Version Compatibility

Reference examples tested with: BioPython 1.83+, htslib/bgzip 1.19+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Compressed Files

Read, write, and randomly access gzip, bzip2, xz, and BGZF compressed sequence files.

"Read a compressed sequence file" -> Open a decompression handle in TEXT mode, then parse with the standard SeqIO interface.

• gzip: gzip.open(path, 'rt') (Python stdlib)
• bzip2: bz2.open(path, 'rt') (Python stdlib)
• xz/LZMA: lzma.open(path, 'rt') (Python stdlib)
• BGZF: bgzf.open(path, 'rt') (BioPython) - BGZF input ONLY

"Make a compressed file randomly accessible" -> Re-compress as BGZF, then index. Only BGZF supports SeqIO.index(), samtools faidx, and tabix on compressed data.

The Governing Principle: BGZF vs plain gzip is asymmetric

A BGZF (Blocked GNU Zip) file IS a valid gzip file - gunzip and zcat read it transparently. The reverse is FALSE: a plain .gz is NOT BGZF, so faidx/tabix/SeqIO.index reject it, and bgzf.open refuses to read it.

The reason is structural. BGZF is a series of concatenated gzip blocks, each <=64 KiB and independently decodable, so any record can be reached by seeking to its block. Plain gzip is one continuous DEFLATE stream with no block boundaries: reaching byte N means decompressing every byte before it (O(n)). Random access therefore REQUIRES BGZF; on a plain .gz, SeqIO.index() would re-decompress huge prefixes on every lookup, which is why Biopython forbids it outright (it raises rather than running slowly).

Consequences the agent must respect:

• Reading sequentially: gzip, bzip2, xz, and BGZF all work via the matching *.open(path, 'rt') handle.
• Random access / indexing: BGZF only. Convert plain gzip to BGZF first.
• bgzf.open() reads BGZF input only. Pointing it at a plain .gz raises ValueError: A BGZF block should start with b'\x1f\x8b\x08\x04'.... To read plain gzip use gzip.open().
• BAM and tabix-indexed files use BGZF natively; bzip2/xz are archive-only (no seekable index).

Required Imports

import gzip
import bz2
import lzma
from Bio import SeqIO
from Bio import bgzf

Reading Compressed Files

Goal: Parse sequence records from a compressed file without decompressing to disk.

Approach: Open a decompression handle in TEXT mode ('rt'), then pass the handle to SeqIO.parse(). The parser is format-agnostic about the underlying compression.

with gzip.open('reads.fastq.gz', 'rt') as handle:
    for record in SeqIO.parse(handle, 'fastq'):
        print(record.id, len(record.seq))

Swap gzip.open for bz2.open (.bz2), lzma.open (.xz), or bgzf.open (.bgz) - the parse loop is identical.

The 'rt' vs 'rb' trap

SeqIO.parse() in Python 3 needs a TEXT handle that yields str. A binary 'rb' handle yields bytes and raises TypeError: a bytes-like object is required (or a decode error). Always use 'rt' for reading and 'wt' for writing through SeqIO. The low-level SimpleFastaParser/FastqGeneralIterator also require text handles.

Writing Compressed Files

Goal: Save records straight to a compressed file with no intermediate plain copy.

Approach: Open a compression handle in TEXT mode ('wt'), then pass it to SeqIO.write().

with gzip.open('output.fasta.gz', 'wt') as handle:
    SeqIO.write(records, handle, 'fasta')

For an indexable result write BGZF instead:

with bgzf.open('output.fasta.bgz', 'wt') as handle:
    SeqIO.write(records, handle, 'fasta')

BgzfWriter.close() (and the with block exit) automatically appends the 28-byte empty-block EOF marker that htslib tools check for; let the context manager close the handle.

Random Access: index a BGZF file

Goal: Pull individual records by id from a large compressed file without a linear scan.

Approach: Compress as BGZF, then build a Biopython offset index. SeqIO.index() keeps virtual offsets in RAM; SeqIO.index_db() stores them in an on-disk SQLite index that persists across sessions and spans multiple files.

records = SeqIO.index('sequences.fasta.bgz', 'fasta')
target = records['gene_042'].seq
records.close()

# Persistent, multi-file, scales beyond RAM:
db = SeqIO.index_db('idx.sqlite', ['a.fasta.bgz', 'b.fasta.bgz'], 'fasta')

SeqIO.index() on a plain .gz raises ValueError: Gzipped files are not suitable for indexing, please use BGZF (blocked gzip format) instead. Convert first (below).

Convert plain gzip to BGZF

"Convert gzip to an indexable format" -> Decompress the gzip stream and re-compress it as BGZF.

CLI (fastest, htslib-native):

# Either decompress then bgzip in place...
gzip -d sequences.fasta.gz && bgzip sequences.fasta      # -> sequences.fasta.gz (now BGZF)
# ...or stream without touching disk:
zcat sequences.fasta.gz | bgzip -@ 4 > sequences.fasta.bgz

# Index a BGZF FASTA for region extraction:
samtools faidx sequences.fasta.bgz                        # writes BOTH .fai and .gzi
samtools faidx sequences.fasta.bgz gene_042:1-200

samtools faidx on a BGZF FASTA writes TWO index files: .fai (record offsets in uncompressed coordinates) AND .gzi (the compressed-to-uncompressed block map). Deleting .gzi breaks region extraction even though .fai survives. Note that bgzip keeps the .gz extension, so a .gz may be EITHER plain gzip or BGZF - check with bgzip -t file.gz (tests for a valid BGZF stream) rather than trusting the suffix.

