> ## Documentation Index
> Fetch the complete documentation index at: https://primerlab-genomic.mintlify.site/llms.txt
> Use this file to discover all available pages before exploring further.

# Off-target Detection

> Feature documentation: Off-target Detection

**v0.3.0** - BLAST-based specificity analysis.

## Overview

PrimerLab checks primer specificity using BLAST alignment against reference databases.

## Features

* **Dual Backend**: BLAST+ or Biopython fallback
* **Parallel Processing**: Multi-threaded for speed (v0.3.2)
* **Caching**: Skip redundant alignments
* **Scoring**: A-F grades based on off-target count

## CLI Usage

### Standalone

```bash theme={null}
primerlab blast -p primers.json -d genome.fasta
```

### Integrated with Design

```bash theme={null}
primerlab run pcr --config my_config.yaml --blast --blast-db genome.fasta
```

### Config-based

```yaml theme={null}
# my_config.yaml
offtarget:
  enabled: true
  database: /path/to/genome.fasta
  mode: auto  # auto, blast, biopython
```

## Scoring System

| Grade | Score  | Off-targets | Risk     |
| ----- | ------ | ----------- | -------- |
| A     | 90-100 | 0-2         | Minimal  |
| B     | 80-89  | 3-5         | Low      |
| C     | 70-79  | 6-10        | Moderate |
| D     | 60-69  | 11-20       | High     |
| F     | \<60   | >20         | Severe   |

## Output

```json theme={null}
{
  "forward": {
    "offtargets": 2,
    "score": 95.0
  },
  "reverse": {
    "offtargets": 1,
    "score": 97.5
  },
  "combined_score": 96.25,
  "grade": "A"
}
```

## Backend Selection

| Mode        | Description                             |
| ----------- | --------------------------------------- |
| `auto`      | BLAST+ if available, else Biopython     |
| `blast`     | Force BLAST+ (faster, requires install) |
| `biopython` | Pure Python (slower, always available)  |

## See Also

* [BLAST Command](/docs/reference/cli/blast) - Detailed CLI options
* [In-silico PCR](/docs/reference/cli/insilico) - Template validation