I always fail to run the FilterSamReads. Even my input is BAM file neither form the HISAT2 nor the CleanSam.
Please guide me on how to deal with this.
The error message as shown below:
Picked up _JAVA_OPTIONS: -Djava.io.tmpdir=/galaxy-repl/main/jobdir/030/032/30032391/_job_tmp -Xmx7g -Xms256m
09:11:59.266 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/cvmfs/main.galaxyproject.org/deps/_conda/pkgs/picard-2.18.2-py36_0/share/picard-2.18.2-0/picard.jar!/com/intel/gkl/native/libgkl_compression.so
Picked up _JAVA_OPTIONS: -Djava.io.tmpdir=/galaxy-repl/main/jobdir/030/032/30032391/_job_tmp -Xmx7g -Xms256m
09:17:25.817 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/cvmfs/main.galaxyproject.org/deps/_conda/pkgs/picard-2.18.2-py36_0/share/picard-2.18.2-0/picard.jar!/com/intel/gkl/native/libgkl_compression.so
ERROR 2020-08-16 09:17:26 FilterSamReads Failed to filter query_sorted_bam.bam
htsjdk.samtools.SAMException: Second record is not marked as second of pair: NS500784:597:HVHV7BGXB:4:11401:10000:19146 1/2 76b aligned read.
at htsjdk.samtools.SamPairUtil.assertMate(SamPairUtil.java:134)
at htsjdk.samtools.filter.FilteringSamIterator.getNextRecord(FilteringSamIterator.java:140)
at htsjdk.samtools.filter.FilteringSamIterator.next(FilteringSamIterator.java:108)
at picard.sam.FilterSamReads.filterReads(FilterSamReads.java:255)
at picard.sam.FilterSamReads.doWork(FilterSamReads.java:345)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:282)
at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:98)
at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:108)
[E::hts_open_format] Failed to open file “/galaxy-repl/main/files/044/077/dataset_44077826.dat” : No such file or directory
[E::hts_open_format] Failed to open file “/galaxy-repl/main/files/044/077/dataset_44077826.dat” : No such file or directory