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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.
Overview
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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.
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44 Own result · 24 Own interpretation · 22 Not checked yet · 8 No basis given · 5 Own method
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In the paper
“However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Abstract
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In the paper
“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).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Sequencing success and statistics
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In the paper
“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).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment I
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In the paper
“The sequencing results for the ten replicates were highly reproducible.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment I
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In the paper
“We did not observe significant differences in sequence abundance among the replicates using different amounts of extracted tissue as a template.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment I
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In the paper
“From the DNA extractions performed with the TissueLyser LT, six more specimens (2.31%) were recovered than when DNA was extracted with liquid nitrogen.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment II
plaindirectn = 6- Asserted: passes — The authors put it forward as true.
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In the paper
“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).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Sequencing success and statistics
plaincorrelationaln = 31- Asserted: passes — The authors put it forward as true.
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In the paper
“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).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Materials and Methods > Primer design and sequencing strategy
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“In experiment II, we did not detect primer bias (t-test, p = 0.41, S4B Fig).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Sequencing success and statistics
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Specimens with a low biomass tend to show relatively high variation in sequence abundance among the ten replicates.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment I
hedgedcorrelationaln = 31- Asserted: passes — The authors put it forward as true.
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- Probably not a substantial claim. 23
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In the paper
“The two independent weight measurements differed in mean by 0.1 mg (SD = 0.03).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment I
plainn = 31- Asserted: passes — The authors put it forward as true.
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In the paper
“The MiSeq runs of experiments I and II yielded 9.63 and 14.07 Gb of read data, respectively.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Sequencing success and statistics
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“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).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment I
plainn = 31- Asserted: passes — The authors put it forward as true.
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In the paper
“The mean normalised sequence abundances had low standard deviations and the linear model fit well (p<0.001, R 2 = 0.65, S8 Fig).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment I
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“For two specimens, we recovered sequence artefacts (see S6 Fig); these did not affect further analyses.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment I
plainn = 2- Asserted: passes — The authors put it forward as true.
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In the paper
“Both MiSeq runs showed good read quality (sequences with Q30 ≥ 85.3% and 83.3%).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Sequencing success and statistics
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Sequences that did not match the target species were similar to sequences derived from a variety of benthic organisms, but also fungi and plants.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment II
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Using our protocol, it is possible to assess community compositions within a week, from sampling to species identification.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
hedgeddirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Furthermore, we used a universal COI primer with a broad target range [41], and only 17% of taxa went undetected.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
What the sources say
- Not checked yetcites [41] O Folmer, 1994, DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates, Mol Marine Biol Biotechnol
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“Of all recovered 213 OTUs, 31 could not be identified using the BOLD database (S9B Fig).”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment II
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“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.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Experiment I: Relationship between specimen biomass and sequence abundance
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Our parallel sequencing strategy leads to an increase in per-site sequence diversity and read quality.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds. It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“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.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Introduction
What the sources say
- Not checked yetcites [11] Merritt RW, Berg MB, Cummins KW. An Introduction to the Aquatic Insects of North America. 2009.
- Not checked yetcites [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 yetcites [13] FC Jones, 2008, Taxonomic sufficiency: The influence of taxonomic resolution on freshwater bioassessments using benthic macroinvertebrates, Environ Rev source
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“The same PCR master mix was used for all reactions to ensure identical PCR conditions for all replicates.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Materials and Methods > Experiment I: Relationship between specimen biomass and sequence abundance
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“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].”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Introduction
What the sources say
- Not checked yetcites [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
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“To ensure reliable hits, only sequences that had a maximum of five mismatches and gaps were included in the analysis.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Materials and Methods > Experiment I: Relationship between specimen biomass and sequence abundance
plain- Asserted: passes — The authors put it forward as true.
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A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“The parallel sequencing approach thus substantially reduced regions of low per-site diversity, improving read quality.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Materials and Methods > Primer design and sequencing strategy
plaindirect- Asserted: passes — The authors put it forward as true.
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None of the sources cited for this claim have been checked yet.
In the paper
“DNA barcoding allows for standardized and accurate species identification [15–18].”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Introduction
What the sources say
- Not checked yetcites [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 yetcites [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 yetcites [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 yetcites [18] BW Sweeney, 2011, Can DNA barcodes of stream macroinvertebrates improve descriptions of community structure and water quality?, J North Am Benthol Soc source
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“frequent identification errors occur and many specimens are not detected in samples [10]”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Introduction
What the sources say
- Not checked yetcites [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
plain- Asserted: passes — The authors put it forward as true.
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What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Introduction
plaindirect- Asserted: passes — The authors put it forward as true.
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- Also stated in: § Abstract · plain
In the paper
“Stein and co-authors showed that ten of 16 assessment metrics had higher statistical power using DNA barcoding than morphological assessment [14].”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Introduction
What the sources say
- Not checked yetcites [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
plaincorrelational- Asserted: passes — The authors put it forward as true.
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In the paper
“For animals, a 658-bp standardized fragment of the mitochondrial gene COI (cytochrome c oxidase subunit 1) is typically used [19].”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Introduction
What the sources say
- Not checked yetcites [19] PDN Hebert, 2003, Biological identifications through DNA barcodes, Proc R Soc Lond B Biol Sci source
plain- Asserted: passes — The authors put it forward as true.
