Problems with nMutations, nMutationRate, and nLength features

[pursellta]

Hello!
I have been trying to utilize some of the new features in the most recent release and am getting a lot of errors. I am analyzing non-human species and using a custom library. I am using App version: 4.5.0; built=Fri Sep 22 05:39:05 PDT 2023; rev=cdb24b4fb7; lib=repseqio.v3.0.1

1. The -nMutations/-aaMutations [and related commands]
   I run the following command:

mixcr exportClones -f --verbose --filter-out-of-frames --filter-stops -nFeature "{VEndTrimmed:DBeginTrimmed}" -nFeature "{DEndTrimmed:JBeginTrimmed}" -nFeature "{FR1Begin:VEndTrimmed}" -nMutations "{FR1Begin:VEndTrimmed}" -aaFeature "{FR1Begin:VEndTrimmed}" -aaMutations "{FR1Begin:VEndTrimmed}" -baseBiochemicalProperties "VRegionTrimmed" epfu.2007.5RACEbulk.TermSpleen.allelic.clns epfu.2007.5RACEbulk.TermSpleen.allelic.VRegionTrimmed.tsv

And I receive the following error:

App version: 4.5.0; built=Fri Sep 22 05:39:05 PDT 2023; rev=cdb24b4fb7; lib=repseqio.v3.0.1
-nMutations: Alignment attached base gene features not allowed (error in VRegionTrimmed)
com.milaboratory.app.ValidationException: -nMutations: Alignment attached base gene features not allowed (error in VRegionTrimmed)
        at com.milaboratory.o.lt.a(SourceFile:71)
        at com.milaboratory.o.lt.a(SourceFile:52)
        at com.milaboratory.o.rC.invoke(SourceFile:1568)
        at com.milaboratory.o.kQ.validateParameters(SourceFile:49)
        at com.milaboratory.mixcr.export.FieldWithParameters.create(SourceFile:314)
        at com.milaboratory.o.kM.createFields(SourceFile:18)
        at com.milaboratory.o.kX.a(SourceFile:80)
        at com.milaboratory.mixcr.cli.CommandExportClones$Cmd.run1(SourceFile:241)
        at com.milaboratory.mixcr.cli.MiXCRCommandWithOutputs.run0(SourceFile:69)
        at com.milaboratory.mixcr.cli.MiXCRCommand.run(SourceFile:37)
        at picocli.CommandLine.executeUserObject(CommandLine.java:1939)
        at picocli.CommandLine.access$1300(CommandLine.java:145)
        at picocli.CommandLine$RunLast.executeUserObjectOfLastSubcommandWithSameParent(CommandLine.java:2358)
        at picocli.CommandLine$RunLast.handle(CommandLine.java:2352)
        at picocli.CommandLine$RunLast.handle(CommandLine.java:2314)
        at picocli.CommandLine$AbstractParseResultHandler.execute(CommandLine.java:2179)
        at picocli.CommandLine$RunLast.execute(CommandLine.java:2316)
        at com.milaboratory.mixcr.cli.Main.registerLogger$lambda-26(SourceFile:447)
        at picocli.CommandLine.execute(CommandLine.java:2078)
        at com.milaboratory.mixcr.cli.Main.main(SourceFile:98)

2. the -nMutationRate/-aaMutationRate

I run the following command:
$mixcr exportClones -f --verbose --filter-out-of-frames --filter-stops -nFeature "{VEndTrimmed:DBeginTrimmed}" -nFeature "{DEndTrimmed:JBeginTrimmed}" -nMutationRate "{FR1Begin:VEndTrimmed}" -aaMutationRate "{FR1Begin:VEndTrimmed}" epfu.2007.5RACEbulk.TermSpleen.allelic.clns epfu.2007.5RACEbulk.TermSpleen.allelic.VRegionTrimmed.TEST.tsv
And I receive the following error:

App version: 4.5.0; built=Fri Sep 22 05:39:05 PDT 2023; rev=cdb24b4fb7; lib=repseqio.v3.0.1
Require tsv file type, got {FR1Begin:VEndTrimmed}

3. the -nLength/-aaLength
   I run the following command:
   mixcr exportClones -f --verbose --filter-out-of-frames --filter-stops -nFeature "{VEndTrimmed:DBeginTrimmed}" -nFeature "{DEndTrimmed:JBeginTrimmed}" -aaLength "{FR1Begin:VEndTrimmed}" -nLength "{FR1Begin:VEndTrimmed}" epfu.2007.5RACEbulk.TermSpleen.allelic.clns epfu.2007.5RACEbulk.TermSpleen.allelic.VRegionTrimmed.TEST.tsv
   And I receive the following error:

App version: 4.5.0; built=Fri Sep 22 05:39:05 PDT 2023; rev=cdb24b4fb7; lib=repseqio.v3.0.1
Require tsv file type, got {FR1Begin:VEndTrimmed}

Thanks for your help!
-Taylor

[mizraelson]

Hi,
I suggest upgrading to the latest MiXCR v4.6, as the first issue you mentioned is resolved in this version. Moreover, the options -aaMutations, -aaMutationRate, and -baseBiochemicalProperties are not compatible with the VEndTrimmed boundary. Instead, you can use FR3End, for example, "{FR1Begin:FR3End}". This limitation is due to the potential inability to fully translate the nucleotide sequence, as the number of trimmed nucleotides is random. We plan to address this issue in the future.

