Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies

To the authors’ knowledge, this is the first report of the use of emulsion-Polymerase chain reaction (e-PCR) coupled with denaturing gradient gel electrophoresis (DGGE) analysis. In the present work the effectiveness of ePCR in improving the power of the DGGE technique for microbial population studies was tested. Our results indicated that ePCR results in uniform amplification of several DNA molecules, overcoming the major limitations of conventional PCR, such as preferential amplification and DNA concentration dependence. Moreover, ePCR-DGGE resulted in higher sensitivity when compared to conventional PCR-DGGE methods used for studying microbial populations in a complex matrix. In fact, compared to conventional PCR, the DGGE profiles of ePCR products permitted the detection of a higher number of the species that were present in the tested sample.

Quantitating primer-template interactions using a deconstructed PCR methodology

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.

Quantitating primer-template interactions using a deconstructed PCR methodology

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.

MethylCal: Bayesian calibration of methylation levels

Bisulfite amplicon sequencing has become the primary choice for single-base methylation quantification of multiple targets in parallel. The main limitation of this technology is a preferential amplification of an allele and strand in the PCR due to methylation state. This effect, known as ‘PCR bias', causes inaccurate estimation of the methylation levels and calibration methods based on standard controls have been proposed to correct for it. Here, we present a Bayesian calibration tool, MethylCal, which can analyse jointly all CpGs within a CpG island (CGI) or a Differentially Methylated Region (DMR), avoiding ‘one-at-a-time' CpG calibration. This enables more precise modeling of the methylation levels observed in the standard controls. It also provides accurate predictions of the methylation levels not considered in the controlled experiment, a feature that is paramount in the derivation of the corrected methylation degree. We tested the proposed method on eight independent assays (two CpG islands and six imprinting DMRs) and demonstrated its benefits, including the ability to detect outliers. We also evaluated MethylCal’s calibration in two practical cases, a clinical diagnostic test on 18 patients potentially affected by Beckwith–Wiedemann syndrome, and 17 individuals with celiac disease. The calibration of the methylation levels obtained by MethylCal allows a clearer identification of patients undergoing loss or gain of methylation in borderline cases and could influence further clinical or treatment decisions.

MethylCal: Bayesian calibration of methylation levels

Bisulfite amplicon sequencing has become the primary choice for single-base methylation quantification of multiple targets in parallel. The main limitation of this technology is a preferential amplification of an allele and strand in the PCR due to methylation state. This effect, known as “PCR bias”, causes inaccurate estimation of the methylation levels and calibration methods based on standard controls have been proposed to correct for it. Here, we present a Bayesian calibration tool, MethylCal, which can analyse jointly all CpGs within a DMR or CpG island, avoiding “one-at-a-time” CpG calibration. This enables more precise modeling of the methylation levels observed in the standard controls. It also provides accurate predictions of the methylation levels not considered in the controlled experiment, a feature that is paramount in the derivation of the corrected methylation degree. We tested the proposed method on eight independent assays (two CpG islands and six imprinting DMRs) and demonstrated its benefits, including the ability to detect outliers. We also evaluated MethylCal’s calibration in two practical cases, a clinical diagnostic test on 18 patients potentially affected by Beckwith-Wiedemann syndrome, and 17 individuals with celiac disease. The calibration of the methylation levels obtained by MethylCal allows a clearer identification of patients undergoing loss or gain of methylation in borderline cases and could influence further clinical or treatment decisions. MethylCal is availability as an R package onhttps://github.com/lb664/MethylCal.

Critical Evaluation of Molecular Monitoring in Malaria Drug Efficacy Trials and Pitfalls of Length-Polymorphic Markers

ABSTRACT Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures. When using length-polymorphic molecular markers, preferential amplification of short fragments can compromise detection of coinfections, potentially leading to misclassification of treatment outcome. We quantified minority clone detectability and competition among msp1, msp2, and glurp amplicons using mixtures of Plasmodium falciparum strains and investigated the impact of template competition on genotyping outcomes in 44 paired field samples. Substantial amplification bias was detected for all three markers, with shorter fragments outperforming larger fragments. The strongest template competition was observed for the marker glurp. Detection of glurp fragments in multiclonal infections was severely compromised. Eight of 44 sample pairs were identified as new infections by all three markers. Ten pairs were defined as new infections based on one marker alone, seven of which were defined by the questionable marker glurp. The impact of size-dependent template competition on genotyping outcomes therefore calls for necessary amendments to the current WHO recommendations for PCR correction of malaria drug trial endpoints. Accuracy of genotyping outcomes could be improved by separate amplification reactions per allelic family and basing results on markers msp1 and msp2 first, with glurp only used to resolve discordant results.