Picard MarkDuplicates error for RNA-Seq

[RockChalkJayhawk]

I am trying to remove PCR duplicates using Picard.
My header looks like this:

@HD	VN:1.0	SO:sorted
@PG	ID:TopHat	VN:1.0.14	CL:/home/guo/bin/tophat -G ../../hg19.GFF3 -g 1 -o Dex -r 160 --solexa1.3-quals -p 4 ../../../../bowtie-0.12.3/indexes/hg19 ../../FASTQ/KUMC_PE_RNASEQ_sample6_1_sequence.txt ../../FASTQ/KUMC_PE_RNASEQ_sample6_2_sequence.txt
@SQ	SN:chr1	LN:249250621
@SQ	SN:chr2	LN:243199373
@SQ	SN:chr3	LN:198022430
@SQ	SN:chr4	LN:191154276
@SQ	SN:chr5	LN:180915260
@SQ	SN:chr6	LN:171115067
@SQ	SN:chr7	LN:159138663
@SQ	SN:chr8	LN:146364022
@SQ	SN:chr9	LN:141213431
@SQ	SN:chr10	LN:135534747
@SQ	SN:chr11	LN:135006516
@SQ	SN:chr12	LN:133851895
@SQ	SN:chr13	LN:115169878
@SQ	SN:chr14	LN:107349540
@SQ	SN:chr15	LN:102531392
@SQ	SN:chr16	LN:90354753
@SQ	SN:chr17	LN:81195210
@SQ	SN:chr18	LN:78077248
@SQ	SN:chr19	LN:59128983
@SQ	SN:chr20	LN:63025520
@SQ	SN:chr21	LN:48129895
@SQ	SN:chr22	LN:51304566
@SQ	SN:chrX	LN:155270560
@SQ	SN:chrY	LN:59373566
@SQ	SN:chrM	LN:16571

But I get this error when I run Picard.

net.sf.picard.sam.MarkDuplicates INPUT=Dex.bam OUTPUT=Dex.NoDup.bam METRICS_FILE=Dex.Metrics REMOVE_DUPLICATES=true ASSUME_SORTED=false    MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP=50000 READ_NAME_REGEX=[a-zA-Z0-9]+:[0-9]:([0-9]+):([0-9]+):([0-9]+).* OPTICAL_DUPLICATE_PIXEL_DISTANCE=100 TMP_DIR=/tmp/shart3 VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false
INFO	2010-08-19 12:25:28	MarkDuplicates	Start of doWork freeMemory: 31023560; totalMemory: 31588352; maxMemory: 620756992
INFO	2010-08-19 12:25:28	MarkDuplicates	Reading input file and constructing read end information.
INFO	2010-08-19 12:25:28	MarkDuplicates	Will retain up to 2463321 data points before spilling to disk.
[Thu Aug 19 12:25:28 CDT 2010] net.sf.picard.sam.MarkDuplicates done.
Runtime.totalMemory()=51314688
Exception in thread "main" java.lang.IllegalArgumentException: No enum const class net.sf.samtools.SAMFileHeader$SortOrder.sorted

Samtools can read it ok and it tells me :

46021417 in total
0 QC failure
0 duplicates
46021417 mapped (100.00%)
46021417 paired in sequencing
23145972 read1
22875445 read2
33385558 properly paired (72.54%)
39196142 with itself and mate mapped
6825275 singletons (14.83%)
0 with mate mapped to a different chr
0 with mate mapped to a different chr (mapQ>=5)

Why can't Picard Read it?

[nilshomer]

The value for the sort order tag "SO" is sorted, whereas the SAM specification lists "unsorted", "queryname" or "coordinate" as allowable values. Picard validates SAM/BAM files, while samtools does not (as much).

Looks like a bug in tophat.

[RockChalkJayhawk]

Originally Posted by nilshomer

The value for the sort order tag "SO" is sorted, whereas the SAM specification lists "unsorted", "queryname" or "coordinate" as allowable values. Picard validates SAM/BAM files, while samtools does not (as much).

Looks like a bug in tophat.

Thanks NIls, I just figured that out. Also, another bug with Tophat is that it doesn't write the MRNM, MPOS, or ISIZE for PEs in the SAM file. So, I will try to re-align them using Bowtie to see if I can overcome this.

Interestingly, I also ran SOAPals, which did the same thing when I converted it to SAM. Is there any RNA-Seq aligner that outputs these data in SAM?

[newbietonextgen]

I am having the same problem. RNA-seq data. Have tried even Splice map and the sam files are just crap cannot use it down stream. Did you find a way around this? If so kindly, let me know.

[RockChalkJayhawk]

We've switched to the updated Tophat2, which seems to work well.

[newbietonextgen]

what version if you can add? I used 2.0.0.4 and my SAM/BAM files were not compatible with GATK. Same data aligned using SHRIMP, no problem works like a charm..

[RockChalkJayhawk]

Originally Posted by newbietonextgen

what version if you can add? I used 2.0.0.4 and my SAM/BAM files were not compatible with GATK. Same data aligned using SHRIMP, no problem works like a charm..

can you post the error message from GATK?