Error while running command buildLibrary java.lang.IllegalArgumentException: Unknown symbol " " #1568

silvia1234567890 · 2024-02-23T13:56:23Z

silvia1234567890
Feb 23, 2024

Hello,

I'm making a library for another species, and I'm getting this error, could you help me please?

Code:

mixcr buildLibrary --debug \
  --v-genes-from-fasta TRAV.fasta \
  --v-gene-feature VExon2 \
  --j-genes-from-fasta TRAJ.fasta \
  --chain TRA \
  --taxon-id 8022 \
  --species Oncorhynchus_mykiss \
  Omykiss-TRA.json.gz

Error message:

Please copy the following information along with the stacktrace:
   Version: 4.6.0; built=Sat Dec 09 20:48:42 CET 2023; rev=c9fafa41fe; lib=repseqio.v4.0
        OS: Mac OS X
      Java: 21.0.2
  Cmd args: buildLibrary --debug --v-genes-from-fasta TRAV.fasta --v-gene-feature VExon2 --j-genes-from-fasta TRAJ.fasta --chain TRA --taxon-id 8022 --species Oncorhynchus_mykiss Omykiss-TRA.json.gz
picocli.CommandLine$ExecutionException: Error while running command buildLibrary java.lang.IllegalArgumentException: Unknown symbol " "
	at com.milaboratory.mixcr.cli.Main.registerExceptionHandlers$lambda-12(SourceFile:395)
	at picocli.CommandLine.execute(CommandLine.java:2088)
	at com.milaboratory.mixcr.cli.Main.main(SourceFile:101)
Caused by: java.lang.IllegalArgumentException: Unknown symbol " "
	at com.milaboratory.o.bJ.dataFromChars(SourceFile:90)
	at com.milaboratory.o.bJ.<init>(SourceFile:31)
	at com.milaboratory.core.sequence.NucleotideSequence.<init>(SourceFile:135)
	at io.repseq.cli.FromFastaActionAbstract.go(SourceFile:87)
	at io.repseq.cli.jcomander.JCommanderBasedMain.main(SourceFile:157)
	at io.repseq.cli.Main.main(SourceFile:94)
	at com.milaboratory.mixcr.cli.CommandBuildLibrary.fromFasta(SourceFile:362)
	at com.milaboratory.mixcr.cli.CommandBuildLibrary.mkLibraryForGeneType(SourceFile:312)
	at com.milaboratory.mixcr.cli.CommandBuildLibrary.run1(SourceFile:381)
	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

Thank you in advance!

Answered by mizraelson

Feb 24, 2024

Hi, could it be that you have some extra space in your command that is not shown here. I have copy pasted your command and it works fine (I only changed the fasta files' names to the one I have).

View full answer

mizraelson · 2024-02-24T00:45:19Z

mizraelson
Feb 24, 2024
Collaborator

Hi, could it be that you have some extra space in your command that is not shown here. I have copy pasted your command and it works fine (I only changed the fasta files' names to the one I have).

0 replies

silvia1234567890 · 2024-02-25T19:28:34Z

silvia1234567890
Feb 25, 2024
Author

Hi, the problem was in the fasta files because I found some blank spaces there. Sorry for the inconvenience!

0 replies

silvia1234567890 · 2024-02-26T12:00:28Z

silvia1234567890
Feb 26, 2024
Author

Hi, one question related to this.
When I run the code above (build a library for TCR alfa), the fact that it does not find any alignment with any of the V segments in the fasta file is not normal, isn't it?
I tried --v-gene-feature VRegion and --v-gene-feature VExon2 and I get the same for every V segment:
For example:

Processing: aOncmyk_Chr8_CM023226.2_TRAV17-3_TRAV_130-35_F_61467747_61468085 (F) TRA
No alignments found.

I send you the command line in pdf and the library I got by executing this command...
Oncorhynchus_mykiss-TRA.json.gz
library_terminal.pdf

And when I run debugLibrary with the library (using --v-gene-feature VRegion):
mixcr debugLibrary Oncorhynchus_mykiss-TRA.json.gz
for each V segment, I get this at the beginning:

aOncmyk_Chr8_CM023226.2_TRAV17-3_TRAV_130-35_F_61467747_61468085 (F) TRA : 8022

WARNINGS: 
Unable to find CDR3 start
VRegion contains a stop codon

And with the library (using --v-gene-feature VExon2), I get this for each V segment:

aOncmyk_Chr8_CM023226.2_TRAV17-3_TRAV_130-35_F_61467747_61468085 (F) TRA : 8022

WARNINGS: 
Unable to find CDR3 start

Maybe I'm using the wrong commands... Could you help me please?
Thank you in advance!

0 replies

mizraelson · 2024-02-27T02:30:34Z

mizraelson
Feb 27, 2024
Collaborator

can you share the FASTA files for V genes?

0 replies

silvia1234567890 · 2024-02-27T08:15:13Z

silvia1234567890
Feb 27, 2024
Author

can you share the FASTA files for V genes?

docs.zip

Hi,
I modified the headers of the fasta files just in case, and I reran the code again and nothing, same problem again... And also for the TRB... I send you the V and J segments for TRA, a copy of the terminal when running the code and the library that I get as output... Please, help me!

Thank you in advance!

