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Research Article

Bisulfite Conversion DNA Visualizer for Designing DNA Methylation Primers

Hernán G. Hernández, Misael Leonardo López-Cepeda, Gustavo A. Contreras-García, Carolina Vargas-Porras and Humberto Arboleda
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Background and Objective: An important guideline for preventing amplification bias in Polymerase chain reaction (PCR) based DNA methylation techniques was recently proposed. However, there is no software that facilitates visualizing and applying such recommendations in PCR primers design. The aim of this work was to provide a web tool to facilitate the visualization of critical sequence elements for the design of primers applying the most recent recommendations for PCR-based methylation techniques. Materials and Methods: BiCVisualizer, a script for the visualization of critical sequence elements was designed using Java/html. The amplification specificity of a set of 10 primer pairs designed with the visual assistance of this web-tool was evaluated in silico PCR using BiSearch software. Results: The in silico PCR test showed a mean of 353 matches per primer and strand (range 84-934) using BiSearch. BiSearch did not find non-specific amplification for any primer pair designed using BiCVisualizer. Conclusion: This script was designed to assist the effective design of primers for methylation-sensitive high-resolution melting, the utility of this tool was supported by in silico evaluation. Users requiring visual construct primers can now spend less time in primer design using BiCVisualizer tool.

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  How to cite this article:

Hernán G. Hernández, Misael Leonardo López-Cepeda, Gustavo A. Contreras-García, Carolina Vargas-Porras and Humberto Arboleda, 2019. Bisulfite Conversion DNA Visualizer for Designing DNA Methylation Primers. Trends in Bioinformatics, 12: 1-6.

DOI: 10.3923/tb.2019.1.6



DNA methylation is considered a main epigenetic mark and has been associated with the pathogenesis of many diseases1-3. As bisulfite treatment changes DNA base composition of non-methylated Cytosines (Cs), it is used to study the methylation status of DNA sequences4,5.

Methylation-Sensitive High-Resolution Melting (MS-HRM) is a bisulfite-based technique that allows for the detection of differentially Methylated Regions (DMRs) based on the assessment of DNA melting temperature. Melting temperature can be measured as sharp decreases in the fluorescence signal of an intercalating dye in real-time thermocyclers6-8. The MS-HRM is widely used for the detection of DMRs due to its high sensitivity, scalability, speed and low cost9. The MS-HRM protocols for the detection of DMRs have shown a measured limit of detection of 1% or even lower6. Therefore, there is a growing interest in the feasibility of using MS-HRM in cancer research and clinical diagnosis6,10. However, bisulfite-based Polymerase Chain Reaction (PCR) techniques including MS-HRM are prone to PCR bias, namely, the preferred amplification of one allele over another without biological meaning usually favoring non-methylated alleles11. To avoid such bias, Wojdacz et al.11 made some recommendations for MS-HRM primers design. According to these recommendations some important critical sequence elements should be considered at the same time; however, current software does not permit the visualization of such elements, thus making the primer design for M S-HRM a time-consuming task5.

Therefore, the objective of the present work was to develop a visualization tool to allow for the easy and quick selection of key-sequence elements in a user-friendly manner for applying the current recommendations of primer designing in PCR-based DNA methylation techniques. To obtain a functional tool that allows for the visualization of the mentioned key elements, the steps required were: Identification of the key elements in the DNA sequence, development of a java-html script to find and highlight the elements in an easy color code, systematically testing a set of primers designed with the developed tool through an in silico analysis and release of the software as a free available web-tool.


Visualizer tool implementation: The first step to develop the web tool was the identification of the key elements required in primer design according the Wojdacz’s recommendations11. A shortened adaptation of the full set of original recommendations12 was displayed in Table 1.

In the designed visualizer software, it considered the linguistic complexity reduction produced by ‘bisulfite conversion’ with disproportional decrease in 'Cs' content, in the original DNA strand or 'As' in the complementary strand. After bisulfite treatment, the methylated CpG dinucleotides represent the only reservoir where Cytosines remain Cytosines; therefore, most Cytosines had been depleted in the DNA sequence13. Consequently, a function for highlighting the high complexity sequences was included in BiCVisualizer. In sum, the following sequence elements were detected and highlighted to be visualized in BiCVisualizer: “CpG dinucleotides”, “Thymines from a bisulfite-converted sequence”, “mononucleotide repeats” and “linguistically complex tetranucleotide sequences”.

