Mpileup And Sam Flag 121 (Read Paired, Mate Unmapped ) [Updated]

Question

[Pierre Lindenbaum]

One of our positive controls was not found using

samtools mpileup  -B ...

when looking at this variation using samtools tview, I can see a clear polymorphism A/G with a depth ~100.

all the reads overlapping the variation have a flag=121:

read paired
mate unmapped
read reverse strand
mate reverse strand
first in pair

does samools mpileup ignore those reads ?

EDIT:

here is the alignment under the variation:

>12
NNN
TRT
tgt
tgt
tat
tat
tgt
tat
tat
tgt
tat
tgt
tgt
tgt
tat
tat
tgt
tat
tat
tat
tat
tgt
tat
tat
tgt
tat
tat
tgt
tat
tat
tat
tgt
tgt
tgt
tat
tat
tat
tat
tgt
tgt
tat
tgt
tat
tat
tat
tat
tgt
tgt
tat
tgt
tat
tgt
tgt
tat
tat
tat
tgt
tgt
tgt
tat
tgt
tat
tat
tgt
tat
tgt
tat
tgt
tgt
tgt
tgt
tat
tat
tgt
tat
tat
tgt
tgt ....

and here are the flags and the qualities of my reads overlapping the mutation:

83    HHHHHHHHHHGHHHHHFIIHIIIIIHIIHHIIIIIIIIIHIIHFEHIIIHIHHHIIIIHIIIIIIIIIIIIIIHHHHHHGGGGGGEEEEEEEEBBBAA???
83    HHHHHHHHHHHHHHHHHHHHHGIIIIHIHIIIIIIIIIIHHIHFHHIIHHHHHHIIIIIHIIIIIIIIIIIIIIHHHHHGGGGGGDDDEEEEDBBBAAAAA
121    HHHFIIIIIHHHHHIHHHEEHEHIHHHIHIIIIIIIIIIIIIIHIHIIIIHIIIHIIHIIIIIHFGGGGGDDDDDDDDA?B?????
121    HHGDIIHIIHGIIIIIIIHEHHHHFHHIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIIHHGGGGGGEDDDEDDDAABAA???
121    FGC=IIIHIIHIIIIIIIHEHEHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHIHIIGGGGGGEEDDEEDDAAB?A???
121    HGEAHDHFGHHGFHHHHHHEH@>GEC-DHGDFEADHGHHGDHHHHHGFHHGGFFFHHHHHFC0BFFFFFFBBBBBB@B@?B?????
121    DEEDEA--CEFDE@-ECE5+>EC@FDCFFCC-8.=@E8-AAAAA9...@-9-@@A=EEAECEAC>A86+C<@9@<<@@==>,==<,
121    IIIHIIHIIHHHIIIIIIIHHEHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGEEEDEDEEAAB?A???
121    HIHHHIHHHHFHHHHIIHFDHHHIIGIIIIIIIHHHIIGIIHIHIIIIIIIIIHHIIIIIIIIHGGGGGGDDDDDDDDA?B?????
121    HFHHHGHHHFCFHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHEBEFHHHHHHHFHFHHFBHFFFFFFDBDDDDDD@?B?????
121    BEFIHHIIIHEBCDHIIHHDHHHFDHGHIIHIHIHHIIIIHIHHIHGHHHHEFDFHIHIHHHFFFFFFFFDDDDDDDD@?B???<<
121    IIIIIIIIIHHHGIHIIIHHHHHIHHIHIHIIIIIIHHIIIIIIIIIIHIIIIIIIIHHHIIIIGGGGGGDDDDDDDDAAB?AAA?
121    FGCAHFHHHFCFHIIHHHHDHEDFHFFHHHHGGIIIIHHIIHHIIIIIHGFFFHHIHIIIHHFHFFFCFFDDDDDDDDA?B?????
121    HHFFIHHHGFHHHHIIIIIHHHHHIIHHHIHHIIIIIIHHHHIIIIIIIIIHIIIIIIIIIIHIFFFFFFDDDDDDDD@?B?????
121    HHHHHHGHFCCFFCCFFFHHHHHHF=DBHGFFEBHHGFHHFDFDHFFHHHHHF?CGCHFFFHFF?FCACFDDDDABBA@?B?????
