Deconstruction of Polymerase Chain Reaction to Reduce Bias Associated with Degenerate Primers

The polymerase chain reaction(PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA templates. To target the high taxonomic and genetic diversity in microbial communities, degenerate primers are frequently used. The use of such primers can lead to inefficient PCR amplification of genes from some taxa, resulting in a distortion of the true relative abundance of target genes. In this thesis, a new mechanism for PCR amplification of genes using degenerate primers was developed and tested to determine if amplification biases could be reduced. PCR yields were analyzed using next-generation sequencing to determine PCR efficiency. Sequence data generated from mock community DNA and unknown environmental genomic DNA were analyzed using custom bioinformatics pipelines to determine the effect of PCR strategy on relative abundance of known and unknown taxa in samples. We demonstrate here that the new two-stage PCR amplification strategy developed here greatly reduces the effect of primer biases in amplification of mock DNA communities of known concentration and abundance, relative to standard amplification reactions. In addition, we show that this method is also able to increase amplification of templates with 3’ mismatches relative to all primers, thereby increasing the potential for primers to target a broader range of taxa. The new method was employed to analyze microbial communities from unknown environmental genomic DNA samples and our analyses demonstrated that the method is effective for analysis of complex microbial communities. The advantages of this strategy touch many areas of research- The method is simple to perform and is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup. We also establish that there is better capture including PCR with degenerate primers, PCR with primers potentially containing mismatches (including single nucleotide polymorphisms,SNPs) to known and unknown templates, multiplex PCR for target capture, and quantitative PCR with degenerate primers

Effect of PEX PCR Stage “A” annealing temperature on observed microbial community structure and primer utilization patterns.

<p>Non-metric multidimensional scaling (NMDS) plot of fecal microbiome, performed at the taxonomic level of family and based on Bray-Curtis similarity (2D stress = 0.05). Samples were rarefied to 1,250 sequences per sample and no transformation was applied. The analysis is based on a single genomic DNA sample, with PEX PCR stage “A” annealing performed at 30°C (down-facing triangles), 35°C (open circles), 40°C (diamonds), 45°C (up-facing triangles), 50°C (closed circles) and 55°C (squares). Symbols are color-coded by the diversity (Shannon Index) of reverse primers (<i>i</i>.<i>e</i>. 806R) utilized in annealing and elongation during stage “A” of PEX PCR. Maximum possible Shannon index for 18 primers in the primer pool is 2.89. Vectors indicate taxa with Pearson correlation of >0.8 with MDS1 and MDS2 axes.</p

Temperature gradient analysis of the PEX PCR and TAS methods using mock community DNA.

<p>The relative abundance of reads mapping to the each of the four target templates (Mock A, Mock B, Mock C and Mock D) is shown for each temperature. The error bars represent standard deviation associated with two to four replicates per sample. <b>(A)</b> Results from PEX PCR and <b>(B)</b> Results from TAS PCR.</p

Types and abundance of DNA fragments found in PCR.

<p><b>(A)</b> Template DNA fragments (containing strands “A” and “B”) are added to PCR reactions and are conserved throughout the reaction. Fragments “A” and “B” serve as templates for copying in each cycle, with hybrid molecules “C” and “D” produced in a linear fashion each cycle. In cycles two and above, “C” and “D” are copied, creating hybrid molecules “E” and “F” in a linear fashion. In cycles three and above, the “E” and “F” fragments generated in prior cycles are copied into inverse complement fragments “F” and “E”, respectively, in an exponential fashion. Red boxes indicate ‘natural’ primer annealing to genomic DNA template or copy of gDNA template. Green boxes indicate ‘artificial’ primer annealing to primer sites that are copies of oligonucleotide primers added to the PCR mixture, and incorporated during previous cycles. <b>(B)</b> The relative abundance of “E” and “F” fragments generated by ‘natural’ template-primer interactions (“C”,”D” → “E”,”F”; shown as solid squares) and by artificial template-primer interactions (“E”,”F” → “F”,”E”; shown as open circles) varies by cycle. At the end of cycle two, all “E” and “F” fragments have been generated only by ‘natural’ template-primer interactions.</p

Schematic of Targeted amplicon sequencing (TAS) and Polymerase Exonuclease (PEX) PCR methods.

<p>Schematic of Targeted amplicon sequencing (TAS) and Polymerase Exonuclease (PEX) PCR methods.</p

Relative abundance of mock DNA templates observed in sequencing of TAS and PEX PCR method reactions.

<p>The error bars represent standard deviation associated with two replicates per sample.</p

Effect of PEX PCR and exonuclease treatment on observed microbial community structure and primer utilization patterns.

<p>Non-metric multidimensional scaling (NMDS) plot of lake sediment microbiome, performed at the taxonomic level of family and based on Bray-Curtis similarity (2D stress = 0.02). Samples were rarefied to 35,500 sequences per sample and no transformation was applied. All reactions were performed with an annealing temperature of 45°C, using PEX PCR with exonuclease (squares), PEX PCR without exonuclease (circles), and TAS PCR (triangles). Symbols are color-coded by the diversity (Shannon Index) of reverse primers (<i>i</i>.<i>e</i>. 806R) detected in the sequences. Maximum possible Shannon index for 18 primers in the primer pool is 2.89. Small, but significant differences in the observed family-level Shannon index (F-SI) were observed between PEX PCRs (with and without exonuclease treatment) and TAS PCRs using a two-tailed ttest (p<0.05).</p