UNCOVER CANCER-SPECIFIC RNA ISOFORMSUSING LONG-READ SEQUENCING

Novel biomarker and drug target discovery with the Iso-Seq® method

Alternative splicing affects protein function in cancer
Alternative splicing (AS) results in the generation of multiple RNA isoforms from a single gene, greatly
increasing both transcriptomic and proteomic diversity. AS controls which introns are removed from premRNAs and which exons are combined to form the final messenger RNA (mRNA).¹GeneProteinsmRNA
isoforms

Cancers display widespread RNA dysregulation, including
alternative splicing of transcripts. These aberrant transcripts
are often translated into cancer-specific proteins and have beenshown to affect cancer initiation, progression, metastasis, anddrug resistance.2 For example, the bcl-x gene expresses twoisoforms: long and short. The short isoform is expressed innormal tissues and regulates apoptosis. The long form is overexpressed in several cancers and blocks apoptosis, promotes
cancer progression, and has been shown to cause resistance tochemotherapy.

Bcl-x pre-mRNA

Bcl-xS mRNA

Bcl-xL mRNA

Bcl-xS

Normal cell

Bcl-xL

Tumor cellGlioblastoma

Much of the cancer isoform
repertoire remains unexplored
While it has been recently reported that cancer-specific isoforms
can be used as novel prognostic biomarkers and serve as anuntapped source of drug targets for immuno-oncology, the vast
majority of cancer-specific isoforms remain unknown.3,4,5 With atleast 95% of genes undergoing alternative splicing and producing
multiple isoforms, much remains to be discovered.1

PI3K isoforms identified
as drug targets1

Breast and OvarianCancer

CD44 isoform linked totumor progression2

Lung CancerMET4 isoform predicts
therapy response4

Gastric Cancer

ARID1A isoform associatedwith poor prognosis5

Prostate Cancer

GLS1 isoform drives
therapy resistance6

Acute myeloid leukemia
WT1 isoform regulates
tumor growth3

Long-read sequencing provides significantly more robust isoform
discovery power
Current short-read sequencing approaches rely on the alignment of sequenced fragments to a reference genome and are only able toreconstruct an estimated 20–40% of the transcriptome.6,7 A recent study showed that a short-read approach captured only a fraction(~41%) of truly novel isoforms identified by PacBio’s long-read approach in cancer samples.¹ These intrinsic limitations in the discovery ofnovel isoforms likely underestimate the impact of isoforms in cancer and result in a partial view of the true biology of cancer.8
ISOFORM DISCOVERYSHORT-READ SEQUENCING
Gene

LONG-READ SEQUENCING
GenemRNA
isoformsmRNA
isoforms

Fragmentedreads

Full-length
reads

Identifying transcripts
is an assembly problem

No assembly required

Partial view of isoform repertoire

Complete view of isoform repertoire

The Iso-Seq method utilizes long-read transcript sequencing to reliably capture full-length transcript isoforms without the need forcomputational assembly. PacBio’s long-read RNA sequencing is also significantly less error-prone than other long-read sequencing
technologies, offering more robust and accurate isoform discovery power.9,10 Future research will continue to explore the complex andmostly unexplored landscape of isoforms across cancer types.

The Iso-Seq method offers an end-to-end
approach for isoform discovery

Library prepSMRT® sequencing

Data analysis

SMRTbell express kitSequel IIe system

SMRT® Link®

®

For a detailed protocol about
RNA sequencing best practices visit
pacb.com/RNAseq-bestpractices

Connect with PacBio for more info:
North America: nasales@pacb.com
South America: sasales@pacb.com
EMEA: emea@pacb.com
Asia Pacific: apsales@pacb.com

KEY REFERENCES
1. Pan, Q., et al. (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40, 1413–1415
2. Pan, Y., et al. (2021) RNA dysregulation: An expanding source of cancer immunotherapy targets. Trends in Pharmacological Sciences, 42(4), 217-312
3. Veiga, D., et al. (2022) A comprehensive long-read isoform analysis platform and sequencing resource for breast cancer. ScienceAdvances, 8(3), doi: 10.1126/sciadv.
abg6711.
4. Huang, K., et al. (2021) Long-read transcriptome sequencing reveals abundant promoter diversity in distinct molecular subtypes of gastric cancer. Genome Biol, 22(4), doi.
org/10.1186/s13059-021-02261-x
5. Oka, M., et al. (2021) Aberrant splicing isoforms detected by full-length transcriptome sequencing as transcripts of potential neoantigens in non-small cell lung cancer.
Genome Biol 22(9), https://doi.org/10.1186/s13059-020-02240-8
6. Tilgner, H., et al. (2014) Defining a personal, allele-specific, and single-molecule long-read transcriptome. PNAS, 111(27), https://doi.org/10.1073/pnas.1400447111
7. Sharon, D., et al. (2013) A single-molecule long-read survey of the human transcriptome. Nat Biotechnol 31, 1009–1014
8. Kahraman, A., et al. (2020) Pathogenic impact of transcript isoform switching in 1,209 cancer samples covering 27 cancer types using an isoform-specific interaction
network. Sci Rep 10(14453), https://doi.org/10.1038/s41598-020-71221-5
9. Wyman, et al. (2019) A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification. bioRxiv. https://doi.org/10.1101/672931.
10. Al’Khafaji, et al. (2021) High-throughput RNA isoform sequencing using programmable cDNA concatenation. https://www.biorxiv.org/content/10.1101/2021.10.01.462818v1
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