[Jane M]

Thank you dpryan,

I think that it's finally working. I ran Picard and I got an non empty file and:

java -jar ./AddOrReplaceReadGroups.jar I=~/../../../data/patient1/s_garma-fibros_converted_sorted.bam O=~/../../../data/patient1/picard_s_garma-fibros_converted_sorted.bam \
> SORT_ORDER=coordinate CREATE_INDEX=true \
> RGPL=illumina RGID=garma-fibros RGSM=garma RGLB=toto RGPU=tata VALIDATION_STRINGENCY=LENIENT
[Wed Jan 18 16:42:12 CET 2012] net.sf.picard.sam.AddOrReplaceReadGroups INPUT=/home/merlevede/../../../data/patient1/s_garma-fibros_converted_sorted.bam OUTPUT=/home/merlevede/../../../data/patient1/picard_s_garma-fibros_converted_sorted.bam SORT_ORDER=coordinate RGID=garma-fibros RGLB=toto RGPL=illumina RGPU=tata RGSM=garma VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=true VERBOSITY=INFO QUIET=false COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_MD5_FILE=false
[Wed Jan 18 16:42:12 CET 2012] Executing as merlevede@U1009-PCJane on Linux 3.1.6-1.fc16.x86_64 amd64; OpenJDK 64-Bit Server VM 1.6.0_22-b22; Picard version: 1.60(1086)
INFO 2012-01-18 16:42:12 AddOrReplaceReadGroups Created read group ID=garma-fibros PL=illumina LB=toto SM=garma

Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1106:7158:91967, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1205:8058:144770, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1206:20528:185225, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1203:4551:95140, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:2205:4442:123188, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1204:17464:45698, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:2208:16832:136911, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1107:17717:4065, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:2104:7277:38433, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1105:16587:151639, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:2108:18278:149545, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1104:12598:60315, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1105:1489:49925, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:2205:16036:86626, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1204:9783:76003, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1103:8346:98868, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1105:8354:181686, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1108:9333:173330, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:2106:5867:60527, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:2202:1737:149838, CIGAR M operator maps off end of reference
Ignoring SAM validation error: ERROR: Read name HWI-ST584_81:4:1203:14352:92986, CIGAR M operator maps off end of reference
[Wed Jan 18 17:23:48 CET 2012] net.sf.picard.sam.AddOrReplaceReadGroups done. Elapsed time: 41,60 minutes.
Runtime.totalMemory()=733675520
[merlevede@U1009-PCJane picard-tools-1.60]$

et la commande de zee donne cette fois quelque chose :

[merlevede@U1009-PCJane patient1]$ samtools view -H picard_s_garma-fibros_converted_sorted.bam | grep ^@RG
@RG ID:garma-fibros PL:illumina PU:tata LB:toto SM:garma

To be sure that my BAM files are correct now, I'm running Picard on my "(tumoral) BAM file".
Then, I will rerun GATK on my two files to do the SomaticIndelDetector analysis.
I will keep you informed if it's definitely ok. I hope so !

[Carlos Borroto]

[merlevede@U1009-PCJane patient1]$ samtools view -H picard_s_garma-fibros_converted_sorted.bam | grep ^@RG
@RG	ID:garma-fibros	PL:illumina	PU:tata	LB:toto	SM:garma

I think you need to be a little more careful than just putting gibberish on some of these fields. I'm in the process of getting familiar with GATK, there are some parts and don't quite understand yet, like the use of these fields.

This two links are the principal source of my information:
Best Practice Variant Detection with the GATK v3 [broadinstitute.org]
Exome sequencing analysis manual [seqanswers.com]

From what I can understand, several of the tools in the GATK pipeline use these fields in order to do their magic. Again I'm not close to be sure what are the dos and don'ts here, but my rule of thumb is to set these three to the same value, sample name(ex. control01, treatment01, etc):
RGLB=String Read Group Library Required.
RGPU=String Read Group platform unit (eg. run barcode) Required.
RGSM=String Read Group sample name Required.

And this one to my platform(ex. illumina):
RGPL=String Read Group platform (e.g. illumina, solid) Required.

This might not be completely correct, but this way at least I'm not confusing the tools telling it two samples are from the same sequencer lane or library preparation, when they are actually not. Which you might be doing if you reuse the same 'gibberish' for more than one BAM file.

I would actually love to find a clear documentation on how to set the correct values. Mainly what information I need to get from the sequencing center to set these values. Are there any documentation I could take a look at?

Regards,
Carlos

[Jane M]

Thanks for the information Carlos. If you find a clear documentation for setting these parameters, I am also interested !


I ran picard on my two datasets and my BAM files seem to be correctly formatted now.
I reran GATK, but I still have an error. It seems that it doesn't accept the mitochondrial chromosome. That's a pity because I was finally at the end of the analysis, after one week of trials

/opt/jdk1.7.0_02/bin/java -Xmx10g -jar GenomeAnalysisTK.jar -R ~/fasta/hg19.fasta -T SomaticIndelDetector --minCoverage 10 -o ~/../../../data/patient1/garma_indels.vcf -verbose indels.txt -I:normal ~/../../../data/patient1/picard_s_garma-fibros_converted_sorted.bam -I:tumor ~/../../../data/patient1/picard_s_garma-296_converted_sorted.bam

