Hello,
I have some Iontorrent pgm sequencing data from mitochondrial primer
capture experiments (with very difficult low quant DNA samples, so reads
are not great). I noticed many duplicates (as in duplicate=reads that start
and end at the same position and are equal in sequence). From this tutorial
https://www.broadinstitute.org/gatk/events/2038/GATKwh0-BP-1-Map_and_Dedup.pdf,
I gather that marking equal CIGAR String reads, is what picard-tools
MarkDuplicates
function does.
I am using picard-tools-1.129, GATKv3.3-0, samtools-0.1.19
so from my bam files I first remove the unmapped reads (bash script):
*samtools-0.1.19/samtools view -b -F 4 $f > $f.mapped.bam*
then I mark the duplicates:
*java -jar picard-tools-1.129/MarkDuplicates.jar
\INPUT=$f.mapped.bam.sorted.bam \OUTPUT=$f.mapped.bam.sorted.bam.dedup.bam
\METRICS_FILE=metrics.txt*
then I build the bai file:
*java -jar picard-tools-1.129BU/BuildBamIndex.jar
\INPUT=$f.mapped.bam.sorted.bam.dedup.bam.bam*
Looking at the data I don't understand why some reads are marked as
duplicate, as example below copied from the sam file (but there are more).
Reads before and after start at other positions.
85MXK:00198:02595 1040 rCRS| 257 67 2S29M1I23M4I66M * 0 0
GTACAGCCGCTTTCCACACAGACATCATAACAAAAAAATTTCCACCAAACCCCCCCCCTCCCCCCCGCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAACCCTAA
;<DE<<:DDD>DD@EEEEF>><;<3==3-33)333333,C@
<CC<?6A5&:::::::::):::::::::A@IBEBFDDDEC
??=C?GBBBDDDDDDFD?C6C6)666)DDD>61:::A:1:<=8C XA:Z:map4-1 ZA:i:125 ZB:i:30
ZC:B:i,189,188,1,0 MD:Z:6A46T64 ZF:i:24 PG:Z:MarkDuplicates
RG:Z:85MXK.IonXpress_016 ZG:i:189 NM:i:7 XM:i:118
ZM:B:s,254,2,322,2,2,268,10,256,254,260,224,516,244,2,2,12,256,28,226,226,2,278,252,222,700,2,2,212,2,4,762,4,482,4,232,4,836,4,26,254,1206,10,4,1012,4,4,4,4,802,4,524,4,46,216,54,214,6,212,6,218,10,60,240,4,4,274,18,66,224,14,4,200,260,2,2,278,2,2,2,2,666,2,52,406,154,6,180,86,220,248,58,0,196,0,92,70,246,14,0,262,236,12,0,518,0,478,0,70,236,300,0,486,38,0,292,0,12,282,1478,270,26,2066,646,18,26,0,546,4,264,0,0,578,24,646,0,102,1398,0,0,256,66,0,0,0,646,0,42,208,312,16,244,208,298,212,182,10,240,0,2,0,252,0,8,254,232,8,38,222,236,24,10,450,578,180,214,0,64,24,446,12,38,246,78,74,218,230,204,172,280,204,74,44,222,24,192,90,44,240,436,190,42,10,0,32
ZP:B:f,0.00519992,0.00619387,9.47046e-07 AS:i:90 XS:i:8
85MXK:01378:02427 1040 rCRS| 257 75 2S53M3I65M * 0 0
GTACAGCCGCTTTCCACACAGACATCATAACAAAAAATTTCCACCAAACCCCCCCCCTCCCCCCGCTTCTGGCCACAGCACTTAAACACATCTCTGCCAAACCCCAAAAACAAAGAACCCTAA
DCCD<<:IDD>EE?DEECC=<;BB:<<:3??0?CCDD?MFAB?::0::&CEIFEIE@
:*::::::?AFGKCC@ECCCBDDB=<988;DBBCEDDD>??<LK;EF:):::::8AAA>:1:::6@
XA:Z:map4-1 ZA:i:123 ZB:i:30 ZC:B:i,189,188,1,0 MD:Z:6A46T64 ZF:i:24
PG:Z:MarkDuplicates RG:Z:85MXK.IonXpress_016 ZG:i:189 NM:i:5 XM:i:118
