DOI: 10.5281/zenodo.15349125
title: Data from: "Comparison of enzymatic- and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses"

description: Abstract

Detection of DNA methylation biomarkers in circulating cell-free DNA (cfDNA) has great clinical potential for cancer management, but method optimization and standardization is needed. Bisulfite conversion is the gold-standard pre-treatment method for methylation analyses, but causes DNA fragmentation and loss. Enzymatic conversion of DNA represents a promising alternative due to the more gentle treatment. The aim of this study was to evaluate and compare enzymatic- and bisulfite conversion to identify the best pre-treatment method for detecting DNA methylation biomarkers in cfDNA from plasma using droplet digital PCR (ddPCR). The performance of the NEBNext Enzymatic Methyl-seq Kit and the EpiTect Plus DNA Bisulfite Kit was evaluated and compared using normal cfDNA and tumor cfDNA samples from colorectal cancer patients. Enzymatic conversion resulted in longer DNA fragments with higher peak fragment sizes compared to bisulfite conversion, but the DNA recovery was considerably lower after enzymatic conversion compared to bisulfite conversion. DNA methylation of the biomarker BCAT1 was detected at similar rates in parallel tumor cfDNA samples pre-treated with either enzymatic- or bisulfite conversion. However, enzymatic conversion resulted in lower number of positive droplets for both target and control ddPCR assays, in line with the lower DNA recovery after conversion. Altogether, bisulfite conversion emerges as the best pre-treatment method due to higher DNA recovery after conversion and higher number of positive droplets in the ddPCR reactions.

 
description: The QIAamp Circulating Nucleic Acid Kit (QIAGEN) was used for isolation of cfDNA from plasma samples. The NEBNext Enzymatic Methyl-seq Kit (full kit) and its sub-component NEBNext Enzymatic Methyl-seq Conversion Module (Conversion module; New England BioLabs) were used for enzymatic DNA conversion according to the manufacturer’s instructions. The EpiTect Plus DNA Bisulfite Kit (QIAGEN) was used for bisulfite conversion of DNA. Droplet digital PCR was performed using the QX200 Droplet Digital PCR System (Bio-Rad). To evaluate enzymatic- and bisulfite conversion efficiency and DNA recovery, the FAM-labeled “Chr3 assay” and VIC-labeled assay for myogenic differentiation 1 (MYOD1) were used. For detection of biomarkers in clinical samples (e.g. proof of concept), CDO1 and BCAT1 were used as target assays (FAM-labelled) and the 4Plex was used as internal control assay (VIC-labelled). 
description: Description of the data files

This dataset consists of raw data from droplet digital PCR (ddPCR) analysis of normal cfDNA, tumor cfDNA from colorectal cancer patients, as well as DNA from the colorectal cancer cell line RKO fragmented to mimic cfDNA. 

The raw ddPCR data was exported from the QuantaSoft Software 1.7.4.0917 (BioRad) in the form of amplitude csv files, and one csv file represents one ddPCR reaction/well. In the csv files, each line indicates the fluorescence amplitude intensity data from one droplet, and the number of lines corresponds to the number of droplets generated for the respective reaction. The first column ("Ch1 Amplitude") indicates the fluorescence amplitude value of FAM-labelled assay (Channel 1) and the second column ("Ch2 Amplitude") indicates the fluorescence amplitude value of the VIC-labelled assay (Channel 2). The third column ("Cluster") indicates which cluster the droplet has originally been assigned to (i.e. double negative, single positive for either the FAM- or VIC-labelled assay, or double positive).

For downstream analysis, all csv files were uploaded to the partition classification algorithm PoDCall - POsitive Droplet CALLer - which automatically dicthomizes positive and negative droplets and returns both non-normalized and normalized concentrations. PoDCall is an R package with an accompagnying shiny graphical user interface (GUI), and is freely availabel on Bioconductor (https://www.bioconductor.org/packages/release/bioc/html/PoDCall.html).

For further information or questions, the corresponding author, Guro E. Lind, can be contacted.