Bridge Amplification: Forward strand

[yapphdorlw]

Hello everyone.
I'm currently trying to figure out how solid-phase/bridge-amplification works. Something still bothering me is, that the amplification step will create both the forward and the complementary strand.
According to slides 19-22 of http://sfgf.stanford.edu/.../general_technology.pdf the reverse strands will be removed (for single read).
But how does one know which one is the forward strand or the reversed strand?

Because we can't rely on the adapters, for identification!
As for slide 15 http://www.broadinstitute.org/.../SamplePrepSlides.pdf
the adapters will be added to the ends of the fragment, but we don't know which ones.

If for example CCCC is the sequence (of the forward strand), A1/A2 are the adapters and A1'/A2' their complements, our template might look like
5': A1 - T - CCCC - A - A2 : 3'
3': A1'- A - GGGG - T - A2' : 5'

or
5': A2'- T - CCCC - A - A1' :3'
3': A2 - A - GGGG - T - A1 :5'

Here A1 marks the forward strand in the first case, but the reversed strand in the second case.

Perhaps you can help me with that question.

[mastal]

The amplification step will create both the forward and the complementary strand.

That's right.

But how does one know which one is the forward strand or the reversed strand?

One doesn't. Except for RNA-Seq strand-specific protocols.

[JamieHeather]

That second link you're providing details the preparation for double stranded (genomic) DNA. You'd be getting both strands in your prep.

If you're interested in stranded information, I'm guessing you're doing RNA-seq, in which case you'd need a protocol that allows differentiation between the strands - figure one of this paper gives some nices ideas how one might do that (Levin et al, Nat methods, 2010).

In terms of the actual sequencing, the forward and reverse strands are dictated effectively by which of the adapter sequences, P5 or P7, that an individual molecule has at its 5'.

Molecules with a 5' P5 are sequenced in the first read, those with a 5' P7 in the paired end read. The trick to getting stranded information is to make libraries where P5 and P7 end up on the appropriate end of your target sequences.