BLAST+ 2.17.0 - User Guide

Cluster: XLence (UNIMI Dipartimento di Scienze Farmacologiche e Biomolecolari) Date: October 12, 2025 Installation: /sw/blast/2.17.0/

📦 Version Information

BLAST+: 2.17.0 Release Date: July 1, 2025 Type: Precompiled binaries (x86_64 Linux)

🚀 Loading the Module

module load blast/2.17.0

# Verify installation
blastn -version

🔍 BLAST Programs

Core BLAST Programs

Program	Description	Query → Database
blastn	Nucleotide BLAST	Nucleotide → Nucleotide
blastp	Protein BLAST	Protein → Protein
blastx	Translated BLAST	Translated Nucleotide → Protein
tblastn	Translated database	Protein → Translated Nucleotide
tblastx	Both translated	Translated Nucleotide → Translated Nucleotide

Additional Tools

makeblastdb: Create BLAST databases from FASTA files
update_blastdb.pl: Download preformatted NCBI databases
blast_formatter: Reformat BLAST archive files
blastdbcmd: Extract sequences from BLAST databases

📖 Basic Usage Examples

1. Nucleotide BLAST (blastn)

module load blast/2.17.0

# Simple query against database
blastn -query sequences.fasta -db nt -out results.txt

# With output format and E-value threshold
blastn -query sequences.fasta -db nt -evalue 1e-10 -outfmt 6 -out results.tsv

# With multiple threads
blastn -query sequences.fasta -db nt -num_threads 8 -out results.txt

2. Protein BLAST (blastp)

# Search protein sequence against protein database
blastp -query protein.fasta -db nr -evalue 1e-5 -out results.txt

# Tabular output with custom fields
blastp -query protein.fasta -db swissprot \
  -outfmt "6 qseqid sseqid pident length evalue bitscore stitle" \
  -out results.tsv

3. Translated BLAST (blastx)

# Translate nucleotide query, search against protein database
blastx -query nucleotide.fasta -db nr -evalue 1e-10 -out results.txt

🗄️ Working with Databases

Download NCBI Databases

module load blast/2.17.0

# List available databases
update_blastdb.pl --showall

# Download a database (e.g., nt, nr, swissprot)
update_blastdb.pl --decompress nt

# Download to specific directory
update_blastdb.pl --decompress nr --blastdb_version 5

Note: Databases are large! Example sizes: - nt (nucleotide): ~100 GB - nr (non-redundant protein): ~250 GB - swissprot: ~500 MB

Create Custom Database

# From FASTA file
makeblastdb -in mysequences.fasta -dbtype nucl -out mydb

# For protein sequences
makeblastdb -in proteins.fasta -dbtype prot -out proteindb

# With taxonomy information
makeblastdb -in sequences.fasta -dbtype nucl -out mydb \
  -parse_seqids -taxid_map taxid.map

Set Database Location

# Option 1: Use environment variable (automatically set by module)
export BLASTDB=/sw/blast/2.17.0/db

# Option 2: Specify with -db flag
blastn -query seq.fasta -db /path/to/database/dbname -out results.txt

📊 Output Formats

Common Output Formats (`-outfmt`)

Format	Description
`0`	Pairwise alignment (default)
`6`	Tabular (TSV)
`7`	Tabular with comment lines
`10`	CSV
`11`	BLAST archive (ASN.1)
`17`	SAM format
`18`	Organism report

Custom Tabular Output

# Specify custom columns
blastn -query seq.fasta -db nt \
  -outfmt "6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore" \
  -out custom_results.tsv

Available fields: - qseqid - Query sequence ID - sseqid - Subject sequence ID - pident - Percentage of identical matches - length - Alignment length - mismatch - Number of mismatches - gapopen - Number of gap openings - qstart qend - Query alignment start/end - sstart send - Subject alignment start/end - evalue - Expect value - bitscore - Bit score - stitle - Subject title

