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Slippage of degenerate primers can cause variation in amplicon length

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@mailys_gauthier @DNAquaNet @leeselab @NatureMetrics @ento_ben @alpineedna @dirch3 @BerndHaenfling @Old_dusty_ @rosiecblackman Smart move ; ) But I actually didn't focus on terrestrial taxa, but all Arthropods. It's BF2 modified in the binding site to reduce primer slippage https://t.co/5pA0TpNJ5S
RT @VascoElbrecht: New @PeerJPreprints: Slippage of degenerate #primers can cause variation in amplicon length! Affects BF1, BF2 & #mlCOIin…
RT @VascoElbrecht: New @PeerJPreprints: Slippage of degenerate #primers can cause variation in amplicon length! Affects BF1, BF2 & #mlCOIin…
335 days ago
RT @VascoElbrecht: New @PeerJPreprints: Slippage of degenerate #primers can cause variation in amplicon length! Affects BF1, BF2 & #mlCOIin…
RT @VascoElbrecht: New @PeerJPreprints: Slippage of degenerate #primers can cause variation in amplicon length! Affects BF1, BF2 & #mlCOIin…
RT @VascoElbrecht: New @PeerJPreprints: Slippage of degenerate #primers can cause variation in amplicon length! Affects BF1, BF2 & #mlCOIin…
335 days ago
RT @VascoElbrecht: New @PeerJPreprints: Slippage of degenerate #primers can cause variation in amplicon length! Affects BF1, BF2 & #mlCOIin…
New @PeerJPreprints: Slippage of degenerate #primers can cause variation in amplicon length! Affects BF1, BF2 & #mlCOIintF primers, species-specific bias (!), but can be mitigated by GC clamps and considering flanking regions in primer design. Please RT =) https://t.co/5pA0TpNJ5S https://t.co/vKvAXNAQ1p
335 days ago
RT @metabar_papers: Slippage of degenerate primers can cause variation in amplicon length https://t.co/PqegcuemJe
Slippage of degenerate primers can cause variation in amplicon length https://t.co/PqegcuemJe
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Supplemental Information

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).

DOI: 10.7287/peerj.preprints.26763v1/supp-1

Table S1: Raw length distribution data and number of sequences used for each taxon and primer

DOI: 10.7287/peerj.preprints.26763v1/supp-2

Scripts S1: R scripts used to analyze primer length distribution

DOI: 10.7287/peerj.preprints.26763v1/supp-3

Manuscript file for providing feedback

Feel free to use this word document with track changes to provide feedback. Thank you!

DOI: 10.7287/peerj.preprints.26763v1/supp-4

Additional Information

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Vasco Elbrecht conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Paul D.N. Hebert authored or reviewed drafts of the paper, approved the final draft.

Dirk Steinke authored or reviewed drafts of the paper, approved the final draft.

Data Deposition

The following information was supplied regarding data availability:

The raw data/code is included in the manuscript (please state where). Raw sequence data is already available from the previous studies (NCBI SRA, under the accession numbers SRR5295658 and SRR5295659 (fwh1 primer set), SRX1619153 (BF/BR primer set), and figshare for the mlCOIintF/jgHCO primer set (R1 direction: https://dx.doi.org/10.6084/m9.figshare.4039821.v1, R2 direction: https://dx.doi.org/10.6084/m9.figshare.4039860.v1))

Funding

This work was supported by the Canada First Research Excellence Fund. It represents a contribution to the ‘Food From Thought’ research program and to the European Cooperation in Science and Technology (COST) Action DNAqua-Net (CA15219). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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