Not getting any modification calls in nanopore reads with dorado basecaller
1
0
Entering edit mode
8 weeks ago
njornet ▴ 20

I want to detect modifications on a run of human DNA without amplification. The sequencing output was fast5 but I'm using dorado, so I first convert the reads to pod5. I then run dorado basecaller with the following command, it is using the model dna_r9.4.1_e8_hac@v3.3 and dna_r9.4.1_e8_hac@v3.3_5mCG_5hmCG@v0:

dorado basecaller hac,5mCG_5hmCG run_16_wo_WGA_pod5/ -r --min-qscore 7 -x > basecall_modifications/bc_output.bam

If I convert that to SAM I can already tell that there are no reads with the tags MM, MN or ML. Anyway, I run modkit on the BAM file with the command:

modkit pileup ../run_16_aligned.bam pileup.bed --log-filepath pileup.log

And I get a bunch of

[src/mod_bam.rs::183][2024-03-21 11:53:55][ERROR] failed to get modbase info for record 3b3459f9-d842-4b67-a224-5fb0fc8dabd9, Skipped: AUX data not found

And a final line with

[src/pileup/subcommand.rs::783][2024-03-21 12:01:08][INFO] Done, processed 0 rows. Processed ~0 reads and skipped ~443600 reads.

The bed file is obviously empty.

Is there anything wrong with this pipeline or is my data wrong in any way?

mofications dorado basecalling nanopore methylation • 1.1k views
ADD COMMENT
1
Entering edit mode

Was the methylation detection turned on in sequencer software when the run was done? If it was not then that may be the problem.

ADD REPLY
0
Entering edit mode

From what I know you don't need to change anything while sequencing, it just changes when basecalling, but maybe I'm wrong.

ADD REPLY
1
Entering edit mode

this is incorrect. at run time, you may specify epigenetic mod calling or not.

probably what you are referring to is the idea that dorado includes ways to account for modified bases in its model no matter what, which is true. this is done in order to avoid base calling errors if modified bases are present in the sample..however it in no way implies that modified bases will be called as such in your sample if you fail to specify that you would like that to be done.

ADD REPLY
1
Entering edit mode

Ok, in that case that's the problem, thank you!

ADD REPLY
1
Entering edit mode

yw. it seems weird the first time around because if it needs to process them anyway, why not just report the data? but there are various answers to that question, like "im not confident my methylation data will mean anything, because I treated my animals a certain way and im not asking that question" or some such.

in that case you might only want to avoid base call errors, but not want to call the variants.

ADD REPLY
0
Entering edit mode

I've looked into it and I can't seem to find any information about this, we also looked at the minKNOW software in the minION but we don't find the option to include modified bases. How do we do that?

ADD REPLY
0
Entering edit mode

Are you using the latest MinKNOW available?

ADD REPLY
0
Entering edit mode

We updated it just before checking

ADD REPLY
0
Entering edit mode

Hi,

I would post this issue in the issues session of the dorado github page: https://github.com/nanoporetech/dorado/issues

Didi you try any of the solutions posted at this link: https://github.com/nanoporetech/dorado/issues/671

ADD REPLY
0
Entering edit mode

I think I'm misunderstanding something. This process would have two steps, first sequence which generates pod5 files and then basecalling which generates fastq/bam as we are basecalling after the run was completed. I basecalled using the options to include modified bases basecalling but I didn't find anything. What you are saying in this comment is that I have to tell the sequencer to detect modified bases in the first or second step?

ADD REPLY
0
Entering edit mode

From what I understand with GridION MinKNOW has an explicit toggle to call methylation. I don't know if the software behaves differently with minION.

ADD REPLY
1
Entering edit mode
8 weeks ago

Have a look at this link

edit: another problem could be that the input file (named with WGA) is a Whole Genome Amplification sample which should not have any methylated bases

ADD COMMENT
0
Entering edit mode

agree. this is a user error for one of three reasons. two are described in this post, the third in GenoMax's comment, above.

ADD REPLY
0
Entering edit mode

Yeah, the name of the input file is incorrect, we had a mix up with the names. We didn't perform amplification in this specific run.

ADD REPLY

Login before adding your answer.

Traffic: 1993 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6