Low DNA yield after adapter ligation

[reprogrammer]

Hi guys,

I am generating RRBS library recently. In the whole process I use Beckman Ampure Xp beads to purify (cleanup and elute) DNA. But only the step of adapter ligation showed a extremely low efficiency of DNA yield (~35%).

The efficiencies of End repair and Add A steps are very good (over 90%). Do you have any experience about that? What should I do to improve the efficiency after adapter ligation?

Thank you so much!

[jdk787]

Hello Reprogrammer,
It is going to be hard to answer this question without more information.

Can you describe the protocol that you are using?
Listing your steps and including the sample and bead volumes that you are using for your cleanups would be a good start.

[luc]

Ligation efficiency will depend a lot on the appropriate adapter concentration. Do you see any adapter dimers after the ligation.

[reprogrammer]

Hello Reprogrammer,
It is going to be hard to answer this question without more information.

Can you describe the protocol that you are using?
Listing your steps and including the sample and bead volumes that you are using for your cleanups would be a good start.

Hi jdk787，

I summarized my protocol and put it in the attachment. Would you help me take a look is there anything wrong, please?
Thank you so much.

[reprogrammer]

I noticed that I made a conceptual mistake. What I am doing is whole-genome DNA methylation but not RRBS since I am using sonication biorupter to fragment genomic DNA.

[jdk787]

Hi jdk787，

I summarized my protocol and put it in the attachment. Would you help me take a look is there anything wrong, please?
Thank you so much.

Hello Reprogrammer,
Sorry for the delayed response, I've been out of town.
One big problem that I see is that you are trying to blunt, phosphorylate, and A-tail your DNA using just Klenow Fragment (3-5 exo)
This enzyme is recommended for A tailing previously blunted and phosphorylated DNA, not blunting and A-tailing.

You need to blunt your DNA first, and then A-tail.

Also, you should check to make sure that your fragmented DNA is in a size range that is compatible with your protocol by either running a portion of it on a gel or BA. If you are over fragmenting your starting material you may be losing it during your cleanups.

For your PCR amplification, I think you can skip the Ampure cleanup after the post conversion column cleanup and just go straight in to PCR.

Finally, you need to make sure that you are using a Uracil literate Taq for your amplification since you will have C -> U conversions in your template.

If you want an easier and more straight forward way to make your libraries, many companies sell kits that will supply you with the protocol and all of reagents that you need to perform WGBS and RRBS library prep. Kapa, Illumina, and Bioo Scientific (where I work) to name a few.

Feel free to PM me if you would like info on the Bioo Scientific kit.

[reprogrammer]

Hello Reprogrammer,
Sorry for the delayed response, I've been out of town.
One big problem that I see is that you are trying to blunt, phosphorylate, and A-tail your DNA using just Klenow Fragment (3-5 exo)
This enzyme is recommended for A tailing previously blunted and phosphorylated DNA, not blunting and A-tailing.

You need to blunt your DNA first, and then A-tail.

Also, you should check to make sure that your fragmented DNA is in a size range that is compatible with your protocol by either running a portion of it on a gel or BA. If you are over fragmenting your starting material you may be losing it during your cleanups.

For your PCR amplification, I think you can skip the Ampure cleanup after the post conversion column cleanup and just go straight in to PCR.

Finally, you need to make sure that you are using a Uracil literate Taq for your amplification since you will have C -> U conversions in your template.

If you want an easier and more straight forward way to make your libraries, many companies sell kits that will supply you with the protocol and all of reagents that you need to perform WGBS and RRBS library prep. Kapa, Illumina, and Bioo Scientific (where I work) to name a few.

Feel free to PM me if you would like info on the Bioo Scientific kit.

Hi jdk787,

Thank you so much for your suggestion. Finally I got the PCR product with your guidance.

First I changed the Taq enzyme to Cx Pfu, I believe this is the major mistake I made. At the beginning I used the PrimeStat which could not recognize U in the template DNA after bisulfite conversion. Second, I over amplified the template DNA with 40 cycles to make sure the product was exist. I am trying to reduce the PCR cycles to 20~25 now.

For the commercial kit, I am contacting with NuGen company which my friend used pretty well with 25 ng DNA input.