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

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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

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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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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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