121    IIIHIIHIIIHHHIHIIHHHHHHIIIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDEDDDAAB?????
121    GCDFHHFFGHHHHFHHHHFE>:HHGFBFHGHHGGHHFFFGHHHHHHHHHHHHHHHDHHFHFFFFFFF>FCBBBDDD@?@?B?????
121    IIHFHHHIHHHFIIIIIHFDHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDDDDDAAB?AAA?
121    IIHFIIHIIIHHHIIIIHIHHHHHGIIIIIIIIIIIHHIIHHIIIIIIIIIIIIIHIIHHIIIIGGGGGGDEDDDEEDA?B?????
121    FHHGIHFHIHGHHHFHHHFDHHHIIIHIHHIIIIIIHHFIHIIIIIIIHHIIIIHIHIIIIIIIGGGGGGDDDDDDDEA?B???A?
121    CCA-FHGFACDGFE-DHGECC>CHGEHHHDFHHFHHHDA?HFHGGHFD?FA9CFEACABFFCE>FACCCFBBA?5+@A@?B?????
121    IHHFIIIIHHIHIIHIIIIHHHHIIHHIIIIIIIIIIIIHHIIHIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDDDDDAAB?AAAA
121    HIHFIHGIHHGIHIIIIIHHHHHIHIIIIIIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIHIIGGGGGGEEDDDDDDAAB?AAAA
121    HHHFIHHHGHGHIIIIIHFDHHHIHHGHHHHHIIHIHIIIIHFIIIIIIIIIIIIIIIIIHHBIGGGGGGDDDDEDDEA?B?????
121    HHHFHIIIHHHIIIIHIIIHHHHIIIIIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDEEEEDDDAAB?????
121    HHFGHHGIHHHHGHHIHGHDHEHHFHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDEDDDEEEAAB?????
121    IIHHIIHIIHHHHHGHHHFHHHHIIHHIIIIIIIIIIHHIIIIIIIIIIIHIIIIHFIIIIIIIGGGGGGDDDDDDDDB?B?????
121    HGIIHHHIHHIHIHHIIHFEHHHIHHFHIHHIHHHIIIIIIIIIIIIIIIIIHHHIIIIIIHFIFFFFFFDDDDDDDDB?B?????
121    HGDFGHHHGFHHFGHHE=FHHDHGA-DFHHFFF?FCFHHHFDBEC=DDFGHHHFHHHHHHHHHF?FC,CFBBBB<<BB@?B?????
121    HHGDIIIHGFCFIIIIIIHHHEHHHHFIHIIIIIIIIIIIIIIHIIIIIIIIIIIIIHHFIHHIGGGGGGDDDDDDDDAABAA?A?
121    HIHHIIIHHHHHHHIHHHFEHHHIIIIHHHIIIIHHHGGIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDDDDDB?B?????
121    HHHFGHHHHFFHHHGGFHHDEEHHHGHHHHHHHHHHHDFHHFDFHFHFGHGFHHGCHHHHFFHHFFCFFFBBA?BDBB@?B?????
121    HHHHIIHIHHIIIHIIIIIHHGHIIIIIIIIIIIIIIHHIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGEEEDEEDDAAB?A?A?
121    IIIHIIHIIIHIHIIIIIHHHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHIIGGGGGGEEEDEEEEA?B?AAA?
121    HHF?HHHFHHFFFFIIIHFDHEHHGHFFHIIHIHIHIIIIIIIIIIIIHIIHHIHHHHIIIIHIFFFFFFDDDDDDDDB?B<?A??
121    IHHGIIIIIIHFHIIIIIHHHHHIIIHIHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGEEEDEEEEAAB?AA??
121    HHFFHIIIIHFHIIIIIIHHHHHHIIIIIIHIIIIIIHHHFIIIIIIIIIIIIIIHIIIHHFFIFFFFFFEDDDDDDDB?B?AA??
121    IIHGIIIIIIHFHIIIIIIHHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHHHIIIIIIGGGGGGDEDDDDDDB?B?????
121    HIHHIIHHHFDFGGIHHGDDEEHHGFIHHHIHHHHGHHFHFHIIIIHIHIIHIIHHIHIIHHFIFFFFFFDDDDDDDD@??,????
