droplet RNAseq data analysis using Kallisto Bustools

[satyam-espci]

Hello Everybody,
I perform single cell sequencing using droplet microfluidics and barcode cells using barcoded hydrogel beads generated by split and pool technique.
The structure of my library is:
Read2 sequencing primer_Barcode A(20bp)_ Barcode B(20bp)_ Barcode C(20bp)_Linker(19bp)_UMI(8bp)_cDNA
Read1 sequencing primer_cDNA

So, my Read 2 contains the barcode, UMI and transcript information
Read 1 contains only transcript information

I have human and mouse cells in the experiment, so have generated mixed index and mixed t2g files.
After generating white list using the bustools, I am running the following command:

kb count -i mixed_index.idx -g mixed_t2g.txt -x 0,0,56:0,75,83:1,0,0 -w ./SPLiT-seq/whitelist.txt -o output --h5ad -t 4 R2.fastq.gz R1.fastq.gz

But the following error is appearing: terminate called after throwing an instance of 'std::bad_alloc'

(sbpython) sbanerje@lbc-bigbi:~/kallisto_ref$ kb count -i mixed_index.idx -g mixed_t2g.txt -x 0,0,56:0,75,83:1,0,0 -w ./SPLiT-seq/whitelist.txt -o output --h5ad -t 4 R2.fastq.gz R1.fastq.gz [2022-09-06 20:20:10,726] INFO [count] Skipping kallisto bus because output files already exist. Use the --overwrite flag to overwrite. [2022-09-06 20:20:10,726] INFO [count] Sorting BUS file output/output.bus to output/tmp/output.s.bus [2022-09-06 20:20:14,550] INFO [count] Inspecting BUS file output/tmp/output.s.bus [2022-09-06 20:20:15,661] INFO [count] Correcting BUS records in output/tmp/output.s.bus to output/tmp/output.s.c.bus with whitelist ./SPLiT-seq/whitelist.txt [2022-09-06 20:20:16,972] ERROR [count] Found 326 barcodes in the whitelist terminate called after throwing an instance of 'std::bad_alloc' what(): std::bad_alloc [2022-09-06 20:20:16,972] ERROR [main] An exception occurred Traceback (most recent call last): File "/home/lbc/sbanerje/.conda/envs/sbpython/lib/python3.9/site-packages/kb_python/main.py", line 1305, in main COMMAND_TO_FUNCTION[args.command](parser, args, temp_dir=temp_dir) File "/home/lbc/sbanerje/.conda/envs/sbpython/lib/python3.9/site-packages/kb_python/main.py", line 550, in parse_count count( File "/home/lbc/sbanerje/.conda/envs/sbpython/lib/python3.9/site-packages/ngs_tools/logging.py", line 62, in inner return func(*args, **kwargs) File "/home/lbc/sbanerje/.conda/envs/sbpython/lib/python3.9/site-packages/kb_python/count.py", line 1079, in count prev_result = bustools_correct( File "/home/lbc/sbanerje/.conda/envs/sbpython/lib/python3.9/site-packages/kb_python/validate.py", line 116, in inner results = func(*args, **kwargs) File "/home/lbc/sbanerje/.conda/envs/sbpython/lib/python3.9/site-packages/kb_python/count.py", line 330, in bustools_correct run_executable(command) File "/home/lbc/sbanerje/.conda/envs/sbpython/lib/python3.9/site-packages/kb_python/dry/init.py", line 25, in inner return func(*args, **kwargs) File "/home/lbc/sbanerje/.conda/envs/sbpython/lib/python3.9/site-packages/kb_python/utils.py", line 203, in run_executable raise sp.CalledProcessError(p.returncode, ' '.join(command)) subprocess.CalledProcessError: Command '/home/lbc/sbanerje/.conda/envs/sbpython/bin/bustools correct -o output/tmp/output.s.c.bus -w ./SPLiT-seq/whitelist.txt output/tmp/output.s.bus' died with <Signals.SIGABRT: 6>.

As our sequencing technology does not match with the listed technologies (kb --list), will I not be able to use Kallisto Bustools for the analysis? I request your kind advice and guidance on the issue.
Thank you for your time and consideration.

[Yenaled]

Barcodes can only be up to 32 bp; see BUStools/bustools#49