Leese, 2015; Vesty et al., 2017), yet our estimates were derived from OTU clustering with singleton sequences excluded, and further removal of OTUs with abundances <5 resulted in very similar…”
BackgroundInfluentialUncovering the complete biodiversity structure in spatial networks: the example of riverine systems
Altermatt, F., Little, C. J., Mächler, E., Wang, S., Zhang, X., & Blackman, R. C. (2020) · Oikos · doi:10.1111/oik.06806 · Cited by 107 · see citing works
“However, due to the poorly conserved region there is often primer bias (Elbrecht and Leese 2015) and identification to species-level is limited to some major taxonomic groups.”
BackgroundMartin, J. L., Santi, I., Pitta, P., John, U., & Gypens, N. (2022) · Metabarcoding and Metagenomics · doi:10.3897/mbmg.6.85794 · Cited by 109 · see citing works
“…issues occurring due to sample preservation (Mäki et al. 2017), DNA extraction (Van der Loos and Nijland 2021), primer choice and specificity (Elbrecht and Leese 2015; Lapeyra Martin et al. 2022; Latz et al. 2022) and ultimately PCR, which under- or overestimate different groups…”
BackgroundEffective mosquito and arbovirus surveillance using metabarcoding
Batovska, J., Lynch, S., Cogan, N. O. I., Brown, K., Darbro, J. M., Kho, E. A., & Blacket, M. J. (2017) · Molecular Ecology Resources · doi:10.1111/1755-0998.12682 · Cited by 108 · see citing works
“The use of universal primers for amplification of a range of species can cause variance in read number among taxa by several orders of magnitude (Elbrecht & Leese, 2015; Pi~nol et al., 2015).”
BackgroundResultComparing eDNA metabarcoding and species collection for documenting Arctic metazoan biodiversity
Leduc, N., Lacoursière‐Roussel, A., Howland, K. E., Archambault, P., Sevellec, M., Normandeau, É., Dispas, A., Winkler, G., McKindsey, C. W., Simard, N. N., et al. (2019) · Environmental DNA · doi:10.1002/edn3.35 · Cited by 108 · see citing works
Lanzén, A., Lekang, K., Jonassen, I., Thompson, E. M., & Troedsson, C. (2016) · Molecular Ecology · doi:10.1111/mec.13761 · Cited by 104 · see citing works
Nakagawa, H., Yamamoto, S., Sato, Y., Sado, T., Minamoto, T., & Miya, M. U. (2018) · Freshwater Biology · doi:10.1111/fwb.13094 · Cited by 104 · see citing works
“In 441 addition, we might reduce the effect of primer-bias (i.e., the variation in amplification 442 efficiency among species) by using multiple universal primers (Elbrecht & Leese, 2015; 443 Vallentini et al., 2016).”
MethodHajibabaei, M., Porter, T. M., Robinson, C. V., Baird, D. J., Shokralla, S., & Wright, M. (2019) · PLoS ONE · doi:10.1371/journal.pone.0225409 · Cited by 104 · see citing works
Macher, J., Salis, R. K., Blakemore, K. S., Tollrian, R., Matthaei, C. D., & Leese, F. (2015) · Ecological Indicators · doi:10.1016/j.ecolind.2015.08.024 · Cited by 102 · see citing works
Self-citationDormontt, E. E., van Dijk, K., Bell, K. L., Biffin, E., Breed, M. F., Byrne, M. M., Caddy‐Retalic, S., Encinas‐Viso, F., Nevill, P., Shapcott, A., et al. (2018) · Frontiers in Ecology and Evolution · doi:10.3389/fevo.2018.00134 · Cited by 103 · see citing works
Vitecek, S., Kučinić, M., Previšić, A., Živić, I. M., Stojanović, K. Z., Keresztes, L., Bálint, M., Hoppeler, F., Waringer, J. A., Graf, W., et al. (2017) · BMC Evolutionary Biology · doi:10.1186/s12862-017-0972-5 · Cited by 97 · see citing works
DNA metabarcoding for high-throughput monitoring of estuarine macrobenthic communities
Lobo, J., Shokralla, S., Costa, M. H., Hajibabaei, M., & Costa, F. O. (2017) · Scientific Reports · doi:10.1038/s41598-017-15823-6 · Cited by 97 · see citing works