ZM:B:s,242,0,284,0,2,252,14,242,222,272,236,496,280,20,6,0,264,4,254,238,16,274,314,226,678,24,0,242,14,0,772,0,470,0,216,26,730,4,0,290,1144,14,0,938,12,16,4,0,804,14,504,20,58,212,110,234,0,206,0,220,46,20,258,4,0,230,0,64,186,42,0,218,244,0,0,254,0,10,0,0,624,0,96,426,126,12,214,98,196,252,42,8,180,0,96,50,272,0,0,276,210,4,2,486,0,466,0,56,232,298,16,452,32,0,280,20,0,264,1430,278,42,1974,592,20,20,0,536,6,224,10,18,608,52,642,0,128,1258,6,2,252,84,4,6,2,612,2,28,274,278,4,288,222,256,252,198,20,242,6,10,6,264,14,6,278,244,6,30,232,226,26,8,458,578,176,200,8,66,8,440,10,28,274,104,76,212,212,168,200,254,176,126,50,226,44,198,86,38,232,426,198,4,12,2,18
ZP:B:f,0.00478832,0.00666637,3.13317e-07 AS:i:99 XS:i:-2147483647
and another example:
85MXK:01331:01601 1024 rCRS| 215 76 79M * 0 0
ATTAATGCTTGTAGGATATAATAATAACAATTGAATGTCTGCACAGCCGCTTTCTACACAGACATCATAACAAAAAATT
II?D?DFDD<??DD>CBEDD@DD@DD<=;?D>BB@CC<===<=<=DC<::::1:@BEDDDDDCCBBB8
;6.-----(/* XA:Z:map4-1 ZA:i:149 ZB:i:30 ZC:B:i,289,289,2,0 MD:Z:16C31A5C24
ZF:i:27 PG:Z:MarkDuplicates RG:Z:85MXK.IonXpress_016 ZG:i:289 NM:i:3 XM:i:79
ZM:B:s,238,0,280,0,6,268,12,250,260,284,246,488,272,0,16,0,242,2,242,220,0,272,248,218,244,0,0,250,0,0,0,0,476,504,0,0,246,0,0,216,26,236,458,190,12,0,12,0,264,2,2,256,2,2,440,268,250,38,6,258,2,2,4,2,234,486,2,2,268,502,2,2,234,4,2,2,520,2,246,418,480,2,178,512,234,18,188,100,248,22,6,44,288,28,2,34,266,20,16,242,18,46,272,0,48,2,0,24,224,-2,286,214,44,50,202,22,6,430,230,14,32,2,-2,0,328,24,18,54,658,42,256,20,198,236,282,10,2,26,30,-4,234,14,266,238,34,14,214,266,6,260,18,256,200,0,-8,0,282,276,18,0,246,456,274,2,-6,1208,24,-8,516,288,-6,-6,258,4,308,410,98,498,-6,112,96,456,48,168
ZP:B:f,0.00484822,0.00638284,7.94061e-06 AS:i:67 XS:i:-2147483647
85MXK:01433:03052 0 rCRS| 215 2 13M103S * 0 0
ATTAATCCTTGTATTGCGCCCGACGCAAAAAACAGCACCACACAGACCACTGGCAGAATCAGCCAGATCTTGGGGACGGCCTTGAGTAACAGCCAGCTCGTCACACCGCTTAGCAC
DDBEBEE?C?CDEEBBBBEF?E=<:?@@@@@*::::::4:?::=CC?=::::3::CC<:<=B?4:=CCCC?CAD8<=>@EAFAEEEDE7<<::3:;;=DFED>==D?=:<=C<=>D
XA:Z:map4-1 ZA:i:116 ZB:i:30 ZC:B:i,229,226,1,0 MD:Z:6G6 ZF:i:27
PG:Z:MarkDuplicates RG:Z:85MXK.IonXpress_016 ZG:i:229 NM:i:1 XM:i:13
ZM:B:s,246,0,294,0,2,264,10,248,232,264,222,494,248,0,20,0,264,0,224,212,0,274,252,226,232,0,0,226,4,0,0,0,470,484,0,0,274,12,466,0,470,34,30,218,32,0,0,0,268,36,0,238,488,22,246,0,22,2,0,0,282,-2,234,694,48,-4,36,236,-6,222,326,232,-8,280,10,14,1352,-2,320,238,-2,16,224,-10,6,300,2,348,-14,4,0,6,410,242,16,286,26,254,326,4,8,240,80,220,-2,14,-4,2,276,42,440,262,10,308,42,50,158,114,352,36,20,72,-16,4,324,236,228,28,30,428,28,68,198,-2,298,-8,0,20,-24,-24,252,274,466,312,44,62,204,234,204,232,72,2,402,860,-12,202,346,34,446,432,434,22,4,262,10,206,54,234,260,26,8,194,470,38,288,222,12,34,250,38,-20,520,6,210,74,248,-14,24,322,20,70,52,230,62,242,224,222,30,244,38,10,104,-10,-12,272,8,300,216,44,482,220,-18,30,320,-8,34,434,4,-32,190,84,24,208,346,18,222,220,32,-26,194,72,-10,248,220,-24,-10,264,26,500,44,4,88
ZP:B:f,0.00478331,0.00679823,1.76384e-07 AS:i:9 XS:i:-2147483647
Somehow think I am missing a point here, can anybody help ?
Thanks,
Mayra
--
Mayra Mayr-Eduardoff
PhD Candidate
Institute of Legal Medicine
Innsbruck Medical University
Müllerstrasse 44
6020 Innsbruck
Austria
Tel:+43512900370624
Mail: mayra.eduard...@i-med.ac.at
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