Quantitating primer-template interactions using a deconstructed PCR methodology
- Subject Areas
- Bioinformatics, Microbiology, Molecular Biology
- PCR bias, primer-template interactions, synthetic DNA, NGS, primer design, degenerate primers
- © 2019 Naqib 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
- 2019. Quantitating primer-template interactions using a deconstructed PCR methodology. PeerJ Preprints 7:e27781v1 https://doi.org/10.7287/peerj.preprints.27781v1
When the polymerase chain reaction (PCR) is used to amplify simultaneously multiple templates, preferential amplification of certain templates (PCR bias) leads to a distorted representation of the original templates in the final amplicon pool. PCR selection, a type of PCR bias, is influenced by mismatches between primers and templates, the locations of mismatches, and the nucleotide pairing of mismatches. Direct measurement of primer-template interactions has not been possible, leading to uncertainty when attempting to optimize PCR reactions and degenerate primer pools. In this study, we developed an experimental system to systematically study primer-template interactions. We synthesized 10 double-stranded DNA templates with unique priming sites, as well as 64 primers with 0, 1, 2 or 3 mismatches with each of the 10 templates. By using a previously described deconstructed PCR (DePCR) methodology, we generated empirical data showing individual primer interactions with templates in complex template-primer amplification reactions. Standard PCR and DePCR amplification protocols were used to amplify templates in a series of 16 experiments in which templates, primers, and annealing temperature were varied. We observed that although perfect match primer-template interactions are important, the dominant type of interactions are mismatch amplifications, and that mismatched primer annealing and polymerase copying starts immediately during the first two cycle of PCR. In reactions with degenerate primer pools, multiple mismatches between primer and template are tolerated, and these do not have a strong effect on observed template ratios after amplification when employing the DePCR methodology. When employing the DePCR methodology, mismatched primer-template interactions were able to amplify source templates with significantly lower distortion relative to standard PCR. We establish here a quantitative experimental system for interrogating primer-template interactions and demonstrate the efficacy of the DePCR method for amplification of complex template mixtures with complex primer pools.
This is a submission to PeerJ for review.
Template and primer utilization profiles for 16 individual experiments conducted in this study
For each study, varying number of primers and templates were used, as described in Table 1. For mMDS plots, samples were color coded by amplification method and different annealing temperatures indicated by shape. Ellipses represent a 95% confidence interval around the centroid. ANOVA was performed to measure differences in measured values by annealing temperature. Intensity scales vary between experiments. All samples were rarefied to 7,000 sequences. Heatmaps are the average of 7-8 technical replicates per condition; all replicates are shown in mMDS plots. (A) For each experiment, primer utilization profiles (PUPs) were generated (left side), and data are presented as mMDS plots (top) and as clustered heatmaps (bottom). Analysis of similarity (ANOSIM) was performed to determine if PUPs were significantly different between TAS and DePCR, regardless of annealing temperature, and within method across annealing temperature. Each slide contains a table showing the percentage of reads with 0, 1, 2 and 3 mismatches between primers and templates, as indicated in experiments with DePCR amplifications. For primer-template interactions with only a single mismatch, percentage of reads with 3’ (-2), middle (-8) and 5’ (-14) mismatches are shown. The average theoretical melting temperature of primers used in each study are shown. (B) Template profiling analyses were performed (right side), and data are presented as mMDS plots (top) and as clustered heatmaps (bottom). In addition to analysis of sequence data, the expected distribution of reads is shown in orange, both in the mMDS plots and in the heatmap. ANOSIM was performed to determine if template profiles were significantly different between TAS and DePCR, regardless of annealing temperature, and within method across annealing temperature. Ideal scores, as described in text, were calculated to determine which method and annealing temperature generated the closest approximation of the expected template distribution.
Effect of PCR methodology and annealing temperature on template profiles in amplification reactions utilizing varying primer pools
One-way clustered heatmaps of untransformed template utilization profiling during amplification of an uneven pooling of synthetic DNA templates and varying primer pools (Figure S17 = C1, C2 and C3 experiments with all ten templates present, and template ST1 at 1/10th the concentration of the other nine templates; Figure S18 = D1, D2, E1 and E2 experiments with four templates). For experiments C1, D1 and E1, only a single primer variant was used (806F_v1), while in experiments C2, D2 and E2, 10 primers were used. In experiment C3, 9 primers were used (806F_v1 was removed). Primer and template details are shown in Table 1. Samples (columns) are color-coded by amplification method (TAS or DePCR), amplification annealing temperature (45°C or 55°C), and average Ideal score. Each column represents the average of 7-8 technical replicates per condition and rarefaction to 7,000 sequences/replicate. Templates (rows) represent all 10 templates (ordered from top to bottom; ST1, ST4, ST6, ST7, ST8, ST11, ST15, ST23, ST39, and ST55). Ideal score comparisons between TAS and DePCR (across both annealing temperatures), within TAS (45°C or 55°C), and within DePCR (45°C or 55°C) are shown in tables. Asterisks indicate significant differences in measured values by annealing temperature (ANOVA, P < 0.01). Intensity scales vary between experiments.
Locus-specific primer sequences used in this study
For each primer, the locus-specific sequence is shown (column B) and a 5' linker sequence (column F) are used. The combined primer sequence used in this study is shown as well (column G). Primer length, estimated melting temperature (Tm; calculated as described in the main text) and mismatches relative to the first 806 primer, 806F_v1, are shown.
Distribution of mismatches between primers and templates used in this study
Locus-specific primer names and primer sequences (columns A and B) are shown next to variant position sequences (column C). Columns F-O represent each of the 10 synthetic DNA templates used in this study, with nucleotide sequences at each potential mismatch position shown in rows 3 and 4. Number of mismatches between templates and primers are colored in columns F-O and rows 5-68. Columns Q-U indicate which primers are used in which series of experiments (1-6). Rows 70-74 indicate which templates are used in which series of experiments (A-E), and are shaded according to relative concentration of each template in each template pool.
Rarefied biological observation matrix (BIOM) for all experiments
Data were rarefied to 7,000 sequences per sample, and each experimental condition has 7-8 replicates. A total of 640 possible interactions are listed (10 templates x 64 primers), and numbers represent the numbers of reads matching each of the combinations. For each row of the BIOM, the number of mismatches between primer and template are shown, along with the position of mismatch, the mismatch sequence pairing, and the theoretical melting temperature of the primer. Reactions conducted with DePCR are highlighted in blue; no highlighting is used for TAS amplification reactions.
Mapping file used for creation of biological observation matrices (BIOMs)
This mapping files is used by the script described in the text and provided in Supplemental Materials 2.
Metadata associated with all samples used in this study
Metadata for each replicate is shown, including the number of raw sequence reads, processed reads, calculated Shannon Index (loge), Ideal Score (calculated as described in the text), and figure(s) where data are shown.
Description of synthetic DNA template design and template sequences
Ten DNA templates were synthesized for this study, and the backbone of the template was derived from the 16S rRNA gene of the bacterium Rhodanobacter denitrificans 2APBS1. The source sequence is shown, along with its inverse complement and region used for gBLOCK synthesis. The design strategy for each gBLOCK is also provided in greater detail. Finally, the exact sequences used for synthesis of each of the templates is shown with primer sites and recognition sequences highlighted.