# BLAST Operations with Bio.Blast

## Overview

Bio.Blast provides tools for running BLAST searches (both locally and via NCBI web services) and parsing BLAST results in various formats. The module handles the complexity of submitting queries and parsing outputs.

## Running BLAST via NCBI Web Services

### Bio.Blast.NCBIWWW

The `qblast()` function submits sequences to NCBI's online BLAST service:

```python
from Bio.Blast import NCBIWWW
from Bio import SeqIO

# Read sequence from file
record = SeqIO.read("sequence.fasta", "fasta")

# Run BLAST search
result_handle = NCBIWWW.qblast(
    program="blastn",           # BLAST program
    database="nt",              # Database to search
    sequence=str(record.seq)    # Query sequence
)

# Save results
with open("blast_results.xml", "w") as out_file:
    out_file.write(result_handle.read())
result_handle.close()
```

### BLAST Programs Available

- **blastn** - Nucleotide vs nucleotide
- **blastp** - Protein vs protein
- **blastx** - Translated nucleotide vs protein
- **tblastn** - Protein vs translated nucleotide
- **tblastx** - Translated nucleotide vs translated nucleotide

### Common Databases

**Nucleotide databases:**
- `nt` - All GenBank+EMBL+DDBJ+PDB sequences
- `refseq_rna` - RefSeq RNA sequences

**Protein databases:**
- `nr` - All non-redundant GenBank CDS translations
- `refseq_protein` - RefSeq protein sequences
- `pdb` - Protein Data Bank sequences
- `swissprot` - Curated UniProtKB/Swiss-Prot

### Advanced qblast Parameters

```python
result_handle = NCBIWWW.qblast(
    program="blastn",
    database="nt",
    sequence=str(record.seq),
    expect=0.001,              # E-value threshold
    hitlist_size=50,           # Number of hits to return
    alignments=25,             # Number of alignments to show
    word_size=11,              # Word size for initial match
    gapcosts="5 2",            # Gap costs (open extend)
    format_type="XML"          # Output format (default)
)
```

### Using Sequence Files or IDs

```python
# Use FASTA format string
fasta_string = open("sequence.fasta").read()
result_handle = NCBIWWW.qblast("blastn", "nt", fasta_string)

# Use GenBank ID
result_handle = NCBIWWW.qblast("blastn", "nt", "EU490707")

# Use GI number
result_handle = NCBIWWW.qblast("blastn", "nt", "160418")
```

## Parsing BLAST Results

### Bio.Blast.NCBIXML

NCBIXML provides parsers for BLAST XML output (the recommended format):

```python
from Bio.Blast import NCBIXML

# Parse single BLAST result
with open("blast_results.xml") as result_handle:
    blast_record = NCBIXML.read(result_handle)
```

### Accessing BLAST Record Data

```python
# Query information
print(f"Query: {blast_record.query}")
print(f"Query length: {blast_record.query_length}")
print(f"Database: {blast_record.database}")
print(f"Number of sequences in database: {blast_record.database_sequences}")

# Iterate through alignments (hits)
for alignment in blast_record.alignments:
    print(f"\nHit: {alignment.title}")
    print(f"Length: {alignment.length}")
    print(f"Accession: {alignment.accession}")

    # Each alignment can have multiple HSPs (high-scoring pairs)
    for hsp in alignment.hsps:
        print(f"  E-value: {hsp.expect}")
        print(f"  Score: {hsp.score}")
        print(f"  Bits: {hsp.bits}")
        print(f"  Identities: {hsp.identities}/{hsp.align_length}")
        print(f"  Gaps: {hsp.gaps}")
        print(f"  Query: {hsp.query}")
        print(f"  Match: {hsp.match}")
        print(f"  Subject: {hsp.sbjct}")
```

### Filtering Results

```python
# Only show hits with E-value < 0.001
E_VALUE_THRESH = 0.001

for alignment in blast_record.alignments:
    for hsp in alignment.hsps:
        if hsp.expect < E_VALUE_THRESH:
            print(f"Hit: {alignment.title}")
            print(f"E-value: {hsp.expect}")
            print(f"Identities: {hsp.identities}/{hsp.align_length}")
            print()
```

### Multiple BLAST Results

For files containing multiple BLAST results (e.g., from batch searches):

```python
from Bio.Blast import NCBIXML

with open("batch_blast_results.xml") as result_handle:
    blast_records = NCBIXML.parse(result_handle)

    for blast_record in blast_records:
        print(f"\nQuery: {blast_record.query}")
        print(f"Hits: {len(blast_record.alignments)}")

        if blast_record.alignments:
            # Get best hit
            best_alignment = blast_record.alignments[0]
            best_hsp = best_alignment.hsps[0]
            print(f"Best hit: {best_alignment.title}")
            print(f"E-value: {best_hsp.expect}")
```

