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.
Profile transcriptome complexity without assembly
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
- Optional size-selection protocols for transcripts > 3 kb
- Compatible with standard target enrichment methods, such as NimbleGen SeqCap EZ Library
- Multiplex transcripts or full transcriptomes with sample barcoding
- Additional training resources:
- SMRT Sequencing with PacBio Systems
- Take advantage of the Sequel System to reduce project costs and generate 7X more reads compared with the PacBio RS II
- Directly sequence full-length transcripts with average read lengths of ~10 kb
- Scale throughput based on project needs
- Scalable throughput
- 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
Featured research: discover new isoforms in a well-characterized cell line
Scientists found a total of 2,428 novel isoforms in the human embryonic stem cell line and 216 novel genes which show evidence of differential expression5.
To learn more about how to profile the complexity of the transcriptome with the Iso-Seq application, contact us.
- 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.
- Au, K.F., et al., (2013) Characterization of the human ESC transcriptome by hybrid sequencing. PNAS. 110, E4821-E4830.
- 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
- Dasari, Surendra et al. (2015) Proteomic detection of immunoglobulin light chain variable region peptides from amyloidosis patient biopsies. Journal of Proteome Research
- You, Xintian et al. (2015) Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity. Nature Neuroscience
- Cavelier, Lucia et al. (2015) Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing. BMC Cancer
- 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
- Pretto, Dalyir I et al. (2015) Differential increases of specific FMR1 mRNA isoforms in premutation carriers. Journal of Medical Genetics
- Kleinman, Claudia L et al. (2014) Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR. Nature Genetics
- Salton, Maayan et al. (2014) Identification by high-throughput imaging of the histone methyltransferase EHMT2 as an epigenetic regulator of VEGFA alternative splicing. Nucleic Acids Research
- Au, Kin Fai et al. (2014) The transcriptome of human pluripotent stem cells. Current Opinion in Genetics & Development
- Schreiner, Dietmar et al. (2014) Targeted combinatorial alternative splicing generates brain region-specific repertoires of neurexins. Neuron
- Chen, Lu et al. (2014) Transcriptional diversity during lineage commitment of human blood progenitors. Science
- Tilgner, Hagen et al. (2014) Defining a personal, allele-specific, and single-molecule long-read transcriptome. Proceedings of the National Academy of Sciences
- Thomas, Sean et al. (2014) Long-read sequencing of chicken transcripts and identification of new transcript isoforms. PLoS One
- Treutlein, Barbara et al. (2014) Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing. Proceedings of the National Academy of Sciences
- Voit, Richard A et al. (2014) Nuclease-mediated gene editing by homologous recombination of the human globin locus. Nucleic Acids Research
- Au, Kin Fai et al. (2013) Characterization of the human ESC transcriptome by hybrid sequencing. Proceedings of the National Academy of Sciences
- Sharon, Donald et al. (2013) A single-molecule long-read survey of the human transcriptome. Nature Biotechnology
- 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.
- Poster: Clark, Tyson A. et al. (2015) Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human diseases.
- Poster: Clark, Tyson A et al. (2015) Single Molecule, Real-Time sequencing of full-length cDNA transcripts uncovers novel alternatively spliced isoforms.
- Poster: Deslattes Mays, A et al. (2014) Complex alternative splicing patterns in hematopoietic cell subpopulations revealed by third-generation long reads.
- 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’
- Ashley, Euan (2016) AGBT Roche and PacBio Workshop: Towards precision medicine
- Ameur, Adam (2016) AGBT Roche and PacBio Workshop: Clinical SMRT Sequencing – from single genese to complete genomes
- Korlach, Jonas (2016) AGBT Conference: Addressing complex disease and hidden heritability with the Sequel System
- Clark, Tyson (2015) Seminar: Iso-seq method – sample prep and experimental design for full-length cDNA sequencing
- Tilgner, Hagen (2014) ASHG PacBio Workshop: Personalized and allele specific long-read transcriptomes
- 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
- 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)
- Nattestad, Maria (2015) ASHG Conference: Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using PacBio long read sequencing
- PacBio Service Providers (2016)
- Case Study: Scientists deconstruct cancer complexity through genome and transcriptome analysis (2016)
- Cancer Brochure: Drive discovery with most complete view of cancer complexity (2016)
- Isoform Sequencing Application Brochure: Read full-length transcripts – no assembly required (2015)
- Sequel System Brochure: Introducing the scalable platform for SMRT Sequencing (2015)
- Human Biomedical Research Brochure: The most comprehensive view of the human genome (2015)
- Case Study: New view of chicken transcriptome offers clues to heart development. (2015)
- Featured Interview: Revolutionize Translational Research – Uppsalas Ulf Gyllensten on how long reads give access to new areas of the genome. (2015)
- Featured Interview: The Rise of Long Reads – Mike Snyder says long-read sequencing is critical to understanding the transcriptome. (2014)