INFO 09:28:04,178 HelpFormatter - ---------------------------------------------------------------------------------
INFO 09:28:04,180 HelpFormatter - The Genome Analysis Toolkit (GATK) v1.4-15-gcd43f01, Compiled 2012/01/12 16:14:10
INFO 09:28:04,180 HelpFormatter - Copyright (c) 2010 The Broad Institute
INFO 09:28:04,180 HelpFormatter - Please view our documentation at http://www.broadinstitute.org/gsa/wiki
INFO 09:28:04,181 HelpFormatter - For support, please view our support site at http://getsatisfaction.com/gsa
INFO 09:28:04,181 HelpFormatter - Program Args: -R /home/merlevede/fasta/hg19.fasta -T SomaticIndelDetector --minCoverage 10 -o /home/merlevede/../../../data/patient1/garma_indels.vcf -verbose indels.txt -I:normal /home/merlevede/../../../data/patient1/picard_s_garma-fibros_converted_sorted.bam -I:tumor /home/merlevede/../../../data/patient1/picard_s_garma-296_converted_sorted.bam
INFO 09:28:04,182 HelpFormatter - Date/Time: 2012/01/20 09:28:04
INFO 09:28:04,182 HelpFormatter - ---------------------------------------------------------------------------------
INFO 09:28:04,182 HelpFormatter - ---------------------------------------------------------------------------------
INFO 09:28:04,195 GenomeAnalysisEngine - Strictness is SILENT
INFO 09:28:04,237 SAMDataSource$SAMReaders - Initializing SAMRecords in serial
INFO 09:28:04,261 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.02
INFO 09:28:04,343 SomaticIndelDetectorWalker - No gene annotations available
INFO 09:28:09,169 TraversalEngine - [INITIALIZATION COMPLETE; TRAVERSAL STARTING]
INFO 09:28:09,169 TraversalEngine - Location processed.reads runtime per.1M.reads completed total.runtime remaining
INFO 09:28:34,684 TraversalEngine - chr1:28598356 2.83e+06 30.0 s 10.6 s 0.9% 54.1 m 53.6 m
INFO 09:29:04,690 TraversalEngine - chr1:60378092 6.04e+06 60.0 s 9.9 s 2.0% 51.3 m 50.3 m
INFO 09:29:34,692 TraversalEngine - chr1:104116800 9.18e+06 90.0 s 9.8 s 3.4% 44.6 m 43.1 m
INFO 09:30:04,697 TraversalEngine - chr1:155165725 1.29e+07 2.0 m 9.3 s 5.0% 39.9 m 37.9 m
INFO 09:30:34,708 TraversalEngine - chr1:186157226 1.64e+07 2.5 m 9.1 s 6.0% 41.6 m 39.1 m
INFO 09:31:04,714 TraversalEngine - chr1:230810753 1.99e+07 3.0 m 9.0 s 7.5% 40.2 m 37.2 m
INFO 09:31:34,716 TraversalEngine - chr2:15519846 2.22e+07 3.5 m 9.5 s 8.6% 40.9 m 37.4 m
INFO 09:32:04,720 TraversalEngine - chr2:61729349 2.57e+07 4.0 m 9.4 s 10.0% 39.8 m 35.8 m
INFO 09:32:34,728 TraversalEngine - chr2:113404649 2.91e+07 4.5 m 9.3 s 11.7% 38.4 m 33.9 m
INFO 09:33:04,729 TraversalEngine - chr2:170063504 3.24e+07 5.0 m 9.3 s 13.5% 36.9 m 31.9 m
INFO 09:33:34,734 TraversalEngine - chr2:204820624 3.59e+07 5.5 m 9.2 s 14.7% 37.5 m 32.0 m
INFO 09:34:04,740 TraversalEngine - chr3:3111817 3.89e+07 6.0 m 9.3 s 16.0% 37.5 m 31.5 m
