The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay

Stadhouders, Ralph; Pas, Suzan D.; Anber, Jeer; Voermans, Jolanda J.C.; Mes, Ted H. M.; Schutten, Martin

doi:10.2353/jmoldx.2010.090035

Cited by 319 publications

(326 citation statements)

References 27 publications

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“…It has to be kept in mind that a typographical error, or just one mismatching base at the 3 ′ -end can make or break a primer (Stadhouders et al, 2010;Piñol et al, 2014). Additionally, primers are often developed on a small set of taxa, and thus might not work well for the ecosystem, geographic region or taxa under study.…”

Section: Primer Success Is Determined By Base Degeneracy and Referencmentioning

confidence: 99%

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

Elbrecht

Leese

2017

Front. Environ. Sci.

233

232

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A central challenge in the present era of biodiversity loss is to assess and manage human impacts on freshwater ecosystems. Macroinvertebrates are an important group for such bioassessments as many taxa show specific responses to environmental conditions. However, generating accurate macroinvertebrate inventories based on primarily larval morphology is difficult and error-prone. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify invertebrates in bulk samples to the species level has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for bioassessment critically relies on carefully evaluated primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 globally most relevant freshwater invertebrate groups for stream assessment. Using these sequence alignments, we developed four primer combinations optimized for freshwater macroinvertebrates. All primers were evaluated by sequencing 10 mock community samples, each consisting of 52 freshwater invertebrate taxa. Additionally, popular metabarcoding primers from the literature and the developed primers were tested in silico against these 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the number of detected taxa, yet, all detected more taxa than standard "Folmer" barcoding primers. Two new primer combinations showed even more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that some primers currently Elbrecht and Leese Freshwater Primer Development and Evaluation used in metabarcoding studies may not be suitable for amplification of freshwater macroinvertebrates. Therefore, careful primer evaluation and more region/ecosystem specific primers are needed before DNA metabarcoding can be used for routine bioassessment of freshwater ecosystems.

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Section: Primer Success Is Determined By Base Degeneracy and Referencmentioning

confidence: 99%

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

Elbrecht

Leese

2017

Front. Environ. Sci.

233

232

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“…2008; Stadhouders et al . 2010). Failure to detect some additional taxa, such as cartilaginous fishes, may be due to known mutations in the primer sites for those taxa (Fig.…”

Section: Resultsmentioning

confidence: 99%

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

et al. 2015

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Preserving biodiversity is a global challenge requiring data on species’ distribution and abundance over large geographic and temporal scales. However, traditional methods to survey mobile species’ distribution and abundance in marine environments are often inefficient, environmentally destructive, or resource‐intensive. Metabarcoding of environmental DNA (eDNA) offers a new means to assess biodiversity and on much larger scales, but adoption of this approach for surveying whole animal communities in large, dynamic aquatic systems has been slowed by significant unknowns surrounding error rates of detection and relevant spatial resolution of eDNA surveys. Here, we report the results of a 2.5 km eDNA transect surveying the vertebrate fauna present along a gradation of diverse marine habitats associated with a kelp forest ecosystem. Using PCR primers that target the mitochondrial 12S rRNA gene of marine fishes and mammals, we generated eDNA sequence data and compared it to simultaneous visual dive surveys. We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 m. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.

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“…Specificity of these primers may be improved, however, by introducing penultimate bases to minimize amplification from the undesired allele. 29 Although we utilized SNPs in the upstream region for designing AS forward primers, SNPs in the downstream region could certainly also be exploited for generating AS reverse primers. To achieve AS amplification from alleles containing A or T, using AS forward and reverse primers may also be an option to be explored.…”

Section: Discussionmentioning

confidence: 99%

CYP2D6 Haplotype Determination Using Long Range Allele-Specific Amplification

Gaedigk

Riffel

Leeder

2015

The Journal of Molecular Diagnostics

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Cytochrome P450 (CYP) 2D6, a major contributor to the metabolism and bioactivation of many clinically used drugs, is encoded by a complex, highly polymorphic gene locus. To aid in the characterization of CYP2D6 allelic variation, we developed allele-specific long-range PCR (ASXL-PCR) to amplify only the allele of interest for further characterization by PCR. This development was achieved utilizing single-nucleotide polymorphisms in the upstream region of CYP2D6 and a universal CYP2D6-specific reverse primer. This approach was assessed and optimized on samples with known genotypes. The application of ASXL-PCR clarified a case with a complex genotype (CYP2D6*2x2/*4Nþ*4) by amplifying the duplicated gene units separately for subsequent analysis. Furthermore, ASXL-PCR and subsequent sequence analysis also resolved genotype discord in a mother/daughter relationship by revealing the presence of the CYP2D6*59 allelic variant in both individuals. Finally, we demonstrated that the 2939G>A single-nucleotide polymorphism present on CYP2D6*59 interfered with the TaqMan genotype assay that detected 2850C>T, causing false genotype assignments. Assay interference was resolved using an alternative TaqMan genotype assay currently available as a custom-made assay. These examples demonstrate the utility of ASXL-PCR for improved CYP2D6 allele/haplotype characterization. This fast, easy-to-perform method is not limited to CYP2D6 but can be adapted to any gene locus for which polymorphic sites are known. (J Mol Diagn 2015, 17: 740e748; http://dx.doi.org/10.1016/j.jmoldx.2015 Cytochrome P450 (CYP) 2D6 is involved in the metabolism and disposition of many clinically used drugs.

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The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay

Cited by 319 publications

References 27 publications

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

Assessing vertebrate biodiversity in a kelp forest ecosystem using environmental DNA

CYP2D6 Haplotype Determination Using Long Range Allele-Specific Amplification

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