AmpliconDesign – an interactive web server for the design of high-throughput targeted DNA methylation assays

ABSTRACT

Targeted analysis of DNA methylation patterns based on bisulfite-treated genomic DNA (BT-DNA) is considered as a gold-standard for epigenetic biomarker development. Existing software tools facilitate primer design, primer quality control or visualization of primer localization. However, high-throughput design of primers for BT-DNA amplification is hampered by limits in throughput and functionality of existing tools, requiring users to repeatedly perform specific tasks manually. Consequently, the design of PCR primers for BT-DNA remains a tedious and time-consuming process. To bridge this gap, we developed AmpliconDesign, a webserver providing a scalable and user-friendly platform for the design and analysis of targeted DNA methylation studies based on BT-DNA, e.g. deep amplicon bisulfite sequencing (ampBS-seq) or EpiTYPER MassArray. Core functionality of the web server includes high-throughput primer design and binding site validation based on in silico bisulfite-converted DNA sequences, prediction of fragmentation patterns for EpiTYPER MassArray, an interactive quality control as well as a streamlined analysis workflow for ampBS-seq.

KEYWORDS:

• DNA methylation
• bisulfite sequencing
• targeted sequencing
• massarray
• epityper
• primer design
• analysis pipeline

List of abbreviations

5mC = 5-methylcytosine	=
ampBS-seq = amplicon bisulfite sequencing	=
BT-DNA = bisulfite-treated DNA	=
CGI = CpG-island	=
DNA = Deoxyribonucleic acid	=
EPIC array = Illumina Infinium MethylationEPIC array	=
MassArray = EpiTYPER MassARRAY	=
PCR = Polymerase chain reaction	=
SNPs = single nucleotide polymorphisms	=

Acknowledgments

We thank all members of the Division of Cancer Epigenomics (DKFZ) for thoughtful discussions related to this study and we especially thank Christoph Plass for supporting this project. We also thank Johannes Beisiegel and the IT Core Facility (DKFZ) for excellent support and technical service related to setting up and hosting the web server.

Authors’ contributions

MS, JaH, PB, YA, DW, PL and DBL designed the study. MS and JaH have written the AmpliconDesign source code. PB implemented the genome annotation database. JL performed experiments. MS, SS, JL and JoH performed data analysis and data interpretation. PL & DBL jointly coordinated and supervised the study. MS, PL and DBL wrote the first draft of the manuscript. All coauthors contributed to the final version of the manuscript.

Availability of data and materials

The source code of the AmpliconDesign software is publicly available under the GNU General Public License v3.0 (https://github.com/MaxSchoenung/AmpliconDesign).

Disclosure statement

The authors declare that they have no competing interests.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This study has in part been supported by funds from Deutsche Krebshilfe [DKH project #70112574 to DBL] and from Deutsche Forschungsgemeinschaft [FOR2674, LI 2492/3-1 to DBL). PL was supported by AMPro Project of the Helmholtz Association [ZT00026].