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Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass--Sequence Relationships with an Innovative Metabarcoding Protocol.

Elbrecht V, Leese F - PLoS ONE (2015)

Bottom Line: The results of both experiments were consistent across replicates.We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads.However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Ecology, Evolution and Biodiversity, Ruhr University Bochum, Universitaetsstrasse 150, D-44801 Bochum, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Results of Experiment I: Dependence of read abundance on specimen biomass.A Specimen weight (y-axis) is plotted against read abundance per specimen (x-axis) for all ten replicates. The linear regression (red line) was highly significant for all replicates with p<0.001. B Ratio of N5LCO/N5HCO sequence abundance for the mixed and four single DNA aliquots. The red line indicates the mean ratio.
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pone.0130324.g002: Results of Experiment I: Dependence of read abundance on specimen biomass.A Specimen weight (y-axis) is plotted against read abundance per specimen (x-axis) for all ten replicates. The linear regression (red line) was highly significant for all replicates with p<0.001. B Ratio of N5LCO/N5HCO sequence abundance for the mixed and four single DNA aliquots. The red line indicates the mean ratio.

Mentions: S1 Table gives an overview of the weights of the 31 specimens. The two independent weight measurements differed in mean by 0.1 mg (SD = 0.03). 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). For two specimens, we recovered sequence artefacts (see S6 Fig); these did not affect further analyses. We did not observe significant differences in sequence abundance among the replicates using different amounts of extracted tissue as a template. 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). We observed a strong negative relationship between specimen weight and variation in sequence abundance, as shown in S5 and S7B Figs. 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.

Elbrecht V, Leese F - PLoS ONE (2015)

Results of Experiment I: Dependence of read abundance on specimen biomass.A Specimen weight (y-axis) is plotted against read abundance per specimen (x-axis) for all ten replicates. The linear regression (red line) was highly significant for all replicates with p<0.001. B Ratio of N5LCO/N5HCO sequence abundance for the mixed and four single DNA aliquots. The red line indicates the mean ratio.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496048&req=5

pone.0130324.g002: Results of Experiment I: Dependence of read abundance on specimen biomass.A Specimen weight (y-axis) is plotted against read abundance per specimen (x-axis) for all ten replicates. The linear regression (red line) was highly significant for all replicates with p<0.001. B Ratio of N5LCO/N5HCO sequence abundance for the mixed and four single DNA aliquots. The red line indicates the mean ratio.
Mentions: S1 Table gives an overview of the weights of the 31 specimens. The two independent weight measurements differed in mean by 0.1 mg (SD = 0.03). 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). For two specimens, we recovered sequence artefacts (see S6 Fig); these did not affect further analyses. We did not observe significant differences in sequence abundance among the replicates using different amounts of extracted tissue as a template. 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). We observed a strong negative relationship between specimen weight and variation in sequence abundance, as shown in S5 and S7B Figs. Specimens with a low biomass tend to show relatively high variation in sequence abundance among the ten replicates.

Bottom Line: The results of both experiments were consistent across replicates.We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads.However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Ecology, Evolution and Biodiversity, Ruhr University Bochum, Universitaetsstrasse 150, D-44801 Bochum, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus