I am processing single-cell RNAseq data which I downloaded using GEO accession number (it was in .sra format which I converted to .bam)
Now I'm trying to run the scRNAseq pipeline and got stuck since it seems like I don't have read groups in the header.
I'm trying to use Picard's AddOrReplaceReadGroups with the following command:
java -Xmx15g picard.jar AddOrReplaceReadGroups \
I=SRR5164436.bam \
O=SRR5164436_RG.bam \
RGID=bam1 \
RGLB=lib1 \
RGPL=illumina \
RGPU=ad_lib_Chow1 \
RGSM=sra36
But I get this error:
Exception in thread "main" htsjdk.samtools.SAMFormatException: SAM validation error: ERROR: Record 1, Read name 1, RG ID on SAMRecord not found in header: 1
I don't understand why it is happening. Can you please help? See below the complete error message.
Also, can you please explain where can I get the RGPU, if I don't have the .fastq file? If I cannot, I'd have to put some arbitrary number?
11:51:55.904 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/cvar/jhlab/Kathy/Drop-seq/picard-2.12.2/picard.jar!/com/intel/gkl/native/libgkl_compression.so
[Mon Oct 23 11:51:55 EDT 2017] AddOrReplaceReadGroups INPUT=SRR5164436.bam OUTPUT=SRR5164436_RG.bam RGID=bam1 RGLB=lib1 RGPL=illumina RGPU=ad_lib_Chow1 RGSM=sra36 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 USE_JDK_DEFLATER=false USE_JDK_INFLATER=false
[Mon Oct 23 11:51:55 EDT 2017] Executing as kushakov@uger-c065.broadinstitute.org on Linux 2.6.32-696.6.3.el6.x86_64 amd64; Java HotSpot(TM) 64-Bit Server VM 1.8.0_121-b13; Deflater: Intel; Inflater: Intel; Picard version: 2.12.2-SNAPSHOT
INFO 2017-10-23 11:51:55 AddOrReplaceReadGroups Created read group ID=bam1 PL=illumina LB=lib1 SM=sra36
[Mon Oct 23 11:51:56 EDT 2017] picard.sam.AddOrReplaceReadGroups done. Elapsed time: 0.00 minutes.
Runtime.totalMemory()=2058354688
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
Exception in thread "main" htsjdk.samtools.SAMFormatException: SAM validation error: ERROR: Record 1, Read name 1, RG ID on SAMRecord not found in header: 1
at htsjdk.samtools.SAMUtils.processValidationErrors(SAMUtils.java:454)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:812)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.(BAMFileReader.java:783)
at htsjdk.samtools.BAMFileReader$BAMFileIterator.(BAMFileReader.java:771)
at htsjdk.samtools.BAMFileReader.getIterator(BAMFileReader.java:474)
at htsjdk.samtools.SamReader$PrimitiveSamReaderToSamReaderAdapter.iterator(SamReader.java:478)
at picard.sam.AddOrReplaceReadGroups.doWork(AddOrReplaceReadGroups.java:141)
at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:268)
at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:98)
at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:108)