tools-only Iso-Seq Analysis - Usage Guide
Overview
Process PacBio Iso-Seq data for full-length transcript discovery and isoform characterization.
Prerequisites
conda install -c bioconda pbccs lima isoseq3 sqanti3 minimap2
Quick Start
- "Process my Iso-Seq data"
- "Discover novel isoforms from PacBio"
- "QC my transcript assembly with SQANTI"
Example Prompts
Processing
"Run the Iso-Seq3 pipeline on my subreads"
"Cluster my refined Iso-Seq reads"
Quality Control
"Classify isoforms with SQANTI3"
"How many novel isoforms did I find?"
Quantification
"Quantify isoform expression from Iso-Seq"
"Compare isoform usage between samples"
What the Agent Will Do
- Generate CCS reads from subreads
- Remove primers and polyA tails
- Cluster into high-quality isoforms
- Classify against reference annotation
- Report novel and known isoforms
Tips
- CCS quality - min-rq 0.9 is standard for Iso-Seq
- Primers - Must match your library prep kit
- SQANTI categories - FSM=known, NIC/NNC=novel
- Collapse - Removes redundancy before quantification
- HQ vs LQ - Use high-quality (HQ) transcripts for analysis