454 sequencing: DETAILED explanation??

Question

[Marvin]

Hello, all over the internet there are explanations that leave out important parts of how things are done (or worse: that are even wrong?). Does anyone have a good source? I am especially interested in this:

How do you make sure, that you assign only one molecule to one bead? If say the adaptor of molecule A hybridises with the probe on the bead, what would keep another molecule B from hybridising with another probe on that bead?

Does the A adaptor anneal to the 5' end and the B adaptor to the 3' end?

Is the probe on the bead complementary to the A or the B adaptor?

Does the bead have only A probes or only B probes annealed to it or both?

How in detail does the amplification on the bead work? I see pictured explanations where on one picture 1 molecule attaches to the bead and on the next picture suddenly there's many of them. And the explanation states "It's PCR". But that's not enough.

I have played through so many scenarios and every time I get stuck somewhere thinking "But that cannot work because of bla". So I would really appreciate a source of a detailed explanation.

Answer 1

[lexnederbragt]

How do you make sure, that you assign only one molecule to one bead? If say the adaptor of molecule A hybridises with the probe on the bead, what would keep another molecule B from hybridising with another probe on that bead?

You try to make a ratio between DNA molecules and beads that ensures there are few beads with multiple templates, after amplification (see answer to last question) you select beads with DNA (i.e. not empty). There will always be beads with multiple templates, but these give very bad reads and are discarded during base-calling.

Does the A adaptor anneal to the 5' end and the B adaptor to the 3' end?

Yes, I think you can say this is true for a single strand molecule.

Is the probe on the bead complementary to the A or the B adaptor?

I don't remember, sorry...

Does the bead have only A probes or only B probes annealed to it or both?

Only one of them. It's not really a probe, it is an adaptor covalently linked to the bead, and this adaptor also serves as the start of synthesis during PCR.

How in detail does the amplification on the bead work? I see pictured explanations where on one picture 1 molecule attaches to the bead and on the next picture suddenly there's many of them. And the explanation states "It's PCR". But that's not enough.

Emulsion PCR: form microdroplets in an oil/water emulsion, each droplet has bead+DNAtemplate+enzymes+dNTPs+buffer etc and becomes a small reaction chamber. Note that a few droplets will get two beads, leading to PCR duplicates among the reads.

Hope that helps!