Error during PicardMarkDuplicates (Illumina paired-ends mapped by Bfast)

[david.tamborero]

I mapped one run of Illumina paired end reads by using Bfast (bwaaln + localalign + postprocess), then converted it to bam, performed the Picard AddOrReplaceReadGroups command plus Samtools sort/index and therefore, when executing Picard MarkDuplicates, I have the following error:

Exception in thread "main" net.sf.picard.PicardException: Value was put into PairInfoMap more than once.  3: grouped_reads:HWUSI-EAS1692_0001:1:1:1841:8839#0
    at net.sf.picard.sam.CoordinateSortedPairInfoMap.ensureSequenceLoaded(CoordinateSortedPairInfoMap.java:124)
    at net.sf.picard.sam.CoordinateSortedPairInfoMap.remove(CoordinateSortedPairInfoMap.java:78)
    at net.sf.picard.sam.DiskReadEndsMap.remove(DiskReadEndsMap.java:61)
    at net.sf.picard.sam.MarkDuplicates.buildSortedReadEndLists(MarkDuplicates.java:285)
    at net.sf.picard.sam.MarkDuplicates.doWork(MarkDuplicates.java:113)
    at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:157)
    at net.sf.picard.sam.MarkDuplicates.main(MarkDuplicates.java:97)

I've noticed that the reads outputted by the bfast-postprocess looks like HWUSI-EAS1692_0001:2:1:1025:14826#0, whereas the original reads indicates the pair-end by either HWUSI-EAS1692_0001:2:1:1025:14826#0/1 or HWUSI-EAS1692_0001:2:1:1025:14826#0/2, I guess it is related, but I do not know how to solve this.

Any help will be appreciated, many thanks in advance!

David

[nilshomer]

Could you create a test case with only the offending reads (in SAM format)?

[david.tamborero]

I've created a file just with the reads pointed out in the MarkDuplicates error, and the same message error appears when running the Picard command. Also, I've created a SAM file with only these reads removed and the same error appears but with another read ID. I would say that MarkDuplicates does not allow more than one mapped position per read in the input file, therefore, I've forced just an unique alignement per read in the bfast postprocess command by changing the value of the "-a" argument:

previously, the command was:

bfast postprocess -i  input.baf -f hg19.fa -a 0 -A 0 -R -O 1 -n 4 -U >  output.sam

I changed it to:

bfast postprocess -i  input.baf -f hg19.fa -a 3 -z -A 0 -R -O 1 -n 4 -U >  output.sam

Now the Picard MarkDuplicates command works fine. Hope it makes sense.