How to use the barcode classification option

[Narmatha99]

I want to use my pod5 files to basecall and eventually to assemble a hybrid genome with unicycler. For sequencing I used the FLO-MIN 114 MinION flow cell in combination with the SQK-RBK114-96 kit. How should I input this in the following command to get the right files (basecalled + barcoded) to continue with assembly: $ dorado basecaller --kit-name > calls.bam

My pod5 files are located in the Downloads file of the mac pro so the path to it would be something like: ~/Downloads/

Thanks!

[malton-ont]

Hi @Narmatha99,

Details of how to run barcoding can be found in the README.md file here.

[Narmatha99]

Hi @malton-ont

Would I then put hac for and the location of my pod5 files for ? also for --kit-name would I say something like: --kit name ?

Thanks

[malton-ont]

@Narmatha99 The readme says:

dorado basecaller <model> <reads> --kit-name <barcode-kit-name> > calls.bam

Replace the bits in <> with your specific values. So it sounds like you want:

dorado basecaller hac ~/Downloads/ --kit-name SQK-RBK114-96 > calls.bam

[Narmatha99]

@malton-ont

Okay, that worked thank you. However maybe another question, I translated the calls.bam file to fastq file with samtools ($ samtools bam2fq SAMPLE.bam > SAMPLE.fastq)

How would I continue in unicycler then? Since this only generated one fastq file?

Thank you

[malton-ont]

I'm not aware of unicycler, it's not one of our products, but I presume you want to demux the barcodes into separate files? You can do this by running either samtools split or dorado demux --no-classify on the barcoded bam file, then converting the results to fastq.