usage-guide.md

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

1. Generate CCS reads from subreads
2. Remove primers and polyA tails
3. Cluster into high-quality isoforms
4. Classify against reference annotation
5. 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