Y adapters different sequences to be annealed to the 5' and 3' ends of each molecule in a library.

The arms of the Y are unique, and the middle part, connected to the DNA fragment, is complementary.

What are the advantages of this?

My take of this over having fully-complementary adapters (ADAPTER1 - - - - - ADAPTER1) is that:

-Upon primer annealing, the primer in turn would bind to the start AND end of the DNA fragment, and thus not allow synthesis to continue to until 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 on the right track?


1 Answer 1


Normally, when you need two unique adapters, say A & B, on either end of unknown insert sequences, cohesive-end ligation is difficult because the insert sequences are "unknown". So you have to do blunt-end adapter ligation, in a reaction containing Unknown Inserts + adapter A + adapter B. This can result in 3 possible versions of insert-ligated product: A-insert-A, B-insert-B and A-insert-B, among which only A-insert-B is the only desired product.

1) Ligation of A-tailed inserts with Y-adapters gives you 100% A-insert-B.

2) Lets now assign a directionality to the insert - say from base 1 to base 400. Following Y-adapter ligation, you will have 2 kinds of insert-ligated products per insert: A-Insert (1-->400)-B and A-Insert(400-->1)-B, both of which are very useful. Each will create a separate clonal cluster and you will get sequence information starting at both base 1 and base 400 of the insert, since in single-read sequencing, the instrument always sequences from Adapter A. However, to know that both these sequences belong to the same insert, one will need to do paired-end sequencing.


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