End Repair + A-Tailing - how do they do it?

[Ingeneious]

Some of the popular library prep kits out there (i.e. NEBNext Ultra and KAPA Hyper) are able to combine end repair and A-tailing into one step. I would like to do the same with minimal cost, but am not sure how they are doing it.

Both NEB and KAPA have the same thermocycler protocol:

• 20°C for 30 min (Activation?)
• 65°C for 30 min (Denaturation?)

End-repair can be done with some or a combination of the following enzymes:

• DNA Polymerase I, Large (Klenow) Fragment (Active at 25°C for 15 min)
• T4 DNA Polymerase (Active at 15°C for 12 min)
• T4 Polynucleotide Kinase (Active at 37°C for 30 min)

A-Tailing can be done with some or a combination of the following enzymes:

• Taq Polymerase (Active at 72°C for 20 min)
• Klenow Fragment (3'→5' exo-) (Active at 37°C for 30 min)

Anyone know how they do it?

[nucacidhunter]

I think they have Taq polymerase in End repair mix. During incubation at 65C Taq actively adds A to 3' ends, while the other mesophilic enzymes used for end repair and 5' phosphorylation are denatured and deactivated.

[Ingeneious]

That is what I am thinking, too, but I have never seen a library prep protocol using it. Everyone seems to be using Klenow 3 - 5 exo for A-tailing and I have seen various combinations of enzymes for end repair.

Could Klenow be working simultaneously with the other enzymes and then they're all denatured?

[luc]

At one point Lucigen claimed to have found a (propietary) enzyme (in hot springs) that is even better at A-tailing than Taq polymerase (in the presence of other nucelotides). This might have been licensed out. But in all likelihood it is simply Taq.

[nucacidhunter]

That is less likely. For end repair (filling in 5’ overhang and removing 3’ overhang) two enzymatic activities are required so it is less likely to add 3’ A while another enzyme is removing 3’ overhangs. This can be tested by omitting incubation at 65C and immediately cleaning up end repair reaction followed by adapter ligation. Lack of 3’ A should significantly reduce ligation efficiency.

[rcook34]

Hey all,

Sorry to reopen this older thread, but I was curious if anyone had anything else to add? I am very interested in this area, since my experiments using both NEB's and an in-house Klenow exo- enzyme for 30min@37 degrees have given mostly negative results in terms of library yield.

Thanks!

[ECO]

IME, Klenow Exo- is unnecessary and only was ever included because the very very first library kits from Solexa had it in there. T4 pol is a monster at end removal and fill in. The blunting phase of the reaction is over in <10 minutes (at least with clean DNA)

The best combination if you really want a one-pot reaction is T4 PNK + T4 Pol + Taq + dNTP. 20C is the ER phase. 65C is the heat-kill + Taq tailing phase. Non-templated addition is better at 65C than at 72C.

You WILL get lower A-tailing efficiency with Taq in the presence of all 4 nucleotides, but it's not that bad and probably worth the time savings.

[zhengz]

The original paper combining several steps into one:

http://nar.oxfordjournals.org/content/38/13/e137.full

[donnyKerabatsos]

This paper describes how to combine these steps in one reaction using individually-ordered (i.e. cheap) enzymes and buffers: T4 PNK, T4 polymerase and taq, same as @ECO suggests.

Library Preparation and Multiplex Capture for Massive Parallel Sequencing Applications Made Efficient and Easy
Mårten Neiman, Simon Sundling, Henrik Grönberg, Per Hall, Kamila Czene, Johan Lindberg , Daniel Klevebring
PLoS One

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048616

[aengstrom]

End-repair at 65 degrees?

Does anyone know of an enzyme that end-repairs at 65 degrees?

[cmbetts]

Any polymerase with 3' Exonuclease activity can do end repair in the presence of nucleotides, which would include many PCR polymerases. Here's a paper using Pfu for old school cloning https://academic.oup.com/nar/article...rectedFrom=PDF. Now heat inactivating them to do subsequent A-tailing would a tall order as the DNA will denature long before the enzyme is killed (and if you don't remove it, it will chew off any new nucleotides you'd want to add). That's why you more typically see a mesophilic enzyme used to polish, which is then heat killed automagically when you A-tail with Taq.

[aengstrom]

Thanks for the answer, appreciated! The reason for me asking is that I am currently using the library prep (and target capture) from Twist Bioscience, exome and custom panel protocol. The current protocol has a combined mix of enzymes for fragmentation, end-repair and a-tailing - incubation at 32 degrees (fragmentation) and 65 degrees (end repair AND a-tailing - this is what a Twist staff claims) - can this be correct? In contrast, for the setup with enzyme mix from Qiagen/Enzymatics, End Repair and A-Tailing are happening at 20°C and 65°C, respectively. Any comments on this? This is important to us since we use degraded FFPE samples and we want to keep the fragmentation time in the Twist protocol as short as possible, but still do efficient end-repair... so I was then thinking how does it happen at 65 degrees and then a-tailing also at the same time!?
Also that has confused me a bit is the enzyme mix from KAPA/Roche (HyperPlus) - here only a 65 degree incubation is included whereas in KAPA/Roche HyperPrep, both 20 degrees and 65 degrees are included. (I have learned that the enzyme mixes are the same for both kits/protocols.) Conclusion - the DNA is not end-repaired in HyperPlus (even though the protocol states that), only a-tailed (since it is enzymatically fragmented)?
Sorry, lots of questions and thoughts... I'm trying to understand this fully.

[nucacidhunter]

Twist Bioscience Exome protocol does not claim end-repair at 65C. It must have been a miscommunication. Most enzymatic library prep kits include fragmentation and end repair at 20-35C and A-tailing at 65C.

[aengstrom]

Thanks for the answer nucacidhunter! I have checked now once again and the reply from Twist is: "I confirm again that both of our End-repair and A-tailing reactions are happening at 65°C. Since our enzyme sources are proprietary, I cannot comment further unfortunately." Any comments on this? What enzymes could it be?

[nucacidhunter]

I can think of following two approaches:

1- Their enzymatic fragmentation leaves blunt end or/and 5’ overhang which can be repaired and A tailed by Taq.

2- Mix of proofreading and non-proofreading polymerases with exonuclease resistant A nucleotide. In this case 3’ overhang can be excised by proofreading polymerase, and 5’ overhang can be filled in by both and A tailed by non-proofreading polymerase. Template 5’ phosphorylation can be done by including PNK in ligation enzyme mix or at fragmentation step.