Flexible Methods for Long-Read RNA Sequencing

RNA analysis has become a vital tool for researchers looking for insight into the study of living things, revealing details about the dynamics of the transcriptome, from single cells to whole tissues.

When applied to medicine, the identification of differentially spliced isoforms and fusion transcripts can assist doctors in providing disease diagnosis and treatment. RNA analysis of a virus allows rapid and effective identification.

In developmental biology, the tracking of transcriptional changes over time helps to resolve the developmental mechanisms at play. Environmental and agricultural science also use transcriptomics to build strategies for pest management amongst other areas.

Direct and PCR-Based cDNA Sequencing Kits

For high-depth sequencing of full-length transcripts, Oxford Nanopore offers both direct and PCR-based cDNA sequencing kits, giving you high-confidence identification as well as accurate quantification of transcripts within one experiment.

Their newly updated Direct and PCR cDNA Sequencing Kits give the highest yields yet for high-depth transcriptome sequencing and greater enrichment for full-length transcripts from lower sample inputs.

The Direct cDNA Sequencing Kit provides a zero amplification library preparation method for high-throughput cDNA sequencing free of PCR bias. Even if the amount of available RNA is limited, the PCR-cDNA Sequencing Kit can still generate the maximum sequencing output from as little as 1 ng of starting material.

RNA Analysis

Although there have been great strides in transcriptome analysis using short-read cDNA sequencing technologies, there are still several drawbacks to these methods.

Transcripts are typically several kilobases in length. A standard human gene contains 12 exons each, with an average length of 236 base pairs, and with alternative splicing seen in 95% of human genes. Short reads only cover a portion of a transcript’s length, causing difficulties with accurate isoform assembly and making it reliant on computational reconstruction. High rates of multi-mapping are also observed with short reads.

Nanopore Sequencing

Nanopore sequencing operates without an upper read length limit: fragmentation is not required and entire transcripts can be sequenced end-to-end in a single read. This enables simple, accurate assembly, as well as the ability to differentiate between highly similar isoforms, highlight novel transcripts, and detect fusions. Multimapping rates are also significantly lower (see figure 1).

Figure 1.

By using spike-in RNA standards (ERCC), accurate isoform quantification is achievable with nanopore sequencing (see Figure 2).

When compared with short-read data, GC bias is all but removed in nanopore cDNA data (see Figure 3), whilst PCR bias is minimal in PCR-cDNA sequencing and completely absent in direct cDNA sequencing (see Figure 4). Furthermore, unlike in other sequencing methods where data analysis only starts after the completion of the sequencing run, nanopore sequencing is performed in real-time: reads can be basecalled and analyzed as sequencing progresses, greatly reducing sample-to-answer time. Runs can also be halted when it is decided sufficient data has been generated – for example, once a coverage target is reached.

Figure 3.

Figure 4.

Oxford Nanopore offers a range of versatile, scalable methods of long-read RNA analysis. In a first for the field, the Direct RNA Sequencing Kit allows the sequencing of full-length native RNA strands without first converting to cDNA, making the analysis of both sequence and base modifications possible.

About Oxford Nanopore Technologies

Oxford Nanopore Technologies Ltd has developed the world’s only real-time, long-read, direct DNA/RNA sequencing technology. Uniquely, it can be scaled from portable formats (MinION and Flongle) that can open up new decentralized models of biological analysis, to ultra-high-throughput devices (GridION and PromethION) that can address population-scale genomics and large genomes or projects.

Commercially available since 2015, the technology is in use globally, across myriad areas of scientific research. These include human genetics, cancer research, disease surveillance, environmental analysis, agriculture and most recently in food safety testing and health applications.

The Company’s goal is to enable the analysis of any living thing, by anyone, anywhere. For more information visit www.nanoporetech.com.


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Last updated: Jul 30, 2019 at 9:43 AM

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