bertrem - 2012-04-11

Using Bowtie2 for my paired-end Illumina data, mapping my reads both single and as pairs with the end-to-end or local alignment algorithm, it has come to my attention that, for instance, in the resulting SAM output file for paired-end, end-to-end mapping the "mapping quality" field is 0 and the same for every reported read, with some outliers.

Does this affect downstream analysis (eg variant detection) with tools like Samtools and/or VarScan? Do these tools calculate their own per read "mapping quality" values? Because, for example, an end-to-end alignment score of 0 represents an excellent alignment while an local alignment with score 0 is a bad alignment in Bowtie2, but if these were real "mapping qualities" then in fact the bad-aligned read would have the best "mapping quality" score.

I am probably missing something here…even after searching the net and/or manuals, it is not clear to me where the real "mapping qualities" are calculated (in bowtie2 or tools like Samtools/VarScan?).

paired-end, end-to-end:
HWUSI-EAS529_0008_FC61GM5AAXX:8:1:1083:19246#ACTTGA 83 chr10 55582547 0 101M = 55582478 -170 TCATCAGTGTTTCACCTTGCCTTATTTCCTCTTTCTCTGTCAAATTTGCCTCTTCAGTTGTAAGCAATGGATTGCTGCTACCTCTGTTGTTTGTACAGATT 5::5AA=>>==+=+:>-??:B9A:B::A?=BA=AB=AB?BC>:?->5??@?==>C@=A:EE=BBEEE:??EEEDEB@D??DBBAAADD3DDDD=D?DEE?A AS:i:-5 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:85T15 YT:Z:CP
HWUSI-EAS529_0008_FC61GM5AAXX:8:1:1083:19246#ACTTGA 163 chr10 55582478 0 82M = 55582547 170 AGAAGTGAGGCCTGGGAAAGCAAAATGAAGAGTCTGAAGAGAGAGATTTCAACTGTTCTGTTCCTTCTATCATCAGTGTTTC EA:AD?DAEEEEA?B?C?=DEA555A>=ABABBDDEEEEE?AE=EE=B?EB??E=DA:EABB=BBBAC,EB@BBDABC?BB= AS:i:0 XN:i:0 XM:i:0 XO:i:0XG:i:0 NM:i:0 MD:Z:82 YT:Z:CP

paired-end, local: here the "mapping quality" is 44 for every mapped pair
HWUSI-EAS529_0008_FC61GM5AAXX:8:1:1171:19773#ACTTGA 83 chr11 76922222 44 50M = 76922101 -171 ACCACAAGTGCACGCGGGAGGAGGTGCTGCAGCTGGGGGCGCTGATCTAC ????B->>:=;0924?BEE9DEDEEEE5EEEEE:7?:DBE=EEE5EDEAE AS:i:100 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:50 YT:Z:CP
HWUSI-EAS529_0008_FC61GM5AAXX:8:1:1171:19773#ACTTGA 163 chr11 76922101 44 55M = 76922222 171 GTGGTAGACCCCGGCGTTGGGGGTCTTGGTGTGGTGGGAAAGGAGCCCACTTCTG EC??B?ED?A5DDA=EE?E:DAA=-DD?D@D@A:);??.5B:CB=E@E:B?C-0A AS:i:110 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:55 YT:Z:CP

single, end-to-end: "mapping quality" = 42
HWUSI-EAS529_0008_FC61GM5AAXX:8:1:1052:5225#ACTTGA 16 chr10 73326495 42 53M * 00 CCNGAATACACCAGTGGGGACGCCCATCTTCATCGTGAATGCCACAGACCCCG ?B#DDDDCECEDDEEBE5DEEDAFGDDGDFFFFFGGFGGDEEGEEBE=DAEDE AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:2A50 YT:Z:UU
HWUSI-EAS529_0008_FC61GM5AAXX:8:1:1054:1877#ACTTGA 0 chr3 46751644 42 58M * 00 ACACTGTGGCAGCAGGAGGGACTCTGTCTGGCCAGGGAGGAGCCCCAGTGGTGAGAAG ?BDEEDDFFEEEBE?D:D;?EACE??=DB=D=D-DEE=AECEBEE@,D5D@:@*@47@ AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:58 YT:Z:UU

single, local: "mapping quality" = 44
HWUSI-EAS529_0008_FC61GM5AAXX:8:1:1173:4430#ACTTGA 16 chr17 18041546 44 83M * 00 TGTTGCTTTCCCCAGGTGAGCCGCAGGCACTGTGTGAGCCTAGTCAGGTCACAGATCTCTCAGCCTCATGTCCTCATCCCACC AB?=?D:=5DD?FFBEEEE=EDAD:DAAAA5A:G?GFFGFGE=EEEDA=FEGDEGGFFFEDGGDGDFFAE?FFEEFEEDBEEB AS:i:166 XN:i:0 XM:i:0 XO:i:0XG:i:0 NM:i:0 MD:Z:83 YT:Z:UU
HWUSI-EAS529_0008_FC61GM5AAXX:8:1:1174:7388#ACTTGA 16 chr17 18070978 44 83M * 00 GGTGTCCAAGCTGGCTTCACTGCAGCATCGCGCCAAGGACCACTTCTACCTGCCAAGCGTGTGAGCATCTGCCCTCCTGCCTC @5@=8A=@:-@?@?-?@=@C=BCB:C5BEDDBC?D@5>?CA;?A>A:>CC5EE5EBCEEDEEEBEBEEDEED??:EE=E=EEE AS:i:159 XN:i:0 XM:i:1 XO:i:0XG:i:0 NM:i:1 MD:Z:54G28 YT:Z:UU