Abstract
The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously ( PrimerSuite ), (ii) screen for primer-primer dimerizing artefacts ( PrimerDimer ), and (iii) support multiplex PCR assays ( PrimerPlex ). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).
Highlights
While the bisulfite method remains the gold standard for quantitative profiling of the methylation status of target DNA at a single base pair resolution, applications which utilize PCR primers to amplify the bisulfite-converted DNA templates have additional design constraints unique to this application
We present here a custom high-throughput web-based program to support bisulfite PCR and multiplex PCR assays called PrimerSuite, which is composed of three modules: PrimerSuite (PS) – for generation of bisulfite and genomic primers from multiple sequences; PrimerDimer (PD) – for prediction of primer dimer formation between single primer pairs or multiple oligonucleotides in a multiplex pool; and PrimerPlex (PP) – for grouping of primers into different pools for multiplex PCR amplification
We have previously described a method for multiplex bisulfite PCR analysis[6] and based on the utility of this method an additional design feature for multiplex PCR assays called PrimerPlex was implemented
Summary
While the bisulfite method remains the gold standard for quantitative profiling of the methylation status of target DNA at a single base pair (bp) resolution, applications which utilize PCR primers to amplify the bisulfite-converted DNA templates have additional design constraints unique to this application. The design considerations which went into the PrimerSuite software were based on several key criteria for bisulfite PCR that were discovered and optimized during two recently published genome-wide methylated biomarker discovery projects, and which required high-throughput validation and primer design for a large numbers of regions[6,7]. This software solution represents the first study where the functionalities of a program has undergone extensive iterative empirical testing, with over 1300 primer pairs designed and screened in the lab to date to ensure the efficiency of the software. The PrimerSuite software package is freely available and its three modules can be accessed online via www.primer-suite.com (PrimerSuite), www.primer-dimer.com (PrimerDimer) and www.primer-plex.com (PrimerPlex)
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