0 replies

mizraelson · 2024-02-29T20:31:37Z

mizraelson
Feb 29, 2024
Collaborator

Hi, so the main issue here is that sequences seem to have some extra nucleotides before the first FR1 position. Thats why FR1 is identified erroneously. You can either fix it in the FASTA, or use {CDR1Begin:VEnd} feature instead.
Oncorhynchus_mykiss-TRA.json

0 replies

silvia1234567890 · 2024-03-04T10:58:32Z

silvia1234567890
Mar 4, 2024
Author

Hi,
I understand.. But, how could I do that? I mean, in the fasta files I just have to identify the extra nucleotides and remove them, right? But how could I do it using that feature {CDR1Begin:VEnd}?

1 reply

mizraelson Mar 6, 2024
Collaborator

Yes, the best way is to just identify and add the missing nucleotides from FR1 or remove extra ones if any. For {CDR1Begin:VEnd} you can try using the files you have as is but it is better to trim your sequences to the very beginning of CDR1 too.

silvia1234567890 · 2024-03-11T14:26:00Z

silvia1234567890
Mar 11, 2024
Author

Hello, I think I tried everything and it's not working...

The V segments are from FR1 to the Cys where FR3 ends and CDR3 region starts.

Code:

mixcr buildLibrary --debug \
  --v-genes-from-fasta TRAVedited.fasta \
  --v-gene-feature {FR1Begin:FR3End} \
  --j-genes-from-fasta TRAJ_edited.fasta \
  --chain TRA \
  --taxon-id 8022 \
  --species Oncorhynchus_mykiss \
  Oncorhynchus_mykiss-TRA.json.gz

Results:

no alignments found for any of the V segments..
WARNING: Unable to find CDR3 end
And this error message at the end:

Please copy the following information along with the stacktrace:
   Version: 4.6.0; built=Sat Dec 09 20:48:42 CET 2023; rev=c9fafa41fe; lib=repseqio.v4.0
        OS: Mac OS X
      Java: 21.0.2
  Cmd args: buildLibrary --debug --v-genes-from-fasta TRAVedited.fasta --v-gene-feature {FR1Begin:FR3End} --j-genes-from-fasta TRAJ_edited.fasta --chain TRA --taxon-id 8022 --species Oncorhynchus_mykiss Oncorhynchus_mykiss-TRA.json.gz
picocli.CommandLine$ExecutionException: Error while running command buildLibrary java.lang.IllegalArgumentException: Can't get feature [[FR3EndCDR3Begin, FR3EndCDR3Begin+3]] from TRAV19-1
	at com.milaboratory.mixcr.cli.Main.registerExceptionHandlers$lambda-12(SourceFile:395)
	at picocli.CommandLine.execute(CommandLine.java:2088)
	at com.milaboratory.mixcr.cli.Main.main(SourceFile:101)
Caused by: java.lang.IllegalArgumentException: Can't get feature [[FR3EndCDR3Begin, FR3EndCDR3Begin+3]] from TRAV19-1
	at com.milaboratory.o.Gh.a(SourceFile:49)
	at com.milaboratory.o.Gh.getFeature(SourceFile:44)
	at io.repseq.core.VDJCGene.getFeature(SourceFile)
	at io.repseq.cli.DebugAction.go(SourceFile:62)
	at io.repseq.cli.jcomander.JCommanderBasedMain.main(SourceFile:157)
	at io.repseq.cli.Main.main(SourceFile:94)
	at com.milaboratory.mixcr.cli.CommandBuildLibrary.run1(SourceFile:390)
	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$lambda-27(SourceFile:514)
	at picocli.CommandLine.execute(CommandLine.java:2078)
	... 1 more
Caused by: com.milaboratory.o.bZ: Can't extract sequence for (0->293), have available (0->290)
	at com.milaboratory.o.bg.a(SourceFile:114)
	at com.milaboratory.o.bg.a(SourceFile:35)
	at com.milaboratory.o.bh.getRegion(SourceFile:107)
	at com.milaboratory.o.ca$a.getRegion(SourceFile:124)
	at com.milaboratory.o.bX.a(SourceFile:119)
	at com.milaboratory.o.bX.getRegion(SourceFile:167)
	at com.milaboratory.o.FX.getSequence(SourceFile:1039)
	at com.milaboratory.o.FK.getFeature(SourceFile:48)
	at io.repseq.core.PartitionedSequenceCached.getFeature$lambda-0(SourceFile:32)
	at java.base/java.util.HashMap.computeIfAbsent(HashMap.java:1228)
	at io.repseq.core.PartitionedSequenceCached.getFeature(SourceFile:31)
	at com.milaboratory.o.Gf.getFeature(SourceFile:1000)
	at com.milaboratory.o.Gh.a(SourceFile:47)
	... 18 more

Could you help me please?
I send you the fasta files if it helps and the library I got running this code.
Thank you in advance!

2 replies

mizraelson Mar 12, 2024
Collaborator

Hi,
I can't download the file you attached. Can you share it again? Alternatively, please send do support@milaboratories.com.

silvia1234567890 Mar 13, 2024
Author

Hi, sent to support@milaboratories.com
Thank you in advance!

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Error while running command buildLibrary java.lang.IllegalArgumentException: Unknown symbol " " #1568

silvia1234567890 Feb 23, 2024

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