Can somebody explain the purpose of Y adapters for paired end preps to me?

[Heisman]

Hey all,

I've tried searching and I just can't figure this out. I'm sure there's a simple explanation that is just eluding me. Why are Y adapters used? If it's Y primers and not Y adapters then I still don't understand, so either way a brief explanation would be wonderful.

[HESmith]

Y adapters allow the addition of different, noncomplementary sequences to the 5' and 3' ends of the library. The arms of the Y are the unique sequences; the stem, which ligates to the insert, is double-stranded (i.e., complementary) DNA.

[Heisman]

Y adapters allow the addition of different, noncomplementary sequences to the 5' and 3' ends of the library. The arms of the Y are the unique sequences; the stem, which ligates to the insert, is double-stranded (i.e., complementary) DNA.

Right, so why do we want/need the arms of the Y adapters to be noncomplementary? Or another way of asking, if the adapters were just linear (complementary), why would that not work?

[ECO]

There is a great diagram of this to be found in the sticky at the top of the forum, which shows you at the base level what happens to each fragment.

[Heisman]

There is a great diagram of this to be found in the sticky at the top of the forum, which shows you at the base level what happens to each fragment.

I looked through that and I see what's going on, but I still don't understand why there has to be a Y adapter. I possibly looked at the wrong thing; it was the attachment in the second post of the middle sticky above. I follow what gets added with each step, but I'm missing the reasoning behind needing a Y adapter. Please excuse my ignorance.

[ECO]

Ok...maybe this will make it more clear.

The goal is obviously the highest efficiency of putting two DIFFERENT adapters on either end of every piece of DNA in the library. This is to enable PCR/clustering/emulsions/etc.

If you just took two totally different standard double stranded adapters (say A and B), and threw them into a ligation, you'd get inserts bounded by A-A, B-B, and only 50% with A-B (or B-A). The A-A and B-B fragments won't amplify in PCR for hopefully obvious reasons. Draw this out if it's not clear. Last I checked this is how ABI/LIFE/Roche do their adapters... just a mix of adapters and reliance on the amplification to pull out the correctly ligated events.

What the Y-adapter enables is the best of both worlds; the use of a single adapter which puts a different sequence on either end of each insert. It's because of this non-complementarity (the Y) that every ligation event results in asymmetric sequence being added to each end.

The most helpful part of that diagram (second post in this thread: http://seqanswers.com/forums/showthread.php?t=1169 ) is what happens to both strands of the initial library molecule upon PCR amplification.

Hope that helps...

[Heisman]

ECO, that clears it up perfectly for me. Thanks a lot!

[ECO]

Woohoo! Glad I was able to sputter that out in a semi-coherent way...

The final wrinkle, talked about at the bottom of that thread I linked, is that the PCR adds additional adapter sequence which is used to cluster.

So...when people want to avoid PCR you need to use modified adapters that already contain that longer tail.

[Heisman]

ECO, I had already figured that last part out, haha. But thanks again. Coming from a chemistry background (mainly physical chemistry at that) it's been a struggle to wrap my head around all of this genetic stuff. Too much biology. And not knowing all of the components of a system (ie, all of the kits present in this field where the specifics of reagents aren't released by the companies that make them) drives me crazy. I'm progressing though!

[james hadfield]

The latest Tru-Seq adapters available from Illumina are full length and allow you to get away with no PCR. They are already modified and have the longer tail Eco mentioned.

[HESmith]

Right, so why do we want/need the arms of the Y adapters to be noncomplementary? Or another way of asking, if the adapters were just linear (complementary), why would that not work?

After any denaturation step (amplification, flow cell loading, cluster formation), complementary ends of single-stranded DNA would self-anneal to form a loop. Self-annealing, being unimolecular, is an efficient process, and those ends would no longer be substrates for the desired reaction.

[carmeyeii]

HESmith,

After any denaturation step (amplification, flow cell loading, cluster formation), complementary ends of single-stranded DNA would self-anneal to form a loop. Self-annealing, being unimolecular, is an efficient process, and those ends would no longer be substrates for the desired reaction.

My thought, actually, is that upon primer annealing, in each cycle the primer in turn would bind to the start AND end of the DNA fragment, and thus not allow synthesis to continue to the end. Therefore, the next primer would not have a template to bind at the start of the fragment...

Y-shaped adapters also allow for paired-end sequencing. Fragments have a unique sequence on either end, which allows for the first "run" to sequence one side of all molecules, then synthesize the reverse and sequence that.

Am I correct?

Carmen

[RenataCAG]

Dear members,

Can you explain the differences between SureSlect Xt adaptors and Roche (a or B) adaptors? Which one is truncated? Can I sequence in the same lane libraries with both types of adapters?