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
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| 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. | |
Iso-Seq Project Calculator
Tell us about your plant or animal RNA sequencing project and receive an estimate on the time and materials needed using the Iso-Seq method on the Sequel System.
Data Release
Iso-Seq Results for Hummingbird and Zebra Finch Brain Tissue
Explore the role of the transcriptome in avian vocal learning with the PacBio Iso-Seq data set generated with the Sequel System.
To learn more and access the data, see our blog post.
Spotlight: 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 13 novel alternatively spliced isoforms, where the previous gene model contained only a single isoform. Explore this research further:
Abdel-Ghany, S.E. et al. (2016) A survey of the sorghum transcriptome using single-molecule long reads. Nature Communications. 7, e11706.
Selected Resources
- Zuo, Chunman et al. (2018) Revealing the transcriptomic complexity of switchgrass by PacBio long-read sequencing. Biotechnology for Biofuels
- Kronenberg, Zev N et al. (2018) High-resolution comparative analysis of great ape genomes. Science
- Zhang, Hangxiao et al. (2018) Transcriptome characterization of moso bamboo (Phyllostachys edulis) seedlings in response to exogenous gibberellin applications. BMC Plant Biology
- Pollard, Martin O et al. (2018) Long reads: their purpose and place. Human Molecular Genetics
- Li, Yao et al. (2018) A survey of transcriptome complexity in Sus scrofa using single-molecule long-read sequencing. DNA Research
- Chao, Yuehui et al. (2018) Analysis of transcripts and splice isoforms in Red Clover (Trifolium pratense L.) by single-molecule long-read sequencing BioRxiv
- Cantsilieris, Stuart et al. (2018) Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family. Proceedings of the National Academy of Sciences of the United States of America
- Zhu, Chunhui et al. (2018) Transcriptome profiling using Illumina- and SMRT-based RNA-seq of hot pepper for in-depth understanding of genes involved in CMV infection. Gene
- Wang, Bo et al. (2018) A comparative transcriptional landscape of maize and sorghum obtained by single-molecule sequencing. Genome Research
- Ren, Panrong et al. (2018) Molecular mechanisms of acclimatization to phosphorus starvation and recovery underlying full-length transcriptome profiling in barley (Hordeum vulgare L.). Frontiers in Plant Science
- Magrini, Vincent et al. (2018) Improving eukaryotic genome annotation using single molecule mRNA sequencing. BMC Genomics
- Workman, Rachael E et al. (2018) Single molecule, full-length transcript sequencing provides insight into the extreme metabolism of ruby-throated hummingbird Archilochus colubris GigaScience
- Tran, Hue T M et al. (2018) Use of a draft genome of coffee (Coffea arabica) to identify SNPs associated with caffeine content. Plant Biotechnology Journal
- Filichkin, Sergei A et al. (2018) Abiotic stresses modulate landscape of poplar transcriptome via alternative splicing differential intron retention, and isoform ratio switching. Frontiers in Plant Science
- Tardaguila, Manuel et al. (2018) SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification. Genome Research
- Makita, Yuko et al. (2018) Construction of Pará rubber tree genome and multi-transcriptome database accelerates rubber researches. BMC Genomics
- Wang, Maojun et al. (2018) A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation. The New Phytologist
- Deiss, Thaddeus C et al. (2017) Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies. Cellular & Molecular Immunology
- Yang, Ting et al. (2017) Transcript profiling of a bitter variety of narrow-leafed lupin to discover alkaloid biosynthetic genes. Journal of Experimental Botany
- Semler, Matthew R et al. (2017) Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques. Immunogenetics
- Cheng, Bing et al. (2017) Long-read sequencing of the coffee bean transcriptome reveals the diversity of full-length transcripts. GigaScience
