Je-demultiplex error - Exit code 1

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1

Hi all,

I have used Je-demultiplex successfully on several occasions for demultiplexing my NGS reads, but this time I run into the following error:
Fatal error: Exit code 1 (Tool exception)

Picked up _JAVA_OPTIONS: -Xmx4G -Xms256m
[Mon Dec 05 19:09:54 CET 2022] org.embl.gbcs.je.jemultiplexer.Jemultiplexer FASTQ_FILE1=/srv/galaxy/shared/database/files/000/463/dataset_463183.dat FASTQ_FILE2=/srv/galaxy/shared/database/files/000/463/dataset_463181.dat BARCODE_FILE=/srv/galaxy/shared/database/files/000/463/dataset_463848.dat BARCODE_READ_POS=BOTH BARCODE_FOR_SAMPLE_MATCHING=BOTH REDUNDANT_BARCODES=false MAX_MISMATCHES=1 MIN_MISMATCH_DELTA=1 MIN_BASE_QUALITY=10 XTRIMLEN=0 ZTRIMLEN=0 CLIP_BARCODE=true ADD_BARCODE_TO_HEADER=false ENSURE_IDENTICAL_HEADER_NAMES=false READ_NAME_REPLACE_CHAR=: QUALITY_FORMAT=Standard OUTPUT_DIR=results KEEP_UNASSIGNED_READ=false METRICS_FILE_NAME=/srv/galaxy/shared/etc/tmpdir/jwd/000/237/237240/outputs/galaxy_dataset_1d2c5a17-af8b-4109-912e-ff02b2345e0e.dat GZIP_OUTPUTS=false STATS_ONLY=false STRICT=false UNASSIGNED_FILE_NAME_1=unassigned_1.txt UNASSIGNED_FILE_NAME_2=unassigned_2.txt FORCE=false WRITER_FACTORY_USE_ASYNC_IO=true USE_EMBASE=false 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
[Mon Dec 05 19:09:54 CET 2022] Executing as galaxy_master@vgcn-condor-worker-1-usegalaxy-be on Linux 3.10.0-1062.12.1.el7.x86_64 amd64; OpenJDK 64-Bit Server VM 11.0.9.1-internal+0-adhoc…src; Picard version: 1.2 JdkDeflater
[Mon Dec 05 19:09:55 CET 2022] org.embl.gbcs.je.jemultiplexer.Jemultiplexer done. Elapsed time: 0.01 minutes.
Runtime.totalMemory()=259522560
Exception in thread “main” htsjdk.samtools.SAMException: Sequence header must start with @: ��BC��]�r�Hr������a싟��˜�����!�{"�HT�����l��qDE�����!˛�G^�����������ȑ�Y��k� at line 1 in fastq /srv/galaxy/shared/database/files/000/463/dataset_463183.dat
at htsjdk.samtools.fastq.FastqReader.readNextRecord(FastqReader.java:98)
at htsjdk.samtools.fastq.FastqReader.(FastqReader.java:64)
at htsjdk.samtools.fastq.FastqReader.(FastqReader.java:52)
at org.embl.gbcs.je.jemultiplexer.Jemultiplexer.doDemultiplexingWork(Jemultiplexer.java:236)
at org.embl.gbcs.je.jemultiplexer.AbstractJemultiplexer.doWork(AbstractJemultiplexer.java:1036)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:209)
at picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:147)
at org.embl.gbcs.je.Je.main(Je.java:109)

I’m not sure what to do with information though. My fastq files have the following format: > >

@A01619:145:HJHMCDSX3:1:1101:28754:1000 2:N:0:CCTATTGG+NCGCTTCT
NACACATGTGTGCCTTTGTGGGTTGACGACTTACTTGTGCTTGTTCATGGGGTAGATGAGGACGGGGCCGCCGGCGCCGGCGGTGCGGCCCNCCAGCACGCCACNGAAGACGCCGCGGTCNNAGTCGGCGATGCGGGACGCCNGCACGAAG
+#FFFF,FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF:#FFFFFF:FFFF:#FFFFFFFFFFFFFFF##FF::FFFFFFFFFFF,:FFF#FFFFF

Any advice would be appreciated.

Thank you in advance, Jonas

2

Hi @JonasDS01

This indicates on a potential issue with the file and datatype:
Exception in thread “main” htsjdk.samtools.SAMException: Sequence header must start with @: ��BC��]�r�Hr������a싟
The tool expects a plain text format, but the file looks binary (gzipped).

Please click on the name of FASTQ file. What value do you see in format field? Is it fastq or fastqsanger? If yes, have you specified the datatype during upload?

If the value is fastq or fastqsanger, try the following: click at Edit Attributes (pencil icon) and in the middle window change the datatype to ‘fastqsanger.gz’.

Tools in Galaxy handle data bases on assigned labels called datatypes. It seems an incorrect datatype was assigned to the file.

Kind regards,

Igor

3

Thanks a lot, Igor! My fastq files were indeed compressed, but after decompressing it worked like a charm!

Thanks again.

Kind regards, Jonas