Whole-Genome Structural Variation and CpG Methylation Analyses Through Long-Read Sequencing
Abstract
Structural Variations (SVs), including DNA base deletion, insertion, inversion, and translocation, are hallmarks of prostate cancer (PCa). PCa is also critically regulated by epigenetic changes, such as DNA methylation. It has been challenging to obtain precise maps of structural variations and DNA methylations across the entire genome, as existing technologies depend on sequencing (e.g., Illumina) methods that generate short reads usually of only 50-300 base pairs long. Short reads have difficulty finding their position on the human reference genome, especially in chromosomal regions of major structural variations or repetitive elements. Further, current methods of detecting DNA methylation depend on bisulfite conversion and are unable to distinguish between DNA cytosine methylation (5mC) and hydroxymethylation (5hmC). For example, current standard whole-genome bisulfite sequencing measures the sum of 5mC and 5hmC as the level of DNA methylation. However, studies have shown that 5mC and 5hmC have opposite roles in regulating PCa gene expression. The goal of the present study is to develop and benchmark an innovative technology called Nanopore Long-Read Sequencing (LRS) for PCa research. Nanopore LRS directly sequences native DNA and is thus able to distinguish between 5mC and 5hmC and provide their precise maps on the same DNA molecules. Nanopore provides long reads in an average of 50 kilobases but up to 4 megabases, if needed, and thus is often able to sequence across the entire region of SVs or repetitive elements, significantly improving accuracy in SV detection. In Aim 1, we will adapt Nanopore LRS technology in PCa cell lines and evaluate its performance in detecting DNA methylations relative to whole-genome bisulfite sequencing. Aim 2 will use LRS to analyze 10 pairs of matched benign and PCa samples and evaluate its ability to detect SV, 5mC, and 5hmC changes that are associated with PCa progression. We will also examine their association with TMPRSS2-ERG gene fusion, which occurs in approximately 50% of all PCa cases and has a lower prevalence in prostate tumors of African American men. Ultimate Applicability of the Research: Once we optimize Nanopore LRS technology in PCa cells and establish all of the bioinformatics analysis pipelines, we will rapidly apply it to a large set of human PCa samples to obtain SV and methylation profiles with unprecedented accuracy. Such analyses will greatly advance our understanding of the biology of lethal PCa and of the genetic and epigenetic bases of health disparity. The project is thus of high relevance to the FY22 PCRP mission of defining the biology of lethal PCa to reduce death, advance health equity, and reduce disparities in PCa.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 04, 2024
- Source ID
- HT94252310006
Entities
People
- Changsheng Zhao
Organizations
- Emory University
- United States Army