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UMI Processing - Usage Guide

Overview

Unique Molecular Identifiers (UMIs) are random sequences added to molecules before PCR amplification. They enable distinguishing PCR duplicates from biological duplicates, crucial for accurate quantification in RNA-seq, targeted sequencing, and single-cell applications.

Prerequisites

conda install -c bioconda umi_tools

Quick Start

Tell your AI agent what you want to do:

• "Extract UMIs from my reads and deduplicate after alignment"
• "Process my UMI-tagged library for accurate quantification"
• "Remove PCR duplicates using UMI information"

Example Prompts

Standard Workflow

"Extract 8bp UMIs from read 1 and move them to the read header"

"Deduplicate my aligned BAM file using UMI information"

Library-Specific

"Process my 10X Genomics library with cell barcodes and UMIs"

"Handle my NEBNext library with 8bp UMIs"

Deduplication Options

"Use directional deduplication method for my RNA-seq data"

"Deduplicate with cluster method for high-error-rate UMIs"

What the Agent Will Do

1. Extract UMIs from reads and add to read headers
2. Pass through alignment with UMI information preserved
3. Deduplicate aligned reads based on UMI + alignment position
4. Generate deduplication statistics
5. Output deduplicated BAM for downstream analysis

UMI Pattern Syntax

Common Library Types

Tips

• Extract UMIs before alignment; they must be in read headers for deduplication
• Directional deduplication is recommended for most RNA-seq applications
• High deduplication rates (>70%) may indicate library over-amplification
• Low deduplication rates (<10%) may indicate under-sequencing
• Check UMI diversity in the deduplication stats to assess library complexity

Resources

• umi_tools Documentation
• umi_tools Publication