Deconstructing the Polymerase Chain Reaction II: An improved workflow and effects on artifact formation and primer degeneracy
- Published
- Accepted
- Subject Areas
- Bioengineering, Microbiology, Molecular Biology
- Keywords
- PCR, chimera, PCR bias, microbiome, primer variants
- Copyright
- © 2019 Naqib et al.
- Licence
- 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
- 2019. Deconstructing the Polymerase Chain Reaction II: An improved workflow and effects on artifact formation and primer degeneracy. PeerJ Preprints 7:e27525v1 https://doi.org/10.7287/peerj.preprints.27525v1
Abstract
Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias (Green et al. 2015). In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.
Author Comment
This is a submission to PeerJ for review.
Supplemental Information
Effect of input gDNA template concentration on microbial community composition and PUPs using DePCR
Analyses were performed on rarefied data sets (8,000 sequences per sample), with five technical replicates for each DNA input level (1.25, 2.5, 5, 10 or 20 ng/µl). (A) Genus-level mMDS ordination of microbial community structure using a distance matrix based on Bray-Curtis similarity. No significant differences were observed between all the concentrations (Global ANOSIM: R=-0.03376, p=0.79). Ellipses represent a 95% confidence interval around the centroid. (B) Primer utilization profiles for all primer variants (RPV1 – RPV18), visualized as a heatmap. (C) A positive correlation between input gDNA (1.25, 2.5, 5, 10, 20 ng/µl) and sequence yield was observed. For all input levels, the same gDNA template was used with five technical replicates. All samples were pooled after stage A of DePCR and amplified together using Illumina P5 and P7 primers. Data were rarefied to 8,000 sequences per sample.