tools-only 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
- Extract UMIs from reads and add to read headers
- Pass through alignment with UMI information preserved
- Deduplicate aligned reads based on UMI + alignment position
- Generate deduplication statistics
- Output deduplicated BAM for downstream analysis
UMI Pattern Syntax
| Pattern | Description |
|---|---|
N |
UMI base |
C |
Cell barcode base |
X |
Base to discard |
NNNNNNNN |
8bp UMI |
CCCCCCCCNNNNNNNN |
Cell barcode + UMI |
Common Library Types
| Library | Pattern |
|---|---|
| NEBNext | NNNNNNNN (8bp in R1) |
| 10X 3' v3 | CCCCCCCCCCCCCCCCNNNNNNNNNNNN (16bp CB + 12bp UMI) |
| TruSeq UMI | NNNNNNNNN (9bp in index) |
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