- Zhang, Shi-Jian et al. (2017) Isoform evolution in primates through independent combination of alternative RNA processing events. Molecular Biology and Evolution
- Wooddell, Christine I et al. (2017) RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Science Translational Medicine
- Li, Yongping et al. (2017) Genome re-annotation of the wild strawberry Fragaria vesca using extensive Illumina-and SMRT-based RNA-seq datasets DNA Research
- Jo, Ick-Hyun et al. (2017) Isoform sequencing provides a more comprehensive view of the Panax ginseng transcriptome. Genes
- Li, Jun et al. (2017) Long read reference genome-free reconstruction of a full-length transcriptome from Astragalus membranaceus reveals transcript variants involved in bioactive compound biosynthesis. Cell Discovery
- Nudelman, German et al. (2017) High resolution annotation of zebrafish transcriptome using long-read sequencing BioRxiv
- Chen, Shi-Yi et al. (2017) A transcriptome atlas of rabbit revealed by PacBio single-molecule long-read sequencing. Scientific Reports
- Gao, Shan et al. (2017) Two novel lncRNAs discovered in human mitochondrial DNA using PacBio full-length transcriptome data. Mitochondrion
- Kim, Hyeongmin et al. (2017) Characterization of the Rosellinia necatrix transcriptome and genes related to pathogenesis by single-molecule mRNA sequencing. The Plant Pathology Journal
- Wang, Taotao et al. (2017) Comprehensive profiling of rhizome-associated alternative splicing and alternative polyadenylation in moso bamboo (Phyllostachys edulis). The Plant Journal
- Zhu, Fu-Yuan et al. (2017) Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings. The Plant Journal
- Ries, Moritz et al. (2017) KIR3DL01 upregulation on gut natural killer cells in response to SIV infection of KIR- and MHC class I-defined rhesus macaques. PLoS Pathogens
- Xu, Qingshan et al. (2017) Transcriptome profiling using single-molecule direct RNA sequencing approach for in-depth understanding of genes in secondary metabolism pathways of Camellia sinensis. Frontiers in Plant Science
- Liu, Haibo et al. (2017) A high-quality annotated transcriptome of swine peripheral blood. BMC Genomics
- Poster: Vierra, M. et al. (2018) Characterizing the pan-genome of maize with PacBio SMRT Sequencing
- Poster: Vierra, Michelle et al. (2018) Full-length transcript profiling with the Iso-Seq method for improved genome annotations
- Poster: Concepion, C., et al. (2018) De novo assembly and preliminary annotation of the Schizocardium californicum genome
- Poster: Tseng, E., et al. (2018) Haplotyping of full-length transcript reads from long-read sequencing can reveal allelic imbalances in isoform expression
- Poster: Vierra, M. et al. (2017) From RNA to full-length transcripts: The PacBio Iso-Seq method for transcriptome analysis and genome annotation
- 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
- Poster: Copetti, Dario et al. (2015) Rapid full-length Iso-Seq cDNA sequencing of rice mRNA to facilitate annotation and identify splice-site variation.
- Pillai, Nisha (2018) Webinar: Survey of transcriptome diversity using Iso-Seq analysis
- Korlach, Jonas (2017) AGBT PacBio Workshop: SMRT Sequencing roadmap: better throughput, lower costs
- (2017) Tutorial: Iso-Seq analysis application [SMRT Link v5.0.0]
- Korlach, Jonas (2017) PAG PacBio Workshop: SMRT Sequencing for complete genomes
- Kuo, Richard (2017) PAG PacBio Workshop: 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
- Application Brief: Long-read RNA sequencing – Best Practices. (2018)
- SMRT Sequencing Brochure: Revolutionize genomics with SMRT Sequencing. (2018)
- 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)
- Plant and Animal Sciences Brochure: A comprehensive view of genetic diversity (2017)
- 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. (2015)
- Certified Service Provider Profile: AGI builds on BAC expertise and SMRT Sequencing for rice and other crop genomes. (2015)
- Case Study: Scientists create gold standard plant and animal genomes with SMRT Sequencing. (2015)