INFO 09:34:34,743 TraversalEngine - chr3:48501244 4.22e+07 6.5 m 9.2 s 17.5% 37.2 m 30.7 m
INFO 09:35:04,744 TraversalEngine - chr3:108117422 4.54e+07 7.0 m 9.2 s 19.4% 36.1 m 29.1 m
INFO 09:35:34,748 TraversalEngine - chr3:148577626 4.88e+07 7.5 m 9.2 s 20.7% 36.2 m 28.7 m
INFO 09:36:04,754 TraversalEngine - chr3:197748351 5.21e+07 8.0 m 9.2 s 22.3% 35.9 m 27.9 m
INFO 09:36:34,757 TraversalEngine - chr4:56325162 5.50e+07 8.5 m 9.3 s 24.1% 35.2 m 26.7 m
INFO 09:37:04,764 TraversalEngine - chr4:106861604 5.84e+07 9.0 m 9.3 s 25.8% 34.9 m 25.9 m
INFO 09:37:34,773 TraversalEngine - chr4:169215011 6.16e+07 9.5 m 9.3 s 27.8% 34.2 m 24.7 m
INFO 09:38:04,775 TraversalEngine - chr5:37665146 6.44e+07 10.0 m 9.3 s 29.7% 33.7 m 23.7 m
INFO 09:38:34,784 TraversalEngine - chr5:94859344 6.77e+07 10.5 m 9.3 s 31.5% 33.3 m 22.8 m
INFO 09:39:04,794 TraversalEngine - chr5:141334425 7.11e+07 11.0 m 9.3 s 33.0% 33.3 m 22.3 m
INFO 09:39:34,806 TraversalEngine - chr6:5109692 7.40e+07 11.5 m 9.3 s 34.5% 33.4 m 21.8 m
INFO 09:40:04,817 TraversalEngine - chr6:41773298 7.73e+07 12.0 m 9.3 s 35.7% 33.6 m 21.6 m
INFO 09:40:34,825 TraversalEngine - chr6:91364545 8.07e+07 12.5 m 9.3 s 37.3% 33.5 m 21.0 m
INFO 09:41:04,826 TraversalEngine - chr6:147249706 8.40e+07 13.0 m 9.3 s 39.1% 33.3 m 20.3 m
INFO 09:41:34,831 TraversalEngine - chr7:23650833 8.69e+07 13.5 m 9.3 s 40.6% 33.2 m 19.7 m
INFO 09:42:04,836 TraversalEngine - chr7:87046856 9.00e+07 14.0 m 9.3 s 42.7% 32.8 m 18.8 m
INFO 09:42:34,838 TraversalEngine - chr7:128220270 9.34e+07 14.5 m 9.3 s 44.0% 33.0 m 18.5 m
INFO 09:43:04,839 TraversalEngine - chr8:16032718 9.64e+07 15.0 m 9.3 s 45.5% 33.0 m 18.0 m
INFO 09:43:34,848 TraversalEngine - chr8:70978649 9.97e+07 15.5 m 9.3 s 47.3% 32.8 m 17.3 m
INFO 09:44:04,849 TraversalEngine - chr8:133141853 1.03e+08 16.0 m 9.3 s 49.3% 32.5 m 16.5 m
INFO 09:44:34,861 TraversalEngine - chr9:36607567 1.06e+08 16.5 m 9.4 s 50.9% 32.4 m 15.9 m
INFO 09:45:04,863 TraversalEngine - chr9:111848126 1.09e+08 17.0 m 9.3 s 53.3% 31.9 m 14.9 m
INFO 09:45:34,870 TraversalEngine - chr10:5788378 1.12e+08 17.5 m 9.4 s 54.5% 32.1 m 14.6 m
INFO 09:46:04,881 TraversalEngine - chr10:63662048 1.15e+08 18.0 m 9.4 s 56.3% 32.0 m 14.0 m
INFO 09:46:34,884 TraversalEngine - chr10:102740355 1.19e+08 18.5 m 9.3 s 57.6% 32.1 m 13.6 m
INFO 09:47:04,885 TraversalEngine - chr11:4967349 1.22e+08 19.0 m 9.4 s 58.8% 32.3 m 13.3 m
INFO 09:47:34,886 TraversalEngine - chr11:47843747 1.25e+08 19.5 m 9.4 s 60.2% 32.4 m 12.9 m
INFO 09:48:04,894 TraversalEngine - chr11:83180407 1.28e+08 20.0 m 9.3 s 61.3% 32.6 m 12.6 m
INFO 09:48:34,902 TraversalEngine - chr11:125325718 1.32e+08 20.5 m 9.3 s 62.7% 32.7 m 12.2 m
INFO 09:49:04,904 TraversalEngine - chr12:25232248 1.35e+08 21.0 m 9.4 s 63.8% 32.9 m 11.9 m
INFO 09:49:34,911 TraversalEngine - chr12:56646266 1.38e+08 21.5 m 9.4 s 64.9% 33.2 m 11.7 m
