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  • Nextera XT without bead normalisation

    I'm using Nextera XT on environmental samples for metagenome sequencing, with only a few samples (2-5) per run. We decided to skip the bead normalisation step in our XT runs and normalise the pooled library manually, following the Nextera DNA protocol. However, the PCR product (CAN) of some samples is at a lower concentration than the 2nM suggested for pooling and denaturing. My current plan is this:
    1. Pool and normalise the libraries by calculating the required volume of each eg
      • 30ul of 0.66nM product
      • 20ul of 1nM product
      • 10ul of 2nM product
    2. Calculate the concentration of the pooled library (in the above example 1nM) and use the equivalent calculated volume for denaturing (ie 20ul of 1nM instead of 10ul of 2nM)
    3. Add the required volume of HT buffer to result in 1000ul at 20pM


    My two main questions are:
    1. What concentration of NaOH should I use? Should I just use the equivalent volume of 0.2 N so that the final concentration is 0.1 N, or should I lower the concentration to match the lower DNA concentration?
    2. Should I rather increase the PCR cycles to try and generate more product?


    Thanks,

    Dylan

  • #2
    Quoting from the MiSeq manual:
    "It is important that the concentration of NaOH is equal to 0.2 N in the denaturation solution and not more than 0.001 (1 mM) in the final solution after diluting with HT1."

    Regardless of how much DNA you have, you need to add an equal amount (by volume) of 0.2 N NaOH to it for the denaturation step to result in a 0.1 N NaOH solution. So if you have 10 ul of DNA solution you need to add 10 ul of 0.2 N NaOH, if you have 20 ul of DNA solution you need to add 20 ul of 0.2 N NaOH.

    The problem is going to be whether you are able to get your NaOH concentration down to 0.001 N in the final solution without diluting your library too much. I think I've tried to do this will libraries at about your concentration and been unable to get it to work out. Additional PCR might be your only option.

    Comment


    • #3
      This is where I start to get confused with the chemistry. Following the MiSeq protocol to create a 20pM DNA solution, 10ul of 0.2N NaOH in 1000ul solution results in 0.002 N, which is already higher than the stated guideline, which means that the DNA needs to be diluted to 10pM to result in 0.001 N NaOH. With my libraries, I should get a final concentration around 0.0025 N NaOH with 12pM DNA. Which way should I lean with adjusting the concentration, considering my libraries have a peak around 1Kbp?

      For now, I'll run the kit again with one extra PCR cycle, and I'll also use less RSB buffer (just enough to cover the Ampure beads) for the final library. That should hopefully get the concentration above 2nM.

      Comment


      • #4
        If you have a low concentration library, add an equal volume of 200mM Tris pH 7.0 after the 5 minute NaOH denaturation to neutralize the NaOH. Then you do not need to worry about NaOH final concentration. This info is from Illumina Tech Support and is what we often do for our libraries and the sequence fine.

        Comment


        • #5
          [QUOTE=microgirl123;104735]Quoting from the MiSeq manual:
          "It is important that the concentration of NaOH is equal to 0.2 N in the denaturation solution and not more than 0.001 (1 mM) in the final solution after diluting with HT1."
          [\QUOTE]

          Actually, Illumina will recommend that you use 0.1N NaOH for your starting concentration instead of 0.2N. We used to have very inconsistent clustering when using the 0.2N and since we've shifted to 0.1N we've had very consistent results. Our FAS actually has told us that the 0.2N is overkill and often leads to clustering variability for a lot of the users he supports so he's been recommending they all switch to using 0.1N.


          As far as the original post goes, we've been doing forgoing the bead normalization for our XT libraries for a while now, and we've had great success with it. We often get sufficient yield after the PCR cleanup step to dilute the samples down to 4nM and then pool them like we would a standard Nextera run. We have, however, had instances were some samples don't give us enough yield to get a 4nM library.

          Depending on how crucial that sample is, we'll either adjust the relative proportion of that sample to others when we pool them together (i.e. for a 2nM library we'd double it's representation in the pool compared to all other samples) or we've also dried the sample down in a SpeedVac and then reconstituted it in water to give us 4nM. I'd be loathe to do more PCR because that means more bias in your final library. You can also do all of the math to figure out that if you start with a 2nM library instead of 4nM, what series of dilutions you would need to make to give you an equivalent 8pM library after denaturing.

          If you want/need more advice about this, feel free to PM me because as I said, we do this all the time with our XT libraries.

          Comment


          • #6
            Mcnelson, thanks for the useful information. We've been considering dropping bead normalization because of variable clustering density. I was wondering how you quant the post PCR cleanup. Do you use Qubit or KAPA qPCR or something else?

            Comment


            • #7
              Originally posted by rwinegar View Post
              Mcnelson, thanks for the useful information. We've been considering dropping bead normalization because of variable clustering density. I was wondering how you quant the post PCR cleanup. Do you use Qubit or KAPA qPCR or something else?
              We do Qubit and run the library on a HS DNA chip on the Bioanalyzer. We then take the average fragment size from the BA output and calculate our nM concentration for the sample based on our Qubit and BA values.

              Comment


              • #8
                Great. Thanks so much. I may have more questions once we start working our way through this.

                Comment


                • #9
                  We're also trying to get more consistent results in our lab, so we tried to omit the beads and go for kapa quantification and manual pooling, but what we got was extremely low amount of clusters. I suspect that it might have been caused by improper denaturation - do you just use NaOH to denature, or follow the nextera protocol and do heat denaturation step with Pooled Amplicon Library? (samples and HT1)?

                  Enclosing a picture of our amplicon runs to illustrate the inconsistency

                  Comment


                  • #10
                    Originally posted by Brunda View Post
                    do you just use NaOH to denature, or follow the nextera protocol and do heat denaturation step with Pooled Amplicon Library? (samples and HT1)?
                    We use 0.1N NaOH, not 0.2N as Illumina states in their standard denaturing protocol. I don't think that heat denaturing would work because the strands will likely re-hybridize in the time between when you do the heat denature and when the fragments get pushed onto the flow cell. With NaOH you have that continually present to inhibit large amounts of re-hybridization but at a low enough concentration to allow the library fragments to hybridize with the anchoring oligos on the flow-cell.

                    Edit:
                    I'll also add that with Nextera, you will have un-even sequencing because of the distribution of fragment sizes. Smaller fragments cluster better than larger ones so if you have samples with a mean fragment size that's smaller than your other samples, it will cluster better and thus give you more data. This is very hard to control, but you can game things a little bit if you look at mean fragment size AND relative abundance of small fragments and then bias your pooling based on that.
                    Last edited by mcnelson.phd; 02-28-2014, 04:39 AM.

                    Comment


                    • #11
                      This is a good point.

                      I asked Illumina tech support about this : " you released a tech note in September with a guideline for pooling libraries and you refer to use this procedure to any Illumina library type. So, if the samples from the original Nextera XT protocol are eluted in NaOH 0.1M and heated to 96 ºC and in Illumina Manual "Preparing DNA Libraries for Sequencing on the MiSeq” you simply advice to pool in NaOH and not to heat. Which one should we follow and get the best results?"
                      Illumina reply:
                      Related to library pooling procedure, heating or no heating?
                      -Heating the samples as suggested helps and has no possible adverse effect to the sample.


                      So.. We use 0.2N NaOH to pool the non-normalized samples (Nextera XT protocol, but normalized after a Bioanalyzer and a qPCR) and dilute 2 times to get a library of 15pM and NaOH of 0.75mM.

                      What's the best option?

                      Comment

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