lima ERROR: [pbcopper] alarm ERROR: cannot write to empty alarm filename about pbbioconda

[JohnsonStev]

Hi I have the same issue

[armintoepfer]

Can you provide a small reproducible example that you can share?

[Tristonli8421]

same issue :(

[armintoepfer]

Does lima v2.9.0 from bioconda / github work?

[JohnsonStev]

I am using lima 2.9.0 and get the error

[armintoepfer]

Someone needs to upload a file to reproduce. https://github.com/PacificBiosciences/pbbioconda?tab=readme-ov-file#file-sharing

[Tristonli8421]

hi, i find that lima works out well if i used the [primer fasta file] (https://github.com/PacificBiosciences/IsoSeq/blob/master/isoseq-clustering.md) provided instead of [kinnex primer file] (https://downloads.pacbcloud.com/public/dataset/Kinnex-full-length-RNA/REF-primers/) provided here. Is a primer format error issue?

[jmattick]

Can you please also share the full set of commands run and the barcode files used?
You can also run lima with this option to see the root cause of the issue:

lima --isoseq --peek-guess sample.hifi_reads.bam barcodes.fa sample.fl.bam --alarms alarms.json

It's possible that the wrong barcode file has been used for the kit that was used. Note that the Kinnex Iso-Seq kit uses different primer sequences than the previous version of Iso-Seq.

[kenneditodd]

Someone needs to upload a file to reproduce. https://github.com/PacificBiosciences/pbbioconda?tab=readme-ov-file#file-sharing

Checking to see if I'm allowed to share the file security wise.

I learned that demultiplexing was already done on this BAM file. Would this potentially cause that error?

[jmattick]

We support demultiplexing at both the library level and cDNA level as long as you use the correct barcodes fasta for each step. If the Kinnex kit was used, read segmentation using skera is required prior to the second round of demultiplexing.
See examples here: https://isoseq.how/clustering/cli-workflow.html

[kenneditodd]

I have permission to share my BAM file now. However, the file drop off link on the README appears to be down.

[armintoepfer]

We've fixed this error and will provide an updated lima version in future.

[SethMagnusJarvis]

Is there a temporary fix for now? If not do you have a timeline for when the new release will happen? I'm having the same issue.

Edit: For anyone else who sees this, I think the issue may come from using the wrong primers so no reads pass the thresholds. I think this because I did some nonsense primers and got the same error.