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.
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 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 – Best Practices
- 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 by using the Sequel System
- 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
- Iso-Seq analysis: output high quality, full-length transcript sequences with no assembly required
- No genome reference required
- Tutorial: Isoform Sequencing Analysis
Featured research: Significantly improved sorghum genome annotation
Splice isoform analysis in the sorghum transcriptome using the Iso-Seq method greatly improved genome annotation, with >77,000 novel splice isoforms and >2,100 novel genes identified. In this example, a gene was discovered to produce 73 novel alternatively spliced isoforms, where the previous gene model contained only a single isoform1.
Explore this research further.
References
Selected Resources
- Workman, Rachael E. et al. (2017) Single molecule, full-length transcript sequencing provides insight into the extreme metabolism of ruby-throated hummingbird Archilochus colubris bioRxiv
- Breaux, Breanna et al. (2017) The Florida manatee (Trichechus manatus latirostris) immunoglobulin heavy chain suggests the importance of clan III variable segments in repertoire diversity. Developmental and comparative immunology
- 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
- Tombácz, Dóra et al. (2016) Full-length isoform sequencing reveals novel transcripts and substantial transcriptional overlaps in a herpesvirus. PLoS One
- Abdel-Ghany, Salah E et al. (2016) A survey of the sorghum transcriptome using single-molecule long reads. Nature Communications
- Wang, Bo et al. (2016) Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing. Nature Communications
- 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
- Kuang, Zheng et al. (2016) The dynamic landscape of fission yeast meiosis alternative-splice isoforms bioRxiv
- Poster: Clark, T et al. (2017) Full-length cDNA sequencing on the PacBio Sequel platform
- 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
- Korlach, Jonas (2017) AGBT PacBio Workshop: SMRT Sequencing roadmap: better throughput, lower costs
- (2017) Tutorial: Iso-Seq analysis application
- Kuo, Richard (2017) PAG PacBio Workshopn: Using the Iso-Seq method to fill in your a_no__tion
- Ware, Doreen (2016) PAG PacBio Workshop: A-maize-ing time for plant science – SMRT Sequencing of the maize genome and transcriptome
- Timp, Winston (2016) PAG Conference: An extreme metabolism: Iso-Seq analysis of the ruby-throated hummingbird transcriptome
- PacBio Certified Service Providers (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: 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)