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Old 08-06-2010, 03:09 PM   #2
kmcarr
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Location: USA, Midwest

Join Date: May 2008
Posts: 1,178
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Dezhi,

There is no --rr because that is the same as --ff, molecularly speaking. The 'f' and 'r' do not refer to the strand which the read aligns to, but the relative orientation of read1 and read2. The relative orientation of the reads 1 and 2 is determined by the method used to generate the paired reads; it should be identical for all read pairs in your data set and you should now what the expected orientation is based on the method used to construct the paired end library. (Let's ignore for now that unexpected pair orientations can signal structural variations.)

The --fr orientation means that the two reads are aligned to opposite strands and are pointed toward each other (i.e. the 3' ends of the reads are closer together than the 5' ends).

Code:
-------------->
===============================================
                                <--------------
The --rf orientation means that the two reads are aligned to opposite strands and are pointed away from each other (i.e. the 5' ends of the reads are closer together than the 3' ends).

Code:
                                -------------->
===============================================
<--------------
The --ff orientation means that both reads are aligned to the same strand and are pointed in the same direction. Further, read1 should always be 5' to read2, relative to the strand they are aligned to. In the two cases above you should have an equal distribution of read1 being the read "on the left".

Code:
read1                           read2
-------------->                 -------------->
===============================================

OR

===============================================
<--------------                 <--------------
          read2                            read1
Read pairs in the --fr orientation are produced using the Illumina paired end protocol. Read pairs in the --rf orientation are produced using the Illumina mate-pair protocol. Read pairs in the --ff orientation are produced in using the SOLiD mate-pair protocol.
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