## Running Local BLAST

### Prerequisites

Local BLAST requires:
1. BLAST+ command-line tools installed
2. BLAST databases downloaded locally

### Running BLAST+ via subprocess

Biopython 1.86 removed `Bio.Application` and the BLAST command-line wrappers in `Bio.Blast.Applications`. Use the standard library `subprocess` module with explicit argument lists. Keep command names and flags fixed, validate file paths before running, and do not interpolate unsanitized user input into shell commands.

```python
import subprocess
from Bio.Blast import NCBIXML

cmd = [
    "blastn",
    "-query", "input.fasta",
    "-db", "local_database",
    "-evalue", "0.001",
    "-outfmt", "5",  # XML format for NCBIXML
    "-out", "results.xml",
]
subprocess.run(cmd, check=True)

# Parse results
with open("results.xml") as result_handle:
    blast_record = NCBIXML.read(result_handle)
```

### Common BLAST+ commands

- `blastn` - nucleotide vs nucleotide
- `blastp` - protein vs protein
- `blastx` - translated nucleotide vs protein
- `tblastn` - protein vs translated nucleotide
- `tblastx` - translated nucleotide vs translated nucleotide
- `makeblastdb` - create local BLAST databases

### Creating BLAST Databases

```python
import subprocess

cmd = [
    "makeblastdb",
    "-in", "sequences.fasta",
    "-dbtype", "nucl",
    "-out", "my_database",
]
subprocess.run(cmd, check=True)
```

## Analyzing BLAST Results

### Extract Best Hits

```python
def get_best_hits(blast_record, num_hits=10, e_value_thresh=0.001):
    """Extract best hits from BLAST record."""
    hits = []
    for alignment in blast_record.alignments[:num_hits]:
        for hsp in alignment.hsps:
            if hsp.expect < e_value_thresh:
                hits.append({
                    'title': alignment.title,
                    'accession': alignment.accession,
                    'length': alignment.length,
                    'e_value': hsp.expect,
                    'score': hsp.score,
                    'identities': hsp.identities,
                    'align_length': hsp.align_length,
                    'query_start': hsp.query_start,
                    'query_end': hsp.query_end,
                    'sbjct_start': hsp.sbjct_start,
                    'sbjct_end': hsp.sbjct_end
                })
                break  # Only take best HSP per alignment
    return hits
```

### Calculate Percent Identity

```python
def calculate_percent_identity(hsp):
    """Calculate percent identity for an HSP."""
    return (hsp.identities / hsp.align_length) * 100

# Use it
for alignment in blast_record.alignments:
    for hsp in alignment.hsps:
        if hsp.expect < 0.001:
            identity = calculate_percent_identity(hsp)
            print(f"{alignment.title}: {identity:.2f}% identity")
```

### Extract Hit Sequences

```python
from Bio import Entrez, SeqIO

Entrez.email = "your.email@example.com"

def fetch_hit_sequences(blast_record, num_sequences=5):
    """Fetch sequences for top BLAST hits."""
    sequences = []

    for alignment in blast_record.alignments[:num_sequences]:
        accession = alignment.accession

        # Fetch sequence from GenBank
        handle = Entrez.efetch(
            db="nucleotide",
            id=accession,
            rettype="fasta",
            retmode="text"
        )
        record = SeqIO.read(handle, "fasta")
        handle.close()

        sequences.append(record)

    return sequences
```

## Parsing Other BLAST Formats

### Tab-Delimited Output (outfmt 6/7)

```python
import subprocess

cmd = [
    "blastn",
    "-query", "input.fasta",
    "-db", "database",
    "-outfmt", "6",
    "-out", "results.txt",
]
subprocess.run(cmd, check=True)

# Parse tabular results
with open("results.txt") as f:
    for line in f:
        fields = line.strip().split('\t')
        query_id = fields[0]
        subject_id = fields[1]
        percent_identity = float(fields[2])
        align_length = int(fields[3])
        e_value = float(fields[10])
        bit_score = float(fields[11])

        print(f"{query_id} -> {subject_id}: {percent_identity}% identity, E={e_value}")
```