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- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Presumably, these morphotaxa contained several taxonomically distant species, which are unequally well amplified with the Folmer primers.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Experiment II: Primer amplification bias between species
hedgedcorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“We used a highly replicated and standardised approach for species detection using DNA metabarcoding”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“show that several technical and logistic problems have to be overcome before this protocol can be used for large-scale monitoring.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Experiment I: Relationship between specimen biomass and sequence abundance
hedgedn = 31- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“However degraded DNA and contamination as in eDNA studies with amplicons is not a concern for organisms collected directly from streams.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: fails — The paper may give no reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.
None of the sources cited for this claim have been checked yet.
In the paper
“Several studies show that DNA-based assessments are superior to morphological assessments in freshwater ecosystems (e.g., [14,25,33])”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion
What the sources say
- Not checked yetcites [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 yetcites [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 yetcites [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: 10.1111/1755-0998.12336</comment> source
plaincorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“but not its abundance because many small organisms generate the same number of sequence reads as a few large organisms.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Thus, it is not possible to accurately estimate species biomass or even abundance in diverse environmental samples using amplification-based sequencing protocols.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
In the paper
“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]”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
What the sources say
- Not checked yetcites [56] P Taberlet, 2012, Towards next-generation biodiversity assessment using DNA metabarcoding, Mol Ecol source
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“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].”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
What the sources say
- Not checked yetcites [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 yetcites [57] Y Ji, 2013, Reliable, verifiable and efficient monitoring of biodiversity via metabarcoding, Ecol Lett source
boosteddirect- Asserted: passes — The authors put it forward as true.
- Checkable: fails — It may be too vague to check against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it. It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We provide a highly reproducible laboratory protocol for processing macroinvertebrate samples in bulk and identified species using metabarcoding with the standard COI region.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Conclusions
boosted- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
In the paper
“Piñol et al. [39] proposed a relationship between biomass and sequence reads, but did not systematically examine this hypothesis.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Experiment I: Relationship between specimen biomass and sequence abundance
What the sources say
- Not checked yetcites [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: 10.1111/1755-0998.12355</comment> source
plaincorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“This is consistent with the primer bias which we already observed between morphotaxa and support the overall findings of strong taxon-specific amplification bias.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Experiment II: Primer amplification bias between species
boostedcorrelational- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“The methods developed in this study can easily be adapted to assess the communities of other ecosystems.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
plain- Asserted: passes — The authors put it forward as true.
- Checkable: fails — It may be too vague to check against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: fails — The paper may give no reason why it holds.
Probably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.
In the paper
“However, while this approach could have comparable taxonomic resolution as the COI barcode, it is currently limited by the lack of reference databases [55].”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
What the sources say
- Not checked yetcites [55] LJ Clarke, 2014, Environmental metabarcodes for insects: in silicoPCR reveals potential for taxonomic bias, Mol Ecol Resour source
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: fails — It may be too vague to check against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. It may be too vague to check against evidence.
- 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?
In the citing paper
“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.
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Results > Experiment II
“We were able to reliably assign OTUs to all 52 taxa included in the extraction.”
§ Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol > Discussion > Implications for large-scale monitoring and future challenges
“Furthermore, we used a universal COI primer with a broad target range [41], and only 17% of taxa went undetected.”
Citations
Cites
R: A Language and Environment for Statistical Computing
R Core Team (2000) · doi:10.32614/r.manuals · Cited by 355,893
QIIME allows analysis of high-throughput community sequencing data
Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., Fierer, N., Peña, A. G., Goodrich, J. K., Gordon, J. I., et al. (2010) · Nature Methods · doi:10.1038/nmeth.f.303 · Cited by 34,887
Cutadapt removes adapter sequences from high-throughput sequencing reads
Martin, M. (2011) · EMBnet.journal · doi:10.14806/ej.17.1.200 · Cited by 33,889
BLAST+: architecture and applications
Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., & Madden, T. L. (2009) · BMC Bioinformatics · doi:10.1186/1471-2105-10-421 · Cited by 22,074
Schloss, P. D., Westcott, S. L., Ryabin, T., Hall, J. R., Hartmann, M., Hollister, E. B., Lesniewski, R. A., Oakley, B. B., Parks, D. H., Robinson, C. J., et al. (2009) · Applied and Environmental Microbiology · doi:10.1128/aem.01541-09 · Cited by 19,549
UPARSE: highly accurate OTU sequences from microbial amplicon reads
Edgar, R. C. (2013) · Nature Methods · doi:10.1038/nmeth.2604 · Cited by 16,770
Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994) · PubMed · doi:10.5281/zenodo.15145663 · Cited by 15,546
Biological identifications through DNA barcodes
Hebert, P. D. N., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003) · Proceedings of the Royal Society of London. Series B: Biological Sciences · doi:10.1098/rspb.2002.2218 · Cited by 13,376
A safe operating space for humanity
Rockström, J., Steffen, W., Noone, K., Persson, Å., Chapin, F. S., Lambin, E. F., Lenton, T. M., Scheffer, M., Folke, C., Schellnhuber, H. J., et al. (2009) · Nature · doi:10.1038/461472a · Cited by 13,333
Ecosystems and Human Well-being: Synthesis
Assessment, M. E. (2005) · Island Press · doi:10.5281/zenodo.18167309 · Cited by 10,665
Cited by
Sorted by citations
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 2,075
“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 1,146
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 884
“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 769
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 700
Metadata
- 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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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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