Split a 10x barcoded sequencing file into barcode-specific BAMs

split_bam_by_barcode.py

"""
MIT License

Copyright (c) 2020 Warren W. Kretzschmar

Permission is hereby granted, free of charge, to any person obtaining a copy
of this software and associated documentation files (the "Software"), to deal
in the Software without restriction, including without limitation the rights
to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
copies of the Software, and to permit persons to whom the Software is
furnished to do so, subject to the following conditions:

The above copyright notice and this permission notice shall be included in all
copies or substantial portions of the Software.

THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE
SOFTWARE.
"""

from pathlib import Path

import pysam


class BamWriter:
    def __init__(self, alignment, barcodes, prefix):
        self.alignment = alignment
        self.prefix = prefix
        self.barcodes = set(barcodes)
        self._out_files = {}

    def write_record_to_barcode(self, rec, barcode):
        if barcode not in self.barcodes:
            return
        if barcode not in self._out_files:
            self._open_file_for_barcode(barcode)
        self._out_files[barcode].write(rec)

    def _open_file_for_barcode(self, barcode):
        self._out_files[barcode] = pysam.AlignmentFile(
            f"{self.prefix}_{barcode}.bam", "wb", template=self.alignment
        )


def main(input_bam, barcodes_file, output_prefix, contigs, barcode_tag):
    """Split a 10x barcoded sequencing file into barcode-specific BAMs

    input:
    barcodes_file can be a file containing barcodes, or a single barcode

    contigs can be '.' for all contigs, 'chr1' for the contig 'chr1',
    or '1-5' for chromosomes 1, 2, 3, 4, and 5
    """
    alignment = pysam.AlignmentFile(input_bam)
    if Path(barcodes_file).is_file():
        with open(barcodes_file, "r") as fh:
            barcodes = [l.rstrip() for l in fh.readlines()]
    else:
        barcodes = [barcodes_file]
        print(f"Extracting single barcode: {barcodes}")
    writer = BamWriter(alignment=alignment, barcodes=barcodes, prefix=output_prefix)
    if contigs == ".":
        print("Extracting reads from all contigs")
        recs = [alignment.fetch()]
    else:
        if "-" in contigs:
            start, end = contigs.split("-")
            print(f"Extracting reads from contigs {start} to {end}")
            recs = (alignment.fetch(str(contig)) for contig in range(start, end + 1))
        elif "," in contigs:
            contigs = contigs.split(",")
            print(f"Extracting reads from contigs {contigs}")
            recs = (alignment.fetch(str(contig)) for contig in contigs)
        else:
            print(f"Extracting reads for one contig: {contigs}")
            recs = (alignment.fetch(c) for c in [contigs])

    for region in recs:
        for rec in region:
            try:
                barcode = rec.get_tag(barcode_tag)
                writer.write_record_to_barcode(rec=rec, barcode=barcode)
            except KeyError:
                pass

if __name__ == "__main__":
    import argparse

    parser = argparse.ArgumentParser(description="Split a 10x barcoded sequencing file into barcode-specific BAMs")
    parser.add_argument("input_bam", help="Input BAM file")
    parser.add_argument("barcodes_file", help="File containing barcodes, or a single barcode")
    parser.add_argument("output_prefix", help="Output file prefix for barcode-specific BAM files")
    parser.add_argument("contigs", help="Contigs to extract reads from: '.' for all contigs, 'chr1' for the contig 'chr1', '1-5' for chromosomes 1, 2, 3, 4, and 5, or '1,2,3' for contigs 1, 2, and 3")
    parser.add_argument("--barcode_tag", default="CB", help="Barcode tag to use for extracting barcodes")


    args = parser.parse_args()

    main(
        input_bam=args.input_bam,
        barcodes_file=args.barcodes_file,
        output_prefix=args.output_prefix,
        contigs=args.contigs,
        barcode_tag=args.barcode_tag,
    )

Thanks for the helpful comment! I agree that this amendment to the script makes a lot of sense.

Hi @winni2k! Thanks for developing this script
I ran the script with a BAM file resulting from cellranger-v7 and wouldn't work. This is because the cell barcode tag is CB:Z instead of CB (as in your script).
An example of one read:

sample_name.96433731	0	chr1	11304	0	84M	*	0	0	GCACTGCAGGGCCCTCTTGCTTACTGTATAGTGGTGGCACGCCGCCTGCTGGCAGCTAGGGACATTGCAGGGTCCTCTTGCTCA	FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF	NH:i:8	HI:i:1	AS:i:82	nM:i:0	RG:Z:GSM4955739:0:1:unknow_flowcell:0	RE:A:I	xf:i:0	CR:Z:CCCAGTTAGCCACCTG	CY:Z:FFFFFFFFFFFFFFFF	CB:Z:CCCAGTTAGCCACCTG-1	UR:Z:GCATTAGCGG	UY:Z:FFFFFFFFFF	UB:Z:GCATTAGCGG

I just changed the line in your script:
barcode = rec.get_tag("CB") --> barcode = rec.get_tag("CB:Z") and worked perfectly.

Thanks for the info @rubenchazarra! There seems to be a bit of demand for this script. I'll see if I can make an update in my spare time.

I had GPT4 change the script so that it now uses argparse and allows the user to define the barcode tag to use on the command line. I have not tested the changes. Please let me know if anything has broken.