Impact of Nanopore Flow Cell Types on DNA Methylation Detection

Area of Science:

• Genomics
• Epigenetics
• Bioinformatics

Background:

• Third-generation sequencing, particularly Oxford Nanopore Technologies (ONT), advances epigenetic studies in human diseases.
• ONT flow cell improvements have increased sequencing accuracy, but variations like R9 vs. R10 exist across studies.
• The impact of different ONT flow cell types on genome-wide DNA methylation detection is not fully understood.

Purpose of the Study:

• To compare the performance of R9 and R10 flow cell types for DNA methylation detection using ONT sequencing.
• To investigate the effects of flow cell type on the accuracy of 5-methylcytosine (5mC) detection in human renal cell carcinoma (RCC).

Main Methods:

• Analysis of 6 human renal cell carcinoma (RCC) samples using both R9 and R10 ONT flow cell types.
• Comparative analysis of genome-wide DNA methylation data obtained from R9 and R10 flow cells.
• Evaluation of sequencing accuracy and detection of 5-methylcytosine (5mC) levels.

Main Results:

• A highly significant correlation was observed between 5mC detection using R9 and R10 flow cells.
• R9 flow cells showed systematic errors, over-estimating 5mC in hypomethylated regions (promoters) and under-estimating in hypermethylated regions (intronic/intergenic).
• These R9 errors, linked to lower sequencing accuracy and issues with homopolymers (CpG islands), were largely corrected by batch-correction software.

Conclusions:

• ONT R10 flow cells demonstrate superior performance and higher accuracy for base sequencing and DNA methylation detection compared to R9.
• R10 flow cells provide more reliable genome-wide DNA methylation profiling.
• Batch correction can improve data comparability between different flow cell types, but R10 offers inherent advantages.