🔧 Advanced Options

Filtering and Thresholds

# E-value threshold
blastn -query seq.fasta -db nt -evalue 1e-10

# Percent identity filter
blastn -query seq.fasta -db nt -perc_identity 90

# Query coverage filter
blastn -query seq.fasta -db nt -qcov_hsp_perc 80

# Maximum number of hits
blastn -query seq.fasta -db nt -max_target_seqs 10

Performance Tuning

# Use multiple threads
blastn -query seq.fasta -db nt -num_threads 16

# Word size (smaller = more sensitive, slower)
blastn -query seq.fasta -db nt -word_size 11

# Use best hit overhang for shorter sequences
blastn -query seq.fasta -db nt -best_hit_overhang 0.1 -best_hit_score_edge 0.1

📝 Example Workflows

Workflow 1: Local Database Search

#!/bin/bash
module load blast/2.17.0

# 1. Create database from FASTA
makeblastdb -in reference.fasta -dbtype nucl -out refdb

# 2. Run BLAST search
blastn -query queries.fasta -db refdb \
  -outfmt "6 qseqid sseqid pident evalue bitscore" \
  -evalue 1e-10 -num_threads 8 \
  -out blast_results.tsv

# 3. Filter results (e.g., >95% identity)
awk '$3 >= 95' blast_results.tsv > high_identity_hits.tsv

Workflow 2: Remote BLAST Search

# Search against NCBI nr database remotely
blastp -query protein.fasta -db nr -remote -out results.txt

# Note: Remote searches are slower but don't require local database

Workflow 3: Batch Processing

#!/bin/bash
module load blast/2.17.0

# Process multiple query files
for query in queries/*.fasta; do
  basename=$(basename $query .fasta)
  blastn -query $query -db nt \
    -outfmt 6 -num_threads 4 \
    -out results/${basename}_blast.tsv
done

🎯 BLAST Strategies

High Sensitivity (slow)

blastn -query seq.fasta -db nt \
  -task blastn \
  -word_size 11 \
  -evalue 1e-10 \
  -num_threads 16

High Speed (less sensitive)

blastn -query seq.fasta -db nt \
  -task megablast \
  -word_size 28 \
  -perc_identity 95 \
  -num_threads 16

Short Sequences (<50bp)

blastn -query short_seq.fasta -db nt \
  -task blastn-short \
  -word_size 7 \
  -evalue 1000

🐛 Troubleshooting

"Database not found"

Check $BLASTDB environment variable: echo $BLASTDB
Verify database exists: ls $BLASTDB
Use full path to database: blastn -db /full/path/to/dbname

"Out of memory"

Reduce -num_threads
Use smaller database or split queries
Increase -max_target_seqs threshold

"Low complexity filter"

BLAST filters low-complexity regions by default
To disable: blastn -dust no (nucleotide) or blastp -seg no (protein)

Slow searches

Use megablast task for similar sequences
Increase word size (-word_size)
Use local database instead of remote

📚 Documentation

Local: - Installation: /sw/blast/2.17.0/ - Binaries: /sw/blast/2.17.0/bin/ - Databases: /sw/blast/2.17.0/db/ (create with update_blastdb.pl)

Online: - BLAST+ Manual: https://www.ncbi.nlm.nih.gov/books/NBK279690/ - BLAST Web Interface: https://blast.ncbi.nlm.nih.gov/ - Database Download: https://ftp.ncbi.nlm.nih.gov/blast/db/ - BLAST Help: https://blast.ncbi.nlm.nih.gov/doc/blast-help/

Command-line help:

blastn -help
makeblastdb -help
update_blastdb.pl --help

📝 Citation

Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T.L. BLAST+: architecture and applications. BMC Bioinformatics 10, 421 (2009)

🔄 Quick Reference

Task	Command
Nucleotide vs nucleotide	`blastn -query seq.fasta -db nt`
Protein vs protein	`blastp -query prot.fasta -db nr`
Translated nucl vs protein	`blastx -query seq.fasta -db nr`
Create nucleotide database	`makeblastdb -in seqs.fasta -dbtype nucl -out dbname`
Create protein database	`makeblastdb -in prots.fasta -dbtype prot -out dbname`
Download NCBI database	`update_blastdb.pl --decompress nt`
Tabular output	`blastn -outfmt 6`
XML output	`blastn -outfmt 5`

For questions contact: Cluster administrators Installation: October 12, 2025 Installed by: Claude Code + Uliano Guerrini