Slippage of degenerate primers can cause variation in amplicon length
- Subject Areas
- Biochemistry, Bioinformatics, Genetics, Molecular Biology
- Primer development, bias, primer slippage, length variation, degeneracy, metabarcoding
- © 2018 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
- 2018. Slippage of degenerate primers can cause variation in amplicon length. PeerJ Preprints 6:e26763v1 https://doi.org/10.7287/peerj.preprints.26763v1
Metabarcoding studies often employ degenerate primers to reduce amplification bias and increase the number of detected taxa. However, degeneracy has the disadvantage of lowering binding specificity although the exact mechanisms and potential biases introduced by such off-target amplification are not fully understood. We examined sequences recovered from the ten most abundant operational taxonomic units (OTUs) in two mock communities to investigate the specificity and binding behavior of five degenerate primer sets. Our results indicate that primers frequently bound 1-2 bp upstream in taxa where a homopolymer region was present in the amplification direction. As well, although less frequent, degeneracy occasionally led to primer binding 1 bp downstream. Some widely used primer sets were severely affected by this slippage effect, while others were not. Our study shows that primer slippage can produce taxon-specific length variation in amplicons and subsequent length variation in recovered sequences. While this variation will only have small impacts on OTU designation by clustering algorithms that ignore terminal gaps, primer sets employed in metabarcoding projects should be evaluated for their sensitivity to slippage. Moreover, steps should be taken to reduce slippage by improving protocols for primer design. For example, the flanking region adjacent to the 3' end of the primer is not considered by current primer development software although GC clamps in this position could mitigate slippage. While degeneracy is important to ensure the universality of a primer, binding in homopolymer regions should be avoided.
Initial version of our manuscript on primer slippage in amplicon studies. Any feedback is apprechiated! Currently looking for a journal to submit this to.
Figure S1: Plots of length variation for six additional primers
Plot of primer binding sites and bar plots depicting length of utilised primers for the 10 most abundant OTUs in mock sample B (sequence data from Elbrecht & Leese 2017). The presence and read abundance for different taxa can vary based on the primer set used, thus while amplifying the same mock samples, the order of and taxa the 10 most abundant specimens is not identical between plots as OTUs are sorted by read abundance. The sample was amplified with the P5_BF1_0 + P7_BR1_4 and P5_BF2_0 + P7_BR1_4 primer set, and the length distribution of the incorporated primers is shown for the BR1 primer (A and B). The percentage of amplicons that incorporated a particular length are shown above each bar. Additional plots for further primer combinations on the next 2 pages (C, D, E).
Table S1: Raw length distribution data and number of sequences used for each taxon and primer
Scripts S1: R scripts used to analyze primer length distribution
Manuscript file for providing feedback
Feel free to use this word document with track changes to provide feedback. Thank you!