Mark Duplicates from Picard does not seem to recognize my sorted BAM files.

Question

[Jordan]

Hi,

I have a BAM file which has chr id's as NC_00000*. I did sorting using samtools sort function.

I wanted to remove duplicates, so I'm using MarkDuplicates.jar from Picard tools to get the job done. But it gives me the following error:

Exception in thread "main" net.sf.picard.PicardException: 13_0501.sorted.bam is not coordinate sorted.
   at net.sf.picard.sam.MarkDuplicates.buildSortedReadEndLists(MarkDuplicates.java:273)
   at net.sf.picard.sam.MarkDuplicates.doWork(MarkDuplicates.java:117)
   at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:158)
   at net.sf.picard.sam.MarkDuplicates.main(MarkDuplicates.java:101)

But I think my bam file is sorted. This is the header of my bam file.

@HD     VN:1.0  SO:unsorted
@SQ     SN:NC_000001    LN:249250621
@SQ     SN:NC_000002    LN:243199373
@SQ     SN:NC_000003    LN:198022430
@SQ     SN:NC_000004    LN:191154276
@SQ     SN:NC_000005    LN:180915260
@SQ     SN:NC_000006    LN:171115067
@SQ     SN:NC_000007    LN:159138663
@SQ     SN:NC_000008    LN:146364022
@SQ     SN:NC_000009    LN:141213431
@SQ     SN:NC_000010    LN:135534747
@SQ     SN:NC_000011    LN:135006516
@SQ     SN:NC_000012    LN:133851895
@SQ     SN:NC_000013    LN:115169878
@SQ     SN:NC_000014    LN:107349540
@SQ     SN:NC_000015    LN:102531392
@SQ     SN:NC_000016    LN:90354753
@SQ     SN:NC_000017    LN:81195210
@SQ     SN:NC_000018    LN:78077248
@SQ     SN:NC_000019    LN:59128983
@SQ     SN:NC_000020    LN:63025520
@SQ     SN:NC_000021    LN:48129895
@SQ     SN:NC_000022    LN:51304566
@SQ     SN:NC_000023    LN:155270560
@SQ     SN:NC_000024    LN:59373566
@PG     ID:0    PN:clcgenomicswb        VN:7.0

The header seems to suggest that it's unsorted. That's what is bothering me.

[Alastair Kerr]

Did you sort by read names rather than chromosomal coordinates in samtools (the -n flag)? If so, this is the problem.

[Jordan]

No, I did not do that. I checked the lines manually in bam file. They all seem to be sorted by coordinates not read names.

[1013002214]

hello,Today,I meet the same question,which is very trouble.Did you give me some tips!thanks

[Jordan]

I did get it fixed. What kind of error do you get?

Answer 1

[Pierre Lindenbaum]

your SAM Header starts with:

@HD     VN:1.0  SO:unsorted

And Picard check that this 'SO:' is set to 'coordinate'. See https://github.com/nh13/picard/blob/master/src/java/net/sf/picard/sam/MarkDuplicates.java

(....)
 if (!ASSUME_SORTED && header.getSortOrder() != SortOrder.coordinate) {
                throw new PicardException("Input file " + f.getAbsolutePath() + " is not coordinate sorted.");
            }

to fix this, use the option

ASSUME_SORTED=true

[Jordan]

Ah.. let me try that and I see. Thanks for the tip!