Pure-Python equivalent (no external tools):

import gzip
from Bio import SeqIO, bgzf

with gzip.open('input.fasta.gz', 'rt') as in_handle:
    with bgzf.open('output.fasta.bgz', 'wt') as out_handle:
        SeqIO.write(SeqIO.parse(in_handle, 'fasta'), out_handle, 'fasta')

Bio.bgzf API and virtual offsets

Bio.bgzf exports open, BgzfReader, BgzfWriter, make_virtual_offset, split_virtual_offset.

A virtual offset packs two coordinates into one 64-bit integer: voffset = coffset << 16 | uoffset, where coffset is the byte position of the block start in the compressed file (top 48 bits) and uoffset is the offset within that block's decompressed data (low 16 bits - 16 bits suffices because a block holds at most 64 KiB).

vo = bgzf.make_virtual_offset(100, 7)        # 6553607
coffset, uoffset = bgzf.split_virtual_offset(vo)   # (100, 7)

with bgzf.open('sequences.fasta.bgz', 'rt') as handle:
    handle.readline()
    saved = handle.tell()        # a VIRTUAL offset, not a byte position
    handle.seek(saved)           # jumps back to the same record

Critical caveat: virtual offsets may be COMPARED for ordering but NEVER SUBTRACTED to get a byte length - they live in two coordinate spaces (compressed position and within-block position), so vo2 - vo1 is meaningless. BgzfReader.tell() returns a virtual offset; seek() consumes one. Text mode forces latin1 and does no newline translation.

Compression Format Comparison

Format	Extension	Random access	Speed	Ratio	Stdlib handle
gzip	.gz	No (O(n) seek)	Fast	Good	gzip.open
BGZF	.bgz / .gz	Yes (block-seekable)	Fast, threadable	Good	bgzf.open (BioPython)
bzip2	.bz2	No	Slow	Better	bz2.open
xz / LZMA	.xz	No	Slowest	Best	lzma.open

When to Use Each Format

Use case	Format	Why
Sequential read/write, sharing	gzip	Universal, fast, every tool reads it
Need faidx/tabix/SeqIO.index	BGZF	Only seekable compressed format
BAM, tabix-indexed VCF/GFF/BED	BGZF	Required natively
Cold archive, max shrink, no random access	xz then bzip2	Highest ratios, slowest
Random access into an existing plain .gz without re-bgzipping	pyfastx	Adds a seek-point index over the gzip stream

pyfastx is the exception that gives random access into a PLAIN gzip FASTA/FASTQ: it builds a seek-point index (via zran from indexed_gzip) plus a SQLite .fxi/.fqi index alongside the file, a different strategy from faidx (which requires the stream itself to be BGZF). Use it when re-compressing a large gzipped genome to BGZF is not an option.

Common Errors

Symptom	Cause	Fix
TypeError: a bytes-like object is required	Handle opened 'rb' instead of 'rt'	Open compressed handles with 'rt'/'wt' for SeqIO
ValueError: Gzipped files are not suitable for indexing, please use BGZF...	SeqIO.index() on a plain .gz	Re-compress as BGZF (`zcat ...
ValueError: A BGZF block should start with b'\x1f\x8b\x08\x04'...	bgzf.open() pointed at a plain gzip file	Read plain gzip with gzip.open(); reserve bgzf.open for BGZF
[bgzf] file ... not BGZF / not compressed with bgzip (htslib)	faidx/tabix given a plain .gz	Convert to BGZF first
[faidx] Failed to read ... / could not load .gzi	.gzi deleted next to a BGZF FASTA	Re-run samtools faidx to regenerate .fai + .gzi
gzip.BadGzipFile / OSError: Not a gzipped file	File is not gzip (wrong suffix / corrupt)	Verify with bgzip -t or file; match handle to real format
UnicodeDecodeError	Non-UTF8 bytes in a text handle	gzip.open(path, 'rt', encoding='latin-1')

Related Skills

• read-sequences - parse vs index vs index_db trade-offs for compressed handles
• write-sequences - write records through a compression handle
• batch-processing - stream many compressed files without loading them into RAM
• filter-sequences - keep R1/R2 in sync when filtering gzipped paired reads
• alignment-files/sam-bam-basics - BAM is BGZF natively; samtools manages the compression

References

• Li H, Handsaker B, Wysoker A, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25(16):2078-2079. (Defines BGZF in the SAM/BAM specification.)
• Bonfield JK. CRAM 3.1: advances in the CRAM file format. Bioinformatics. 2022;38(6):1497. (Per-column block compression beyond BGZF.)
• Du L, Liu Q, Fan Z, et al. Pyfastx: a robust Python package for fast random access to sequences from plain and gzipped FASTA/Q files. Briefings in Bioinformatics. 2021;22(4):bbaa368.