DNAscent: src/event_handling.cpp:547: void normaliseEvents(DNAscent::read&, bool): Assertion `et.n > 0' failed.

[ZHOUXY-QH99]

Hi , bam file and reference are ok when I use the DNAscent detect parameter, but this error occurs at runtime. Like this :

Importing reference... ok.
Opening bam file... ok.
Scanning bam file...ok.
DNAscent: src/event_handling.cpp:547: void normaliseEvents(DNAscent::read&, bool): Assertion `et.n > 0' failed.
Aborted (core dumped)

Any suggestions on what this could be? Thank you!

[MBoemo]

Any chance you've overwritten the minimum mapping length?

[ZHOUXY-QH99]

Thank you for your reply!
This is my test code, and I'm not overwrrite the minimum mapping length

~/software/Dorado/dorado-0.8.2-linux-x64/bin/dorado aligner ~/data/13_lignin_2024_10_31/07_dorado_flye/02_flye_contig/contig_96.fa ~/data/13_lignin_2024_10_31/011_dorado/barcode01/calls_2024-11-11_T03-20-56.fastq > barcode01_aligned.bam

~/software/DNAscent/bin/DNAscent index -f ~/data/13_lignin_2024_10_31/00_pod5/barcode01 -o barcode01_index.dnascent

~/software/DNAscent/bin/DNAscent detect -b barcode01_aligned.bam -r ~/data/13_lignin_2024_10_31/07_dorado_flye/02_flye_contig/contig_96.fa -i barcode01_index.dnascent -o barcode01.detect -t 20 --GPU 0

[MBoemo]

Can I check which commit you're using? There were a few early problems with v4.0.3 and bam parsing but these have since been fixed and the singularity image updated.

[ZHOUXY-QH99]

Sorry, I didn't understand what's commit ,how can i get it ?
I was using the v4.0.3 version of DNAscent, and dorado was using the latest version.

When running detect, my output file barcode01.detect is not empty.

Importing reference... ok.
Opening bam file... ok.
Scanning bam file...ok.
[> ] 0% 18/2179 2hr53min25sec failed: 2

The program will stop at this point with the error message
DNAscent: src/event_handling.cpp:547: void normaliseEvents(DNAscent::read&, bool): Assertion `et.n > 0' failed.
Aborted (core dumped)

[ThDef]

Hi,

I just got the same issue using the v.4.0.3, pod5 files and Dorado basecalled data. The DNAscent version used was built from scratch today and the .bam file was produced by Minimap2.

[=================> ] 51% 44/86 0hr 0min14sec failed: 20
[=================> ] 51% 44/86 0hr 0min14sec failed: 20
[==================> ] 52% 45/86 0hr 0min14sec failed: 20
[==================> ] 52% 45/86 0hr 0min14sec failed: 20
DNAscent: src/event_handling.cpp:547: void normaliseEvents(DNAscent::read&, bool): Assertion `et.n > 0' failed.

/path/DNAscent_4.0.3/bin/DNAscent detect -b alignment/chr13_header.sorted.bam -r /path/genome.fasta -i DNAscent_output/index.dnascent -o DNAscent_output/chr13.detect -t 10' died with <Signals.SIGABRT: 6>

[MBoemo]

Shouldn't have failed so I'll take a look, but on v4.0.3 I'd recommend generating the bam file with Dorado unless there's a very good reason not to.

[ZHOUXY-QH99]

Thank for your reply!
I have soved this problem with changed the basecalling model.
l find that DNAscent's workflow recommends Dorado basecalling model for v4.0.3 is dna_r10.4.1_e8.2_400bps_fast@v5.0.0 , so I change the model ,and detect succesfully!

[MBoemo]

I'm surprised that fixed it - which model were you using before?

[ZHOUXY-QH99]

I was also surprised to solve this problem in this way. I used the latest version of dorado before, and the model selection is high-precision identification, as follows： dorado basecaller hac pod5s > calls.bam I suspected it was a coincidence, but I had no way to verify it. ***@***.*** From: Michael A. Boemo Date: 2024-11-15 16:18 To: MBoemo/DNAscent CC: ZHOUXY-QH99; Author Subject: Re: [MBoemo/DNAscent] DNAscent: src/event_handling.cpp:547: void normaliseEvents(DNAscent::read&, bool): Assertion `et.n > 0' failed. (Issue #76) I'm surprised that fixed it - which model were you using before? — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you authored the thread.Message ID: ***@***.***>

[ThDef]

Shouldn't have failed so I'll take a look, but on v4.0.3 I'd recommend generating the bam file with Dorado unless there's a very good reason not to

The .bam was not generated with Dorado because the pipeline analysis was done through a custom Snakemake that was originally made for R.9 data and the v.3 of DNAscent. It was then modified to accomodate the changes in commandline of the v.4 but the alignment part was left untouched with Minimap2.

Using Dorado to do the alignment is also not ideal when your data is only a part of a barcoded run

On my side, the basecalling was done with the superior model because part of the run was for assembly purposes but as my .bam file was then generated with Minimap2, I don't know if it is relevant ?

[MBoemo]

Unfortunately I haven't yet been able to reproduce this. @ThDef which version of Dorado are you using and which basecalling model?

[ThDef]

I used the dna_r10.4.1_e8.2_400bps_sup@v5.0.0 model with the version 0.7.2 of Dorado for the basecalling.

[ZHOUXY-QH99]

As for the detection results of detect, I noticed that the probability that the thymidine is actually BrdU is very low. I would like to know what probability is considered as BrdU

[ZHOUXY-QH99]

I used the dna_r10.4.1_e8.2_400bps_sup@v5.0.0 model with the version 0.7.2 of Dorado for the basecalling.

I recommend that the file format you generate during basecalling is bam file