Picard flag issue

Question

[mw]

Hi all,

I get the following error message using picard FixMateInformation:

Found two records that are paired, not supplementary, and first of the pair: E00489:426:H23VNCCX2:7:1101:10003:10169

I've extracted all the entries for this read in my bamfile. It looks like this:

98291879:E00489:426:H23VNCCX2:7:1101:10003:10169        **99**      chr1H   112553443       60      127M    =       112553774       481     CACTGAAAAGAATATCGGAGAGGCTATTTTTGGTACATTGTTCGACATAGATGGGAAGACAAAGGATAATACTAAGGCTAGAGTCGATCAAGAGACCCTATGCCATAGACTGTTACAAAACATGCGA FJJJJJJJJJJ<FJAJJJJJJJJJJ<FJJJJJJFFJJJFJJJJJJJJJJJJJJFJ<FJFJA7FJJJJFJJJJJJJJJJFJJJJJJJJJFJJJJJJJJJAJJFFFFJJJJJAF-FFJJJJJFAFJJJF RX:Z:CAGCCGAGTCTCTTTA   QX:Z:AAFFFAJFJJFJFJJJ   TR:Z:CAATGGT    TQ:Z:FJJJ<JJ    BC:Z:CGTATCGG   QT:Z:AAAFAAAF   XS:i:-10        AS:i:-2 XM:A:0  AM:A:1  XT:i:0  BX:Z:CAGCCGAGTCTCTTTA-1 DM:Z:0.785714   RG:Z:SA2Morex2019_WGS:LibraryNotSpecified:1:unknown_fc:0        OM:i:60 MI:i:4337815
98291880:E00489:426:H23VNCCX2:7:1101:10003:10169        **1123**    chr1H   112553443       60      127M    =       112553774       481     CACTGAAAAGAATATCGGAGAGGCTATTTTTGGTACATTGTTCGACATAGATGGGAAGACAAAGGATAATACTAAGGCTAGAGTCGATCAAGAGACCCTATGCCATAGACTGTTACAAAACATGCGA FJJJJJJJJJJ<FJAJJJJJJJJJJ<FJJJJJJFFJJJFJJJJJJJJJJJJJJFJ<FJFJA7FJJJJFJJJJJJJJJJFJJJJJJJJJFJJJJJJJJJAJJFFFFJJJJJAF-FFJJJJJFAFJJJF RX:Z:CAGCCGAGTCTCTTTA   QX:Z:AAFFFAJFJJFJFJJJ   TR:Z:CAATGGT    TQ:Z:FJJJ<JJ    BC:Z:CGTATCGG   QT:Z:AAAFAAAF   XS:i:-10        AS:i:-2 XM:A:0  AM:A:1  XT:i:0  BX:Z:CAGCCGAGTCTCTTTA-1 DM:Z:0.785714   RG:Z:SA2Morex2019_WGS:LibraryNotSpecified:1:unknown_fc:0        OM:i:60 MI:i:4337815
98292078:E00489:426:H23VNCCX2:7:1101:10003:10169        **147**     chr1H   112553774       60      150M    =       112553443       -481    TAATGCCGGTGATGTTACATGACTTTATTCCTGAGGATGAATGGCTAGTACTGGCGGAGCTGAGCTATTTCTTCTGTGTTCTTTGTGCGAAAGAATTGTCGCCTGGCGTGGTAGCAGACATGGAGGAGTTTGCGCCGGAGTTGTTGTGCA  <FF-<<AFJJJJJJJJJJJFJJJJFJJJAJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJFJJJFJJJFAJFJJJJJJJJJJJFJJJJJJJFJJJJJJJJJ7JJJJJJJJJJFFF-JJJFF<JJFJJJJJJJJJJJJJJFFAAAF-FF<AA  RX:Z:CAGCCGAGTCTCTTTA   QX:Z:AAFFFAJFJJFJFJJJ   BC:Z:CGTATCGG   QT:Z:AAAFAAAF   XS:i:-12        AS:i:-2 XM:A:0  AM:A:1  XT:i:0  BX:Z:CAGCCGAGTCTCTTTA-1 DM:Z:0.785714   RG:Z:SA2Morex2019_WGS:LibraryNotSpecified:1:unknown_fc:0        OM:i:60 MI:i:4337815
98292079:E00489:426:H23VNCCX2:7:1101:10003:10169        **1171**    chr1H   112553774       60      150M    =       112553443       -481    TAATGCCGGTGATGTTACATGACTTTATTCCTGAGGATGAATGGCTAGTACTGGCGGAGCTGAGCTATTTCTTCTGTGTTCTTTGTGCGAAAGAATTGTCGCCTGGCGTGGTAGCAGACATGGAGGAGTTTGCGCCGGAGTTGTTGTGCA  <FF-<<AFJJJJJJJJJJJFJJJJFJJJAJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJFJJJFJJJFAJFJJJJJJJJJJJFJJJJJJJFJJJJJJJJJ7JJJJJJJJJJFFF-JJJFF<JJFJJJJJJJJJJJJJJFFAAAF-FF<AA  RX:Z:CAGCCGAGTCTCTTTA   QX:Z:AAFFFAJFJJFJFJJJ   BC:Z:CGTATCGG   QT:Z:AAAFAAAF   XS:i:-12        AS:i:-2 XM:A:0  AM:A:1  XT:i:0  BX:Z:CAGCCGAGTCTCTTTA-1 DM:Z:0.785714   RG:Z:SA2Morex2019_WGS:LibraryNotSpecified:1:unknown_fc:0        OM:i:60 MI:i:4337815

The reads with the flags "1123" and "1171" are PCR duplicates. The mate pair reads with the flags "99" and "147" should be matched to each other. I don't see why the error message occurs. Can someone help me? Thanks in advance.

