Oxford Nanopore integrates “Remora”: a tool to enable real-time, high-accuracy epigenetic insights with nanopore sequencing software MinKNOW

Oxford Nanopore has today released Remora, a high-performance tool for methylation analysis, into its operating software, MinKNOW, broadening access to direct, PCR-free nanopore sequencing that captures methylation across the whole genome.

From today, Oxford Nanopore users have easy access to precise whole genome methylation detection from PCR-free nanopore sequencing using Remora. By integrating Remora into MinKNOW the analysis of epigenetic modifications becomes seamless as it now runs in parallel to standard basecalling. This release complements the simplicity of native DNA sample preparation, which can be done in just 10 minutes using the same run, so at no additional cost. Nanopore sequencing is now the most comprehensive technology for characterising  methylation, with the first releases aimed at targeting all CpG areas.

Native sequencing for greater epigenetic insight

Methylation detection has traditionally been dominated by using bisulphite treatment with short-read sequencing. Whilst this method has opened up the discovery of methylation sites, it also has limitations, increasing the cost and complexity of sequencing, owing to the requirement to repeat the run for essential comparison. Bisulphite cannot easily differentiate between methylation types such as 5mC/5hmC, or indeed add the detection of other modification types such as 6mA.

The Oxford Nanopore solution for detecting CpG methylation provides a number of advantages. No additional, complex sample preparation is required and epigenetic modification analysis can be performed across the whole genome during the experiment. Therefore, no additional toxic chemistry is needed, and the phasing of base modifications is put in genomic context with the ability to sequence long fragments of DNA. Nanopore technology enables base-modification analysis to be performed alongside nucleotide sequencing on a single read basis, so without the need to repeat the run.

Remora models demonstrate improved performance from a significantly simplified machine learning training process and they improve signal scaling which results in higher detection accuracy and quality filtered calls achieving 99.8% accuracy for 5mC in CpG contexts. This means that Remora delivers industry-leading methylation detection accuracy from a single read and provides biological insight into eukaryotic samples at only 20X coverage. Nanopore sequencing opens up the possibility of looking at methylation directly on individual DNA molecules, therefore representing how the DNA strands were in the cell, potentially the best and “most true” way of looking at DNA.

Marcus Stoiber, Senior Data Analyst, Oxford Nanopore commented:

“Detection of methylation from raw nanopore signal is an incredible strength of nanopore technology, but it has long been a cumbersome task to extract this information at the highest accuracy. Remora changes this and delivers epigenetic modifications at the same time as the highest accuracy canonical basecalls. As part of the team who developed Remora, we are delighted that it is now fully integrated and accessible to all Nanopore users and we’re very excited see how the research community puts it to work in their groundbreaking research efforts.”

Nanopore sequencing provides one platform for all your biology

These new tools will enable users to choose to sequence short fragments of DNA and perform methylation analysis of those fragments in real time for greater insights. DNA methylation is a process by which a methyl group is added to the DNA molecule, thereby modifying its behaviour but not altering the sequence. This modification of the DNA and its genetic expression is studied in epigenetics. The greater epigenetic insight that methylation analysis reveals is enabling discoveries across a breadth of genomic applications. Here are two examples from the Nanopore community:

Ian Henderson, University of Cambridge, and his research group investigated Arabidopsis thaliana (a small flowering plant) centromeres and found a ‘striking suppression of recombination around the centromere, as well as enrichment of epigenetic marks, like DNA methylation’. This was remarkable as it’s only been since the advent of long-read sequencing that the centromeres can be studied in detail; previously, these regions have been too repetitive to sequence using short-read sequencing. Full presentation here.

Luna Djirackor, Oslo University Hospital, used nanopore analysis to show the future potential to classify brain tumours using methylation in as little as 91 minutes – quick enough for results to be returned to the operating table during brain surgery. Strikingly, in 60% of the samples sequenced in the study, the information obtained would have altered the pre-planned surgical strategy, demonstrating how this approach has the potential to significantly improve surgical outcomes in future. Full presentation here.

From today, users of Oxford Nanopore sequencing devices will be able to use Remora fully integrated and accessible in MinKNOW — the operating software for nanopore sequencing. Remora can be accessed here and a dataset is available in EPI2ME labs for users to download here.

Further information from Marcus Stoiber, Senior Data Analyst, Oxford Nanopore Technologies: https://nanoporetech.com/resource-centre/video/ncm21/nanopore-methylation-better-way-mods

Direct DNA and RNA sequencing for methylation analysis with nanopore sequencing (animation): https://www.youtube.com/watch?v=7PraIiiN_Gc&list=PLxpxXZj-IgXyR0it4QjoBETC5JeEanW-o&index=17