Is end repair and 5’phosphate necessary for the adaptor ligation?

[sunnymercy]

Hi,everyone! I’m new here and this forum has helped me a lot,thanks.
We are now hoping to carry out NGS of PCR amplified DNA fragment. The DNA fragments have amplified using the Clontech Advantage 2 polymerase mix(which is combined Taq polymerase and a minor amount of a proofreading polymerase) and the PCR products do have a 3’A overhang. We have used the NEBnext Ultra library prep kit to prepare the library. As the PCR amplified DNA fragments have a A overhang, so we skip the end repair and dA-tailing procedure(which generate 5’phosphate and add a 3’A overhang), just do the adaptor ligation,beads purification and 8 cycles library PCR .But the QPCR shows that the libray concentration that have both adapters on the two ends is very low. Is end repair and 5’phosphate necessary for the adaptor ligation? The adaptors were bought form NEB and the 5’ end has already phosphate.
And advice well help us a lot.

[nucacidhunter]

Yes, it is. Look at this thread and others in the forum:
http://seqanswers.com/forums/showthr...ght=end+repair

[pmiguel]

Even with a "minor" amount of proofreading polymerase, the 3' A-tails will likely be removed during PCR

--
Phillip

[sunnymercy]

Why is the 5' phosphate important for the adaptor ligation ? I really don't know how it works during the adaptor ligation.

[cmbetts]

Why is the 5' phosphate important for the adaptor ligation ? I really don't know how it works during the adaptor ligation.

T4 ligase needs a 5' phosphate to ligate to a 3' hydroxyl. With Y adapters (typical TruSeq already phosphorylated), you need a 5' phosphate on the desired insert or else only half of the Y will ligate and you library won't amplify or sequence.