Bambu cannot identify parameters

[Choeun-fran]

Hi, thanks for your great effort to make bambu for single-cells. But I'm getting errors with bambu and bambu_EM.

I think my most serious problem is bambo cannot identify the parameters.
I tried to '--clusters' and '--barcodeMap' with cluster.csv file and barcodeMAP.csv file

My command pasted below:
/home/home/dlchdms0109/.local/bin/nextflow run GoekeLab/bambu-singlecell-spatial
--bams /home/node200data/dhkim1674/Ouroseq_Samsung_Alzheimer/SMC041/OuroSeq_Samsung_SMC041/OuroSeq_Samsung_SMC041.tagged.bam
--genome /home/home/dhkim1674/refdata-gex-GRCh38-2024-A/fasta/genome.fa
--annotation /home/home/dhkim1674/refdata-gex-GRCh38-2024-A/genes/genes.gtf
--ncore 16 --outdir /home/home/dlchdms0109/Bambu_result_with_cluster/
--processByChromosome TRUE
--clusters /home/node200data/dhkim1674/Ouroseq_Samsung_Alzheimer/SMC041/OuroSeq_Samsung_SMC041/SMC041_clusters.csv

[Detailed description of my situations]
when I got the first error which related to read identification,

[error statements]
Error: BiocParallel errors
1 remote errors, element index: 1
0 unevaluated and other errors
first remote error:
Error in bambu.readsByFile(bam.file = reads[i], genomeSequence = genomeSequence, : No reads left after filtering.
Execution halted

I realized that the problem caused by read format. My read bamfile had different readnames without'#', cell barcode tags CB instead of CR, and UMI tags UB instead of UR.

So, I made barcodeMAP.csv file matched with the reference types. It wasn't working..

The situation repeated again when I had an error related to clusters.

Even though I made barcodeMAP.csv and cluster.csv with --barcodeMap and --clusters parameters. Bambu did not identify the files... So, I'm still having the same problem.

[lingminhao]

Hi @Choeun-fran, sorry for the late reply. Do you still have the issue with you ? I can help to have a look if you can share with me the command you ran with --barcodeMap and --clusters arguments

[Choeun-fran]

Thank you for your response.
I solved the problem by replacing the tags and setting --clusters none. I think some parameters set already null or none can't change by myself.. But it was not a big problem for me cause I finally ran Bambu and got the result files.
However, I have one more questions. When I search the parameters of Bambu, I find 'trackReads' to track where the individual reads are assigned into transcripts. Can I use this parameters in single cell mode as well?

[lingminhao]

Hi @Choeun-fran , you may use this parameter in single cell mode if you run Bambu independently (but it may be very memory intensive). If you use this pipeline, you might have to intervene the code manually for now since it's still in beta. In future, we plan to allow the arguments that work on Bambu transferable to Bambu-Pipe.

Best Regards,
Min Hao