Download Full Text (383 KB)


Targeted Bisulfite Pyrosequencing & Amplicon Bisulfite Sequencing Epigenetic Analysis

Charles Tran, Dept. of Biology, with Dr. Karolina Aberg, VCU School of Pharmacy

Background: The Great Smoky Mountain Study is a longitudinal study that started in 1992 and includes 1,420 participants that were 9 to 13 years at intake and have since been revisited ~ every 2 years. Participants (and their parents) provided detailed assessment of stressors and health outcomes as well as blood samples at each interview. In a recent methylome-wide association study the samples were used to identify methylation marks associated with childhood trauma. In the current work, we present an investigation to replicate these methylation marks in an independent sample. Objective: Our objective is to optimize and apply epigenomic-specific protocols in order to replicate trauma associated methylation biomarkers in an independent study sample. Materials and Methods: We will use DNA samples extracted from saliva from The Young Adolescent Project, another longitudinal study which has obtained relevant information related to childhood trauma. In this sample we will perform replication of top findings using targeted amplicon bisulfite sequencing in saliva samples where amplicons are amplified with JUNO sequencing platform or Pyromark PCR pyrosequencing. Forward and reverse primers are first designed using Pyromark Assay Design software. Primer set candidates are chosen based off of a score of 100; scores are determined by potential for mispriming, likelihood for primer dimers, etc. Higher scores correlate to better PCR performance. Then, BiSearch, an online primer-design algorithm and search tool is used to check primer sets in order to ameliorate PCR efficiency by avoiding non-specific PCR products due to genomic repetition. PCR product is then examined with 2% agarose gel electrophoresis and Agilent Bioanalyzer chip-based capillary electrophoresis in order to determine if amplicons of the correct size were obtained. (Once primers of sufficient efficacy are designed, they are subject to 5’ biotin tag modifications—this makes purification of proteins and other target molecules easier while utilizing streptavidin-coupled Dynabeads). Methylation sites incompatible with JUNO due to amplicon sites exceeding 200 base pairs would instead be analyzed using Pyromark Pyrosequencing Assay for which it is easier to design assays but is more costly and lower throughput: the output of resulting data being similar in quality. Results: We attempted to design assays for 60 loci. Of these we have designed and validated the quality of 23 assays for JUNO and 3 for the Pyromark Q96 sequencing and quantification platform. PCR analysis followed afterwards. We were not able to design assays for 34 sites due to: amplicon sites having exceeded 200 base pairs, forming of hair pins, forming of primer dimers, amplicon sites being too far from target region, or formation of multiple PCR products, as determined by IDT analysis. The 3 primer sets were incompatible with JUNO due to formation of primer dimer and hairpin formations when 5’ tags were added therefore Pyromark Q96 assay was optimal. Conclusion: In conclusion we have optimized and evaluated 23 assays for the JUNO sequencing platform and 3 primers for Pyromark Q96 that, in the next step, will be used to assess the replication of loci of interest in trauma associated methylation biomarkers from saliva samples.

Publication Date


Faculty Advisor/Mentor

Karolina Aberg, Ph.D.


Virginia Commonwealth University. Undergraduate Research Opportunities Program

Is Part Of

VCU Undergraduate Research Posters


© The Author(s)

Targeted Bisulfite Pyrosequencing & Amplicon Bisulfite Sequencing Epigenetic Analysis