Alternative splicing occurs when exons from the same gene combine in different ways, resulting in different (but related) RNA transcript isoforms, which in turn create different proteins with distinct structures and functions. Despite being “alternative,” this process isn’t rare. Up to 95% of genes in the human genome undergo alternative splicing. The resulting RNA diversity plays a key role in human biology, as different isoforms can be drivers of disease.
For this reason, full-length isoform sequencing is a powerful tool that allows investigators to look at how alternative splicing disrupts gene function in ways that lead to disease. Long-read RNA sequencing using PacBio HiFi reads enables researchers to sequence complete isoforms.
Enter: Kinnex. New Kinnex kits from PacBio supercharge this ability, breaking the throughput barrier and allowing for long-read RNA sequencing at much higher throughput than ever before. Using a method called MAS-Seq, the Kinnex kits concatenate smaller amplicons into larger fragments to take advantage of long HiFi read lengths. Now, you can get full-length RNA transcript sequences at a scale that even population-level studies can use.
Here, we showcase key applications of Kinnex in three acts, featuring real stories from real people, so you can see the power of Kinnex in action. Hear from three researchers who are using full-length RNA sequencing to visualize the transcriptome changes at play in human disease.
Act I: Unraveling the mysteries of rare disease with Kinnex
In this example, Dr. Carolina Jaramillo Oquendo (@Carolinaj215), a Research Fellow at the University of Southampton (@unisouthampton), presents her analyses of rare disease samples, in collaboration with the University Hospital Southampton NHS Foundation Trust.
Dr. Oquendo uses the new full-length Kinnex RNA kit to investigate splicing variants in rare disease pathogenesis. In her work, Kinnex data helped to uncover transcriptional differences in rare disease cases, especially those where short-read sequencing did not provide an explanation.
“It looked like it might be an intron retention event… but we couldn’t really tell” using short-read sequencing, Dr. Oquendo says. In contrast, the PacBio Kinnex data showed multiple transcripts covering the intron in question, confirming the alternative splicing event.
Dr. Oquendo explains how the Kinnex RNA kit also delivers advantages in data processing compared to nanopore technology:
“The [Kinnex] data itself was really clean, and the size of the data was a huge advantage because it saved computational time and resources. I’ve worked with Oxford Nanopore data before; it’s really large and it took weeks to process one sample. PacBio data was so much smaller and such high quality.”
— Dr. Carolina Jaramillo Oquendo, University of Southampton
Act II: Quantifying isoforms using Kinnex data
Accurate quantification of RNA transcript isoforms is critical for complete transcriptome analyses, and can shed light on the molecular mechanisms behind disease. In this example, David Wissel at the University of Zurich (@UZH_en) shares how he quantifies isoforms using Kinnex full-length RNA kits.
As Wissel explains, it’s well-established that PacBio long-read RNA-Seq performs extremely well for isoform discovery. But how well does it do for quantification and downstream tasks, like differential transcript expression?
The answer: “Quite well,” according to Wissel.
He compares Kinnex long-read RNA data to short reads and finds that Kinnex quantification shows comparable replicability to short-read data. Wissel concludes that Kinnex RNA-Seq enables isoform discovery and quantification within one dataset—a major benefit.
“Before Kinnex, PacBio had the highest read length and quality. Now, with Kinnex, RNA-Seq data from PacBio is also comparable in yield to RNA-Seq data from Illumina.”
—David Wissel, University of Zurich
He also demonstrates how open-source tools are easy to run on Kinnex datasets, and explains how to use community-supported analysis tools to quantify isoforms and identify differentially expressed genes and transcripts.
Watch the full webinar here
Or, read our application note to learn more about using the Kinnex full-length kits for isoform sequencing.
Act III: Revealing regional isoform switching events in spatial transcriptomics for cardiovascular disease
Because different isoforms behave differently, changes in expression from one isoform to another have consequences for disease biology. Isoform switching refers to when a cell switches from expressing one RNA isoform and starts expressing another. Different cells within the same tissue can have a different composition of isoforms, which is why single-cell resolution is needed to see these gene expression changes.
Long-read spatial transcriptomics with full-length isoform resolution is transforming how we understand heart development, function, and disease. In this example, Dr. Christoph Dietrich at University Hospital Heidelberg and his team use PacBio single-cell RNA sequencing to look at isoform switching in cardiac tissue after heart attacks.
Using MAS-Seq (the method shared by Kinnex kits), Dr. Dietrich and team identified 117 regional isoform switching events post-heart attack. Depending on the region of cardiac tissue in question, the isoform abundance and expression patterns change considerably for individual genes. The team found that PacBio sequencing could detect many different types of isoforms, including novel ones, as well as regional isoform switching.
“It’s very valuable information and a very rich information set.”
—Dr. Christoph Dietrich, University Hospital Heidelberg
Supercharge your RNA research with Kinnex kits
Learn more about Kinnex kits and how you can get scalable, cost-effective RNA sequencing for your transcriptome research. Want to watch more Kinnex webinars? Learn from tutorials? See how it’s being used by other researchers? Check out the Kinnex YouTube playlist.