Oxford Nanopore Technologies: Detecting Isoforms and RNA Modifications with PCR-Free Direct RNA Sequencing

Specifiche del prodotto

• Tecnologia: Oxford Nanopore sequencing
• Applicazione: Direct RNA sequencing
• Caratteristiche: Detecting isoforms, RNA modifications, gene expression
• Risoluzione: Single-base resolution for m6A methylation detection
• Compatibilità: PromethIONTM sequencing device

Istruzioni per l'uso del prodotto

Estrazione:

1. Obtain high-quality RNA by following the extraction protocol recommendations for your sample type available at nanoporetech.com/extraction-methods.
2. Enrich 3′-polyadenylated (poly(A)) transcripts from total RNA using the Invitrogen DYNAL Dynabeads mRNA Purification Kit. For non-polyadenylated transcripts, use NEB E. coli Poly(A) Polymerase reagent to add poly(A) tails.
3. Assess RNA quality with a Nanodrop instrument and check for degradation using the Agilent Bioanalyzer RNA Analysis Kit before proceeding to library preparation.

Preparazione della biblioteca:

• Prepare native RNA samples by following the Oxford Nanopore library preparation guidelines available at nanoporetech.com/prepare.
• Perform reverse transcription adapter annealing and ligation for 100 minutes, followed by reverse transcription for 35 minutes.
• Attach sequencing adapters to the RNA sampmeno.

Sequenziamento:
Generate high outputs of long native RNA reads by using PromethION sequencing devices. For optimal results, use poly(A)+ samples as input and basecall in high accuracy (HAC) mode.

Analisi:

1. Access the dedicated EPI2ME workflow wf-transcriptomes through the EPI2METM application for accurate RNA transcript detection and methylation calling.
2. EPI2ME workflows simplify data analysis with an intuitive interface suitable for all levels of expertise. These workflows can be run on local compute, clusters, cloud services, or directly on your nanopore sequencing device.

INTRODUZIONE

• Accurately capturing the range of RNA diversity — including gene expression, isoforms, and RNA methylation — can help elucidate the molecular mechanisms of disease and functional roles of RNA modifications. For example, RNA modifications, such as
  n6-methyladenosine (m6A) impacts RNA metabolism1 and plays a significant role in the initiation and progression of cancers2,3, as well as neurological disorders, including Alzheimer’s disease4 and Huntington’s disease5.
• Traditional short-read sequencing technology typically generates 50–100 bp reads, making it challenging to accurately assemble and quantify transcript isoforms and difficult to measure poly(A) tail length. Additionally, the reverse transcription and amplification steps used to prepare these libraries erase RNA modifications and can introduce PCR bias.
• Oxford Nanopore sequencing is the only available technology that directly reads native RNA transcripts, enabling quantitation of gene and isoform expression without PCR bias. It also provides resolution of full-length isoforms6 and accurately estimates poly(A) tail length. Furthermore, direct RNA nanopore sequencing provides direct detection of m6A methylation7 for the DRACH motif at single-base resolution without additional sampla preparazione.
• Here, we present a simple workflow for RNA modification analysis from a human blood research sample, using direct RNA sequencing on PromethION™.

Estrazione

Obtaining high-quality RNA

• To ensure high outputs of long reads from Oxford Nanopore sequencing, it is important to select an extraction method that preserves native RNA transcripts and minimises chemical contamination. For total RNA extraction from blood research samples, we recommend using the QIAGEN PAXgene Blood RNA Kit, followed by globin depletion using the Invitrogen GLOBINclear-Human Kit. If starting with human cell lines, we recommend using Invitrogen TRIzol RNA Isolation Reagent. When isolating RNA, we recommend working in an RNase-free environment to minimise degradation during extraction.
• To maximise output, 3’-polyadenylated (poly(A)) transcripts can be enriched from total RNA using the Invitrogen DYNAL Dynabeads mRNA Purification Kit. For non-polyadenylated transcripts, we recommend the use of NEB E. coli Poly(A) Polymerase reagent to add poly(A) tails for library preparation compatibility. Before proceeding to library preparation, we recommend assessing RNA quality via a Nanodrop instrument and degradation using the Agilent Bioanalyzer RNA Analysis Kit.
• 

Library preparation

Preparing native RNA samples
For direct sequencing of native RNA transcripts and modifications, prepare your libraries with the Direct RNA Sequencing Kit. This method does not require fragmentation or amplification, preserving long transcripts and RNA modifications. Starting from either total RNA or a poly(A)-enriched (poly(A)+) sample, adapters are ligated onto the RNA strand before a second complementary DNA (cDNA) strand is synthesised during reverse transcription. The cDNA strand is not sequenced, but it helps increase the sequencing output of native RNA molecules. Sequencing adapters are then attached to the RNA-cDNA hybrid.

