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hello,I am trying do snp calling for RNA-seq data ,when I do "Split'N'Trim",I get a trouble with it

zjwangzjwang ChinaMember
the code I ran as follow
gatk SplitNCigarReads \
-R /public/home/zjwang/rna-seq/rice_index/all.con \
-I /public/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.bam \
-O /public/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.split.bam \
-RF ReassignOneMappingQuality \
-RMQF 255 -RMQT 60 \

report an error 'Argument '[R, reference]' cannot be specified more than once', I hope to get your help. thank you .

Best Answers


  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    HI @zjwang

    Can you please post the entire error log and the version of gatk you are using.

  • zjwangzjwang ChinaMember
    HI @bhanuGandham
    thank you for your answer
    the version of gatk is ''
    the entire error log as follow:
    Using GATK jar /public/home/software/opt/bio/software/GATK/
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdkasync_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /public/home/software/opt/bio/software/ SplitNCigarReads -R /public/home/zjwang/rna-sce_index/all.con -I /public/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.bampublic/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.split.bam -RF ReassignOningQuality -RMQF 255 -RMQT 60
    USAGE: SplitNCigarReads [arguments]

    A USER ERROR has occurred: Argument '[R, reference]' cannot be specified more than once.

    Set the system property GATK_STACKTRACE_ON_USER_EXCEPTION (--java-options '-DGATK_STACKTRACE_ON_USER_TION=true') to print the stack trace

    I guess it is a version update, the code and parameter have changed now. I hope I can get your advice.
  • Tiffany_at_BroadTiffany_at_Broad Cambridge, MAMember, Administrator, Broadie, Moderator admin

    Maybe you had a typo when you originally ran the command? In your log the input looks pretty peculiar, no? It is missing -o between "bampublic" -I /public/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.bampublic/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.split.bam

  • zjwangzjwang ChinaMember
    @bhanuGandham :

    gatk SplitNCigarReads -R /public/home/zjwang/rna-seq/rice_index/all.con -I /public/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.bam -O /public/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.split.bam
    Using GATK jar /public/home/software/opt/bio/software/GATK/
    java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -jar /public/home/software/opt/bio/software/GATK/ SplitNCigarReads -R /public/home/zjwang/rna-seq/rice_index/all.con -I /public/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.bam -O /public/home/zjwang/rna-seq/2018_WH/rna-seq_gatk/star_2pass/allign/YY-1.dedup.split.bam
    21:34:33.651 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/public/home/software/opt/bio/software/GATK/!/com/intel/gkl/native/libgkl_compression.so

    21:34:35.387 INFO SplitNCigarReads - HTSJDK Version: 2.18.1
    21:34:35.387 INFO SplitNCigarReads - Picard Version: 2.18.16
    21:34:35.387 INFO SplitNCigarReads - HTSJDK Defaults.COMPRESSION_LEVEL : 2
    21:34:35.387 INFO SplitNCigarReads - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
    21:34:35.387 INFO SplitNCigarReads - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
    21:34:35.387 INFO SplitNCigarReads - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
    21:34:35.387 INFO SplitNCigarReads - Deflater: IntelDeflater
    21:34:35.388 INFO SplitNCigarReads - Inflater: IntelInflater
    21:34:35.388 INFO SplitNCigarReads - GCS max retries/reopens: 20
    21:34:35.388 INFO SplitNCigarReads - Requester pays: disabled
    21:34:35.388 INFO SplitNCigarReads - Initializing engine
    21:34:35.395 INFO SplitNCigarReads - Shutting down engine
    [August 20, 2019 9:34:35 PM CST] org.broadinstitute.hellbender.tools.walkers.rnaseq.SplitNCigarReads done. Elapsed time: 0.03 minutes.
    java.lang.IllegalArgumentException: File is not a supported reference file type: /public/home/zjwang/rna-seq/rice_index/all.con
    at htsjdk.samtools.reference.ReferenceSequenceFileFactory.lambda$getFastaExtension$0(ReferenceSequenceFileFactory.java:249)


    it can not work ,whether the reference has some trouble ?
  • zjwangzjwang ChinaMember
    @bhanuGandham @mshand

    extremely grateful ,I only change the name of 'all.con' into 'all.fasta'. and the content do not change. the problem has resolve.

    thank you for your help
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