Question regarding command-line basecalling

[hwangumn]

Hi,
I initially tried using MinKNOW for basecalling, but the process took too long on my PC (i9-10850k, GTX3080). I am now using the A100 on our supercomputing institute for basecalling. I am wondering how to convert .bam files into .fastq files with dorado. The demultiplexed output from MinKNOW consists of individual folders (named with barcodes) containing .fastq files. Will the command-line code below produce similar results?
dorado basecaller hac <Path_to_Pods5> --kit-name SQK-NBD114-96 --output-dir /<Path_to_Output_Folder> > calls.bam

And have the barcodes been removed from the output files?

Thanks,

[HalfPhoton]

Hi @hwangumn,
Use --emit-fastq to create the fastq directly and the barcodes are automatically trimmed.

Best regards,
Rich

[hwangumn]

Hi @hwangumn, Use --emit-fastq to create the fastq directly and the barcodes are automatically trimmed.

Best regards, Rich

Thanks for the info. Do I also change calls.bam to reads. fastq? Will the output be in folders named with barcode (i.e, barcode01, barcode02, barcode03, etc)?

[HalfPhoton]

To split barcodes into files in this way please pipe the output from basecaller into demux as explained here

dorado basecaller <model> <reads> --emit-fastq ... | dorado demux --kit-name <kit-name> --output-dir <output-dir> 

Rich

[hwangumn]

To split barcodes into files in this way please pipe the output from basecaller into demux as explained here

dorado basecaller <model> <reads> --emit-fastq ... | dorado demux --kit-name <kit-name> --output-dir <output-dir> 

Rich

Hi Rich:
Thank you so much for your help! However, most of the reads seemed unclassified after basecaller and demux. Any suggestions? I am using the Rapid Barcoding 96 kit for bacterial isolates.
Here are the codes I was using:
dorado basecaller hac /home/ishiis/wang4637/ONT/Isolates/raw_pod5 --kit-name SQK-NBD114-96> calls.bam
dorado demux --emit-fastq --no-classify --output-dir /home/ishiis/wang4637/ONT/Isolates/Output calls.bam

[hwangumn]

update - it seems like I should use SQK-RBK114-96 instead of SQK-NBD114-96. I will see if that resolve the issue.
Thanks.