Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Picard MarkDuplicates error for RNA-Seq

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

  • #2
    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.

    Comment


    • #3
      Originally posted by nilshomer View Post
      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?

      Comment


      • #4
        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.

        Comment


        • #5
          We've switched to the updated Tophat2, which seems to work well.

          Comment


          • #6
            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..

            Comment


            • #7
              Originally posted by newbietonextgen View Post
              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?

              Comment

              Latest Articles

              Collapse

              • seqadmin
                Strategies for Sequencing Challenging Samples
                by seqadmin


                Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                03-22-2024, 06:39 AM
              • seqadmin
                Techniques and Challenges in Conservation Genomics
                by seqadmin



                The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                Avian Conservation
                Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                03-08-2024, 10:41 AM

              ad_right_rmr

              Collapse

              News

              Collapse

              Topics Statistics Last Post
              Started by seqadmin, Yesterday, 06:37 PM
              0 responses
              8 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, Yesterday, 06:07 PM
              0 responses
              8 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-22-2024, 10:03 AM
              0 responses
              49 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-21-2024, 07:32 AM
              0 responses
              66 views
              0 likes
              Last Post seqadmin  
              Working...
              X