Dorado in PowerShell on Windows

Question

[sbissi102]

Hello Everyone,

I used P2 Solo to perform genome sequencing on 21 bacterial strains. I applied the "--one-to-one" option of the pod5 conversion pipeline to convert hundreds of fast5 files to pod5. I then carried out basecalling and demultiplexing (I'm working on WSL).

dorado-1.1.1-linux-x64/bin/[dorado][1] basecaller --no-trim hac /mnt/c/data/bacteria/pod5_out  > --output-dir my_output

dorado-1.1.1-linux-x64/bin/dorado demux --kit-name SQK-RBK114-96 --emit-fastq --output-dir demux_out calls.bam

The issue is that we worked with 21 barcodes (5 to 25), yet I received 96 fastq files in demux_out that corresponded to the 96 barcodes. Additionally, I'm unsure if I should re-trim the fastq files prior to doing any downstream analysis (e.g. Porechop).

Suggestions appreciated. Thanks

[jared.andrews07]

If you're using WSL, PowerShell has nothing to do with this. It also sounds like a better question for your sequencing facility/provider rather than anyone here. Are all of your actual supposed barcodes found?

[sbissi102]

Yes, I found all the barcodes, but could working on WSL affect the basecalling results?

Answer 1

[GenoMax]

yet I received 96 fastq files in demux_out that corresponded to the 96 barcodes. Additionally, I'm unsure if I should re-trim the fastq files prior to doing any downstream analysis (e.g. Porechop).

dorado demux puts out files for the entire set of barcodes that are in a kit. If you check the files you will see that only the barcodes in use should have significant amounts of data in corresponding files. Rest of the files may have a few (or no) reads.

There is a samplesheet option (https://github.com/nanoporetech/dorado/blob/release-v1.1/documentation/SampleSheets.md ) that you can use if you want to focus on specific indexes ( did not use this myself).

dorado basecaller will automatically trim the adapters unless you use the option not to do so. Since you used that option you will need to deal with those.

The --no-trim option will prevent the trimming of detected barcode sequences as well as the detection and trimming of adapter and primer sequences.