Please try running the command bellow with V 4.6 and let me know if it works:

mixcr exportClones -f --verbose --filter-out-of-frames --filter-stops -nFeature "{VEndTrimmed:DBeginTrimmed}" -nFeature "{DEndTrimmed:JBeginTrimmed}" -nMutations "{FR1Begin:VEndTrimmed}" -aaFeature "{FR1Begin:FR3End}" -aaMutations "{FR1Begin:FR3End}" -baseBiochemicalProperties "VRegion" -nMutationRate "{FR1Begin:VEndTrimmed}" -aaMutationRate "{FR1Begin:FR3End}" -aaLength "{FR1Begin:VEndTrimmed}" -nLength "{FR1Begin:VEndTrimmed}" epfu.2007.5RACEbulk.TermSpleen.allelic.clns epfu.2007.5RACEbulk.TermSpleen.allelic.VRegionTrimmed.TEST.tsv

[pursellta]

I'm sorry but I have done as you've suggested and it still doesn't work. Here is the code I used:
~/Applications/mixcr-4.6.0/mixcr exportClones -f --verbose --filter-out-of-frames --filter-stops -nMutationsRate "{FR1Begin:VEndTrimmed}" epfu.2007.5RACEbulk.TermSpleen.allelic.clns epfu.2007.5RACEbulk.TermSpleen.allelic.VRegionTrimmed.TEST.tsv

And this is the error I got:

picocli.CommandLine$ExecutionException: Error while running command exportClones java.lang.NullPointerException
        at com.milaboratory.mixcr.cli.Main.registerExceptionHandlers$lambda-12(SourceFile:395)
        at picocli.CommandLine.execute(CommandLine.java:2088)
        at com.milaboratory.mixcr.cli.Main.main(SourceFile:101)
Caused by: java.lang.NullPointerException
        at com.milaboratory.o.Ha.b(SourceFile:53)
        at com.milaboratory.o.Ha.a(SourceFile:39)
        at com.milaboratory.o.GU.c(SourceFile:87)
        at com.milaboratory.o.GV.a(SourceFile:151)
        at com.milaboratory.o.ca$a.b(SourceFile:111)
        at com.milaboratory.o.ca$a.getRegion(SourceFile:123)
        at com.milaboratory.o.bX.a(SourceFile:119)
        at com.milaboratory.o.bX.getRegion(SourceFile:167)
        at com.milaboratory.o.ur.e(SourceFile:1782)
        at com.milaboratory.o.ur.a(SourceFile:19773)
        at com.milaboratory.o.ue.invoke(SourceFile:2112)
        at com.milaboratory.o.lt.extractValue(SourceFile:1216)
        at com.milaboratory.mixcr.export.FieldWithParameters$create$2.invoke(SourceFile:583)
        at com.milaboratory.mixcr.export.FieldWithParameters$create$2.invoke(SourceFile:582)
        at com.milaboratory.o.lE.a(SourceFile:25)
        at com.milaboratory.o.mp.put(SourceFile:40)
        at cc.redberry.pipe.CUtils.drain(CUtils.java:82)
        at cc.redberry.pipe.util.PipeExtensionsKt.drainToAndClose(PipeExtensions.kt:155)
        at com.milaboratory.mixcr.cli.CommandExportClones$Cmd.run1$runExport(SourceFile:322)
        at com.milaboratory.mixcr.cli.CommandExportClones$Cmd.run1(SourceFile:369)
        at com.milaboratory.mixcr.cli.MiXCRCommandWithOutputs.run0(SourceFile:69)
        at com.milaboratory.mixcr.cli.MiXCRCommand.run(SourceFile:37)
        at picocli.CommandLine.executeUserObject(CommandLine.java:1939)
        at picocli.CommandLine.access$1300(CommandLine.java:145)
        at picocli.CommandLine$RunLast.executeUserObjectOfLastSubcommandWithSameParent(CommandLine.java:2358)
        at picocli.CommandLine$RunLast.handle(CommandLine.java:2352)
        at picocli.CommandLine$RunLast.handle(CommandLine.java:2314)
        at picocli.CommandLine$AbstractParseResultHandler.execute(CommandLine.java:2179)
        at picocli.CommandLine$RunLast.execute(CommandLine.java:2316)
        at com.milaboratory.mixcr.cli.Main.registerLogger$lambda-27(SourceFile:514)
        at picocli.CommandLine.execute(CommandLine.java:2078)
        ... 1 more

It does not throw an error with the -aaMutationRate "{FR1Begin:FR3End}" though.

[mizraelson]

This error indicates a missing fasta file from the library. Did you use a custom reference library for the analysis?