Metabarcoding of lake benthic diatoms: from structure assemblages to ecological assessment
Rivera, S. F., Vasselon, V., Jacquet, S., Bouchez, A., Arizteguí, D., & Rimet, F. (2017) · Hydrobiologia · doi:10.1007/s10750-017-3381-2 · Cited by 96 · see citing works
“…choice of a targeted gene for DNA barcode (Kermarrec et al., 2013; Valentini et al., 2016), the choice of the set of primers for DNA metabarcoding (Elbrecht & Leese 2015), the choice of the PCR amplification protocol (Kebschull & Zador, 2015), the choice of the sequencing technology (Quail et…”
MethodSato, H., Sogo, Y., Doi, H., & Yamanaka, H. (2017) · Scientific Reports · doi:10.1038/s41598-017-14978-6 · Cited by 94 · see citing works
Deagle, B. E., Clarke, L. J., Kitchener, J. A., Polanowski, A. M., & Davidson, A. T. (2017) · Molecular Ecology Resources · doi:10.1111/1755-0998.12740 · Cited by 94 · see citing works
Smith, K. F., Kohli, G. S., Murray, S. A., & Rhodes, L. L. (2017) · New Zealand Journal of Marine and Freshwater Research · doi:10.1080/00288330.2017.1298632 · Cited by 94 · see citing works
“Biases caused by primer-associated PCR efficiency, even when using ‘universal primers’, can lead to highly varied read numbers, affecting both abundant and rare taxa, and can result in false negatives (Elbrecht & Leese 2015; Zhan &MacIsaac 2015).”
BackgroundResulteDNA in subterranean ecosystems: Applications, technical aspects, and future prospects
Saccò, M., Guzik, M. T., van der Heyde, M., Nevill, P., Cooper, S. J., Austin, A. D., Coates, P. J., Allentoft, M. E., & White, N. E. (2022) · The Science of The Total Environment · doi:10.1016/j.scitotenv.2022.153223 · Cited by 93 · see citing works
A DNA metabarcoding approach to characterize soil arthropod communities
Oliverio, A., Gan, H., Wickings, K. G., & Fierer, N. (2018) · Soil Biology and Biochemistry · doi:10.1016/j.soilbio.2018.06.026 · Cited by 92 · see citing works
“approaches are not a valid proxy for abundances or relative abundances due to body size differences and given that DNA can be shed in frass (Elbrecht and Leese, 2015).”
Background
- Authors
- Elbrecht, V., & Leese, F.
- Affiliations
- Ruhr University Bochum (DE)
- Corresponding author
- Leese, Florian
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2015-07-08
- Volume
- 10
- Issue
- 7
- Pages
- e0130324
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC4496048
- OpenAlex
- W1551951435
- Semantic Scholar
- 5b5925709b20821df1f956c4c737e5fadb1e8b43
- Cited by
- 765
- References
- 60
- Citations by year
- 2026: 75 · 2025: 73 · 2024: 49 · 2023: 69 · 2022: 71 · 2021: 91 · 2020: 75 · 2019: 73 · 2018: 108 · 2017: 51 · 2016: 26 · 2015: 3
- FWCI
- 56.82
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Topics
- Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
- Life Sciences › Agricultural and Biological Sciences › Ecology, Evolution, Behavior and Systematics › Invertebrate Taxonomy and Ecology
- Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
- Keywords
- DNA metabarcoding, primer bias, species abundance estimation, COI barcoding, freshwater macroinvertebrates, Plecoptera, MiSeq sequencing, biodiversity assessment
- MeSH terms
- Animals, Electron Transport Complex IV, Diptera, DNA, Fresh Water, Haplotypes, Species Specificity, Reproducibility of Results, Polymerase Chain Reaction, Ecosystem, DNA Primers, Biomass, Biodiversity, DNA Barcoding, Taxonomic, High-Throughput Nucleotide Sequencing, Odonata
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