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. 2024 Aug 8:12:e17787.
doi: 10.7717/peerj.17787. eCollection 2024.

Quantitating primer-template interactions using deconstructed PCR

Jeremy Kahsen #  1 , Sonia K Sherwani #  1 , Ankur Naqib  2 , Trisha Jeon  1 , Lok Yiu Ashley Wu  1 , Stefan J Green  1
Affiliations

Quantitating primer-template interactions using deconstructed PCR

Jeremy Kahsen et al. PeerJ. .

Abstract

When the polymerase chain reaction (PCR) is used to amplify complex templates such as metagenomic DNA using single or degenerate primers, preferential amplification of templates (PCR bias) leads to a distorted representation of the original templates in the final amplicon pool. This bias can be influenced by mismatches between primers and templates, the locations of mismatches, and the nucleotide pairing of mismatches. Many studies have examined primer-template interactions through interrogation of the final products of PCR amplification with controlled input templates. Direct measurement of primer-template interactions, however, has not been possible, leading to uncertainty when optimizing PCR reactions and degenerate primer pools. In this study, we employed a method developed to reduce PCR bias (i.e., Deconstructed PCR, or DePCR) that also provides empirical data regarding primer-template interactions during the first two cycles of PCR amplification. We systematically examined interactions between primers and templates using synthetic DNA templates and varying primer pools, amplified using standard PCR and DePCR protocols. We observed that in simple primer-template systems, perfect match primer-template interactions are favored, particularly when mismatches are close to the 3' end of the primer. In more complex primer-template systems that better represent natural samples, mismatch amplifications can dominate, and heavily degenerate primer pools can improve representation of input templates. When employing the DePCR methodology, mismatched primer-template annealing led to amplification of 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 DePCR for amplification of complex template mixtures with complex primer pools.

