Real-time analysis of DNA base modifications, particularly methylation, is essential for rapid decision-making in contexts such as forensics, and clinical contexts specifically combined with selective/adaptive sequencing. Traditional methods like bisulfite sequencing, while accurate, are limited by their need for large DNA samples and the inability to capture long-range methylation patterns. Nanopore sequencing, with its ability to generate long reads and detect base modifications through electrical signal analysis, offers a promising alternative.
realfreq is a lightweight framework for retrieving real-time base modification frequencies while sequencing on nanopore sequencing devices. realfreq is composed of two primary components: realfreq-pipeline, a modular script that manages (monitors raw signals directory, basecalls raw signals, and aligns to a reference genome) data flow from the sequencing device to base modification detection, and realfreq-prog, a C-based application that performs base modification calling based on information retrieved from alignment files and maintains an in-memory map of base modifications. Additionally, we developed realfreq-server within realfreq-prog using simple socket connections, that provide an interface to query base modification information in real-time.
We demonstrate realfreq’s ability to keep up with the output data stream of the Oxford Nanopore Technologies (ONT) PromethION. The base modification calling algorithm we developed for realfreq is separately bundled with Minimod, a simple base modification analysis tool we developed.
realfreq and Minimod are open source and publicly available at https://github.com/imsuneth/realfreq, https://github.com/warp9seq/minimod.