Sequenziamento

Generating high outputs of long native RNA reads.

• For high-output sequencing of long native RNA transcripts and m6A methylation detection, RNA-specific flow cells must be used. RNA sequencing using nanopore technology can be scaled to your requirements with the PromethION family, which features the high-throughput PromethION 24 sequencing device, with the capacity to run up to 24 high-output flow cells. For lower throughput requirements, the compact PromethION 2 Solo and PromethION 2 Integrated devices allow sequencing on up to two independent flow cells. A single direct RNA PromethION Flow Cell can generate up to 30 million reads.
• For the highest sequencing output and accuracy, we recommend using poly(A)+ samples as input and basecalling in high accuracy (HAC) mode.

Analisi

Accurate RNA transcript detection and methylation calling.g

• The workflow wf-transcriptomes8 is accessible through the EPI2METM application and simplifies data analysis with an intuitive interface. EPI2ME workflows enable nanopore data analysis for all levels of expertise: the pre-configured analysis packages are free to access from an intuitive interface or the command line8. They can be run on local compute, via a cluster or cloud service, or on your nanopore sequencing device, such as GridION™ or PromethION.
• The dedicated wf-transcriptomes pipeline identifies RNA transcripts, assembles, annotates transcriptomes, detects gene fusions, and identifies differential gene expression and transcript usage.
• Direct RNA basecalling is enabled by Dorado, an optimised basecaller for nanopore sequencing, integrated into the device software, MinKNOWTM. Poly(A) tail length estimation and m6A modified RNA basecalling are currently available through the standalone version of Dorado9 and will soon be integrated into MinKNOW, enabling seamless RNA analysis for immediate access to results.

PER MAGGIORI INFORMAZIONI

View the direct RNA sequencing protocol: nanoporetech.com/direct-rna-sequencing-protocol

Riferimenti:

1. Shafik, A.M. et al. Genome Biol. 22:1–19 (2021). DOI: https://doi.org/10.1186/s13059-020-02249-z
2. Bradner, J.E., Hnisz, D. and Young, R.A. Cell 168(4):629–643 (2018). DOI: https://doi.org/10.1016/j.cell.2016.12.013
3. Barbieri, I. and Kouzarides, T. Nat. Rev. Cancer 20(6):303–322 (2020). DOI: https://doi.org/10.1038/s41568-020-0253-2
4. Jiang, X. et al. Sig. Transduct. Target Ther. 6:74 (2021). DOI: https://doi.org/10.1038/s41392-020-00450-x
5. Malla, B. et al. Front. Genet. 12 (2021). DOI: https://doi.org/10.3389/fgene.2021.751033
6. Glinos, D.A. et al. Nature 608(7922):353–359 (2022). DOI: https://doi.org/10.1038/s41586-022-05035-y
7. Gleeson, J. et al. bioRxiv (2024). DOI: https://doi.org/10.1101/2024.01.31.578088
8. GitHubWf-transcriptomess. Available at: https://github.com/epi2me-labs/wf-transcriptomes [Accessed 15 Aug 2025]
9. GitHub. Dorado. Available at: https://github.com/nanoporetech/dorado [Accessed 15 Aug 2025]

INFORMAZIONI SULL'AZIENDA

• telefono +44 (0)845 034 7900
• e-mail support@nanoporetech.com
• oxford-nanopore-technologies
• @nanopore
• nanoporetech.com
• www.nanoporetech.com

Domande frequenti

Can this technology accurately detect RNA modifications like m6A?

Yes, Oxford Nanopore sequencing can directly detect m6A methylation at single-base resolution without additional sampla preparazione.

How can I maximize the sequencing output?

To maximize output, enrich 3'-polyadenylated (poly(A)) transcripts from total RNA and use poly(A)+ samples as input for sequencing on PromethION devices.

Are there specific recommendations for assessing RNA quality?

Before proceeding to library preparation, it is recommended to assess RNA quality using a Nanodrop instrument and checking for degradation with the Agilent Bioanalyzer RNA Analysis Kit.

Documenti / Risorse

Oxford Nanopore Technologies Detecting Isoforms and RNA Modifications with PCR Free Direct RNA Sequencing [pdf] Guida utente Detecting Isoforms and RNA Modifications with PCR Free Direct RNA Sequencing, Isoforms and RNA Modifications with PCR Free Direct RNA Sequencing, RNA Modifications with PCR Free Direct RNA Sequencing, PCR Free Direct RNA Sequencing

Riferimenti

• Manuale d'uso