High proportion of "couldn't calibrate" for short RNA reads

[kaltinel]

Hi,

I would like ask a question similar to issue #540.

I am having a very similar problem with my short RNA samples (they are 90 bp with polyA: so I am left with ~70 nt)

[post-run summary] total reads: 140905, unparseable: 0, qc fail: 0, could not calibrate: 135904, no alignment: 4986, bad fast5: 0

After nanopolish event-align scale-events, I am left with 18 reads..
I see that nanopolish has problems with calibrating and needs longer length: but then how do I ever work with my short length input?
Do I have to ligate them?
Should I use different parameters? Or a different tool you might suggest?

I would appreciate your comments!
Thank you.

[jts]

Unfortunately, these reads are too short for nanopolish currently. If possible ligating them would be a good thing to try.

Jared

[kaltinel]

Thank you for your reply.
May I ask, what would be the minimum length for nanopolish to work with direct RNA reads (1D + 1D2 )?
It would be a useful information for my ligation experiment.

[jts]

There isn't a hard threshold, I'd suggest at least 500bp but longer would be better (I think the typical length is about 1kb).

[kaltinel]

Hmm.. I use nanopolish for my 5S rRNA data which is ~120 bps, and it works okay..
Do you think these data would be faulty and I should not trust?

[jts]

I wouldn't say they are necessarily unreliable - if nanopolish was able to align and calibrate the signals the results are probably fine.

[kaltinel]

Thank you, I see.. And, do you have any suggestions for the maximum length?

[jts]

There's no maximum

[kaltinel]

Thanks for your reply.
However, I do not understand certain aspects..

I have my RNA data in various lengths, one set is 120 bp (5S), the other set is 2kb (18S) and the last one is 4.5kb (28S rRNA).
In my event align outputs, I can only see that 18S is aligned...
I have no alignments for others..

If 5S is too short, I don't understand what is the issue with 28S..

Can you elaborate on this please?

[jts]

Hi,

Sorry for the slow reply, Do you have reads aligned to 28S in the bam file?

Jared

[kaltinel]

Hi,

Apologies for my late reply too!
Yes, my alignment file has lots of 28S reads ( and 18S, 5S and 5.8S). They are quite high in coverage as well.

However only 18S are seen in the nanopolish eventalign output..