121    IHIHIIIIHIHFIIIIIIIHHHHHGIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIHFFIGGGGGGEDDDDDDDB?B?AA??
121    EEBCEEECCEFFE@FFFFDECDCFCFFFFECC8.8-@A8ECBFFGFGFFDCCDEFDEECEEEBCECCCCC<5<5++=++</<5==<
121    FHGFHHHHFHHGGGHHHHFEEHHHFDHHHHHHHFHFFHHHHFFHHHHHHHHHHHHFC?HHFEEFFFFFFFBBBBBBB?59?5????
121    IHHGHIIIIHHHHHHGHGFEHHHHFGHIHIIIIIHHGFIIIIIIIHIIIIIIIIHHFCIHHIHHGGFGGFDBDDDDDDB?B??A??
121    IIHHIIIIHHHHHHIIIIHHHHHHHGFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGEEEDDEDDA?B?????
121    IIHHIIIIHIHHHIIHIHIHHHHIIIHIIIIHIIIIIIIIIIIIIIIIIIIIHIIIIHHIIIIHGGGGGGDEDDDDDDAAB?????
121    HHHHHHHHHHHHHHGDFGFDHHHHHHFHHHHGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHFFEFFFFFBBDDDDDDD@?B?????
121    FFEGFHHHHHFHHHHGEF?CHEHHGGEHHHHGFGHGHGDGCFGFGHGHHHHHFFHHHHHHHFFEFFCFFFBBBDDDBA@?B??<??
121    IIHIIIHIHHGHGIIIHHHEHHHIHHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDEDDDA?B?????
121    IHFHHIIIHIIIIIIIHHFDHHHIHHGIGIIIHIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDDDDD@?B?A???
121    IGF?HGDFC5-HFDHGDHFED@HFHFHHHGHHGHHHIHFHHIIHGHHFHHFHHFFHF;FFFC>BFCFFFFDBDDDDBA@??<<5<<
121    HHGFHHHHGFHHGHHHHHHEDEHHHDDFHHHHHHHHHHHHHHGFHHHHHHHHHHHHHHHHHFFCFFFFFFDDDDDEDD@?B?????
121    FHGFCHGFHFGFCFGECEECHHFGGFHGGFFGIHHHGFFGHFHIIIHIIIHGHHGIIHIHIHHFCFFFFFDBDDDDDDB?B?????
121    FHFGFE?E-C-FCGE-FBFEHHHHGEFGEBC=GHHHHFFHHF@E=CGE0DGDEEFHHFEC-HFFCC>FCFDDDDDD@B@<?<????
121    HHGCHHIHGHHHHHIIIHIHHHHIIIIIIIIIIIHIIIIHHIIIIIIIHIIIIIIIIIIIIIIHGGGGGGEEDDDDDDA?BA????
121    IHGFIIIIIIIIHIHGGHHEHEHHHIIHIIIIIIHIIIHHHIIIIHHIIIIIIIHHIIIHIIHIFFFFFFDDDDDDDDAAB?????
121    IIIHIIIIIIHGHHHFHHHHHHHIIHIIHIIIIIIHIIIIIIIIIHHHHHFDFIIHIHHFIGIIGGGGGGDDDDDDDDAAB?????
121    IIHHIIIIHIHIIIIIIIHHHHHIIIIIIIIIIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDDDDDA?B?A???
121    FFDFHHHGGCGHGDGHHHHEHHHHHHGHGDFDHHHHFGHGFCHHGHFHHHHFDHHHHHF@HFFFFFFCCFDDBBDDDD@?B?????
121    FGD=HGFHHHGBFHHHHHFE>@EFDHHHHGGEFGDHHGFFFHGHHHHHHHGFFDHFHHHFEC0HFCFCFFDDDB<BDD@?B????<
121    HHHHIIIHHHHFIIIHHHDCHEHHIHIIIIFIIIIIIHIIIIIIHIIIHIIIHHHHHFIHHHFFFFFFFFDDDDDDDD@?B?????
121    IIIHIIIIIIIIIIIIIIIHHHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDEEDD@AB?AA??
121    IIHHIIIIIIIIIIHIIHFEEDHHHHHHIIIIIIIIIIIHHIGIIIHIIIIIIIIIIIIHIHIIGGGGGGDDDDDDDD@?B<???<
121    GFHGFHHFFFEDHGFGGF@HHHCHHHFHHFHHHHHHHHFFDFFDHFEGGHFHFDFHHFBHFA>FF?FFFFDDDADB=?=?B?????
121    HGDGIIIIHHGHFIHIIHIHHDHHIIHIHHIIIIIIIHHHFIIIIIIIIIIIIIIIIIIHIHHIGGGGGFDDDDDEDDB?B?AAA?
