RNA sequencing

Reveal novel biology that other sequencing methods miss with full-length transcripts using the Iso-Seq method

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RNA sequencing

With long and accurate HiFi reads, you can characterize the full diversity of the transcriptome — that are tens of kilobases long. That’s enough to capture most transcripts in a single sequencing read, with no downstream bioinformatic assembly required. This became known as the Iso-Seq® method because it enabled full-length isoform RNA sequencing, which can be used to explore a transcriptome in its entirety or in a targeted fashion to examine individual genes.

The Iso-Seq method can be used in RNA sequencing to:

  • See alternative start and end sites
  • Characterize splicing events
  • Find fusion transcripts
  • Identify allele-specific isoforms
  • Discover non-coding RNAs
  • Profile expression at isoform resolution
  • Predict full-length open read frames and more

Iso-Seq method

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Detecting cancer-related RNA dysregulation with long-read sequencing

Full-length, single-cell RNA data provides critical insights into the understanding of cancer transcriptomic features, such as isoforms, fusions, and expressed mutations. However, until recently, cell typing based on gene expression has been challenging with long reads due to insufficient sequencing depth.

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Human RNA sequencing

Human transcriptome studies

Explore how changes in isoform usages contribute to phenotypic differences between health and disease.

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RNA sequencing — plant + animal

Plant + animal transcriptome studies

Advance breeding programs and basic research with full-length cDNA sequencing that does not require a reference genome.

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Single-cell transcriptome studies

Visualize biology at an even higher resolution by capturing a comprehensive and complete transcript sequence from individual cells.

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“More than 50% of isoforms robustly detected using Iso-Seq could not be readily recapitulated using standard RNA-seq, highlighting the advantage of long-read sequencing for characterizing isoform diversity.”1

Iso-Seq application workflows at a glance

Genome annotation Whole transcriptome Single-cell transcriptome
Goal High-quality genome annotation Sample-specific reference transcriptome for isoform expression analysis with or without related short read data Identify cell type-specific isoforms
Library prep Iso-Seq protocol Iso-Seq protocol MAS-Seq protocol
Sequencing 1 SMRT Cell 8M (recommend multiplex up to 12 tissues) 1 SMRT Cell 8M per sample 1 SMRT Cell
Analysis Iso-Seq Analysis (SMRTLink) or command line ( Iso-Seq Analysis followed by community tools (SQANTI3, tappAS, Kallisto) Read segmentation + single-cell Iso-Seq analysis (SMRT Link) followed by tertiary tools (Seural, Scanpy, Kana)

RNA sequencing datasets

Application Dataset Download literature Technology Sequencing system
MAS-Seq single cell Homo sapiens - PBMC 10x Chromium Single Cell 3' libraries MAS-Seq for single-cell isoform sequencing HiFi long read Sequel II and Revio systems
Whole transcriptome Homo sapiens– brain with Alzheimer’s disease Bulk and single-cell isoform sequencing for human disease research HiFi long read Sequel II system
Whole transcriptome Homo sapiens – universal human reference RNA (UHRR) Bulk and single-cell isoform sequencing for human disease research HiFi long read Sequel II system

More datasets

RNA sequencing with Iso-Seq analysis in action

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Scalable RNA isoform sequencing using intra-molecular multiplexed cDNAs

For scalable full-length RNA sequencing, we have developed multiplexed arrays sequencing…

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Read the recap on the latest Iso-Seq research that is pushing the frontiers of cutting-edge science

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Researchers use Iso-Seq data to predict open reading frames integrated into a proteogenomics workflow to discovery novel protein isoforms to reveal the functional impacts of alternative splicing

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Did you know we have a comprehensive library of articles, reports, papers, and videos related to RNA sequencing with the Iso-Seq method?

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The MAS-Seq for 10x Single Cell 3’ kit (based on the MAS-Seq method by Al’Khafaji et al.) takes advantage of long and accurate HiFi read lengths to increase single-cell Iso-Seq throughput by 16-fold through concatenated arrays of single-cell cDNA molecules.

Learn more about the MAS-Seq kit now at the single-cell Iso-Seq webpage.

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Iso-Seq Method delivers full-length transcript isoforms, no assembly required

Unlike short-read RNA-seq methods, Iso-Seq method reads span the whole transcript, capturing even untranslated regions.

  • Detect alternative polyadenylation
  • Detect differential isoform usages (DIU)
  • Obtain isoform-level phasing information

Iso-seq method chart showing SNPs are inherited with Ki11 in only one version of the hybrid and that SNPs are inherited at 50/50 ratio despite the type of hybrid - PacBio

Wang, B. et al. (2020) Variant phasing and haplotypic expression from long-read sequencing in maize. Commun Biol.

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iso-method comprehensive - PacBio

Figure 3e from Leung et al.(2021)

Iso-Seq Data is comprehensive

Characterize whole transcriptome isoform diversity that can reveal complex alternative splicing, fusion events, and transcriptional readthroughs.

Iso-Seq Analysis offers unparalleled insight at the single cell level

Use any single-cell platform to generate full-length cDNA as input for SMRT sequencing and achieve all the same benefits of Iso-Seq on bulk cDNA — complete transcript sequence, isoform-level phasing, and isoform profiling — at single-cell resolution.

Joglekar brain region graph - PacBio

Joglekar A, et al. (2021) A spatially resolved brain region and cell type-specific isoform atlas of the postnatal mouse brain. Nat Commun.

Common questions about PacBio Iso-Seq method and RNA sequencing

The Iso-Seq method refers to sequencing full-length cDNA using PacBio SMRT sequencing technology. Full-length cDNAs can be generated from eukaryotic RNA, bacterial RNA, or even viral RNA. Because the full-length cDNA is sequenced in a single HiFi read, no assembly is required.

The Iso-Seq method and bioinformatics workflow outputs high-quality, full-length transcript sequences of 10kb or longer. The high accuracy of the HiFi reads ensure that SNPs can be identified, as well as UMIs and barcodes used for single-cell studies.

The Iso-Seq bioinformatics analysis workflow does not require a reference genome, although if a reference genome is available, it can be used to map the full-length transcripts back to the genome.

The Iso-Seq method has been used to detect cancer fusion genes. Examples include identifying the IGH-DUX4 fusion in B-cell acute lymphoblastic leukemia and 3-hop fusions in SKBR3 cell line.

The single-cell Iso-Seq (scIso-Seq) method has been applied to various single-cell platforms that generate full-length single-cell molecules and have uncovered cell-type specific isoforms not detectable with gene-level short read information in samples such as postnatal mouse brain and Down Syndrome aging brains. With the MAS-Seq for 10x Single Cell 3’ kit, full-length isoform sequencing at the single-cell level is readily accessible on the PacBio long read sequencing systems.”

The Iso-Seq method has been shown to have concordant gene-level expression with matching short read data, while being able to identify differential isoform usage (DIU) that is not observable using short read based transcript assemblies. With existing RNA-Seq data, Iso-Seq method can provide a sample-specific isoform reference transcriptome that improves isoform quantification.


Iso-Seq analysis provides insights into feats of physiology of hibernating bears

Washington State University researchers use Iso-Seq analysis to shed light on the metabolic mystery of brown bears.

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