Read and Write Sequence Files

SeqIO is the I/O workhorse of Biopython. Three functions cover 90% of the work: parse (streaming), index (random access by ID), and write (single or batch). The remainder is convert for the times you don't need to touch the records. This skill is the cheat sheet I keep open.

When to use

• Streaming reads from FASTA/FASTQ/GenBank/EMBL.
• Random access to a record by ID (huge files).
• Writing records back out, optionally after modification.
• Format conversion without touching record content.

When NOT to use

• BAM/SAM/CRAM/VCF → use pysam (see ors-bioinformatics-sequence-pysam-genomics).
• gzipped BAM/CRAM with random access → pysam.AlignmentFile.
• For >10 GB files, consider pyfastx (faster than Biopython for FASTA).

Prerequisites

• biopython>=1.83
• For indexed random access on BGZF FASTA, Biopython 1.83+ supports index() on .bgz.

Format strings (cheat sheet)

| Format | String | Notes | |--------|--------|-------|" | FASTA | "fasta" | Sequence only, single-line or wrapped | | FASTQ (Sanger / Illumina 1.8+) | "fastq" | Phred+33 | | FASTQ (Illumina 1.3-1.7) | "fastq-illumina" | Phred+64 | | FASTQ (Solexa) | "fastq-solexa" | Solexa+64 | | GenBank | "genbank" | Annotation + sequence | | EMBL | "embl" | EBI submission format | | SwissProt | "swiss" | UniProt curated | | UniProt XML | "uniprot-xml" | EBI download | | GFF3 | "gff3" | Genome features, not sequence records | | GTF | "gtf" | Gene transfer format | | ABI | "abi" | Sanger trace | | Phylip | "phylip" | Alignment | | Clustal | "clustal" | Alignment | | Stockholm | "stockholm" | Pfam/Rfam | | Tab-delimited | "tab" | ID, seq, optional qualifiers |

Code patterns

Stream parse

from Bio import SeqIO

for rec in SeqIO.parse("input.fasta", "fasta"):
    # rec.id, rec.description, rec.seq
    ...

List parse (small files only)

records = list(SeqIO.parse("small.fasta", "fasta"))

Indexed random access (huge FASTA)

from Bio import SeqIO

db = SeqIO.index("large.fasta", "fasta")
seq = db["target_id"].seq
db.close()

For persistent index (no rebuild on next open):

db = SeqIO.index_db("large.idx", "large.fasta", "fasta")

Indexed BGZF random access

db = SeqIO.index("indexable.fasta.bgz", "fasta")

Write records (single file, multiple records)

from Bio import SeqIO

SeqIO.write(records, "out.fasta", "fasta")

The return value is the number of records written.

Write one record at a time

from Bio import SeqIO
from Bio.SeqRecord import SeqRecord
from Bio.Seq import Seq

with open("out.fasta", "w") as fh:
    rec = SeqRecord(Seq("ACGT"), id="seq1", description="example")
    SeqIO.write(rec, fh, "fasta")

Append records across formats (streaming)

from Bio import SeqIO

def stream_to_fasta(in_path, in_fmt, out_path):
    with open(out_path, "w") as out:
        for rec in SeqIO.parse(in_path, in_fmt):
            # modify rec if needed
            SeqIO.write(rec, out, "fasta")

Convert without touching records

from Bio import SeqIO
n = SeqIO.convert("in.gb", "genbank", "out.fasta", "fasta")
print(f"Converted {n} records")

Handle FASTA with wrapped multi-line sequences

Biopython auto-handles wrapped FASTA in parse(); record.seq is always the full sequence.

Parse GenBank with features

from Bio import SeqIO

for rec in SeqIO.parse("annotation.gb", "genbank"):
    for feat in rec.features:
        if feat.type == "CDS":
            gene = feat.qualifiers.get("gene", ["?"])[0]
            print(gene, feat.location)

Parse BLAST tab output

from Bio import SeqIO
for qresult in SeqIO.parse("blast.tab", "blast-tab"):
    for hsp in qresult:
        ...

blast-tab parses the tabular -outfmt 6.

Truncate IDs to first whitespace

def short_id(rec):
    rec.id = rec.id.split()[0]
    rec.description = ""
    return rec

Common pitfalls

• Mixing parse() with index() and forgetting to close the index. Index files hold file handles; use a context manager or explicit close().
• Indexed access on a .fasta.gz (plain gzip) fails. Convert to BGZF first.
• Write format mismatch. If you parsed with "fastq-illumina" and wrote with "fastq", Biopython re-encodes qualities. Usually fine, but worth being explicit.
• Mismatched record count from SeqIO.convert. Returns 0 silently if source format is wrong. assert n > 0.
• Header pollution. FASTA identifiers stop at first whitespace; multi-word descriptions go into description. If you write a record with both id="seq1 chr1" and a description, downstream tools will see seq1 as the ID.

Validation

• n_records_in == n_records_out for round-trips.
• For indexed access: db["known_id"].seq == expected_seq.
• For GenBank round-trips: len(features_in) == len(features_out), modulo per-format feature filtering.

Open alternatives

References

• Biopython SeqIO: https://biopython.org/docs/latest/api/Bio.SeqIO.html
• Format list: https://biopython.org/wiki/SeqIO#File_Formats
• Companion: ors-bioinformatics-sequence-format-conversion, ors-bioinformatics-sequence-compressed-sequence-files.

Changelog

• 1.0.0 (2026-06-10): Initial adaptation by Pradyumna Jayaram from bio-read-sequences and bio-write-sequences (bioSkills-main/sequence-io/).