Whole-transcriptome sequencing

Gain a comprehensive view of whole transcriptomes with high-output nanopore sequencing of full-length transcripts. Unambiguously identify and quantify isoforms and detect fusion genes. Discover new biology with the power of single-cell sequencing. Detect base modifications and avoid PCR bias with nanopore direct RNA sequencing — the only technology enabling direct sequencing of native RNA molecules.


What is whole-transcriptome sequencing?

Whole-transcriptome sequencing enables the analysis of the RNA transcripts present in a sample from an organism of interest. The method provides a dynamic view of the cellular activity at the point of sampling, allowing characterisation of gene expression and identification of isoforms. However, accurate analysis of transcripts using traditional short-read sequencing technology can be challenging, as RNA samples must be fragmented, sequenced in short sections, then reassembled, leading to potential multi-mapping — where short sequencing reads align to multiple locations.

With long nanopore reads, full-length transcripts can be sequenced end-to-end, facilitating their accurate, unambiguous analysis. Researchers are using whole-transcriptome sequencing to shed light on developmental biology, study the mechanisms underlying diseases such as cancers, and identify potential future disease-associated biomarkers.

Why nanopore technology for whole-transcriptome sequencing?

Nanopore technology enables complete, isoform-level transcriptome characterisation — at up to single-cell resolution.

Using nanopore sequencing, you can:

  • Characterise and quantify full-length transcripts
  • Unambiguously identify splice variants and fusion transcripts
  • Reveal gene expression heterogeneity with single-cell transcriptomics
  • Explore epigenetic modifications through direct RNA sequencing
  • Enhance viral identification from metagenomic samples

Experimental approach to whole-transcriptome sequencing

Sequencing kits are available for the preparation of both RNA and cDNA libraries. The Direct RNA Sequencing Kit allows native RNA molecules — including base modifications — to be sequenced directly. Enabling high-output sequencing of whole transcriptomes from low input amounts, the cDNA-PCR Sequencing Kit is ideal for identifying and quantifying full-length transcripts at the isoform level.

Sequence RNA molecules directly and preserve base modifications Identification and quantification of full-length transcripts with highest output
Direct RNA Sequencing Kit cDNA-PCR Sequencing Kit
Preparation time 105 min ~210 min + PCR
Input requirement 50 ng poly(A)+ RNA or 500 ng total RNA 4 ng poly(A)+ RNA or 200 ng total RNA
Reverse transcription required Optional Yes
PCR required No Yes
Read length Equal to RNA length Enriched for full-length cDNA
Typical throughput 1/3 3/3
Multiplexing options In development Yes
Methylation included Yes No
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Find out more about whole-transcriptome sequencing

Learn more about the benefits of sequencing full-length transcripts with long nanopore reads and get best-practice recommendations for single-cell sequencing.

Sequencing devices

Which device for whole-transcriptome sequencing?

From powerful, portable Flongle and MinION devices, suitable for low-pass whole-transcriptome sequencing, to the flexible GridION and high-output PromethION platforms, ideal for isoform-level differential expression analysis — scale your RNA sequencing to match your specific research requirements.

* Theoretical max output (TMO). Assumes system is run for 72 hours (or 16 hours for Flongle) at 420 bases / second. Actual output varies according to library type, run conditions, etc. TMO noted may not be available for all applications or all chemistries.
Recommended for whole-transcriptome sequencing

PromethION 2 and PromethION 2 Solo

Offering the flexibility of two independent, high-output PromethION Flow Cells, the compact PromethION 2 devices bring the benefits of high-coverage, real-time nanopore sequencing to every lab. Ideal for low-cost access to highly accurate whole transcriptomes.

Analysis techniques for whole-transcriptome sequencing

Analysis solutions

Oxford Nanopore provides end-to-end bioinformatics workflows for the analysis of whole transcriptomes. The workflow wf-transcriptoms enables de novo or reference-guided transcript assembly from either cDNA or direct RNA reads, and provides differential gene expression and differential transcript usage analysis. For single-cell transcriptome analysis, the workflow wf-single-cell, a research pipeline, provides outputs including barcode and unique molecular identifier (UMI) tags, gene x cell expression matrices, and cell x transcript expression matrices. Both workflows are available in EPI2ME Labs.

Find out more about analysing nanopore whole-transcriptome and single-cell transcriptomic sequencing data.

Featured whole-transcriptome sequencing workflow

For high-throughput analysis of whole human transcriptomes, we recommend the following:

Inspiration for whole-transcriptome sequencing

Discover more about applying nanopore whole-transcriptome sequencing to your area of research.

Research areas


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