Iso-Seq

Iso-Seq (Isoform Sequencing) is a long-read RNA sequencing technology developed by Pacific Biosciences (PacBio). that enables the identification and characterization of full-length transcript isoforms without the need for computational assembly.

Overview

Iso-Seq uses PacBio's Single Molecule, Real-Time (SMRT) sequencing technology to generate full-length cDNA sequences from the 5' UTR to the 3' poly-A tail . Unlike traditional short-read RNA sequencing methods, Iso-Seq eliminates errors that occur with short-read sequencing and complex transcript reconstruction using isoform-inference algorithms (Iso-seq) PacBio Isoform Sequencing

Technology

The method is based on sequencing full-length cDNA using PacBio SMRT sequencing technology RNA sequencing. Full-length cDNAs can be generated from eukaryotic RNA, bacterial RNA, or even viral RNA . The technology produces HiFi (High-Fidelity) reads RNA sequencing with high accuracy through circular consensus sequencing (CCS).

Workflow

The Iso-Seq workflow consists of several key steps:

1. CCS generation: Each sequencing run is processed by ccs to generate one HiFi read from productive ZMWs
2. Primer removal and demultiplexing: Removal of primers and identification of cDNA barcodes is performed using lima isoseq: Introduction
3. Refinement: Trimming of poly(A) tails and identification and removal of artificial concatemers
4. Clustering: Grouping similar reads to identify distinct isoforms
5. Polishing: Generating high-quality consensus sequences

Capabilities

• The Iso-Seq method and bioinformatics workflow output high-quality, full-length transcript sequences of 10 kb or longer
• Version 4.0 adds a new cluster2 tool that enables clustering of hundreds of millions of HiFi reads
• Starting with version 3.4, support for UMI and cell barcode based deduplication has been added

Applications

Iso-Seq has been used for:

• Characterizing alternative splicing events
• Discovering novel isoforms and genes
• Single-cell transcriptomics
• Identifying fusion genes in cancer research
• Creating comprehensive transcriptome atlases

Software

IsoSeq contains the newest tools to identify transcripts in PacBio single-molecule sequencing data. Starting in SMRT Link v6.0.0, those tools power the IsoSeq GUI-based analysis application. The software is available through Bioconda and maintained on GitHub by Pacific Biosciences.

See also

• RNA sequencing
• Pacific Biosciences
• Long-read sequencing
• Alternative splicing

External links

• Official Iso-Seq documentation
• PacBio IsoSeq GitHub repository

References

{{Draft categories| [[Category:RNA]] }} {{Draft categories| [[Category:Sequencing techniques]] }} {{Draft categories| [[Category:Bioinformatics]] }} {{Draft categories| [[Category:Transcriptomics]] }}

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