Assertion failed in align.c while running f5c eventalign on Nanopore Direct RNAseq

[Euchiz]

Hello,

I encountered an error while running f5c eventalign on my Nanopore Direct RNAseq data. The error message is as follows:

f5c: src/align.c:457: int32_t align(AlignedPair*, char*, int32_t, event_table, model_t*, uint32_t, scalings_t, float): Assertion `outIndex < (int)(n_events * 2)' failed.

The command line that I used was:

f5c eventalign -t 64 --pore rna004 --rna -b input.bam -g gencode.v46.transcripts.fa -r input.fastq --slow5 input.blow5 \
    --signal-index --scale-events

Details:

• BAM Input: Output from dorado-v0.7.2, aligned to GENCODEv46 transcripts by minimap2.
• BLOW5 Input: Converted from raw POD5 (rna004) files and merged using bluecrab and slow5tools.
• f5c Version: Latest build from release/binaries.
• Error: The error occurred after generating approximately 300GB of events. There are no other error messages or read ID provided except the assertion failure mentioned above.

Could you please advise on what might be causing this error and how I can resolve it?

Thank you for your assistance!

[hasindu2008]

Hello,

This rare corner case that seems to happen with rna004 was fixed a couple of months ago and is still in the dev branch. Would you be able to compile the dev branch and try if it works for you?

Regards

[Euchiz]

Thank you for your prompt response!

I cloned the development branch as you suggested, compiled it, and reran the eventalign function on my Nanopore direct RNA-seq data. I am happy to report that the issue is now resolved, and everything worked smoothly!

[hasindu2008]

Great, I have released v1.5 that incorporates this fix.