[Pierre Lindenbaum]

what is the version of picard ?

[mw]

I've tried it with the newest: 2.21.8-SNAPSHOT

[mw]

Similar problem here (error message: Value was put into PairInfoMap more than once. 1: RGE00489:426:H23VNCCX2:7:1220:10257:31107):

2557:E00489:426:H23VNCCX2:7:1220:10257:31107    145     chr1H   18015  5       7S143M  chr1H_2 128015184       0       GGGGGGGGGGGGGGGGGGGGGGCCAACCCACCCCGGGGAAGCCCATAGGCATTGGGGGAGCCACACCAGCCCTTAGTGGGCTGGTGGGACAGCCCACAAGTGCCCAATGCGCCAAGAGAAGAAAAATCAAGAGGAAAGAAAAAAAAAGGA        -JJJJJJJJJJJJJJFA)A)-))7A7FFF)FJA-JJJFJ7JJJJJFJJJJFAFJJJJJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJ<JJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA RX:Z:AGCCTAATCCTGTAGA   QX:Z:AAFFFJJJJJJJJJJJ   BC:Z:ATGCCTTA   QT:Z:AAFFFJJJ   XS:i:-15        AS:i:-14        XM:A:0  AM:A:0  XT:i:0 BX:Z:AGCCTAATCCTGTAGA-1 RG:Z:SA2Morex2019_WGS:LibraryNotSpecified:1:unknown_fc:0        OM:i:5 2558:E00489:426:H23VNCCX2:7:1220:10257:31107    1169    chr1H   18015  5       7S143M  chr1H_2 128015184       0       GGGGGGGGGGGGGGGGGGGGGGCCAACCCACCCCGGGGAAGCCCATAGGCATTGGGGGAGCCACACCAGCCCTTAGTGGGCTGGTGGGACAGCCCACAAGTGCCCAATGCGCCAAGAGAAGAAAAATCAAGAGGAAAGAAAAAAAAAGGA        -JJJJJJJJJJJJJJFA)A)-))7A7FFF)FJA-JJJFJ7JJJJJFJJJJFAFJJJJJFJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJ<JJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA RX:Z:AGCCTAATCCTGTAGA   QX:Z:AAFFFJJJJJJJJJJJ   BC:Z:ATGCCTTA   QT:Z:AAFFFJJJ   XS:i:-15        AS:i:-14        XM:A:0  AM:A:0  XT:i:0 BX:Z:AGCCTAATCCTGTAGA-1 RG:Z:SA2Morex2019_WGS:LibraryNotSpecified:1:unknown_fc:0        OM:i:5 
331851620:E00489:426:H23VNCCX2:7:1220:10257:31107       97      chr1H_2 128015184       0       127M    chr1H   18015   0       AACCCACAGGGTCTGCACACTTAAGGTTCGACGTTGTTTTATGCGTATTTGAGTTATATGGTTGGTTACCAAATGTTGTTCGGAGTCCCGGATGAGATCACGGACGTCACGAGGGTTTCCGGAATGG JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJFJJJAAFJJJJJJJJJ RX:Z:AGCCTAATCCTGTAGA   QX:Z:AAFFFJJJJJJJJJJJ   TR:Z:GTTCTCG    TQ:Z:JJJJJJJ    BC:Z:ATGCCTTA   QT:Z:AAFFFJJJ   XS:i:-16        AS:i:-14        XM:A:1  AM:A:0  XT:i:0  BX:Z:AGCCTAATCCTGTAGA-1 RG:Z:SA2Morex2019_WGS:LibraryNotSpecified:1:unknown_fc:0        OM:i:0 
331851621:E00489:426:H23VNCCX2:7:1220:10257:31107       1121    chr1H_2 128015184       0       127M    chr1H   18015   0       AACCCACAGGGTCTGCACACTTAAGGTTCGACGTTGTTTTATGCGTATTTGAGTTATATGGTTGGTTACCAAATGTTGTTCGGAGTCCCGGATGAGATCACGGACGTCACGAGGGTTTCCGGAATGG JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJFJJJAAFJJJJJJJJJ RX:Z:AGCCTAATCCTGTAGA   QX:Z:AAFFFJJJJJJJJJJJ   TR:Z:GTTCTCG    TQ:Z:JJJJJJJ    BC:Z:ATGCCTTA   QT:Z:AAFFFJJJ   XS:i:-16        AS:i:-14        XM:A:1  AM:A:0  XT:i:0  BX:Z:AGCCTAATCCTGTAGA-1 RG:Z:SA2Morex2019_WGS:LibraryNotSpecified:1:unknown_fc:0        OM:i:0