Hi All,

I'm using BWA-MEM to align my reads back to my assembly, and despite using the -M flag to call shorter reads secondary alignments (>= 256 in second column), I find that I am still getting multiple primary alignments for some of my reads. Can someone explain what is causing these duplicate primary alignments?

A01346:38:HFMFMDRXY:2:2142:12762:13855  369 NODE_1_length_763630_cov_81.931622  50  18  215H36M NODE_335_length_61409_cov_82.645557 300 0   ACAGCTCCCACACTGAACAGTGTTTGCTGAAAGAAA    ,F,,:,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF    NM:i:0  MD:Z:36 MC:Z:194H57M    AS:i:36 XS:i:31 SA:Z:NODE_1268906_length_247_cov_191.600000,2,-,40S76M6I20M2I71M36S,18,15;NODE_112769_length_1204_cov_1.001857,1012,+,172S79M,2,2;  XA:Z:NODE_3_length_618943_cov_101.508943,-500138,215S36M,1;
A01346:38:HFMFMDRXY:2:2142:12762:13855  177 NODE_335_length_61409_cov_82.645557 300 60  194S57M NODE_1268906_length_247_cov_191.600000  2   0   CCCCCCCCCCCCCCCCACACGCCATCGCTCCCTCCCCCTCACACTCCCTCTCGATGTTGCTCCCCATTTTTTTTCTACCCTCCTACCCGGTTCGGTGTGGGCGCGGTGGCGCCTGCGCGTGGCTGCTACTGCGTTTCATCGGTCAACCTCTCTTCTCCCCCCGCCGCCGCCCCCGCCCCCCCGCCCGGCCCCCCCAGCTCCCACAGGGGGTTGGTGGTGGGTCAGTTGAATCGGTGTGTGTTTTGGAGTCA ,:,,,:,:,,,F,F:,,:,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,:,,,,,,,:,,,,,,,,:,,,,,,,,,:,,,,,,,,,,,,,,,,,:,,:F,,,,,,,,,,:,,,,,:,,,::,,,:,:,,,,,,,,,,,:,,,,:,,,,,,,,,,,,,:,,,,F:,::,F:,,F,F,,F:,:,F:,:,,:,F,,:,::,FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NM:i:0  MD:Z:57 MC:Z:40S76M6I20M2I71M36S    AS:i:57 XS:i:34 SA:Z:NODE_900496_length_426_cov_0.632107,31,-,156S30M65S,0,0;
A01346:38:HFMFMDRXY:2:2142:12762:13855  353 NODE_112769_length_1204_cov_1.001857    1012    2   172H79M NODE_335_length_61409_cov_82.645557 300 0   GGGGGGGGGGGGGTGGGGGGGGGGGGGGGGGCGGGGGGGTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG ,,,,:F,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,:,:,,,,,:,,,,,:,,,,,,:,,:,,:,,,,,,F,: NM:i:2  MD:Z:39G0C38    MC:Z:194H57M    AS:i:69 XS:i:66 SA:Z:NODE_1268906_length_247_cov_191.600000,2,-,40S76M6I20M2I71M36S,18,15;NODE_1_length_763630_cov_81.931622,50,-,215S36M,18,0; XA:Z:NODE_38215_length_2230_cov_6.684261,+2082,172S13M2D66M,3;NODE_707261_length_458_cov_1.024169,-24,65M186S,0;NODE_1240700_length_381_cov_1.464567,-26,65M186S,0;NODE_1178802_length_389_cov_0.874046,-3,60M191S,0;
A01346:38:HFMFMDRXY:2:2142:12762:13855  433 NODE_900496_length_426_cov_0.632107 31  0   156H30M65H  NODE_1268906_length_247_cov_191.600000  2   0   CCCCCCGCCGCCGCCCCCGCCCCCCCGCCC  ,,:,,,,F:,::,F:,,F,F,,F:,:,F:,  NM:i:0  MD:Z:30 MC:Z:40H76M6I20M2I71M36H    AS:i:30 XS:i:29 SA:Z:NODE_335_length_61409_cov_82.645557,300,-,194S57M,60,0;
A01346:38:HFMFMDRXY:2:2142:12762:13855  113 NODE_1268906_length_247_cov_191.600000  2   18  40S76M6I20M2I71M36S NODE_335_length_61409_cov_82.645557 300 0   CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCACCCCCCCGCCCCCCCCCCCCCCCCCACCCCCCCCCCCCCACCCCCCCCCCTCCCCCCCCCCCCCCCCCCCCCCCCCGGGCCGCCCCCCGCCCCCCCGCCCCCGGCCCCCCCGCCCCCCCCCCCCCCACCCCCGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCACAGCTCCCACACTGAACAGTGTTTGCTGAAAGAAA :,F,,,,,,:,,:,,:,,,,,,:,,,,,:,,,,,:,:,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,F:,,,,,,::,:,:,,,,,,,,,,::FFFFFF:FF:,,:F:,,,,,,,,,,,:,,,,,:,,,:,,,,,,,,:,::,,:,,F,,F,,,,,,,,,,,,,,,,,,,,::::,:,,,,::,,,,,:F:::,F,:F:F,,::::,F:,F,,:,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NM:i:15 MD:Z:7C17C13C10C31C7C12C63  MC:Z:194S57M    AS:i:112    XS:i:100    SA:Z:NODE_112769_length_1204_cov_1.001857,1012,+,172S79M,2,2;NODE_1_length_763630_cov_81.931622,50,-,215S36M,18,0;  XA:Z:NODE_8956_length_5979_cov_14.419002,-2871,40S75M4I85M47S,14;NODE_47535_length_1966_cov_14.032083,+1804,135S116M,4;NODE_50207_length_1907_cov_1.469663,+1774,137S63M4I47M,5;NODE_68274_length_1609_cov_1.117409,-1,30S34M2I47M9I88M41S,22;