BiCVisualizer Java-html tool and on-line use: BiCVisualizer tool for primer design was written for Java-html version 1.2. For public access the tool was made freely available online at For using BiCVisualizer, the input should be introduced in plain text or FASTA format with no characters other than A, G, C and T in DNA sequence.

The BiCVisualizer input sequence can be accepted in lowercase, uppercase, mixed lowercase/uppercase and with spaces or newline characters. In addition, practical user instructions are available at the BiCVisualizer’s web site. Script designing and improving took the equivalent to 6 months of intensive work. Designing the testing primers took less than 30 min per primer pair.

The BiCVisualizer output is a bisulfate converted and highlighted version of the input sequence that is susceptible of being copied or modified in a text editor. The output sequence was intended to support users in designing primers according to the current recommendations (Fig. 1). The output sequence was designed for displaying a straightforward color code, assisting users to distinguish the following critical sequence elements:

Table 1:Summary of current Wojdacz’s primer design recommendations and rationale
Source: Wojdacz et al.11

Fig. 1:Overall functions of BiCVisualizer
  The diagram displayed the tool processes arrangement for the color/style code construction to make visible critical sequence elements for the design of the primers

Fig. 2:Screen-shot of BiCVisualizer DNA visualizer for MS-HRM primer design web site interface
  Upper square allows pasting sequence of interest in FASTA or raw format. Capital case letters, as well as lower case letters are allowed in the input, even if the letters are scattered or in separate lines. The bottom box shows the output bisulfite converted sequence. CpG sites are shown without conversion as if they were methylated in red boldcapital letters; the non-CpG site converted Thymines are shown in green capital letters, mono-nucleotide tracks of more than 4 nucleotides appear as strike-through text and complex tetranucleotides as underlined bold text

Cytosine-guanine dinucleotide sites (CpG): The CpG are potentially methylated or non-methylated DNA tracks at the 5th carbon of the cytosine. To control PCR bias, one or two CpGs should be included in the primer design, as far as possible from the 3’oligonucleotide extreme. Non-methylated alleles of each CpG included in the primer will generate a mismatch near the 5’ oligonucleotide extreme, thus reducing the efficiency of amplification of non-methylated allele and equilibrating the bias11. Including three CpG sites is allowable but it should be done cautiously because it could redirect the bias toward methylated alleles11. In BiCVisualizer output the CpG sites remain unconverted appearing in red and bold for easy visualization (Fig. 2).

Thymines generated by conversion process: After bisulfite treatment, all Cytosines from non-CpG sites will be transformed into Uracils and the subsequent PCR amplification replaces them with Thymines (Ts). In order to increase the specificity of the amplification for completely converted sequences it is important to include at least one Thymine derived from a converted Cytosine, as close as possible to the 3’ end4,14.

Table 2:Primers designed with BiCVisualizer and corresponding in silico specificity evaluation
Matches: Primer sequences matches of each primer in the bisulfite converted genome (mean by strand) using Bisearch/Simple-search tool as a measure of specificity, HG38 position: Genomic position of amplicon in Hg38 human genome annotation CpG sites in bold capital letters, the non-CpG site converted Thymines are shown as capital letters and complex tetranucleotides as underlined bold text

BiCVisualizer output colors include differential highlighting of Cs located in non-CpG sites after replacing them with Ts. A green color and bold capital letters distinguish those new Ts from the unconverted nucleotides in the original sequence (Fig. 2).

Visualizing complexity: Complexity sequences were emphasized in two ways: low complexity mono-nucleotide repeats and complex nucleotide sequences.

Mono-nucleotide repeats (>5 nt): Mono-nucleotide repeats, defined here as mono-nucleotide tracks of five or more single nucleotide repeats become even more frequent after bisulfite conversion(for Ts or As nucleotides). Long stretches of nucleotides in primer sequence affect PCR specificity; therefore, in BiCVisualizer’s output mono-nucleotide sequences are shown in cross-out letters to warn users about their presence.

Complex tetranucleotides: For primer designing, linguistically complex sequences would be preferred because they are less likely to be repeated in the genome, producing a specific amplification. In other words, linguistically complex nucleotides are relatively infrequent targets of unspecific amplification. In BiCVisualizer output these sequences were shown as underlined letters to facilitate their inclusion in the 3’ end of the designed oligonucleotides (Fig. 2).

