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  • jassirishi
    replied
    Hi, I know it is the old post.

    I wanted to know have you been sucessful doing end repair and dA tailing in one step?

    I tried using a combination of
    1. Large Klenow fragment
    2. T4 DNA polymerase
    3. Klenow exo
    4. T PNK

    but it didn't work do you think replacing klenow exo with taq polymerase will work?

    Leave a comment:


  • Honey Sharma
    replied
    Kapa hyperplus end repair and A tailing

    Originally posted by aengstrom View Post
    KAPA hyper plus buffer mix contains both dideoxy ATP and other deoxyNTPs (A, T, G, C) along with the enzymes. how do the end repair and A tailing happen in the same step by selecting only the dideoxy A for A tailing(not the dATP (Deoxy ATP))?

    Leave a comment:


  • luc
    replied
    Yep, the enzymatic fragmentation products generated by kits from from several vendors do not require any end-repair.

    Leave a comment:


  • nucacidhunter
    replied
    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.

    Leave a comment:


  • aengstrom
    replied
    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?

    Leave a comment:


  • nucacidhunter
    replied
    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.

    Leave a comment:


  • aengstrom
    replied
    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.

    Leave a comment:


  • cmbetts
    replied
    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.

    Leave a comment:


  • aengstrom
    replied
    End-repair at 65 degrees?

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

    Leave a comment:


  • donnyKerabatsos
    replied
    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
    During the recent years, rapid development of sequencing technologies and a competitive market has enabled researchers to perform massive sequencing projects at a reasonable cost. As the price for the actual sequencing reactions drops, enabling more samples to be sequenced, the relative price for preparing libraries gets larger and the practical laboratory work becomes complex and tedious. We present a cost-effective strategy for simplified library preparation compatible with both whole genome- and targeted sequencing experiments. An optimized enzyme composition and reaction buffer reduces the number of required clean-up steps and allows for usage of bulk enzymes which makes the whole process cheap, efficient and simple. We also present a two-tagging strategy, which allows for multiplex sequencing of targeted regions. To prove our concept, we have prepared libraries for low-pass sequencing from 100 ng DNA, performed 2-, 4- and 8-plex exome capture and a 96-plex capture of a 500 kb region. In all samples we see a high concordance (>99.4%) of SNP calls when comparing to commercially available SNP-chip platforms.
    Last edited by donnyKerabatsos; 05-24-2018, 11:21 AM.

    Leave a comment:


  • zhengz
    replied
    The original paper combining several steps into one:

    Leave a comment:


  • ECO
    replied
    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.

    Leave a comment:


  • rcook34
    replied
    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!

    Leave a comment:


  • nucacidhunter
    replied
    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.

    Leave a comment:


  • luc
    replied
    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.

    Leave a comment:

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