Error when running findAlleles

[pursellta]

I am processing 10x 5' chemistry BCR enriched single cell data with a custom reference for a non-human species. When I run the following code:

(base) tpursell@yellowbird:$ ~/Applications/mixcr-4.4.2-1-dev/mixcr findAlleles -f --output-template Mufu_0dpi1.allelic.clns  --export-alleles-mutations Mufu_0dpi1-allele_stats.tsv ~/Applications/mixcr-4.4.2-1-dev/Ferret/MuFu_0dpi-1-BCR-V2.clns

I get the following output/error:

Step 1 of 7: count diversity for dataset: 0%
Step 2 of 7: grouping by the same J gene: 0%
Step 2 of 7: Searching for J alleles: 0%
Step 3 of 7: grouping by the same V gene: 0%
Step 3 of 7: Searching for V alleles: 15.7%
Step 3 of 7: Searching for V alleles: 99.1%  ETA: 00:00:00
Step 4 of 7: grouping by the same J gene: 0%
Step 4 of 7: Searching for J alleles: 25%
Step 5 of 7: grouping by the same V gene: 0%
Step 5 of 7: Searching for V alleles: 15.7%
Error on searching alleles for IGKV_74
4.4.2-1-develop; built=Sun Jul 30 01:49:05 PDT 2023; rev=e69e9041ed; lib=repseqio.v3.0.1
picocli.CommandLine$ExecutionException: Error while running command findAlleles java.lang.NullPointerException
        at com.milaboratory.mixcr.cli.Main.registerExceptionHandlers$lambda-12(SourceFile:323)
        at picocli.CommandLine.execute(CommandLine.java:2088)
        at com.milaboratory.mixcr.cli.Main.main(SourceFile:98)
Caused by: java.lang.NullPointerException
        at com.milaboratory.mixcr.alleles.AllelesBuilder.a(SourceFile:1026)
        at com.milaboratory.mixcr.alleles.AllelesBuilder.a(SourceFile:676)
        at com.milaboratory.mixcr.alleles.AllelesBuilder.a(SourceFile:503)
        at com.milaboratory.mixcr.alleles.AllelesBuilder.a(SourceFile:474)
        at com.milaboratory.mixcr.alleles.AllelesBuilder.a(SourceFile:30213)
        at com.milaboratory.o.hq.process(SourceFile:66)
        at cc.redberry.pipe.blocks.ParallelProcessor$Worker.run(ParallelProcessor.java:308)
        at java.base/java.lang.Thread.run(Thread.java:829)

I was using the 4.4.1 version until I found a thread where someone was getting a similar error and was able to use the 4.4.2-dev version, but the error remains. Any advice/help would be amazing! Thank you!

[gnefedev]

Hello!

In IGKV_74 gene absent gene feature {CDR3Begin:VEnd} - GermlineVCDR3Part.

How you build your custom reference? Is there VEnd in the anchor points? Or maybe it's equal to CDR3Begin?

[pursellta]

I followed the instructions on the MiXCR website and used repseqio (v1.8.0-4). Looking into the reference file, it looks like this gene only has FR1Begin and VEnd anchor points. Should this type of V gene be removed during the debug step of the reference build?

[Alex-Davydov]

Hi, can you send the nucleotide sequence to us, we will try to find the anchor points manually.

[pursellta]

Of course, but would it cause trouble if I just removed it from the current reference?

[Alex-Davydov]

Yes, you can remove it