INFO 09:50:04,920 TraversalEngine - chr12:102038451 1.41e+08 22.0 m 9.4 s 66.3% 33.2 m 11.2 m
INFO 09:50:34,922 TraversalEngine - chr13:21429694 1.44e+08 22.5 m 9.4 s 68.0% 33.1 m 10.6 m
INFO 09:51:04,924 TraversalEngine - chr13:77736127 1.47e+08 23.0 m 9.4 s 69.9% 32.9 m 9.9 m
INFO 09:51:34,930 TraversalEngine - chr14:34395037 1.50e+08 23.5 m 9.4 s 72.2% 32.6 m 9.1 m
INFO 09:52:04,936 TraversalEngine - chr14:76644221 1.54e+08 24.0 m 9.4 s 73.5% 32.6 m 8.6 m
INFO 09:52:34,947 TraversalEngine - chr15:32450575 1.57e+08 24.5 m 9.4 s 75.6% 32.4 m 7.9 m
INFO 09:53:04,951 TraversalEngine - chr15:62991093 1.60e+08 25.0 m 9.4 s 76.6% 32.7 m 7.7 m
INFO 09:53:35,534 TraversalEngine - chr16:81725 1.64e+08 25.5 m 9.4 s 77.8% 32.8 m 7.3 m
INFO 09:54:05,545 TraversalEngine - chr16:48204121 1.67e+08 26.0 m 9.3 s 79.4% 32.8 m 6.7 m
INFO 09:54:35,749 TraversalEngine - chr17:69409 1.70e+08 26.5 m 9.4 s 80.8% 32.8 m 6.3 m
INFO 09:55:05,756 TraversalEngine - chr17:28791767 1.74e+08 27.0 m 9.3 s 81.7% 33.1 m 6.1 m
INFO 09:55:35,767 TraversalEngine - chr17:56399591 1.77e+08 27.5 m 9.3 s 82.6% 33.3 m 5.8 m
INFO 09:56:05,781 TraversalEngine - chr18:8370781 1.80e+08 28.0 m 9.3 s 83.7% 33.5 m 5.5 m
INFO 09:56:35,800 TraversalEngine - chr18:70149611 1.83e+08 28.5 m 9.3 s 85.7% 33.3 m 4.8 m
INFO 09:57:05,803 TraversalEngine - chr19:31039619 1.87e+08 29.0 m 9.3 s 86.9% 33.4 m 4.4 m
INFO 09:57:35,805 TraversalEngine - chr19:55119393 1.90e+08 29.5 m 9.3 s 87.7% 33.7 m 4.1 m
INFO 09:58:05,806 TraversalEngine - chr20:34599131 1.93e+08 30.0 m 9.3 s 88.9% 33.8 m 3.7 m
INFO 09:58:35,812 TraversalEngine - chr21:34721693 1.97e+08 30.5 m 9.3 s 91.0% 33.5 m 3.0 m
INFO 09:59:05,817 TraversalEngine - chr22:38336813 2.00e+08 31.0 m 9.3 s 92.6% 33.5 m 2.5 m
INFO 09:59:35,822 TraversalEngine - chrX:64028096 2.03e+08 31.5 m 9.3 s 95.1% 33.1 m 96.7 s
INFO 10:00:05,834 TraversalEngine - chrY:5605887 2.06e+08 32.0 m 9.3 s 98.3% 32.6 m 34.0 s
INFO 10:00:10,787 GATKRunReport - Uploaded run statistics report to AWS S3
##### ERROR ------------------------------------------------------------------------------------------
##### ERROR A USER ERROR has occurred (version 1.4-15-gcd43f01):
##### ERROR The invalid arguments or inputs must be corrected before the GATK can proceed
##### ERROR Please do not post this error to the GATK forum
##### ERROR
##### ERROR See the documentation (rerun with -h) for this tool to view allowable command-line arguments.
##### ERROR Visit our wiki for extensive documentation http://www.broadinstitute.org/gsa/wiki
##### ERROR Visit our forum to view answers to commonly asked questions http://getsatisfaction.com/gsa
##### ERROR
##### ERROR MESSAGE: Badly formed genome loc: Parameters to GenomeLocParser are incorrect:Unknown contig chrMt
##### ERROR

