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
- Iso-Seq sample preparation
- Prepare full-length transcripts from as low as 1 ng of poly A+ RNA or 2 ng of total RNA
- Sequel System loading protocols reduce need for size selection for transcripts <4 kb
- Optional size-selection protocols for transcripts >4 kb
- Compatible with standard target enrichment methods, such as NimbleGen SeqCap EZ Library or IDT xGen Lockdown Probes
- Multiplex transcripts or full transcriptomes with sample barcoding
- Additional training resources:
- Application Brief: Long read RNA Sequencing
- Tutorial: Isoform Sequencing (Iso-Seq) Overview
- Webinar: Sample Prep and Experimental Design for Full-Length cDNA Sequencing
- SMRT Sequencing with PacBio Systems
- Simplify sample prep workflow and reduce project costs when using the Sequel System
- Directly sequence full-length transcripts with average read lengths of ~10 kb
- Scale throughput based on project needs
- Profile multiplexed transcripts in a single SMRT Cell
- Survey transcriptomes in 1–2 SMRT Cells on the Sequel System
- Increase sequencing depth for more comprehensive transcriptome characterization
- Data analysis with SMRT Analysis or PacBio DevNet
- Iso-Seq analysis: output high quality, full-length transcript sequences with no assembly required
- No genome reference required
- Tutorial: Isoform Sequencing Analysis
Featured research: 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
- Deininger, Prescott et al. (2016) A comprehensive approach to expression of L1 loci. Nucleic Acids Research
- Karl, Julie A et al. (2016) Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities bioRxiv
- Vassoler, Fair M et al. (2016) Transgenerational attenuation of opioid self-administration as a consequence of adolescent morphine exposure. Neuropharmacology
- Shi, Lingling et al. (2016) Long-read sequencing and de novo assembly of a Chinese genome. Nature Communications
- 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
- Weirather, Jason L et al. (2015) Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing. Nucleic Acids Research
- Gao, Qingsong et al. (2015) Predominant contribution of cis-regulatory divergence in the evolution of mouse alternative splicing. Molecular Systems Biology
- Macaulay, Iain C et al. (2015) G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nature Methods
- You, Xintian et al. (2015) Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity. Nature Neuroscience
- Dasari, Surendra et al. (2015) Proteomic detection of immunoglobulin light chain variable region peptides from amyloidosis patient biopsies. Journal of Proteome Research
- Cavelier, Lucia et al. (2015) Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing. BMC Cancer
- Pretto, Dalyir I et al. (2015) Differential increases of specific FMR1 mRNA isoforms in premutation carriers. Journal of Medical Genetics
- Vollmers, Christopher et al. (2015) Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing. PLoS One
- 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
- Presentation: Nattestad, Maria et al. (2015) Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using PacBio long read sequencing
- Poster: Clark, T. et al. (2015) Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human cancer
- Poster: Tseng, Elizabeth et al. (2015) Full-length isoform sequencing of the human MCF-7 cell line using PacBio long reads.
- Poster: Baybayan, Primo et al. (2015) SMRT Sequencing of DNA and RNA samples extracted from formalin-fixed and paraffin embedded tissues using adaptive focused acoustics by Covaris.
- Korlach, Jonas (2017) AGBT PacBio Workshop: SMRT Sequencing roadmap: better throughput, lower costs
- (2017) Tutorial: Iso-Seq analysis application
- 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
- Pandya, Chetanya (2016) AGBT Virtual Poster: Comparative analysis of somatic fusion gene detection using short read and long read sequencing
- Pandya, Chetanya (2016) Customer Experience: SMRT Sequencing for cancer research is the ‘Way to go’
- Nattestad, Maria (2015) ASHG Conference: Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using PacBio long read sequencing
- Gyllensten, Ulf (2015) AGBT Virtual Poster: Clinical sequencing using Pacific Biosciences RS II for HLA typing and monitoring of drug resistance in chronic myeloid leukemia (CML)
- Racacho, Lemuel (2015) Customer Experience: For long, noncoding RNAs, SMRT Sequencing offers better view
- Tseng, Elizabeth (2015) Seminar: Iso-seq analysis & beyond – advanced bioinformatics for transcriptome sequencing using long reads
- Clark, Tyson (2015) Seminar: Iso-seq method – sample prep and experimental design for full-length cDNA sequencing
- PacBio Certified Service Providers (2017)
- Cancer Brochure: Discover the hidden landscape of cancer variants (2017)
- Human Biomedical Research Brochure: The most comprehensive view of the human genome (2017)
- Application Brief: Long-read RNA sequencing – Best Practices (2017)
- SMRT Sequencing Brochure: Revolutionize genomics with SMRT Sequencing (2017)
- Isoform Sequencing Application Brochure: Read full-length transcripts – no assembly required (2016)
- Case Study: Scientists deconstruct cancer complexity through genome and transcriptome analysis (2016)
- Case Study: New view of chicken transcriptome offers clues to heart development. (2015)
- Featured Interview: Revolutionize Translational Research – Uppsala’s Ulf Gyllensten on how long reads give access to new areas of the genome. (2015)