Abstract 4364: Establishment and validation of a breast cancer panel using targeted next generation bisulfite sequencing (tNGBS)
Authors: Poulin Matthew L. , Drennan Ryan , Miller Andrew , Miller Andrew , Meyer Ann , Rauscher Garth H. , Yan Liying 
Published: July 2017

Abstract
Aberrant DNA methylation (e.g., global reduction in DNA methylation and differential methylation of promoter regions) is a hallmark of cancer and may function in various ways to influence transcription. We developed a panel of bisulfite specific methylation sequencing assays in order to assess the methylation status of large number of genes using pyrosequencing. By combining these pyrosequencing assays into a targeted NextGen bisulfite sequencing panel, we can screen both a large number of genes and a large number of samples simultaneously. We first assembled the 40 pyrosequencing assays and sensitivity and reproducibility were analyzed on pilot DNA samples isolated from FFPE tissue. Assays were specifically designed to work in highly fragmented and cross-linked DNA from formalin-fixed, clinical samples. Initial pyrosequencing on these pilot samples showed that 24 of the 40 assays gave quality validation and sample results. Reasons that some assays were not successful may be due to the A/T or G/C content within the amplicon, PCR Bias, low amplification efficiency, or strong cross-linking from fixation within the assay to name a few. The initial 40 assays were next assembled in a combination of 7 multiplex PCRs in which gradient PCRs were performed and analyzed for optimal multiplex amplification using a bioanalyzer. Final assay multiplexing combinations were established and PCR bias testing was performed by tNGBS on a sample set of high and low methylated DNA mixed at different ratios. Assays with an R-square of less than 0.9 between the expected and generated methylation from this mixing were removed, leaving us with 21 validated assays in our panel (9 of 24 assays that were previously validated in pyrosequencing and 12 of 19 assays that could not be validated in pyrosequencing). In the present study we looked at DNA methylation changes in breast cancer development using paired adjacent normal, invasive and in situ tissue from a 30 patients with breast cancer as well as 11 various normal tissues other than breast, and 12 cell lines for a total of 83 samples in a panel of 21 genes. The results of the tNGBS were compared with the pyrosequencing results. This process of panel development, where small amounts of bisulfite treated DNA can be amplified in a target specific manner, prior to sequencing, makes investigating a large number of samples on a large panel of genes possible in a small amount of time. We observed a high correlation between the pyrosequencing and tNGBS sequencing results, and we show that that there are several genes that are either hypermethylated or hypomethylated in tumor tissue when compared to adjacent normal tissue.
Citation Format: Matthew L. Poulin, Ryan Drennan, Andrew Miller, Andrew Miller, Ann Meyer, Garth H. Rauscher, Liying Yan. Establishment and validation of a breast cancer panel using targeted next generation bisulfite sequencing (tNGBS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4364. doi:10.1158/1538-7445.AM2017-4364