PrimerMiner: an R package for development and in silico validation of DNA metabarcoding primers
- Subject Areas
- Bioinformatics, Ecology, Genetics, Molecular Biology, Zoology
- primer bias, in silico PCR, Primer development, high throughput sequencing, DNA barcoding, data mining, monitoring, primer evaluation, ecosystem assessment, PrimerMiner
- © 2016 Elbrecht et al.
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2016. PrimerMiner: an R package for development and in silico validation of DNA metabarcoding primers. PeerJ Preprints 4:e2352v1 https://doi.org/10.7287/peerj.preprints.2352v1
1) DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that serve as a backbone for primer design is currently available.
2) We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico, which are in our opinion more realistic then the strategy employed in another available software for that purpose, ecoPCR.
3) We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both databases, we were able to increase the amount of reference data 249-fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner.
4) With PrimerMiner we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU based reference alignments generated with PrimerMiner can be used for manual primer design, or processed with bioinformatic tools for primer development.
Shortened "Application" article for consideration in Methods in Ecology and Evolution. Was previously part of this preprint but we were requested in the review process to split the article in two; one describing the R package (this manuscript) and one showing experimental data; peerj.com/preprints/2044/ We are ahppy to considder any fedback on both artcles!