Single-cell mRNA isoform sequencing with Oxford Nanopore Technologies

The transcription of genetic information into (m)RNA and the translation of mRNA into proteins regulate cellular functions and differentiation of cells into various tissues. By analyzing the type and amount of mRNA, gene expression studies are crucial in health and disease.

NGI tested different methods for long-read sequencing of single-cell gene expression with Oxford Nanopore Technologies (ONT) and release the results in a technical note.

Short reads can miss the bigger picture

10x Genomics Chromium Single Cell Gene Expression is a widespread and robust method for analyzing gene expression on a cellular level. The method starts with capturing full-length mRNA molecules, but subsequent steps needed for Illumina type short-read sequencing involve fragmentation. While the obtained data is suitable for quantification of expressed genes, the fragmentation erases the variation found in the full-length mRNA.
In fact, different versions of mRNA molecules can be produced from the same gene, allowing for a higher number of proteins from the same source. These are called isoforms. In order to analyze this complexity of mRNA isoforms, long-read sequencing of unfragmented mRNA is necessary.

Methods for single-cell mRNA isoform sequencing

We evaluated the performance of three different methods for adding long-read sequencing of single-cell mRNA isoform sequencing to the 10x Chromium Single Cell Gene Expression protocol: One publicly available method with detailed protocols shared by a research team in Australia (FLT-seq, Tian et al. 2021), and two protocols released by ONT.
All three methods performed similarly well in our test and are suitable for mRNA isoform analysis. The “FLT-seq” method was preferred over the other methods for its simpler protocol and cost-effectiveness.

Read more on bioRxiv: https://doi.org/10.1101/2025.08.12.668929

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References
Tian,  L., Jabbari,  J.S., Thijssen,  R. et al. (2021). Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing. Genome Biol 22, 310.doi:10.1186/s13059-021-02525-6p

Last Updated: 17th September 2025