What is Isoform Sequencing (Iso-Seq)?

Isoform Sequencing (Iso-Seq) is a full-length transcriptome sequencing method developed by PacBio (Pacific Biosciences). Unlike short-read sequencing, which often struggles with reconstructing transcript isoforms, Iso-Seq provides complete transcript structures in a single read—capturing transcription start sites (TSS), exon-exon junctions, alternative splicing, and polyadenylation sites without the need for assembly.

Overview

• Platform: PacBio Single Molecule Real-Time (SMRT) Sequencing

• Read Length: Full-length transcripts (up to ~10 kb+)

• Purpose: To identify and quantify all transcript isoforms in a sample

• Key Output: High-confidence, full-length, non-concatemer (FLNC) transcripts

How Iso-Seq Works

1. Total RNA Extraction
   High-quality RNA is extracted from the biological sample.

2. mRNA Enrichment or cDNA Synthesis
   Poly-A selection is used to enrich for mRNA or full-length cDNA is synthesized using oligo-dT primers.

3. cDNA Amplification
   Full-length cDNA is amplified using PCR.

4. Library Preparation
   SMRTbell adapters are ligated to the cDNA ends to form circular templates.

5. PacBio SMRT Sequencing
   Long reads are generated by the PacBio sequencer using real-time imaging of DNA polymerase.

6. Bioinformatics Pipeline
   PacBio’s Iso-Seq analysis pipeline removes concatemers, clusters similar transcripts, and generates high-quality isoform sequences.

Key Features of Iso-Seq

Why Use PacBio for Isoform Sequencing?

• Accuracy with HiFi Reads: Uses consensus sequences for >99.9% base accuracy.

• Structural Resolution: Unambiguous reconstruction of splice variants and novel isoforms.

• Single-Molecule Long Reads: Eliminates the need for complex assembly algorithms.

• Tissue-Specific Insights: Enables comparison of isoform expression across tissues or conditions.

Applications of Iso-Seq

• Alternative Splicing Analysis
  Discovers novel and known splice variants missed by short-read technologies.

• Gene Annotation Improvement
  Enhances the accuracy and completeness of genome annotations.

• Cancer Transcriptome Profiling
  Detects tumor-specific isoforms that may act as biomarkers or therapeutic targets.

• Neurobiology
  Captures complex isoforms involved in neural regulation and brain development.

• Plant and Animal Genomics
  Used in species with incomplete or complex genomes to discover novel transcripts.

• Single-Cell Isoform Sequencing
  Enables isoform detection at the single-cell level (via technologies like MAS-Seq).

Advantages of Iso-Seq Using PacBio

• Captures true transcript diversity

• Detects fusion transcripts and read-through events

• High-resolution transcriptome mapping for non-model organisms

• Ideal for low-expression genes due to sensitive detection

• Cross-species compatible with minimal annotation dependency

Limitations and Considerations

• Cost: PacBio sequencing is more expensive than short-read methods.

• Throughput: Lower than Illumina in terms of number of reads.

• Input Requirements: High-quality, high-quantity RNA needed.

• Read Depth: May require more sequencing to capture low-abundance isoforms.

• Complexity: Bioinformatics analysis may require specialized tools or support.

Popular Tools for Iso-Seq Data Analysis

• PacBio SMRT Link (Iso-Seq pipeline) – Official processing and filtering pipeline

• IsoSeq3 – Improved toolkit for FLNC detection and clustering

• TALON – Identifies known and novel transcript models from Iso-Seq data

• SQANTI3 – Structural classification and quality assessment of isoforms

• FLAIR – Isoform-level analysis and quantification from long-read data

• Cupcake ToFU – Collapses redundant isoforms for clean transcriptome output

Comparison: Iso-Seq vs Other RNA-Seq Techniques