inefficient NEB DNA library prep with plenty of starting material

[nsmackler]

Here's the short description:
We want to ligate adaptors and indexes to 1-3μg of sheared DNA and, in the end, have at least 500ng of adaptor-ligated (and indexed) DNA. It would be great if we could do and still minimize the number of PCR cycles (even just 2 cycles would be great). Does anyone have any advice on how we can do this in the most efficient way possible?

Longer story:
We've been using the NEB Next Ultra DNA kits. We shear 10μg of DNA down to 400bp and size select using beads. At this step we have ~5μg of sheared and size-selected DNA (400bp). We then run it through the NEB DNA Ultra library prep kit using a 6X PCR. After this, in a perfect world (perfect ligation, no loss during cleanups, and 100% efficient PCR), we would expect have 320μg (5 x 2^6) of adaptor ligated and indexes libraries. The problem is that we don't even come close to getting that. In fact, we usually end up with ~200-600ng of adaptor-ligated and indexed DNA.

So my question is: What is going on here? We'd prefer not to use so much starting material and we shouldn't need to. Even if we started with just 1μg and then did 2-4x PCR, we should still have much more than 500ng in our final library. We called NEB and they weren't of much help, so I'm putting this out to the seqanswers community.

Any thoughts?

[nucacidhunter]

So my question is: What is going on here?

Short answer:
The reactions are designed for up to 1 ug input. By using more input the reactions efficiency drops due to insufficient reagents. The expectation is that by PCR step at least 50% of fragments have been lost due to size-selection as well. The results would be better if you set up 5-10 reactions by dividing 5 ug DNA input into multiple reactions and pooling libraries after PCR.

[nsmackler]

Short answer:
The reactions are designed for up to 1 ug input. By using more input the reactions efficiency drops due to insufficient reagents. The expectation is that by PCR step at least 50% of fragments have been lost due to size-selection as well. The results would be better if you set up 5-10 reactions by dividing 5 ug DNA input into multiple reactions and pooling libraries after PCR.

Thanks. We've actually already thought of that and ran some reactions with only 1ug of input. Using the rule of thumb in your post (50% lost by PCR step), we should have 500ng of DNA before the PCR, right? Then, after a perfectly efficient 6x PCR, we should have 500 x 2^6, but we still end up with only ~300-500ng. Any thoughts on this?

Thanks for the response!

[nucacidhunter]

There are limited amount of primers and nucleotides in each PCR reaction which violates exponential amplification assumptions. I would suggest quantifying DNA after ligation clean-up and setting up each PCR reaction with 50-100 ng input. Also note that only input DNA fragments with ligated adapters in both ends are PCR amplifiable.

[nsmackler]

There are limited amount of primers and nucleotides in each PCR reaction which violates exponential amplification assumptions. I would suggest quantifying DNA after ligation clean-up and setting up each PCR reaction with 50-100 ng input. Also note that only input DNA fragments with ligated adapters in both ends are PCR amplifiable.

Thanks. I had heard that the primers could limit the reaction and then, when they run out, that the 3'-5' exonuclease can chew up the DNA. I'm just curious why NEB would suggest that 1ug would be good for the kit and then the reaction wouldn't efficiently ligate adaptors to a small portion of that 1ug (and, yes, these are the only fragments that will amplify in PCR).

Thanks for the thoughts. If anyone else has thoughts on a PCR-free way of ligating adaptors efficiently to 1ug of DNA then I'm all ears!

[nucacidhunter]

If anyone else has thoughts on a PCR-free way of ligating adaptors efficiently to 1ug of DNA then I'm all ears!

NEB kit that you are using requires PCR because NEB adapters are partial. Missing motives are added during PCR. If you want PCR free libraries you may try Illumina's TruSeq PCR free or Bioo Scientific PCR free kits.

[nsmackler]

NEB kit that you are using requires PCR because NEB adapters are partial. Missing motives are added during PCR. If you want PCR free libraries you may try Illumina's TruSeq PCR free or Bioo Scientific PCR free kits.

We're using this as a first step in a probe-based pulldown protocol and there will be more PCRs later on (after the pull-down), so I don't think we're concerned about the getting the final bits of index/adaptor on there at this step. Just getting adaptor on enough of the fragments.

[nucacidhunter]

I do not know your experiment aim and if you are doing sequence capture. Using PCR free libray for standard hybridization based pull down will decrease capture efficiency because of non-amplifiable captured fragments and most likely will increase PCR duplication level after amplifying post-capture library as well.

[lilichen]

Hi, I got the same issue here. I started with 1ug of cDNA samples, by following the NEBNext Ultra DNA Library Prep kit, the final PCR product I got is only 50ng/ul. I know I would lost some DNA in the clean-up step, so I gave the 15 cycles. Did I use too much starting materials or PCR cycles? Did you resolve this problems yet?

[Olivia16]

Hi, I got the same issue here. I started with 1ug of cDNA samples, by following the NEBNext Ultra DNA Library Prep kit, the final PCR product I got is only 50ng/ul. I know I would lost some DNA in the clean-up step, so I gave the 15 cycles. Did I use too much starting materials or PCR cycles? Did you resolve this problems yet?

I spoke with NEB about the efficiency of their NEBNext Ultra DNA Library Kit and this is what NEB said:

If you start with 1 microgram of input DNA (no size selection done) you should get 350 ng yield after 3 PCR cycles.

I hope this is helpful!

