Re: [Samtools-help] trouble with rmdup

[Shankar A. S. <sha...@gm...>]

Thanks, Heng.

I tried option 2 that you suggested.
fixmate didn't work on the name-sorted bam when leaving in the
trailing read1 & read2 identifiers - "/[12]" . Only the strand flag
remains set.

Shankar

On Mon, Mar 22, 2010 at 5:30 PM, Heng Li <lh...@sa...> wrote:
> Fixmate tells read1 and read2 from the tailing "/[12]" in read names. As
> you do not have this information, fixmate cannot do the job and rmdup
> will fail. It is recommended to use an aligner that generates proper
> FLAGs or to keep "/[12]" in the read names. Manually fixing flags is
> often problematic.
>
> Heng
>
> On Mon, Mar 22, 2010 at 08:46:16PM +0000, Shankar Ajay Subramanian wrote:
>> Hi all,
>>
>> I've been trying to get a predictable outcome with rmdup but I can't seem to.
>>
>> Here are the steps that I've gone through to try and remove duplicate
>> read pairs:
>>
>> Step 1: Name sorted bam file
>>
>> 510.1.1.1204.14 16      chr12   132047285       91      100M    *
>>  0       0
>> CCTTGCCAAGACTTGACNNNNTNNTTCCTCCTCCCCATACAATCACTTATCTTTTGTAAATTAATATGTATTAATGTGGAGTCCTAATTAGGGAAAAAGA
>>    ###########################?*894<==GHGGHFFBFFFDFG;HHECHHHHHHHHHHFHHHHHHHHHHHHHHHHFHHHHHHHHHHEEHHHHHH
>> 510.1.1.1204.14 0     chr12   132047018       72      1S84M15S
>> *       0       0
>> NCAAACTACAAAGACCAAANTGANNTGACATTAAAAATACCTTAATTAGNTTATTTACGTGCATAATTTTTAAAAACTGAGTCTACNNNNNNNNNANNNN
>>    #++**7::50FFFFF+++'#+*+##+++**FFF<FFFF<7FFFFF+++*#+*+'&FF=FFFF==<?8;=?##############################
>> 510.1.1.1214.14997      16     chr12   132047285       91      100M
>> *       0       0
>> CCTTGCCAAGACTTGACNNNNTNNTTCCTCCTCCCCATACAATCACTTATCTTTTGTAAATTAATATGTATTAATGTGGAGTCCTAATTAGGGAAAAAGA
>>    ###########################?*894<==GHGGHFFBFFFDFG;HHECHHHHHHHHHHFHHHHHHHHHHHHHHHHFHHHHHHHHHHEEHHHHHH
>> 510.1.1.1214.14997      0     chr12   132047018       72      1S84M15S
>>        *       0       0
>> NCAAACTACAAAGACCAAANTGANNTGACATTAAAAATACCTTAATTAGNTTATTTACGTGCATAATTTTTAAAAACTGAGTCTACNNNNNNNNNANNNN
>> #++**7::50FFFFF+++'#+*+##+++**FFF<FFFF<7FFFFF+++*#+*+'&FF=FFFF==<?8;=?##############################
>>
>> Step 2: samtools fixmate test.nameSrt.bam test.nameSrt.fxm.bam
>>
>> 510.1.1.1204.14 16      chr12   132047285       91      100M    =
>>  132047018       -367
>> CCTTGCCAAGACTTGACNNNNTNNTTCCTCCTCCCCATACAATCACTTATCTTTTGTAAATTAATATGTATTAATGTGGAGTCCTAATTAGGGAAAAAGA
>>    ###########################?*894<==GHGGHFFBFFFDFG;HHECHHHHHHHHHHFHHHHHHHHHHHHHHHHFHHHHHHHHHHEEHHHHHH
>> 510.1.1.1204.14 32     chr12   132047018       72      1S84M15S
>> =       132047285       367
>> NCAAACTACAAAGACCAAANTGANNTGACATTAAAAATACCTTAATTAGNTTATTTACGTGCATAATTTTTAAAAACTGAGTCTACNNNNNNNNNANNNN
>> #++**7::50FFFFF+++'#+*+##+++**FFF<FFFF<7FFFFF+++*#+*+'&FF=FFFF==<?8;=?##############################
>> 510.1.1.1214.14997      16      chr12   132047285       91      100M
>>  =       132047018       -367
>> CCTTGCCAAGACTTGACNNNNTNNTTCCTCCTCCCCATACAATCACTTATCTTTTGTAAATTAATATGTATTAATGTGGAGTCCTAATTAGGGAAAAAGA
>> ###########################?*894<==GHGGHFFBFFFDFG;HHECHHHHHHHHHHFHHHHHHHHHHHHHHHHFHHHHHHHHHHEEHHHHHH
>> 510.1.1.1214.14997      32     chr12   132047018       72
>> 1S84M15S        =       132047285       367
>> NCAAACTACAAAGACCAAANTGANNTGACATTAAAAATACCTTAATTAGNTTATTTACGTGCATAATTTTTAAAAACTGAGTCTACNNNNNNNNNANNNN
>>  #++**7::50FFFFF+++'#+*+##+++**FFF<FFFF<7FFFFF+++*#+*+'&FF=FFFF==<?8;=?##############################
>>
>> Step 3: samtools rmdup test.nameSrt.fxm.bam test.nameSrt.fxm.uniq.bam
>>
>> 510.1.1.1204.14 16      chr12   132047285       91      100M    =
>>  132047018       -367
>> CCTTGCCAAGACTTGACNNNNTNNTTCCTCCTCCCCATACAATCACTTATCTTTTGTAAATTAATATGTATTAATGTGGAGTCCTAATTAGGGAAAAAGA
>>    ###########################?*894<==GHGGHFFBFFFDFG;HHECHHHHHHHHHHFHHHHHHHHHHHHHHHHFHHHHHHHHHHEEHHHHHH
>> 510.1.1.1204.14 32      chr12   132047018       72      1S84M15S
>>  =       132047285       367
>> NCAAACTACAAAGACCAAANTGANNTGACATTAAAAATACCTTAATTAGNTTATTTACGTGCATAATTTTTAAAAACTGAGTCTACNNNNNNNNNANNNN
>> #++**7::50FFFFF+++'#+*+##+++**FFF<FFFF<7FFFFF+++*#+*+'&FF=FFFF==<?8;=?##############################
>> 510.1.1.1214.14997      16      chr12   132047285       91      100M
>>  =       132047018       -367
>> CCTTGCCAAGACTTGACNNNNTNNTTCCTCCTCCCCATACAATCACTTATCTTTTGTAAATTAATATGTATTAATGTGGAGTCCTAATTAGGGAAAAAGA
>> ###########################?*894<==GHGGHFFBFFFDFG;HHECHHHHHHHHHHFHHHHHHHHHHHHHHHHFHHHHHHHHHHEEHHHHHH
>> 510.1.1.1214.14997      32      chr12   132047018       72
>> 1S84M15S        =       132047285       367
>> NCAAACTACAAAGACCAAANTGANNTGACATTAAAAATACCTTAATTAGNTTATTTACGTGCATAATTTTTAAAAACTGAGTCTACNNNNNNNNNANNNN
>>  #++**7::50FFFFF+++'#+*+##+++**FFF<FFFF<7FFFFF+++*#+*+'&FF=FFFF==<?8;=?##############################
>>
>> Changing the flags from 16 to 83 and 32 to 163 didn't seem to work
>> either. Is there something fundamentally wrong with what I am doing?
>>
>> Any help will be much appreciated.
>>
>> Thanks,
>> Shankar
>>
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