FASTQ MP to PE orientation

[Guest]

If we want to get from the MP orientation to the PE orientation (by ourselves), is it correct to do the complement of the actual sequence line while the quality (orientation) remains the same? I just want to be sure...

<---- ----> orientation
@ID 1:N:0
CAGCTC
+
??A??B
@ID 2:N:0:
AGGACA
+
????AB

----> <---- orientation
@ID 1:N:0
GTCGAG
+
??A??B
@ID 2:N:0:
TCCTGT
+
????AB

Thanks!

[lorendarith]

...or do the reads have to be turned "physically":

MP
3' <--- 5' 5' ---> 3'
just reverse ORDER (no complement) of nucleotides and quality scores?
5' ---> 3' 3' <--- 5'

:/

[kmcarr]

If we want to get from the MP orientation to the PE orientation (by ourselves), is it correct to do a reverse complement of the actual sequence line while the quality (orientation) remains the same? I just want to be sure...

<---- ----> orientation
@ID 1:N:0
CAGCTC
+
??A??B
@ID 2:N:0:
AGGACA
+
????AB

----> <---- orientation
@ID 1:N:0
GTCGAG
+
??A??B
@ID 2:N:0:
TCCTGT
+
????AB

Thanks!

In your text you say "reverse complement" but the example you showed only complemented the sequence, you did not reverse it. And yes, if your reverse the sequence, then you need to reverse the quality as well to ensure that the quality scores remain associated with their original base.

Done correctly it is:


<---- ----> orientation
@ID 1:N:0
CAGCTC
+
??A??B
@ID 2:N:0:
AGGACA
+
????AB

----> <---- orientation
@ID 1:N:0
GAGCTG
+
B??A??
@ID 2:N:0:
TGTCCT
+
BA????

[lorendarith]

In your text you say "reverse complement" but the example you showed only complemented the sequence, you did not reverse it. And yes, if your reverse the sequence, then you need to reverse the quality as well to ensure that the quality scores remain associated with their original base.

Yes I made a mistake. As in the example I was just referring just to the complement.

So we have to do a reverse complement to get the reads back in the 5'->3' orientation in the file by reversing the order, but the actual "biological" orientation (MP to PE) changes by converting the read to the complementary strand, right?

Sorry I'm just confused...

[kmcarr]

Yes I made a mistake. As in the example I was just referring just to the complement.

So we have to do a reverse complement to get the reads back in the 5'->3' orientation in the file by reversing the order, but the actual "biological" orientation (MP to PE) changes by converting the read to the complementary strand, right?

Sorry I'm just confused...

Well you've managed to confuse me now too.

Let's try another approach. Why do you want to manipulate the read data?

[lorendarith]

Well you've managed to confuse me now too.

Let's try another approach. Why do you want to manipulate the read data?

I just want to turn the outward facing MP reads to inward facing reads as with normal PE data so that I could map them, as some mappers do not have the option for specifying the inward/outward orientation.
:/