Keywords: Next-generation sequencing; PCR; PCR bias; Primer design; Primer-template interactions.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Schematic of Deconstructed PCR (DePCR) workflow.
CS1, common sequence 1 linker sequence. CS2, common sequence 2 linker sequence. BC, barcode. F, Forward primer. R, Reverse primer, P5/P7, Illumina primers, PE1/PE2, Fluidigm Access Array Barcode Library Illumina adapters. In stage 1 (linear copying only), individual samples are cycled for four cycles with locus-specific primers and Fluidigm barcoded primers. Subsequently, all reactions are pooled and purified together, and then amplified with Illumina P5 and P7 primers in stage 2 (exponential amplification with primers targeting linker sequences). During stage 1, linear copying of templates leads to products which contain Illumina sequencing adapters, sample-specific barcodes, and the locus-specific region of interest. Only fragments with Illumina adapters and barcodes are exponentially amplified in stage 2. Locus-specific primer sequences can be modified as needed.
Figure 2
Figure 2. Schematic of primers, template, and mechanism for determining perfect match and mismatch annealing.
Sixty-four unique oligonucleotide primers were synthesized in this study of which ten are shown here (V0–V9). Primers were identical except for three positions at -2, -8 and -14 positions relative to the 3′ ends. Variant bases have been indicated by color (“C”, Blue, “T”, Red, “A”, Green, and “G”, Black). A schematic of the ten synthetic DNA templates used in this study (ST0 to ST9) are also shown. Each template was identical except for the 806F priming site and the 12-base recognition sequence. Each unique priming site sequence is linked with a unique recognition sequence. A total of 640 potential primer-template interactions can occur in this system (10 templates × 64 primers), of which two are shown here. Shown are primer-template interactions indicating the annealing of a perfectly matched primer (template ST0 and primer V0) and a primer with a single mismatch (template ST1 and primer V0). Perfect match and mismatch annealing are determined by comparing the inferred primer site based on the recognition sequence to the observed primer sequence for each sequencing reaction. Only reactions conducted using the DePCR methodology retain the sequence of the primer annealing to the source DNA templates.
Figure 3
Figure 3. Head-to-head competition between perfect match primers and single-mismatch primers when amplifying a single template.
Synthetic template ST0 was amplified using DePCR in reactions with two primers only: a perfectly matching primer (V0) and a primer with a single mismatch. Perfect match and mismatch primers were input into the reaction at equimolar levels, and amplifications were performed using 45 °C or 55 °C annealing during the 1st stage of DePCR. For each primer combination, four technical replicates were performed. Using the scheme depicted in Fig. 2, the sequenced amplicons were then evaluated to determine which primer annealed to the ST0 template during the first two cycles of the DePCR reaction and to determine the level of perfect match and single mismatch annealing and elongation. Control reactions were conducted wherein only perfectly matching primers (V0) were used. The percentage of sequences with mismatch primers are shown in (A) (55 °C) and (B) (45 °C). Experiments conducted with primers containing mismatches towards the 5′ end of the primer are colored blue, while those with mismatches towards the 3′ end of the primer are colored red. Experiments with mismatches in the middle position (‘M’) are colored yellow. The mismatch pairing (template:primer) is shown above each column. The ratio of mismatch-to-perfect-match primer usage by mismatch location is shown in (C) (55 °C) and (D) (45 °C). An ANOVA was run for each annealing temperature followed by Tukey’s post hoc test for pairwise comparison between mismatch positions. The exact values of the mismatch-to-match ratios are shown in the table (E), with significance values from Welch’s two-sample- t-test comparing results from experiments with different annealing temperatures. Significance levels: ‘ns’ (p ≥ 0.05), ‘*’ (p < 0.05), ‘**’ (p < 0.01), ‘***’ (p < 0.001), ‘****’ (p < 0.0001).
Figure 4
Figure 4. Amplification and sequencing of template pools with primer pools of varying complexity.
Ten synthetic DNA templates (ST0 through ST9) were pooled equimolarly and amplified using standard PCR and DePCR with different primer combinations. These conditions included: a single primer perfectly matching the ST0 template (primer V0; ‘B1’ experiment), ten primers each perfectly matching one of the STs (primers V0–V9; ‘B10’ experiment), and 27 primers with one to three mismatches with each of the ten templates and no perfect matches (primers V10–V36; ‘B27’ experiment). Each condition was performed with eight replicates. (A) For each experiment (B1, B10, B27) and for each template (ST0 through ST9), the distribution of primers annealing to each template are shown for PCR and DePCR at 45 °C (bottom) and 55 °C (top) annealing temperature. The distributions are color-coded by the type of primer-template interaction, including perfect matches (black) and single, double or triple mismatches (color coded by locations of mismatches). The dotted line represents the 10% relative abundance of each ST added to the reaction. Metrics for evaluating how evenly the primers were utilized and templates amplified are shown at the top of each panel and in tables below. These include the Ideal Score for primers (IPS; black) and the Ideal Score for templates (ITS; grey). For both ITS and IPS, values range from 0 (perfect representation of input) to 200. IPS and ITS values are shown in tables D and E respectively, with significant differences by annealing temperature or by PCR method indicated. Ratios of mismatch amplifications to perfect match amplifications (mismatch-to-match ratio) are shown in table C. Mismatch-to-match calculations could not be performed for the B27 experiment due to the absence of perfect match primers (n/0 = Inf). Visualization of the recovered template profiles relative to the known input template composition was performed with a multi-dimensional scaling plot ordination (B). The green circle represents the input template composition, and the data points closer to the green circle indicate closer representation of the experimental results compared to the input templates. Data from the B1, B10 and B27 experiments are shown for standard PCR and DePCR at 45 °C and 55 °C annealing temperatures. The red numbers indicate the average ITS score for each condition. All p-values were calculated with Welch’s-two-sample- t-test. Significance levels: ‘ns’ (p ≥ 0.05), ‘*’ (p < 0.05), ‘**’ (p < 0.01), ‘***’ (p < 0.001), ‘****’ (p < 0.0001).
Figure 5
Figure 5. Amplification and sequencing of a single template with primer pools of varying complexity.
A single synthetic DNA template (ST0) was amplified using standard PCR and DePCR with either ten primers (primers V0–V9; 1 perfect match and nine single mismatch primers; ‘A10’ experiment) or 64 primers (primers V0–V63; 1 perfect match and 63 primers with one to three mismatches; ‘A64’ experiment). All experiments were conducted with eight replicates. (A) For each experiment, the distribution of perfect match and mismatch amplifications is shown with color coding of bar charts to indicate type of mismatch amplification. For example, perfect matches are shown in black, while triple mismatch amplifications (27 possible combinations) are shown in brown. The number of possible mismatch types are indicated above each column (one, three, nine, or 27 possible combinations). (B) The ratio of mismatch amplifications of any type to perfect match amplifications is shown in the table below. (C) For experiment A64, the distribution of the usage of all 64 primers is shown for standard PCR and DePCR at 45 °C (bottom) and 55 °C (top). (D) The primer distributions in the sequencing output were compared to the equimolar input of the 10 (experiment A10) or 64 primers (experiment A64) by calculating the Ideal Score for Primers (IPS) for each replicate and subsequently averaged. Large IPS values represent more selective utilization of primers (i.e., more uneven), while small IPS values represent more broad utilization of all primers provided. Primer distributions from standard PCR do not represent primer-template interactions but represent the less informative primer-amplicon interactions. The primer distributions in DePCR represent the primers annealing to templates during the first two cycles of template copying. Welch’s two-sample t-test was used to calculate significance values for annealing temperature effects and separately for PCR method effects. Significance levels: ‘ns’ (p ≥ 0.05), ‘*’ (p < 0.05), ‘**’ (p < 0.01), ‘***’ (p < 0.001), ‘****’ (p < 0.0001).
Figure 6
Figure 6. Estimation of polymerase error rates in standard PCR and DePCR using proofreading and non-proofreading polymerases.
Synthetic template ST0 was amplified using a perfectly matching primer (primer V0) in standard PCR and DePCR reactions with three different PCR mastermixes. The three mastermixes included MT (non-proofreading), DT (non-proofreading) and QB (proofreading). DT and QB reactions were conducted with primers containing phosphorothioate modifications to prevent exonuclease activity of proofreading polymerases. Reactions were conducted at 45 °C (bottom), and 55 °C (top) annealing temperatures with seven to eight replicates. Error rates were measured by mapping sequence data against the ST0 reference and the number of mismatches between reference and sequence averaged across the first 20 bases (location of the primer), the next 20 bases (positions 21–40), and the next 60 bases (positions 41–100). Mean and standard deviation are shown. The first 20 bases (primer site) had significantly lower error rates in standard PCR relative to the next 20 bases (P < 2.93e−03). Conversely, the first 20 bases had significantly higher error rates in DePCR relative to the next 20 bases (p < 1.46e−07).
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