Short- vs Long-Read RNA-Seq

RNA sequencing has transformed our understanding of transcriptomes, from quantifying gene expression to uncovering complex isoforms and RNA modifications. This is due in part to advancements in sequencing technologies. With multiple sequencing strategies available, researchers must ask: Which platform is right for your experiment?

Classic RNA-seq with Illumina Sequencing

The most traditional method for RNA-seq uses Illumina short-read sequencing (typically 2x150bp configuration). Either total RNA or mRNA is converted into cDNA and then fragmented and sequenced in 150bp fragments. These reads are then mapped to a reference genome, counted for each gene, and then compared to provide insights into differential gene expression.

Short-read RNA-seq has become the workhorse of methods due to its ease of use and low cost compared to other methods. If you are looking for differential gene expression from a well annotated genome, then this is the approach for you!

Long-read RNA Sequencing Technologies

Recent advancements to long-read RNA sequencing have led to diversification of methods and kits. One of the most frequent questions we get is about the differences and when to use each.

All commercially available long-read workflows are currently poly-A–based. Sequencing non-polyadenylated RNA with long reads requires additional upstream modifications (e.g., enzymatic polyadenylation), which are not standard in commercial kits. Below we describe the four most common kits used for long-read RNA sequencing:

PacBio Iso-Seq – This is the classic long-read RNA-seq method which simply coverts poly-adenylated RNA molecules into cDNA before ligation of adapters and full-length RNA.

PacBio Kinnex RNA – Since most RNA molecules are < 2 Kb, sequencing individual RNA fragments on PacBio does not take advantage of modern PacBio SMRT-cells capacity to generate reads up to 25Kb sequencing. To fully utilize the capacity, PacBio developed the Kinnex workflow, which concatenates barcoded cDNA before sequencing resulting is six times the yield of Iso-Seq; most Revio SMRT-cell runs generate > 60 million full-length transcripts.

Oxford Nanopore cDNA-PCR – This workflow is similar to PacBio Iso-Seq but yields results akin to PacBio Kinnex. One PromethION flow cell can generate > 60 million full length transcripts. The major difference between this and PacBio Kinnex is the lower per based accuracy. However, Oxford Nanopore cDNA-PCR can sequence large RNA transcripts (e.g., > 15Kb) more effectively.

Oxford Nanopore Direct RNA – This first-of-its-kind technique directly sequences RNA molecules instead of cDNA, enabling examination of modifications on the RNA molecules (e.g., m6A, pseudouridine) because the native RNA remains intact. Oxford Nanopore Direct RNA is poly-A based and has much lower yield than other long-read RNA sequencing methods for a higher cost. This workflow is ideal if you are looking for RNA modifications or want to avoid cDNA conversion.

Which RNA-Seq Method is Right for You?

Interested in RNA modifications? Choose Nanopore Direct RNA. It is currently the only widely accessible method that sequences native RNA molecules.

Interested in isoform discovery? PacBio Kinnex or Oxford Nanopore cDNA-PCR are good fits for isoform detection if you do not need RNA modification information. These methods can still provide high sequencing depth of full-length RNA at a reasonable price.

Focused on differential gene expression? Illumina short-read RNA-seq provides the best value of high sequencing depth at an affordable cost. There is also lower RNA size bias since all RNAs are fragmented.

Working with non–polyadenylated RNA? Illumina short-read RNA-seq with rRNA depletion offers an easy way to sequence non-polyadenylated RNA (e.g., lncRNA and prokaryotic RNA). This method uses random hexamer primers to convert all RNAs into cDNA followed by the removal of rRNA fragments using rRNA probes so that there is no wasted sequencing of common rRNAs. This workflow makes it easy to target RNAs that are not poly-adenylated starting with total RNA and is only slightly more expensive than Illumina poly-A based library prep.

De novo transcriptome assembly? Long-read sequencing (especially PacBio Kinnex) is increasingly favored for eukaryotic transcriptome assembly. The full-length transcripts simplify assembly, isoforms are directly resolved, and structural annotation is more accurate. Hybrid strategies (long-read structure + short-read depth) are also becoming common.

Below is a practical comparison of how these methods stack up in pricing, sequencing depth, and what they are best at.

Both short-read and long-read sequencing have distinct strengths. Rather than thinking in terms of “better or worse,” consider what biological question you’re trying to answer:

• Need statistical power and depth? Go with short-read.
• Need transcript structure and isoform resolution? Go with long-read.
• Need both? Consider a hybrid approach.

Want to learn more about RNA-Seq? Check out our RNA Sequencing eBook. Ready to take the next step? GENEWIZ offers these services and more. Request a free consultation or quote.