paired end mapping with strand

Question

[mangfu100]

Hi

I have a question about paired end mapping with strand.

The question is below detailed.

Let's assume that the reference sequence is ATTGCCCATGAT and I have a one paired end sequence

that ATT _ _ _ _ _ _ GAT with FR strand. (F- forward , R reverse).

More details, the paired end sequence should be formed the following sequence because it is FR strand.

5' ATT _ _ _ _ _ _ 
3'        _ _ _ _ _ _  G A T.

So I start to align the sequence and the alignment result is below.

(5') ATTGCCCATGAT  (3')     - ref.seq
(5') ATT_ _ _ _ _         (3')     - paired. end seq
(3')                    GAT  (5')

Though about my concept of alignment, I can't understand the alignment with strand....

the second order of paired end sequence is GAT and it's complement DNA is CTA. but it does not match the

reference seq( ref seq is GAT). SO I am very confused...

I am looking forward to your help

Thank you.

[Ashutosh Pandey]

I didnt get your question completely but let me still answer it. An aligner aligns a read to both reference sequence and its reverse complement sequence that represents the other strand. For a "fr" pair, if a read aligns to the forward strand of reference sequence then the other read will be aligned to the reverse strand (reverse complement) of reference sequence for most of the cases OR vice versa. An exception case will be an inversion of a region in the genome of interest wrt to reference genome.

Answer 1

[Pierre Lindenbaum]

For your example, the sequences would be ATT and ATC (NOT GAT)

The alignment is:

ATTGCCCATGAT
ATT          > FORWARD
         CTA < REVERSE

to align the second read, you need to reverse-complement its sequence = reverse strand.