Nanopolish calling methyaltion

[xyy-xiao]

Hi,
I got pod5 files and fastq file, and want to call methylation. I know Nanopolish does not support pod5 files, so I used blue-crab to convert pod5 files to blow5 files. Then I used slow5tools to convert the blow5 files to fast5 files. But I got errors:
[readdb] num reads: 5561098, num reads with path to fast5: 392000
fast5 files could not be located for 5169098 reads
[post-run summary] total reads: 375421, unparseable: 0, qc fail: 0, could not calibrate: 0, no alignment: 0, bad fast5: 5309727
Is there something wrong with my convert files?

[hasindu2008]

@xyy-xiao

This is R10 or RNA004 data I believe? Nanopolish only supports R9 and that could be why. You may try f5c. The command line and output will be the same as nanopolish.

[Tang-pro]

@hasindu2008
hi,
so Can f5c also calculate polyA length?

[hasindu2008]

Unfortunately, f5c only supports methylation calling and eventalign, not polyA length.

[Tang-pro]

@hasindu2008
Okay ,thank you!
If it is RNA004, what methods can be used to calculate the polyA length?

[hasindu2008]

I think newer versions of ONT's Dorado have a polyA length programme built-in - note I have not tried this.

[Tang-pro]

@hasindu2008

Yes, But this step is coducted by sequecing company, because I don't have GPU.
Okay, thank you again!