Specificity assessment: After BiCVisualizer implementation, PCR specificity of 10 primers designed with the visual assistance of this tool was evaluated using the ePCR/BiSearch software tool to assess the number of primer pairs present and the number (or the absence) of unspecific amplicons15. Previous observations recommended less than 3000 primers match and the absence of in silico amplicons in BiSearch5. The absence of unspecific amplicons is indicative of total specificity5,15.

Although BiSearch/ePCR is the only one available tool to perform the specificity assessment of this class of primers through in silico PCRs in the human genome, BiSearch does not assist MS-HRM primer desinging15.

The pairs of testing primers used in this work were designed in a semi-manual manner using BiCVisualizer according to the recommendations, directed to regions of epigenetic importance from MBP, APP, FMR1, CAPN10 and CLU genes (Table 2).


In the individual specificity analysis of testing primer designed using BiCVisualizer based on in silico search of matches in BiSearch/ePCR tool, less than 1000 matches per primer were found in a range of 84-934 matches (mean±SD; 353.6±302) (Table 2). This range of results had clearly shown a satisfactory individual specificity measurements according to previous recommendation of less than 3000 matches in the genome per primer5.

Regarding the primer pairs specificity assessment, only one specific amplicon and no unspecific amplicons were found for each of the five in in silico PCRs analyses (Table 2). This results indicated the complete specificity of the evaluated primer pairs according to the in-silico gold standard specificity measurement measurement15.

The present software was developed to facilitate MS-HRM primers design following current recommendations and additionally to assist in the location of linguistically complex sequences in this process. Current recommendations for primers design became a guideline useful for PRCs in many biomedical research fields including oncology16, neurology17 and pregnancy18. BiCVisualizer tool is available as a free easy-to-access web-tool for any scientific interested in studying DNA methylation.

Finding the high complexity sequences may be critical for PCR success, as was found in the in silico PCR evaluation. Sequences with known biological function such as genes, conserved regions, regulatory domains and exons have been associated with linguistic complexity19,20 and previous software that measured this parameter were developed to address questions of biological importance20-22. For example, Fast PCR a commercially available software has used sequence complexity criteria for primer selection according to the alphabet capacity method22. However, Fast PCR is not comparable with BiCVisualizer, because it is not intended to assist MS-HRM primers design with current recommendations22.

The present software, BiCVisualizer, is a bisulfite converted DNA visualizer for researchers interested in MS-HRM and other PCR based DNA methylation techniques. Other primer designing programs for bisulfite-based PCR techniques including Methprimer and BiSearch have different functions15,23,24. However, BiCVisualizer is the first available tool that allows for the visualization of CpG sites, converted Thymines, mononucleotide tracks and complex sequences at the same time, in an efficient and rapid manner. Furthermore, the primer pairs designed using BiCVisualizer primer only showed one amplicon per primer pair, indicating high specificity in the in silico evaluation tool15.

Since Bisulfite-converted methylated sequences including MS-HRM, amplify less efficiently than non-methylated sequences due the lower melting temperature of non-methylated templates8; only if new primers designing recommendations are included12, the increasing annealing temperature can improve the amplification of methylated templates and the decreasing annealing temperature may increases the amplification of non-methylated targets6. Therefore, PCR bias can be controlled in MS-HRM assays11. In this setting, BiCVisualizer has provided visual insights for designing HRM primers not available in other software15,23,24.

Finally, it is recommended (as for any other kind of primer-design) to evaluate conventional primer parameters such as dimer stability and hairpin stability before experimental usage. This could be a limitation of the present work because the evaluation of the designed primers should be completed using additional software. On the other hand, this work has presented a reliable, straightforward and efficient software for primer designing providing a DNA methylation study tool for the scientific community interested in the field. The present work provides to the scientific community the BiCVisualizer, a previously unavailable software to visually assist with DNA methylation primer design including MS-HRM, one of the most used and cost-effective quantitative DNA methylation assays5.


BiCVisualizer software is a tool for specific locus DNA methylation primer designing, easy to use in MS-HRM. Using BiCVisualizer is possible to obtain reliable primers with satisfactory specificity in a short time.


The work of the authors was supported in part by Universidad Santo Tomás, Bucaramanga, Colombia, Universidad Nacional de Colombia, Bogotá, Colombia and the Universidad Industrial de Santander, Bucaramanga, Colombia.

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