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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

The future of RNA sequencing is with long reads! The Iso-Seq method sequences the entire cDNA molecules – up to 10 kb or more – without the need for bioinformatics transcript assembly, so you can characterize novel genes and isoforms in bulk and single-cell transcriptomes and further:

  • Characterize alternative splicing (AS) events, including alternative start sites, end sites, intron retention, and exon-skipping events
  • Find gene fusions in tumor samples
  • Identify allele-specific isoforms
  • Detect differentially expressed isoforms and isoform switching events
  • Predict functional impact of novel isoforms through open reading frame (ORF) prediction

Isoform discovery

Isoform discovery

On-demand webinar

UNDERSTANDING CLONAL EVOLUTION USING GAME THEORY AND SINGLE-CELL LONG-READ ISOFORM ANALYSIS

See how cancer researchers use the MAS-Seq for 10x Single Cell 3’ kit (now the Kinnex single-cell RNA kit) to identify driver mutations and trace clonal populations over time in a patient’s research sample who progressed from MDS (Myelodysplastic syndrome) to AML (acute myeloid leukemia).

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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 rna sequencing white icon

Single-cell
transcriptome studies

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

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“Long-read sequencing results in more accurate measurements than any obtained from short-read RNA-seq to determine the presence or absence of specific isoforms.”

The right solution for your RNA applications

Genome annotation Whole transcriptome Single-cell transcriptome
Goal Comprehensive, high-quality genome annotation for plant + animal organisms

- Isoform discovery in disease cohorts

- Differential isoform expression analysis in disease vs normal samples

 Cell-type specific, allele-specific isoform and variant characterization in single-cell studies
Library prep Kinnex full-length RNA kit Kinnex full-length RNA kit Kinnex single-cell RNA kit
Sequencing recommendation 1 Revio SMRT Cell for 40 million reads total (5-10 million reads per tissue) 1 Revio SMRT Cell for 40 million reads total (5-10 million reads per sample) 1 Revio SMRT Cell for 80-100 million reads for one single-cell library
Analysis SMRT Link followed by tertiary tools SMRT Link followed by tertiary tools SMRT Link followed by tertiary tools

RNA sequencing datasets

Application Dataset Download literature Technology Sequencing system
Kinnex single-cell Homo sapiens - PBMC 10x Chromium Single Cell (5' and 3' libraries) Kinnex single-cell application note HiFi long-read Sequel II and Revio systems
Kinnex single-cell Homo sapiens - HG002 (10x 5') Kinnex single-cell RNA application note HiFi long-read Revio system
Kinnex full-length RNA Homo sapiens – UHRR Kinnex full-length RNA application note HiFi long-read Sequel II and Revio systems
Kinnex full-length RNA Homo sapiens – HG002 Kinnex full-length RNA application note HiFi long-read Revio system
Kinnex full-length RNA Homo sapiens - Heart Kinnex full-length RNA application note HiFi long-read Revio system
Kinnex full-length RNA Homo sapiens - Cerebellum Kinnex full-length RNA application note HiFi long-read Revio system

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Long-read RNA sequencing in action

Article

ISO-SEQ METHOD DIFFERENTIATES PSEUDOGENES ASSOCIATED WITH DISEASES

Read how researchers at University College London used the Iso-Seq method to accurately map and characterize the parent-pseudogene pair GBA-GBAP1 which is implicated in Gaucher’s disease and Parkinson’s disease.

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Pseudogenes - Stylized image of a broken mirror, representing a faulty replication

Targeted capture for long transcripts using Samplix Xdrop

See how researchers at Radboud use Samplix Xdrop technology to capture ultra-long (18.9kb) transcripts.

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Null

ISO-SEQ METHOD OUTPERFORMS OTHER LONG-READ METHODS IN BENCHMARKETING CONSORTIUM STUDY

The LRGASP consortium published their assessment of long-read RNA sequencing using different sequencing platforms and analysis tools.

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Protein isoform discovery - Stylized image representing protein isoform discovery on pink to magenta gradient background

PROTEIN ISOFORM DISCOVERY ENHANCED BY ISOFORM SEQUENCING

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

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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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FULL-LENGTH RNA SEQUENCING SPOTLIGHT

KINNEX FULL-LENGTH RNA KIT ENABLES SCALABLE, COST-EFFECTIVE ISOFORM SEQUENCING

The Kinnex full-length RNA kit produces full-length isoforms up to 10 kb. Combine with the Iso-Seq express 2.0 kit and SMRT Link analysis to characterize and compare known and novel isoforms for your RNA research.

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COMMON QUESTIONS ABOUT PACBIO ISO-SEQ METHOD AND KINNEX KITS FOR 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 output high-quality, full-length transcript sequences of 10 kb 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 Kinnex kits offer scalable, cost-effective RNA sequencing solutions. The Kinnex full-length RNA kit (together with Iso-Seq express 2.0 kit) offers an end-to-end solution for whole transcriptome RNA sequencing. The Kinnex single-cell RNA kit can be used to sequence single-cell cDNA such as those from 10x Chromium Single Cell kits. The Kinnex 16S rRNA kit offers full-length 16S rRNA sequencing.

Long-read RNA sequencing can offer full-length isoform information, whereas traditional short reads rely on computational transcript assembly that cannot unambiguously resolve isoform structures. The Kinnex full-length RNA kit can sequence full-length cDNA generated using the Iso-Seq express 2.0 kit to characterize and compare isoforms with extraordinary throughput and accuracy.

Long-read single-cell RNA sequencing can offer isoform information at the single-cell level, whereas traditional short reads can only offer gene information. The Kinnex single-cell RNA kit can sequence full-length single-cell cDNA such as those from the 10x Chromium system for unprecedented insight into single-cell RNA biology.

Long reads can identify full-length transcript structure of cancer fusion genes, which may express multiple isoforms. The Iso-Seq method has been used to identify cancer fusion genes. Examples include identifying the IGH-DUX4 fusion in B-cell acute lymphoblastic leukemia, 3-hop fusions in SKBR3 cell line, and an ovarian cancer patient-specific fusion that was mis-classified by short read data.

The Iso-Seq method has been shown to have concordant gene-level expression with matching short read data, while being able to identify differential transcript usage (DTU) that is not observable using short read data. Examples include identifying DTU in developing brain, isoform switching events in hibernating brown bears, and cell type differences in aging down syndrome brains.

The SMRT Link Iso-Seq and Single-cell Iso-Seq workflows fully support long-read RNA-Seq data generated on PacBio systems. Our https://isoseq.how/ wiki page further details command line usages. Finally, our Bioinformatics tools for full-length isoform sequencing application brief has a recommended list of community tools for tertiary analyses.

Shaping the future

A HiFi movement: Isoform-resolved cell atlases

A shift to higher-quality, isoform-resolved cell atlases with the increasing adoption of full-length RNA sequencing with PacBio.

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FEATURED LONG-READ SEQUENCING SYSTEMS

Achieve highly accurate full-length cDNA sequencing with PacBio long-read sequencers.

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