Picard MergeBamAlignment failure

Question

[dec986]

Hello,

I'm trying to go through the drop-seq pipeline, but unfortunately I'm stuck on a seemingly insurmountable technical issue:

I enter this command to merge BAM files:

[Mon Mar 06 13:23:14 EST 2017] picard.sam.MergeBamAlignment UNMAPPED_BAM=./unaligned_mc_tagged_polyA_filtered.bam ALIGNED_BAM=[./aligned.sorted.bam] OUTPUT=./merged.bam REFERENCE_SEQUENCE=/home/con/Alex/mm10/mm10.fasta PAIRED_RUN=false INCLUDE_SECONDARY_ALIGNMENTS=false    CLIP_ADAPTERS=true IS_BISULFITE_SEQUENCE=false ALIGNED_READS_ONLY=false MAX_INSERTIONS_OR_DELETIONS=1 READ1_TRIM=0 READ2_TRIM=0 ALIGNER_PROPER_PAIR_FLAGS=false SORT_ORDER=coordinate PRIMARY_ALIGNMENT_STRATEGY=BestMapq CLIP_OVERLAPPING_READS=true ADD_MATE_CIGAR=true VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json

which generates the output:

[Mon Mar 06 13:23:14 EST 2017] Executing as con@gen on Linux 3.16.0-77-generic amd64; OpenJDK 64-Bit Server VM 1.7.0_121-b00; Picard version: 1.138(89618e408692ff6288c7c880658f32f16fcbec53_1441135673) IntelDeflater
INFO    2017-03-06 13:23:14 SamAlignmentMerger  [./aligned.sorted.bam]
[Mon Mar 06 13:23:14 EST 2017] picard.sam.MergeBamAlignment done. Elapsed time: 0.01 minutes.
Runtime.totalMemory()=2022703104
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
Exception in thread "main" java.lang.NullPointerException
    at picard.sam.AbstractAlignmentMerger.createNewCigarIfMapsOffEndOfReference(AbstractAlignmentMerger.java:631)
    at picard.sam.AbstractAlignmentMerger.createNewCigarsIfMapsOffEndOfReference(AbstractAlignmentMerger.java:654)
    at picard.sam.AbstractAlignmentMerger.updateCigarForTrimmedOrClippedBases(AbstractAlignmentMerger.java:686)
    at picard.sam.AbstractAlignmentMerger.transferAlignmentInfoToFragment(AbstractAlignmentMerger.java:514)
    at picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:410)
    at picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:138)
    at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:248)
    at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:206)
    at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:95)
    at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:105)

I asked this question GATK, but couldn't find a solution, and neither could the Drop-Seq group. I don't even understand what the underlying issue is.

As an aside, I've tried the latest version, but I get errors like this:

 Unsupported major.minor version 52.0

Answer 1

[Pierre Lindenbaum]

Unsupported major.minor version 52.0

Your version of java is out of date; java 8 is required.

If you're able to run your picard with a non-java8 then you're using a very old version of picard. Update picard. Current version is 2.9 : https://github.com/broadinstitute/picard/releases

Answer 2

[dec986]

Hi Pierre,

thanks, but I get another error with a message I don't see how to solve.

