Universal and blocking primer mismatches limit the use of high‐throughput DNA sequencing for the quantitative metabarcoding of arthropods

Piñol, J.; Mir, G.; Gomez‐Polo, P.; Agustí, N.

doi:10.1111/1755-0998.12355

Cited by 425Environmental DNA in Biodiversity StudiesInvertebrate Taxonomy and EcologyGenomics and Phylogenetic Studies

Universal and blocking primer mismatches limit the use of high‐throughput DNA sequencing for the quantitative metabarcoding of arthropods

Piñol, J., Mir, G., Gomez‐Polo, P., Agustí, N.·2015·Molecular Ecology Resources

DOI 10.1111/1755-0998.12355

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Abstract

The quantification of the biological diversity in environmental samples using high‐throughput DNA sequencing is hindered by the PCR bias caused by variable primer–template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator‐specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high‐throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer–template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer–template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region.

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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.”
From Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol (2015) · cites this paper as [39]
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.”
From Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol (2015) · cites this paper as [39]
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].”
From Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol (2015) · cites this paper as [39]
Piñol 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.”
From Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol (2015) · cites this paper as [39]

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Data from: Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods
Piñol, J., Mir, G., Gomez‐Polo, P., & Agustí, N. (2014) · Data Archiving and Networked Services (DANS) · doi:10.5061/dryad.2p51n
Self-citation
Data from: The choice of universal primers and the characteristics of the species mixture determines when DNA metabarcoding can be quantitative.
Piñol, J., Senar, M. À., & Symondson, W. O. C. (2018) · Data Archiving and Networked Services (DANS) · doi:10.5061/dryad.q2r3b1f
Self-citation
The choice of universal primers and the characteristics of the species mixture determine when DNA metabarcoding can be quantitative
Piñol, J., Senar, M. À., & Symondson, W. O. C. (2018) · Molecular Ecology · doi:10.1111/mec.14776 · Cited by 262 · see citing works
Self-citation
Characterizing mitochondrial copy number variation and PCR amplification bias as sources of quantitative constraints in DNA metabarcodingPreprint
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Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding samples
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Authors: Piñol, J., Mir, G., Gomez‐Polo, P., & Agustí, N.
Affiliations: Universitat Autònoma de Barcelona (ES)
CRAG CSIC IRTA UAB UB Cerdanyola del Vallès 08193 Spain
Institute of Agrifood Research and Technology (ES)
Corresponding author: Piñol, J.
ORCID: Piñol, J.
Gomez‐Polo, P.
Agustí, N.
Venue: Molecular Ecology Resources
Publisher: Wiley
Published: 2014-12-23
Volume: 15
Issue: 4
Pages: 819-830
ISSN: 1755-098X (ISSN-L)
1755-0998
DOI: 10.1111/1755-0998.12355
OpenAlex: W2051331361
Cited by: 423
References: 48
Citations by year: 2026: 37 · 2025: 42 · 2024: 37 · 2023: 34 · 2022: 45 · 2021: 53 · 2020: 40 · 2019: 31 · 2018: 53 · 2017: 22 · 2016: 20 · 2015: 9
FWCI: 14.76
Type: journal article
Language: en
Open access: closed
Licence: onlinelibrary.wiley.com/termsAndConditions#vor (vor)
Locations: Molecular Ecology Resources (publishedVersion)
PubMed (publishedVersion)
DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion)
Funders: MINECO (CGL2010-18182, AGL2011-24349)
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
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords: PCR bias, primer-template mismatch, arthropods, COI barcoding, mock communities, amplification efficiency, annealing temperature, dietary assessment, species relative abundance
MeSH terms: Animals, Arthropods, Diagnostic Errors, Molecular Sequence Data, Polymerase Chain Reaction, Sequence Analysis, DNA, DNA Primers, Base Pair Mismatch, DNA Barcoding, Taxonomic, High-Throughput Nucleotide Sequencing

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Universal and blocking primer mismatches limit the use of high‐throughput DNA sequencing for the quantitative metabarcoding of arthropods

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