### Custom Output Formats

```python
import subprocess

# Specify custom columns (outfmt 6 with custom fields)
cmd = [
    "blastn",
    "-query", "input.fasta",
    "-db", "database",
    "-outfmt", "6 qseqid sseqid pident length evalue bitscore qseq sseq",
    "-out", "results.txt",
]
subprocess.run(cmd, check=True)
```

## Best Practices

1. **Use XML format** for parsing (outfmt 5) - most reliable and complete
2. **Save BLAST results** - Don't re-run searches unnecessarily
3. **Set appropriate E-value thresholds** - Default is 10, but 0.001-0.01 is often better
4. **Handle rate limits** - NCBI limits request frequency
5. **Use local BLAST** for large-scale searches or repeated queries
6. **Cache results** - Save parsed data to avoid re-parsing
7. **Check for empty results** - Handle cases with no hits gracefully
8. **Consider alternatives** - For large datasets, consider DIAMOND or other fast aligners
9. **Batch searches** - Submit multiple sequences together when possible
10. **Filter by identity** - E-value alone may not be sufficient

## Common Use Cases

### Basic BLAST Search and Parse

```python
from Bio.Blast import NCBIWWW, NCBIXML
from Bio import SeqIO

# Read query sequence
record = SeqIO.read("query.fasta", "fasta")

# Run BLAST
print("Running BLAST search...")
result_handle = NCBIWWW.qblast("blastn", "nt", str(record.seq))

# Parse results
blast_record = NCBIXML.read(result_handle)

# Display top 5 hits
print(f"\nTop 5 hits for {blast_record.query}:")
for i, alignment in enumerate(blast_record.alignments[:5], 1):
    hsp = alignment.hsps[0]
    identity = (hsp.identities / hsp.align_length) * 100
    print(f"{i}. {alignment.title}")
    print(f"   E-value: {hsp.expect}, Identity: {identity:.1f}%")
```

### Find Orthologs

```python
from Bio.Blast import NCBIWWW, NCBIXML
from Bio import Entrez, SeqIO

Entrez.email = "your.email@example.com"

# Query gene sequence
query_record = SeqIO.read("gene.fasta", "fasta")

# BLAST against specific organism
result_handle = NCBIWWW.qblast(
    "blastn",
    "nt",
    str(query_record.seq),
    entrez_query="Mus musculus[Organism]"  # Restrict to mouse
)

blast_record = NCBIXML.read(result_handle)

# Find best hit
if blast_record.alignments:
    best_hit = blast_record.alignments[0]
    print(f"Potential ortholog: {best_hit.title}")
    print(f"Accession: {best_hit.accession}")
```

### Batch BLAST Multiple Sequences

```python
from Bio.Blast import NCBIWWW, NCBIXML
from Bio import SeqIO

# Read multiple sequences
sequences = list(SeqIO.parse("queries.fasta", "fasta"))

# Create batch results file
with open("batch_results.xml", "w") as out_file:
    for seq_record in sequences:
        print(f"Searching for {seq_record.id}...")

        result_handle = NCBIWWW.qblast("blastn", "nt", str(seq_record.seq))
        out_file.write(result_handle.read())
        result_handle.close()

# Parse batch results
with open("batch_results.xml") as result_handle:
    for blast_record in NCBIXML.parse(result_handle):
        print(f"\n{blast_record.query}: {len(blast_record.alignments)} hits")
```

### Reciprocal Best Hits

```python
def reciprocal_best_hit(seq1_id, seq2_id, database="nr", program="blastp"):
    """Check if two sequences are reciprocal best hits."""
    from Bio.Blast import NCBIWWW, NCBIXML
    from Bio import Entrez

    Entrez.email = "your.email@example.com"

    # Forward BLAST
    result1 = NCBIWWW.qblast(program, database, seq1_id)
    record1 = NCBIXML.read(result1)
    best_hit1 = record1.alignments[0].accession if record1.alignments else None

    # Reverse BLAST
    result2 = NCBIWWW.qblast(program, database, seq2_id)
    record2 = NCBIXML.read(result2)
    best_hit2 = record2.alignments[0].accession if record2.alignments else None

    # Check reciprocity
    return best_hit1 == seq2_id and best_hit2 == seq1_id
```

## Error Handling

```python
from Bio.Blast import NCBIWWW, NCBIXML
from urllib.error import HTTPError

try:
    result_handle = NCBIWWW.qblast("blastn", "nt", "ATCGATCGATCG")
    blast_record = NCBIXML.read(result_handle)
    result_handle.close()
except HTTPError as e:
    print(f"HTTP Error: {e.code}")
except Exception as e:
    print(f"Error running BLAST: {e}")
```
