Bad fast5 error with nanopolish eventalign

[balberti]

Hello,

I'm running nanopolish index and eventalign on a puc19 dataset downloaded on SRA with the accession SRR5219626. I have downloaded the fast5 files and I used a fast5 to fastq converter (https://github.com/rrwick/Fast5-to-Fastq) to get the fastq corresponding files. (I cannot run guppy on my own) The ref plasmid is coming from New England Biolabs.

I ran nanopolish index -d gdna_fast5_files/ gdna.fastq (without the sequencing summary file because I don't have it) I have the following warnings But index, index.fai, index.gzi, and index.readdb are created and not empty.

Then, when I'm running eventalign I get (The bam file was generated with minimap2 and samtools by mapping gdna.fastq on the reference plasmid)

Does this error occur because I'm not using a basecaller on the fast5 files ? Is the sequencing summary file mandatory for the indexing ? Is it possible to recreate manually this file or to get around this error ?

Thank you in advance for your time Best regards, Baptiste Alberti.

[jts]

Hi,

I think that data is very old so the format is probably no longer supported by nanopolish. If it is R9 data, you may try ONT's tools to reformat it (single-to-multi). If it is R7 data you will have to try to use an older version of nanopolish.

Jared

[balberti]

Thank you for your quick answer. I will try to reformat it as you indicate. I'll let you know if the error disappears after that.

Best, Baptiste.

[balberti]

Hello Jared,

I'm sorry, but I tried both solutions to counter my problem. Sadly, the error is still there even with multi fast5 files. I also ran index and eventalign commands with nanopolish version from the current one to the v0.5 but nothing worked.

Is the issue coming from the dataset itself or from the fact that the fastq I have was not created by guppy?

Thanks and have a nice day, Baptiste.