PCR effects of melting temperature adjustment of individual primers in degenerate primer pools

Deep sequencing of small subunit ribosomal RNA (SSU rRNA) gene amplicons continues to be the most common approach for characterization of complex microbial communities. PCR amplifications of conserved regions of SSU rRNA genes often employ degenerate pools of primers to enable targeting of a broad spectrum of organisms. One little noticed feature of such degenerate primer sets is the potential for a wide range of melting temperatures between the primer variants. The melting temperature variation of primers in a degenerate pool could lead to variable amplification efficiencies and PCR bias. Thus, we sought to adjust the melting temperature of each primer variant individually. Individual primer modifications were used to reduce theoretical melting temperature variation between primers, as well as to introduce inter-cluster nucleotide diversity during Illumina sequencing of primer regions. We demonstrate here the suitability of such primers for microbial community analysis. However, no substantial differences in microbial community structure were revealed when using primers with adjusted melting temperatures, though the optimal annealing temperature decreased

Atopic dermatitis and food sensitization in South African toddlers: Role of fiber and gut microbiota

The pathogenesis of atopic dermatitis (AD) is complex and related to allergic responses and defects in skin barrier function. In common with many atopic diseases, the prevalence of AD has been increasing across the world.1 One of the theories for this increase is increased hygiene and urbanization-related changes in the environment, which can affect the human microbiome.2 Previous studies have found associations between the composition of the early gut microbiome and development of atopic conditions, including AD.3 Although the rate of atopic conditions, including AD and food allergy, is increasing on all continents, the prevalence of these diseases is still lower in African countries.1 This is especially interesting because individuals of African origin who live in Western countries, such as African Americans, are at a higher risk for severe AD.4 This variation places Africa in a special position; studying African populations is necessary not only to find ways to prevent increases of allergy conditions in African countries but also to provide important clues to the causes of this global increasing of allergic conditions. Young children who have developed AD in African communities with a low incidence of atopic disease might be the transitional group. In the current study, we have, for the first time to our knowledge, analyzed the fecal microbiota composition of a group of young black African children aged 12 to 36 months old with and without AD living in the same community in Cape Town, South Africa. Our primary goal was to examine whether toddlers with AD and control toddlers from Cape Town have different microbiomes in terms of bacterial richness and diversity. We also aimed to investigate the differences in the relative abundance for different operational taxonomic units between these 2 groups. In our subgroup analyses, we further tested the effect of multiple environmental factors on the gut microbiome in these children

Evaluating PCR Bias Through Experimental Investigations of Complex Primer-Template Interactions

The polymerase chain reaction (PCR) is a well-established tool for amplification of regions of DNA and is used in a broad range of biological studies. PCR bias, in which some templates within a mixture of templates are preferentially amplified, is a well-known phenomenon. Despite substantial effort invested into correcting such bias, PCR-based studies continue to generate data that distort underlying template ratios. A major source of PCR bias is from primer-template interactions, leading to PCR selection favoring certain templates. Motives of this study were to understand better the causes of selection bias in PCRs with complex templates and complex degenerate primer pools, and to develop novel strategies to decrease bias. An experimental system was developed to reduce PCR bias by separating linear copying of templates from exponential amplification of amplicons (Deconstructed PCR or ‘DePCR’), and this system also provides a mechanism to quantify primer-template interactions (Primer utilization profiles or ‘PUPs’). DePCR was used to interrogate mock DNA communities and complex environmental samples, and all reactions were compared to standard PCR workflows. Experiments with annealing temperature gradients demonstrated a strong negative correlation between annealing temperature and the evenness of primer utilization in complex pools of degenerate primers. Critically, shifting primer utilization patterns mirrored shifts in observed microbial community structure. In experiments with mock DNA templates, DePCR demonstrates that although perfect match primer-template interactions are abundant, the dominant type of primer-template interactions are mismatch interactions, and mismatch amplification starts immediately during the first cycle of PCR. Furthermore, in DePCR reactions involving multiple mismatches, no strong effect on template profiles was observed. DePCR allows improved representation of templates, greater tolerance for mismatches between primers and templates, and greater success in amplifying complex templates with low complexity primer pools. In addition, PUPs are empirical quantitative data derived from primer interactions with genomic DNA templates, and are a novel form of biological information that can be acquired only with DePCR. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, has applicability to amplicon-based microbiome studies, and may also serve in other PCR-based protocols where primers and templates have mismatches

Deconstructing the polymerase chain reaction: understanding and correcting bias associated with primer degeneracies and primer-template mismatches.

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 template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3-12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled "Polymerase-exonuclease (PEX) PCR", in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3' end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible

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 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

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