The uncertainty of transcriptome reconstruction
Because most human genes are alternatively spliced1, knowing which isoform is expressed in a sample is critical for accurate research and analysis. A single gene may code for a surprising number of proteins — in some cases with opposing biological functions2. Splicing mutations have been associated with a variety of disease phenotypes, and numerous human diseases have been linked to changes in levels of alternative spliced isoforms3. However, tools for reconstructing complete isoforms from short-read data lack sensitivity and specificity, which complicates the interpretation of RNA sequencing data4.
Single Molecule, Real-Time (SMRT) Sequencing delivers the long reads needed to capture intact isoform information without assembly or complicated algorithms. The Iso-Seq application identifies transcripts and reveals novel gene isoforms, including gene fusions and full-length mRNA and lncRNA transcripts. You can use the Iso-Seq method to:
- Directly sequence full-length transcripts and eliminate the need for transcript reconstruction
- Perform broad or targeted surveys of transcript diversity to obtain key information about the frequency and types of alternative transcription
- Observe allele-specific gene expression
- Differentiate isoform expression between cells, tissues, and disease states
Workflow: from RNA to full-length transcripts
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| Iso-Seq Sample Preparation
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| SMRT Sequencing with PacBio Systems
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 | Data Analysis with SMRT Analysis or PacBio DevNet
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| *Read lengths, number of reads, data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size, among other factors. | |
Spotlight: Identify novel full-length isoforms in castration-resistant prostate cancer (CRPR)
Scientists used long-read sequencing to identify and quantify full-length androgen receptor (AR) isoforms expressed in CRPC. They identified a truncated isoform, AR-V9, which shows potential to be a more predictive biomarker than AR-V7 of primary resistance to AR-targeted therapies.5
Explore this research further
References
- Pan Q., et al., (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nature Genetics. 40, 1413-1415.
- Schwerk, C. and Schulze-Osthoff, K. (2005) Regulation of apoptosis by alternative pre-mRNA splicing. Molecular Cell. 19(1), 1-13.
- http://www.eurasnet.info/scientists/alternative-splicing-and-disease/list-of-diseases, accessed on 8/3/2015
- Korf, I. (2013) Genomics: the state of the art in RNA-seq analysis. Nature Method. 10(12), 1165-1166.
- Clark, T., et al., (April, 2017) SMRT Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants. Poster presented at American Association for Cancer Research. Washington D.C.
Selected Resources
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- Bruijnesteijn, Jesse et al. (2018) Human and rhesus macaque KIR haplotypes defined by their transcriptomes. Journal of Immunology
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- Karl, Julie A et al. (2016) Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities BioRxiv
- O'Grady, Tina et al. (2016) Global transcript structure resolution of high gene density genomes through multi-platform data integration. Nucleic Acids Research
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- Criscione, Steven W et al. (2016) Genome-wide characterization of human L1 antisense promoter-driven transcripts. BMC Genomics
- Mangul, Serghei et al. (2016) HapIso: An accurate method for the haplotype-specific isoforms reconstruction from long single-molecule reads
- Singh, Neetu et al. (2016) IsoSeq analysis and functional annotation of the infratentorial ependymoma tumor tissue on PacBio RSII platform. Meta Gene
- Chi, Kelly Rae et al. (2016) Finding function in mystery transcripts. Nature
- Tevz, Gregor et al. (2016) Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer. Molecular and Cellular Endocrinology
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- Macaulay, Iain C et al. (2015) G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nature Methods
- Poster: Liqing, Tian et al. (2018) Allelic specificity of immunoglobulin heavy chain (IGH@) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing
- Poster: Ashby, M. et al. (2018) Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts
- Poster: Ho, Yeung et al. (2018) The role of androgen receptor variant AR-V9 in prostate cancer
- Poster: Ekholm, J. et al. (2017) Screening for causative structural variants in neurological disorders using long-read sequencing
- Poster: Ashby, M. et al. (2017) Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel platform
- Poster: Clark, Tyson A. et al. (2017) SMRT Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants
- Poster: Kujawa, Steve et al. (2017) A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing
- Poster: Ashby, Meredith et al. (2017) Using the PacBio IsoSeq method to search for novel colorectal cancer biomarkers
- Poster: Tassone, F. et al. (2016) Alternative splicing in FMR1 premutations carriers
- Poster: Kujawa, S. et al. (2016) A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing
- Poster: Ekholm, Jenny et al. (2016) Candidate gene screening using long-read sequencing
- Poster: Ekholm, Jenny et al. (2016) Enrichment of unamplified DNA and long-read SMRT Sequencing in unlocking the underlying biological disease mechanisms of repeat expansion disorders
- Poster: Baybayan, Primo et al. (2016) Application specific barcoding strategies for SMRT Sequencing
- Pillai, Nisha (2018) Webinar: Survey of transcriptome diversity using Iso-Seq analysis
- Ashby, Meredith (2017) Webinar: Chasing Alternative Splicing in Cancer: Simplified Full-length Isoform Sequencing
- Korlach, Jonas (2017) AGBT PacBio Workshop: SMRT Sequencing roadmap: better throughput, lower costs
- Auvignen, Petra (2017) AGBT Virtual Poster: Interspecies interation amoung meat spoilage-related lactic acid bacteria
- Ashby, Meredith (2017) AGBT Virtual Poster: Using the PacBio Iso-Seq method to search for novel colorectal cancer biomarkers
- (2017) Tutorial: Iso-Seq analysis application [SMRT Link v5.0.0]
- Tassone, F. and Olaby, R. and Tang, H. and Hickey, L and Tseng, E. (2016) ASHG Virtual Poster: Alternative splicing in FMR1 premutations carriers
- Korlach, Jonas (2016) AGBT Conference: Addressing complex disease and hidden heritability with the Sequel System
- Ameur, Adam (2016) AGBT Roche and PacBio Workshop: Clinical SMRT Sequencing – from single genes to complete genomes
- Ashley, Euan (2016) AGBT Roche and PacBio Workshop: Towards precision medicine
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- Pandya, Chetanya (2016) Customer Experience: SMRT Sequencing for cancer research is the ‘Way to go’
- Application Brief: Long-read RNA sequencing – Best Practices. (2018)
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- PacBio Certified Service Providers (2018)
- Case Study: SMRT sequencing brings clarity to HIV vaccine and transplant research at the Wisconsin national primate research center. (2017)
- Cancer Brochure: Discover the hidden landscape of cancer variants (2017)
- Human Biomedical Research Brochure: The most comprehensive view of the human genome (2017)
- SMRT Analysis Brochure: Gain a deeper understanding of your sequencing data (2017)
- Case Study: Scientists deconstruct cancer complexity through genome and transcriptome analysis. (2016)