> ## 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.

# BLAST Command

> CLI reference for the BLAST Command command

Off-target primer specificity check using BLAST+ or Biopython fallback.

## Quick Start

```bash theme={null}
# Basic usage with comma-separated primers
primerlab blast -p "ATGACCATGATTACG,GCAACTGTTGGGAAG" -d genome.fasta

# Using primer file (FASTA or JSON)
primerlab blast -p primers.fasta -d database.fasta

# With target filtering
primerlab blast -p primers.json -d genome.fasta --target my_gene
```

## Arguments

| Argument     | Short | Description                                                 |
| ------------ | ----- | ----------------------------------------------------------- |
| `--primers`  | `-p`  | Primer input (JSON, FASTA, or comma-separated) **required** |
| `--database` | `-d`  | Path to FASTA or BLAST database **required**                |
| `--target`   | `-t`  | Expected target sequence ID                                 |
| `--output`   | `-o`  | Output directory (default: `blast_output`)                  |
| `--json`     |       | Output JSON only                                            |
| `--mode`     |       | Alignment mode: `auto`, `blast`, `biopython`                |

## Input Formats

### Comma-separated

```bash theme={null}
primerlab blast -p "ATGACCATGATTACG,GCAACTGTTGGGAAG" -d db.fasta
```

### JSON File

```json theme={null}
{
  "forward": "ATGACCATGATTACG",
  "reverse": "GCAACTGTTGGGAAG"
}
```

### FASTA File

```fasta theme={null}
>forward_primer
ATGACCATGATTACG
>reverse_primer
GCAACTGTTGGGAAG
```

## Output

### Console

```
🔬 Off-target Check (v0.3.0)
   Database: genome.fasta
   Forward:  ATGACCATGATTACG
   Reverse:  GCAACTGTTGGGAAG

✅ Specificity Score: 95.0 (Grade: A)
   Forward: 2 off-targets
   Reverse: 1 off-target

✅ Primers are specific!

📁 Output: blast_output
   • blast_result.json
   • specificity_report.md
```

### JSON Output (`blast_result.json`)

```json theme={null}
{
  "forward": {
    "offtargets": 2,
    "score": 92.5
  },
  "reverse": {
    "offtargets": 1,
    "score": 97.5
  },
  "combined_score": 95.0,
  "grade": "A",
  "is_specific": true
}
```

## Scoring

| Grade | Score  | Interpretation        |
| ----- | ------ | --------------------- |
| A     | 90-100 | Excellent specificity |
| B     | 80-89  | Good specificity      |
| C     | 70-79  | Acceptable            |
| D     | 60-69  | Low specificity       |
| F     | \<60   | Poor specificity      |

## Alignment Modes

* **auto** (default): Uses BLAST+ if installed, otherwise Biopython
* **blast**: Force BLAST+ (requires installation)
* **biopython**: Force Biopython pairwise aligner

## Integration with Design

Use `--blast` flag after primer design:

```bash theme={null}
# Design and check in one command (if config has offtarget enabled)
primerlab run --config my_config.yaml --blast --blast-db genome.fasta
```

Or enable in config:

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

## See Also

* [In-silico PCR](insilico) - Validate primers against template
* [PCR Workflow](/docs/guides/pcr-design) - Full primer design pipeline