Sequence I/O

Read, write, and manipulate biological sequence files (FASTA, GenBank, FASTQ).

When to Use This Skill

This skill should be used when:

• Reading or writing sequence files (FASTA, GenBank, FASTQ)
• Converting between sequence file formats
• Manipulating sequences (complement, reverse complement, translate)
• Extracting sequences from large indexed FASTA files (faidx)
• Calculating sequence statistics (GC content, molecular weight, Tm)

When NOT to Use This Skill

• NGS alignment files (SAM/BAM/VCF) → Use pysam
• BLAST searches → Use gget (quick) or blat-integration (large-scale)
• Multiple sequence alignment → Use msa-advanced
• Phylogenetic analysis → Use etetoolkit
• NCBI database queries → Use pubmed-database or gene-database

Tool Selection Guide

Quick Start

Installation

uv pip install biopython pysam

Read FASTA

from Bio import SeqIO

for record in SeqIO.parse("sequences.fasta", "fasta"):
    print(f"{record.id}: {len(record.seq)} bp")

Convert GenBank to FASTA

from Bio import SeqIO

SeqIO.convert("input.gb", "genbank", "output.fasta", "fasta")

Random Access with faidx

import pysam

# Create index (once)
pysam.faidx("reference.fasta")

# Random access
fasta = pysam.FastaFile("reference.fasta")
seq = fasta.fetch("chr1", 1000, 2000)  # 0-based coordinates
fasta.close()

Sequence Operations

from Bio.Seq import Seq

seq = Seq("ATGCGATCGATCG")
print(seq.complement())
print(seq.reverse_complement())
print(seq.translate())

Reference Documentation

Consult the appropriate reference file for detailed documentation:

references/biopython_seqio.md

• Bio.Seq object and sequence operations
• Bio.SeqIO for file parsing and writing
• SeqRecord object and annotations
• Supported file formats
• Format conversion patterns

references/faidx.md

• Creating FASTA index with pysam.faidx()
• pysam.FastaFile for random access
• Coordinate systems (0-based vs 1-based)
• Performance considerations for large files
• Common patterns (variant context, gene extraction)

references/utilities.md

• GC content calculation (gc_fraction)
• Molecular weight (molecular_weight)
• Melting temperature (MeltingTemp)
• Codon usage analysis
• Restriction enzyme sites

references/formats.md

• FASTA format specification
• GenBank format specification
• FASTQ format and quality scores
• Format detection and validation

Coordinate Systems

Biopython: Uses Python-style 0-based, half-open intervals for slicing.

pysam.FastaFile.fetch():

• Numeric arguments: 0-based (fetch("chr1", 999, 2000) = positions 999-1999)
• Region strings: 1-based (fetch("chr1:1000-2000") = positions 1000-2000)

Common Pitfalls

1. Coordinate confusion: Remember which tool uses 0-based vs 1-based
2. Missing faidx index: Random access requires .fai file
3. Format mismatch: Verify file format matches the format string in SeqIO.parse()
4. Iterator exhaustion: SeqIO.parse() returns an iterator; convert to list if multiple passes needed
5. Large files: Use iterators, not list(), for memory efficiency