Isoform Sequencing (full-length mRNA-seq)

Overview
Analysis Principle
Specifications
Data Analysis
Sample Requirements
Frequently Asked Questions

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Overview

Isoform sequencing (Iso-Seq) analysis utilizes PacBio’s long-read sequencing technology to provide comprehensive full-length RNA analysis.
This approach reads the entire mRNA molecule in a single pass, making it particularly effective for detailed analyses of splicing variants and discovery of novel isoforms, which are challenging with short-read sequencing.

With the latest updates to sequencing platforms and reagent kits, throughput has significantly improved, enabling not only qualitative but also quantitative data acquisition.

* Library preparation requires cDNA synthesis using Oligo-dT, limiting analysis to polyA RNA.

Analysis Principle

Libraries are constructed without fragmenting the mRNA, and cDNA molecules are sequenced repeatedly in the nanowells (ZMW) of a SMRT Cell.
Consensus sequences are generated from the reads, representing the full-length transcript sequences.

Specifications

Platform	PacBio Revio
Reagent	Kinnex full-length RNA kit
Data Amount	5 million HiFi reads/sample or 10 million HiFi reads/sample

* Choose from two scales: 5 million HiFi reads or 10 million HiFi reads per sample. Analyses are pooled, allowing use from a minimum of one sample.

Data Analysis

・Raw Aata Processing and Analysis

Generates full-length transcript sequences from raw reads using PacBio’s SMRT Link software.

・Alignment with Reference Genome

Aligns full-length transcript sequences to a specified reference genome.

・Functional Annotation of Transcripts

Predicts the functionality of obtained transcript sequences by performing similarity searches against established databases (e.g., NR, KOG/COG, Swiss-Prot).

・Gene Structure Analysis

Examines alternative splicing, alternative polyadenylation (APA), and novel gene/transcript sequences. Annotation of novel sequences is also performed.

・Gene/Transcript Expression Level Analysis

Calculates expression levels for genes and transcripts using the reference genome.

・Differential Gene/Transcript Expression Analysis

Evaluates expression variations between genes or transcripts based on expression level data.

・GO Enrichment Analysis (optional)

For analyses involving two or more groups, identifies enriched Gene Ontology (GO) terms for differentially expressed genes.

・Pathway Enrichment Analysis (optional)

For analyses involving two or more groups, identifies enriched pathways associated with differentially expressed genes.

Sample Requirements

Sample Type	Total Amount	Concentration	Volume
Total RNA	≥ 1.5 µg	≥ 50 ng/µL	≥ 30 µL

Frequently Asked Questions

Q: Can I request analysis for a single sample?
Yes. Analyses are pooled on PacBio Revio, and you can choose between 5 million or 10 million HiFi reads starting from one sample.

Q: Can I customize the data amount?
Currently, only two scales―5 million or 10 million HiFi reads―are available. Customization is not supported.

Q: Are there recommended RNA extraction kits?
We do not specify a particular kit. We recommend using commercially available RNA extraction kits (e.g., QIAGEN RNeasy) along with DNase treatment.
Quality metrics: RIN ≥ 7, A260/280 between 1.8 and 2.0, A260/230 between 1.3 and 2.5.

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