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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.


Overview of taxa recovery in experiment II.Sequence abundances for the 52 morphologically identified taxa is shown in rows and the ten replicates used in the experiment in columns. Sequence abundance was normalised across the ten replicates and the amount of tissue used in each extraction. Sequence abundance of each specimens (morphotaxon) of the ten replicates is visualised by different shades of blue. If a field is i.e. half filled (50%) with the mid blue shade (= 1% of total sequences), the respective specimen represented 0.5% (50% of 1%) of the total sequences in that replicate. When no sequences or only a few sequences (below 0.003% of total abundance per replicate) were found for a specimen, it was scored as "No Hit," as indicated by an orange asterisk. On the right, K2P-corrected neighbour-joining (NJ) trees for each taxon, based on the most abundant sequence obtained for each specimen (calculated with MEGA6.06), are shown. MOTUs are defined by a 2% sequence difference based on the NJ tree.
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pone.0130324.g003: Overview of taxa recovery in experiment II.Sequence abundances for the 52 morphologically identified taxa is shown in rows and the ten replicates used in the experiment in columns. Sequence abundance was normalised across the ten replicates and the amount of tissue used in each extraction. Sequence abundance of each specimens (morphotaxon) of the ten replicates is visualised by different shades of blue. If a field is i.e. half filled (50%) with the mid blue shade (= 1% of total sequences), the respective specimen represented 0.5% (50% of 1%) of the total sequences in that replicate. When no sequences or only a few sequences (below 0.003% of total abundance per replicate) were found for a specimen, it was scored as "No Hit," as indicated by an orange asterisk. On the right, K2P-corrected neighbour-joining (NJ) trees for each taxon, based on the most abundant sequence obtained for each specimen (calculated with MEGA6.06), are shown. MOTUs are defined by a 2% sequence difference based on the NJ tree.

Mentions: S2 Table gives an overview of specimen weights of the 52 tissues parts, used in each of the ten replicates. We were able to reliably recover 83% (43) of the 52 taxa included in experiment II. We recovered many of the typical bio-indicator taxa such Ephemeroptera, Plecoptera, Trichoptera, and Diptera (Table 1). 34 taxa were recovered in all ten replicates (Fig 3). From the DNA extractions performed with the TissueLyser LT, six more specimens (2.31%) were recovered than when DNA was extracted with liquid nitrogen. Furthermore, we did not observe substantial differences in recovery rates when different numbers of PCR products were pooled.


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)

Overview of taxa recovery in experiment II.Sequence abundances for the 52 morphologically identified taxa is shown in rows and the ten replicates used in the experiment in columns. Sequence abundance was normalised across the ten replicates and the amount of tissue used in each extraction. Sequence abundance of each specimens (morphotaxon) of the ten replicates is visualised by different shades of blue. If a field is i.e. half filled (50%) with the mid blue shade (= 1% of total sequences), the respective specimen represented 0.5% (50% of 1%) of the total sequences in that replicate. When no sequences or only a few sequences (below 0.003% of total abundance per replicate) were found for a specimen, it was scored as "No Hit," as indicated by an orange asterisk. On the right, K2P-corrected neighbour-joining (NJ) trees for each taxon, based on the most abundant sequence obtained for each specimen (calculated with MEGA6.06), are shown. MOTUs are defined by a 2% sequence difference based on the NJ tree.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130324.g003: Overview of taxa recovery in experiment II.Sequence abundances for the 52 morphologically identified taxa is shown in rows and the ten replicates used in the experiment in columns. Sequence abundance was normalised across the ten replicates and the amount of tissue used in each extraction. Sequence abundance of each specimens (morphotaxon) of the ten replicates is visualised by different shades of blue. If a field is i.e. half filled (50%) with the mid blue shade (= 1% of total sequences), the respective specimen represented 0.5% (50% of 1%) of the total sequences in that replicate. When no sequences or only a few sequences (below 0.003% of total abundance per replicate) were found for a specimen, it was scored as "No Hit," as indicated by an orange asterisk. On the right, K2P-corrected neighbour-joining (NJ) trees for each taxon, based on the most abundant sequence obtained for each specimen (calculated with MEGA6.06), are shown. MOTUs are defined by a 2% sequence difference based on the NJ tree.
Mentions: S2 Table gives an overview of specimen weights of the 52 tissues parts, used in each of the ten replicates. We were able to reliably recover 83% (43) of the 52 taxa included in experiment II. We recovered many of the typical bio-indicator taxa such Ephemeroptera, Plecoptera, Trichoptera, and Diptera (Table 1). 34 taxa were recovered in all ten replicates (Fig 3). From the DNA extractions performed with the TissueLyser LT, six more specimens (2.31%) were recovered than when DNA was extracted with liquid nitrogen. Furthermore, we did not observe substantial differences in recovery rates when different numbers of PCR products were pooled.

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.