[bilyl]

Here's the short description:
We want to ligate adaptors and indexes to 1-3μg of sheared DNA and, in the end, have at least 500ng of adaptor-ligated (and indexed) DNA. It would be great if we could do and still minimize the number of PCR cycles (even just 2 cycles would be great). Does anyone have any advice on how we can do this in the most efficient way possible?

Longer story:
We've been using the NEB Next Ultra DNA kits. We shear 10μg of DNA down to 400bp and size select using beads. At this step we have ~5μg of sheared and size-selected DNA (400bp). We then run it through the NEB DNA Ultra library prep kit using a 6X PCR. After this, in a perfect world (perfect ligation, no loss during cleanups, and 100% efficient PCR), we would expect have 320μg (5 x 2^6) of adaptor ligated and indexes libraries. The problem is that we don't even come close to getting that. In fact, we usually end up with ~200-600ng of adaptor-ligated and indexed DNA.

So my question is: What is going on here? We'd prefer not to use so much starting material and we shouldn't need to. Even if we started with just 1μg and then did 2-4x PCR, we should still have much more than 500ng in our final library. We called NEB and they weren't of much help, so I'm putting this out to the seqanswers community.

Any thoughts?

Just use the Kapa Hyper Prep kit with your own adapters. Crank up the adapter concentration and you can even go PCR-free for downstream assays.

[nsmackler]

Just use the Kapa Hyper Prep kit with your own adapters. Crank up the adapter concentration and you can even go PCR-free for downstream assays.

This a great idea. I hadn't heard of this kit before. We will give it a shot and I'll keep you posted.

Thanks!

[lilichen]

I spoke with NEB about the efficiency of their NEBNext Ultra DNA Library Kit and this is what NEB said:

If you start with 1 microgram of input DNA (no size selection done) you should get 350 ng yield after 3 PCR cycles.

I hope this is helpful!

Thanks for your help. Here is my experimental summary:
I got the 300-500ng of clean PCR product after following the protocol from NEB. I took 300ng of PCR procuct to run the 2nd time PCR with 6 cycles. Then I get about 15 ug of 2nd time PCR product. I tested the Ampure XP beads clean up step by using those 2nd time PCR product. Then I got nothing ( alomost no DNA) from the clean up. Even I start from the beginning with 300-500 input of DNA, I got a lout of PCR product, but after last step wash up, nothing left ( nothing in Nanodrop test, no band in 2% Gel ). But when I check the un-cleaned up PCR product, I found the size is around 150bp.
I was stucked with last step clean up now.
1. My starting material is cDNA from Qiagen REPLI-g WTA single-cell kit, the size of my cDNA should be like 2,00bp-70,000bp. Looks huge in the gel.
2. My suggestion is use 300-500ug DNA as the input material, run 4-6 cycles.
3. I didn't run the size selection step, just clean DNA, run PCR, then clean again. For the 1st clean-up by beads, I got 90% yiels, but for the clean up after PCR cycles, I lost all the PCR product. My 1st time beads clean up means my clean up step is OK. I am totally confused about the 2nd clean up. I tested the PCR product by Nonodrop, 400ng/ul, good quality, but after clean-up by beads, nothing came out.?????????
4. Nano drop used for testing the final PCR product is realiable? 2% gel showed nothing either. I have no Bioanalyzer.
Thanks.

[Manna]

If you don't have a bioanalyzer, and you don't see anything on a gel, I would suggest a fluorescent nucleic acid detection set up such as Qubit.

[Olivia16]

Thanks for your help. Here is my experimental summary:
I got the 300-500ng of clean PCR product after following the protocol from NEB. I took 300ng of PCR procuct to run the 2nd time PCR with 6 cycles. Then I get about 15 ug of 2nd time PCR product. I tested the Ampure XP beads clean up step by using those 2nd time PCR product. Then I got nothing ( alomost no DNA) from the clean up. Even I start from the beginning with 300-500 input of DNA, I got a lout of PCR product, but after last step wash up, nothing left ( nothing in Nanodrop test, no band in 2% Gel ). But when I check the un-cleaned up PCR product, I found the size is around 150bp.
I was stucked with last step clean up now.
1. My starting material is cDNA from Qiagen REPLI-g WTA single-cell kit, the size of my cDNA should be like 2,00bp-70,000bp. Looks huge in the gel.
2. My suggestion is use 300-500ug DNA as the input material, run 4-6 cycles.
3. I didn't run the size selection step, just clean DNA, run PCR, then clean again. For the 1st clean-up by beads, I got 90% yiels, but for the clean up after PCR cycles, I lost all the PCR product. My 1st time beads clean up means my clean up step is OK. I am totally confused about the 2nd clean up. I tested the PCR product by Nonodrop, 400ng/ul, good quality, but after clean-up by beads, nothing came out.?????????
4. Nano drop used for testing the final PCR product is realiable? 2% gel showed nothing either. I have no Bioanalyzer.
Thanks.

LiliChen - It would be nice to bioanalyze the sample that way you could actually see if there is anything there at all....

I would check your sample on the Qubit to check the quantity of your sample, it is more accurate than looking at the nanodrop. Also if you have a small amount of sample you probably won't be able to see if on the gel, so even if you do have some sample there, the gel just may not be sensitive enough to such a small amount of sample that you may not be able to see it. I can see my samples on the bioanalyzer but I know I wouldn't be able to see some of them on the gel.

It also sounds like something weird is going on with your clean up or size selection. What size are you aiming for? Are you shearing your samples? Your cDNA is huge (bp) so make sure you are making your samples are size selected for the appropriate insert size. Hope that helps!