RNA sequencing with the Iso-Seq method

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

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Human transcriptome studies

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

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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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Introduction to RNA sequencing using the Iso-Seq method

With long and accurate HiFi reads, you can characterize the full diversity of the transcriptome:

  • See alternative start and end sites
  • Characterize splicing events
  • Identify allele-specific isoforms
  • Profile expression at isoform resolution

  • Detect fusion genes
  • Predict full-length open read frames
  • …and more

Iso-Seq method

“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

– Leung, et al, (2021)

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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Iso-Seq analysis enthusiasts share research wins at virtual social event

Dozens of self-proclaimed Iso-Seq analysis geeks and other curious researchers gathered…

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Isoform-level transcriptome reference helps shrimp ward off disease

Shrimp are one of the fastest growing global foods, but also one of the most vulnerable.

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EXPLORE

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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SHAPING THE FUTURE

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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Iso-Seq application workflows at a glance

Genome annotation Whole transcriptome Targeted Iso-Seq Single-cell transcriptome
Goal High-quality genome annotation Sample-specific reference transcriptome for isoform expression analysis with or without related short read data Quantitative characterization of isoforms of select genes Identify cell type-specific isoforms
Library prep Iso-Seq protocol Iso-Seq protocol Coming soon Single-cell Iso-Seq protocol
Sequencing 1 SMRT Cell 8M (recommend multiplex up to 12 tissues) 1 SMRT Cell 8M per sample 1 SMRT Cell 8M (recommend multiplex up to 24 samples) 1 SMRT Cell 8M (suggest adding matching short read data)
Analysis Iso-Seq Analysis (SMRTLink) or command line (isoseq.how) Iso-Seq Analysis followed by community tools (SQANTI3, tappAS, Kallisto) Iso-Seq Analysis followed by community tools (SQANTI3, tappAS, Kallisto) Single-cell analysis
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Advantages of RNA sequencing with the
Iso-Seq method

Powered by HiFi reads, the Iso-Seq method uncovers biology that other approaches miss.

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.

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Mas-Iso-Seq image - PacBio

TECHNOLOGY DEVELOPMENT SPOTLIGHT

MAS-ISO-Seq method delivers >15× throughput through concatenation

Our technology is constantly evolving. MAS-ISO-Seq data takes advantage of long and accurate HiFi read lengths to increase Iso-Seq throughput by >15-fold through multiplexed arrays of cDNA molecules.

Watch the MAS-ISO-Seq talk at ASHG or read the preprint.

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.

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.
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Sequel II Systems

Sequel II systems can generate longer reads with greater accuracy and throughput, at a significantly lower cost. Explore our sequencing platforms for full-length cDNA sequencing.

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