I don't know if I should remove this chromosome from my fasta file or my bam files. I guess that it's present in both...

Here is my fasta.fai file:

...
chr21 48129895 2837230949 50 51
chr22 51304566 2886323449 50 51
chrX 155270560 2938654113 50 51
chrY 59373566 3097030091 50 51
chrM 16571 3157591135 50 51

Do you know what should be done from this mitochondrial chromosome?

[gringer]

Picard is reporting 'chrMt', while your fasta index file suggests 'chrM'. These are different, but they shouldn't be. You should be using exactly the same FASTA file that was used for the mapping.

[Jane M]

Thanks for pointing it out ! I haven't noticed that the name are different
I cannot use the same FASTA file that was used for the mapping, because the mapping was done by a allowance, and they didn't give us this file... So, I will change the name chrM into chrMt in my fasta files.

[gringer]

I cannot use the same FASTA file that was used for the mapping, because the mapping was done by a allowance, and they didn't give us this file... So, I will change the name chrM into chrMt in my fasta files.

Okay, but take a little bit of care in interpreting the results. There may be slight differences in sequences / lengths for different FASTA files.

[Jane M]

Ok. I finally managed to run GATK completely
I guess my input files are correct now! I have some trouble with the output, but it will be a new topic!

Thanks everybody for your help!

[aforntacc]

hello all please i need some help
i am trying to add readgroup to my bam files and i got this error or exception
please can some one explain and what should i do.
Exception in thread "main" net.sf.samtools.SAMFormatException: SAM validation error: ERROR: Record 52142073, Read name HWI-ST132_0461:3:1201:1275:7049#GTCCTA, MAPQ should be 0 for unmapped read.
at net.sf.samtools.SAMUtils.processValidationErrors(SAMUtils.java:448)
at net.sf.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:506)
at net.sf.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:487)
at net.sf.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:446)
at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:641)
at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:619)
at net.sf.picard.sam.AddOrReplaceReadGroups.doWork(AddOrReplaceReadGroups.java:98)
at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:177)
at net.sf.picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:119)
at net.sf.picard.sam.AddOrReplaceReadGroups.main(AddOrReplaceReadGroups.java:66)

[dpryan]

@aforntacc: Try setting the stringency to "Lenient", GATK can be rather picky.

[Jane M]

Well, I have something similar, when using Picard with VALIDATION_STRINGENCY to lenient. It's not really an error in my case, since it's working afterwards (thanks to lenient maybe) :

Ignoring SAM validation error: ERROR: Record 104416918, Read name HWI-ST584_0081:4:2202:1737:149838#AGTCAA, MAPQ should be 0 for unmapped read.
Ignoring SAM validation error: ERROR: Record 104416919, Read name HWI-ST584_0081:4:2205:16036:86626#AGTCAA, MAPQ should be 0 for unmapped read.
Ignoring SAM validation error: ERROR: Record 104416920, Read name HWI-ST584_0081:4:2207:1759:176116#AGTCAA, MAPQ should be 0 for unmapped read.
INFO 2012-09-05 15:13:24 FixMateInformation Sorting by queryname complete.
INFO 2012-09-05 15:13:24 FixMateInformation Output will be sorted by coordinate
INFO 2012-09-05 15:13:24 FixMateInformation Traversing query name sorted records and fixing up mate pair information.
INFO 2012-09-05 15:13:34 FixMateInformation Processed 1,000,000 records. Elapsed time: 00:00:10s. Time for last 1,000,000: 10s. Last read position: chr14:74,489,555

[zillur]

Hi,
I was trying to use picard using the following lines. But it is always showing me unrecognised option.
MacBook-Pro:Strain_Naushon ZILLURRAHMAN$ java -Xmx16g -jar /Users/ZILLURRAHMAN/Desktop/PhD/GATK/picard-tools-1.119/AddOrReplaceReadGroups.jar I=Naushon.sorted.bam O=Naushon.picard.bam \ PL=illumina ID=$RANDOM SM=mysample
ERROR: Unrecognized option: PL

java -Xmx16g -jar /Users/ZILLURRAHMAN/Desktop/PhD/GATK/picard-tools-1.119/AddOrReplaceReadGroups.jar I=Naushon.sorted.bam O=Naushon.picard.bam \ RGPL=illumina RGID=$RANDOM RGSM=mysample
ERROR: Unrecognized option: RGPL


MacBook-Pro:Strain_Naushon ZILLURRAHMAN$ java -Xmx16g -jar /Users/ZILLURRAHMAN/Desktop/PhD/GATK/picard-tools-1.119/AddOrReplaceReadGroups.jar I=Naushon.sorted.bam O=Naushon.picard.bam \ CREATE_INDEX=true \ RGPL=illumina RGID=$RANDOM RGSM=mysample
ERROR: Unrecognized option: CREATE_INDEX


Anyone help me.

[sklages]

Remove the backslashes \ from your command. Why did you put them there?

And please do not hijack (old) threads .. just open a new one ..