   con@e:~/Alex2/DS3-1-43468516$ java -jar ../picard.jar MergeBamAlignment UNMAPPED_BAM=./unaligned_mc_tagged_polyA_filtered.bam ALIGNED_BAM=./aligned.sorted.bam OUTPUT=./merged.bam REFERENCE_SEQUENCE=/home/con/Alex/mm10/mm10.fasta PAIRED_RUN=false INCLUDE_SECONDARY_ALIGNMENTS=false    CLIP_ADAPTERS=true IS_BISULFITE_SEQUENCE=false ALIGNED_READS_ONLY=false MAX_INSERTIONS_OR_DELETIONS=1 READ1_TRIM=0 READ2_TRIM=0 ALIGNER_PROPER_PAIR_FLAGS=false SORT_ORDER=coordinate PRIMARY_ALIGNMENT_STRATEGY=BestMapq CLIP_OVERLAPPING_READS=true ADD_MATE_CIGAR=true VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json
    [Tue Mar 07 09:12:12 EST 2017] picard.sam.MergeBamAlignment UNMAPPED_BAM=./unaligned_mc_tagged_polyA_filtered.bam ALIGNED_BAM=[./aligned.sorted.bam] OUTPUT=./merged.bam REFERENCE_SEQUENCE=/home/con/Alex/mm10/mm10.fasta PAIRED_RUN=false CLIP_ADAPTERS=true IS_BISULFITE_SEQUENCE=false ALIGNED_READS_ONLY=false MAX_INSERTIONS_OR_DELETIONS=1 READ1_TRIM=0 READ2_TRIM=0 ALIGNER_PROPER_PAIR_FLAGS=false SORT_ORDER=coordinate PRIMARY_ALIGNMENT_STRATEGY=BestMapq CLIP_OVERLAPPING_READS=true INCLUDE_SECONDARY_ALIGNMENTS=false ADD_MATE_CIGAR=true VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json    ATTRIBUTES_TO_REVERSE=[OQ, U2] ATTRIBUTES_TO_REVERSE_COMPLEMENT=[E2, SQ] UNMAP_CONTAMINANT_READS=false MIN_UNCLIPPED_BASES=32 MATCHING_DICTIONARY_TAGS=[M5, LN] UNMAPPED_READ_STRATEGY=DO_NOT_CHANGE
    [Tue Mar 07 09:12:12 EST 2017] Executing as con@e on Linux 4.4.0-65-generic amd64; OpenJDK 64-Bit Server VM 1.8.0_121-8u121-b13-0ubuntu1.16.04.2-b13; Picard version: 2.9.0-1-gf5b9f50-SNAPSHOT
    INFO    2017-03-07 09:12:12 SamAlignmentMerger  Processing SAM file(s): [./aligned.sorted.bam]
    [Tue Mar 07 09:12:12 EST 2017] picard.sam.MergeBamAlignment done. Elapsed time: 0.01 minutes.
    Runtime.totalMemory()=124780544
    To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
    Exception in thread "main" java.lang.IllegalArgumentException: Do not use this function to merge dictionaries with different sequences in them. Sequences must be in the same order as well. Found [1, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2, 3, 4, 5, 6, 7, 8, 9, CHR_MG117_PATCH, CHR_MG132_PATCH, CHR_MG153_PATCH, CHR_MG184_PATCH, CHR_MG3231_PATCH, CHR_MG3561_PATCH, CHR_MG3562_PATCH, CHR_MG3609_PATCH, CHR_MG3829_PATCH, CHR_MG3833_PATCH, CHR_MG3835_PATCH, CHR_MG4136_PATCH, CHR_MG4151_PATCH, CHR_MG4180_PATCH, CHR_MG4209_PATCH, CHR_MG4211_PATCH, CHR_MG4212_PATCH, CHR_MG4213_PATCH, CHR_MG4214_PATCH, CHR_MG4222_MG3908_PATCH, CHR_MG4248_PATCH, CHR_MG4249_PATCH, CHR_MG4254_PATCH, CHR_MG4255_PATCH, CHR_MG4259_PATCH, CHR_MG4261_PATCH, CHR_MG4264_PATCH, CHR_MG4265_PATCH, CHR_MG4266_PATCH, CHR_MMCHR1_CHORI29_IDD5_1, MT, X, Y, JH584299.1, ...
...abbreviated for character limit...
Un_GL456239, Un_GL456359, Un_GL456360, Un_GL456366, Un_GL456367, Un_GL456368, Un_GL456370, Un_GL456372, Un_GL456378, Un_GL456379, Un_GL456381, Un_GL456382, Un_GL456383, Un_GL456385, Un_GL456387, Un_GL456389, Un_GL456390, Un_GL456392, Un_GL456393, Un_GL456394, Un_GL456396, Un_JH584304, X, X_GL456233_random, Y, Y_JH584300_random, Y_JH584301_random, Y_JH584302_random, Y_JH584303_random].
        at htsjdk.samtools.SAMSequenceDictionary.mergeDictionaries(SAMSequenceDictionary.java:305)
        at picard.sam.SamAlignmentMerger.getDictionaryForMergedBam(SamAlignmentMerger.java:197)
        at picard.sam.AbstractAlignmentMerger.mergeAlignment(AbstractAlignmentMerger.java:346)
        at picard.sam.SamAlignmentMerger.mergeAlignment(SamAlignmentMerger.java:181)
        at picard.sam.MergeBamAlignment.doWork(MergeBamAlignment.java:282)
        at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:205)
        at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:94)
        at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:104)

Perhaps there is another alternative to Picard's MergeBamAlignment?

[Pierre Lindenbaum]

The mesage is clear:

Do not use this function to merge dictionaries with different sequences in them. Sequences must be in the same order as well. Found [1, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2, 3, 4, 5, 6, 7, 8, 9, CHR_MG117_PATCH, CHR_MG132_PATCH, CHR_MG153_PATCH, CHR_MG184_PATCH, CHR_MG3231_PATCH, CHR_MG3561_PATCH, CHR_MG3562_PATCH, CHR_MG3609_PATCH, CHR_MG3829_PATCH, CHR_MG3833_PATCH, CHR_MG3835_PATCH, CHR_MG4136_PATCH, CHR_MG4151_PATCH, CHR_MG4180_PATCH, CHR_MG4209_PATCH, CHR_MG4211_PATCH, CHR_MG4212_PATCH, CHR_MG4213_PATCH, CHR_MG4214_PATCH, CHR_MG4222_MG3908_PATCH, CHR_MG4248_PATCH, CHR_MG4249_PATCH, CHR_MG4254_PATCH, CHR_MG4255_PATCH, CHR_MG4259_PATCH, CHR_MG4261_PATCH, CHR_MG4264_PATCH, CHR_MG4265_PATCH, CHR_MG4266_PATCH, CHR_MMCHR1_CHORI29_IDD5_1, MT, X, Y, JH584299.1, ...