121    DGDGHHHHFFADHHHHHFHE>DEFDDFHHHDHHHHHHHFHFDGFHHHGHGHHFC?CA>HFHHBFFFFFFFDDDADBDD@?B?????
121    HIHGHHHHHHFHHIIIHHFEHHHIHHHHIIIIIIIIHHHIHHIIIIIIIIIHIHHIIIIIIIIHGGGGFFDDDDDDDD@?B?????
121    FHHHHHHHHHHHHHHHHHDEHHHHHHFHHHHHHHHHHHHHHHHHHHGHHHHHHHHFHHHHHHHFFFFFFFBDDDDDDD@?B?????
121    HHFGIHHHFHFHHIHIIIIHHEHHIIIIHHHHIIIIIHIIIIIHIIIIIIIHIIHFIIIIIIIIGGGGGGDEDDDDDD@?B?????
121    =E>-C5-DEEFFEFAFFE5+>ECAA-E@CFF=AFFC-A-FEA-9A@F=CFE@EEFFCEE>ECA8-CC>;C+@@@@@@@=</,====
121    HFHHGHGEFFCGHHHFHHDCHEEGHDHFHHHHHHHHHHHGFFDGFFHHGHFHFDBFECFFHFHHFFFFFFBDBDDDDD@?B?????
121    HIHHEE=HFFEFFDHDGHFFCHHHHFIHHHIHHFEDFGHIIHHFFHDF=HHGHFFHHFFFHHFHGGGGGGDBDBA+DDB?B?????
121    IIHHIHFHEFDHHHIHGIHHHDHIIHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHIGGGGGGDDDDEEEEAABAAAAA
121    HHGFIIIIHHGHHIIIHHHHHFHIHHFIIHIHIIIIHHIHHFIIHHHIIIIIIIHIIIIIIHHIGGGGGGDDDDDDEDBAB?????
121    IIIHHHHIHFDGHHIHHIHHHHHHHHHIHIIIIIHIIHFHHHHHHIIHIIIIIHIIIIIHIHHHFFFFFFDDDDDDDDB?B?????
121    IIIIIIHHHIIHHHHIIHHEHHHIHIIHHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDDDDDDEEAAB?A???
121    IIIHIIIIIIHGIIIIIIHHHHHIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGDEEEEEEDAABAA???
121    HHGHIIIIHHIGIIIIIIIHHHHIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGGGEDDDDDEEAAB?AAA?
(...)

mpileup in the region: As far as I can see thos read flags=121, haven't been used.

samtools mpileup  -s -f ref.fasta -r region my.bam 
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000
mychrom    pos-1    T    2    ,,    GG    ]]
mychrom    pos    G    2    ,,    GG    ]]
mychrom    pos+1    T    2    ,,    ED    ]]

[Damian Kao]

Could it be low mapping quality?

[Pierre Lindenbaum]

I'm away from the lab but I think the quals are OK.

[Biomonika (Noolean)]

I experienced samtools not to call my variant in case that the reads at that position were not uniquely mapped (white reads in IGV).

Answer 1

[Pierre Lindenbaum]

As far as I can see, using the option -A in mpileup, fixes the problem:

   -A           count anomalous read pairs

[Biomonika (Noolean)]

What are anomalous read pairs, please?

[Pierre Lindenbaum]

in the code using option 'A', sets a variable:

 use_orphan=1

[Christof Winter]

I think anomalous read pairs are those where only one read is mapped, but not both. Without the -A option, your pileup only contains bases from reads that have a mapped mate. If you want to include reads that have an unmapped mate, use -A.