Obtain a more comprehensive set of transcript isoforms
A single gene may code for a surprising number of proteins, each with a distinct biological function. This is especially true in higher organisms. Short-read RNA sequencing (RNA-seq) works by breaking up transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest.
Capture full-length transcripts with no assembly required
The PacBio Isoform Sequencing (Iso-Seq) method employs long reads to sequence transcript isoforms of up to 10 kb in their entirety. Surveying this transcript diversity, either broadly or in a targeted fashion, reveals key information about the frequency and types of alternative transcription, improving genome annotation and gene discovery.
Single Molecule, Real-Time (SMRT) Sequencing enhances RNA sequencing, allowing you to:
- Make direct, evidence-based genome annotations
- Discover novel genes and isoforms, even in well-characterized samples
- Identify promoters and splice sites in comprehensive gene models to understand gene regulation
- Improve accuracy of RNA-seq quantification with isoform-level resolution for gene expression studies
- Distinguish important stress response, developmental, and tissue-specific isoforms
Workflow: from RNA to accurate gene models
- 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: discovery of thousands of new isoforms in chicken
Scientists at the Gladstone Institutes used PacBio Iso-Seq data to improve the annotation of the chicken genome. They identified more than 9,000 novel transcript isoforms, including these examples of an alternate transcription start site, a heart-specific isoform, and an antisense transcription event1.
Explore this research further.
To learn more about how to profile the complexity of the transcriptome with the Iso-Seq method, contact us.
- Thomas, S., et al., (2014) Long-read sequencing of chicken transcripts and identification of new transcript isoforms. PLoS One. 9 (4), e94650.
- Gao, Shan et al. (2016) PacBio full-length transcriptome profiling of insect mitochondrial gene expression. RNA Biology
- Van't Hof, Arjen E et al. (2016) The industrial melanism mutation in British peppered moths is a transposable element. Nature
- Mangul, Serghei et al. (2016) HapIso: an accurate method for the haplotype-specific isoforms reconstruction from long single-molecule reads
- Kuang, Zheng et al. (2016) The dynamic landscape of fission yeast meiosis alternative-splice isoforms bioRxiv
- Dong, Lingli et al. (2015) Single-molecule real-time transcript sequencing facilitates common wheat genome annotation and grain transcriptome research. BMC Genomics
- Lu, Tingting et al. (2015) Transcriptome-wide investigation of circular RNAs in rice. RNA
- Westbrook, Catherine J et al. (2015) No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing. Human Immunology
- Minoche, André E et al. (2015) Exploiting single-molecule transcript sequencing for eukaryotic gene prediction. Genome Biology
- Gordon, Sean P et al. (2015) Widespread polycistronic transcripts in fungi revealed by single-molecule mRNA sequencing. PLoS One
- Gao, Qingsong et al. (2015) Predominant contribution of cis-regulatory divergence in the evolution of mouse alternative splicing. Molecular Systems Biology
- Xu, Zhichao et al. (2015) Full-length transcriptome sequences and splice variants obtained by a combination of sequencing platforms applied to different root tissues of Salvia miltiorrhiza and tanshinone biosynthesis. The Plant Journal
- Tombácz, Dóra et al. (2015) Characterization of novel transcripts in pseudorabies virus. Viruses
- Poster: Tseng, Elizabeth et al. (2016) Reconstruction of the spinach coding genome using full-length transcriptome without a reference genome
- Poster: Baybayan, Primo et al. (2016) Application specific barcoding strategies for SMRT Sequencing
- Poster: Clark, Tyson et al. (2016) Full-length cDNA sequencing for genome annotation and analysis of alternative splicing
- Poster: Clark, Tyson A et al. (2015) Single Molecule, Real-Time sequencing of full-length cDNA transcripts uncovers novel alternatively spliced isoforms.
- Poster: Copetti, Dario et al. (2015) Rapid full-length Iso-Seq cDNA sequencing of rice mRNA to facilitate annotation and identify splice-site variation.
- Timp, Winston (2016) PAG Conference: An extreme metabolism: Iso-Seq analysis of the ruby-throated hummingbird transcriptome
- Smith, Tim (2015) Customer Experience: Long-read assemblies correct problems, advance research in livestock
- Clark, Tyson (2015) Seminar: Iso-seq method – sample prep and experimental design for full-length cDNA sequencing
- Tseng, Elizabeth (2015) Seminar: Iso-seq analysis & beyond – advanced bioinformatics for transcriptome sequencing using long reads
- PacBio Service Providers (2016)
- Certified Service Provider Profile: Institute for Genome Sciences expands long-read sequencing services with new Sequel System (2016)
- Case Study: First comprehensive view of maize genome reveals regulatory and structural mechanisms (2016)
- Case Study: With SMRT Sequencing for genomes, transcriptomes, and epigenomes, scientists are overcoming barriers in plant and animal research (2016)
- Certified Service Provider Profile: AGI builds on BAC expertise and SMRT Sequencing for rice and other crop genomes (2016)
- Isoform Sequencing Application Brochure: Read full-length transcripts – no assembly required (2015)
- Sequel System Brochure: Introducing the scalable platform for SMRT Sequencing (2015)
- Plant and Animal Sciences Brochure: A comprehensive view of genetic diversity (2015)
- Case Study: Scientists create gold standard plant and animal genomes with SMRT Sequencing. (2015)
- Case Study: New view of chicken transcriptome offers clues to heart development. (2015)