Separate single cell BAM file by the cell barcode

Question

[zbidav]

Dear Biostar community,

I am a bit new to Dropseq analysis (10x sequenced files, if not mistaken). I followed the standard CellRanger protocol and received an aligned BAM file of the samples and the files for downstream analysis with Seurat.

I wonder if there is an efficient way to separate the resulting BAM file into multiple files by the Cell Barcode. (it is a special attribute in the bam file - "CB"). I tried to do it using samtools, but due to the large file number, it was not so efficient.

If summarized: "My input is a single-cell BAM file, and the output is separated bam files - one for each cell barcode. Do you know a tool that can do it or an efficient way to do so?

Much appreciated!

Answer 1

[GenoMax]

10x makes a tool available: https://github.com/10XGenomics/subset-bam

Also: https://github.com/timoast/sinto

[zbidav]

Hi, thanks for the answer! Sadly this not quite what I am trying to do :( if I may quote from the manual (of subset-bam):

subset-bam ... takes a 10x Genomics BAM file, a CSV file defining the subset of cells you want to isolate, and produces a new BAM file with only alignments associated with those cells.

This tool is very useful in creating pseudo-bulk files from multiple cells. In my case, as I need to separate each single cell to a different file. I can create a temporary file of a single csv, but I wonder if that approach is indeed efficient (compared to the naive one using samtools)

[GenoMax]

Looks like sinto is multi-threaded so it may be more performant: https://timoast.github.io/sinto/basic_usage.html#filter-cell-barcodes-from-bam-file

How many cell barcodes were you planning to use?

[zbidav]

Hmmm... all of them :) To be more exact - all the barcodes that resulted from CellRanger. I think in average ~8,000-10,000 barcodes.

[Ram]

You may want to run subset-bam using GNU parallel where each temporary single line csv is read in through parallel's ::: option. I am no expert in GNU parallel so take my advice with a grain of salt.

[zbidav]

This is a bit late response - but Sinto worked really well - it does require a barcode list and (maybe it is a system limitation) could work only in 500 cell batches, but it indeed worked and was the most efficient way.

[Ukesh]

Hi there, Could you please share how exactly you did it with sinto? I have multiple scRNA bam files and cell barcode list I want to extract and the way I am doing it will take ages. So, please share it.

[zbidav]

Definitely! I am so sorry for not seeing this on time, I hope this is still relevant. I will provide the code that works in my case:

input_bam="possorted_genome_bam.bam"
output_dir="splitedFile/"
mkdir -p $output_dir
out_cell_barcodes_annot="cell_barcodes_tabular_sinto.tsv"
sinto filterbarcodes --bam $input_bam --cells $out_cell_barcodes_annot --outdir $output_dir --nproc 20

The usual sinto input requires:

• Bam file
• Cell annotation file

The cell annotation for example:

TTGGATGTCGCACGAC-1      epithelial
TTGGGCGAGTGACCTT-1      endothelial
TTGGGTATCTGGCCAG-1      endothelial
TTGTGTTCAACCAACT-1      endothelial

I changed to be:

TTGGATGTCGCACGAC-1      TTGGATGTCGCACGAC-1 
TTGGGCGAGTGACCTT-1      TTGGGCGAGTGACCTT-1
TTGGGTATCTGGCCAG-1      TTGGGTATCTGGCCAG-1
TTGTGTTCAACCAACT-1      TTGTGTTCAACCAACT-1

It seems that Sinto can do it only in batches of ~100-500, depending on your system.