Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Nextera XT sample prep from genomic samples - question about QC along the way

    Hello,

    The newest version of the XT kit does not include the suggestions to do QC along the way anywhere - which makes me very nervous.

    The older protocol had you check via a BioAnalyzer chip for the size distribution, and quantify with Qubit.

    Now they just have the library normalization step with the beads, does anyone have experience with this step - is it consistently successful? I have a hard time blindly trusting the protocol!

    Any information or experiences you've had would be very helpful

  • #2
    It's still possible to check your PCR product on the Bioanalyzer. I've heard some mixed results regarding the bead normalization step. If you only have a few samples, it's still easier to pool manually than using the beads.

    Comment


    • #3
      Thanks.

      I have 24 samples to pool, so I am very tempted to use the beads. We also don't have our own bioanalyzer, so I have to use another labs - which I can, but I just feel bad.

      I will look in to using it though.

      Comment


      • #4
        I'm the one who's probably complained the most about the bead normalization of the XT kits, so I might as well chime in...

        When we first tested the XT kit, we did QC on the Bioanalyzer after the tagmentation and PCR cleanup steps. The post-tagmentation samples showed nothing, possibly because it was too little DNA at that point, while the post-PCR cleanup samples looked good although the peak looked nothing like Illumina shows in their documentation. For our first tests, we did the bead normalization and denaturing following the XT protocol, but before sequencing we did a ssDNA qubit which only worked about half the time.

        Our first test run worked perfectly, but cluster density was around 500K. Our second attempt we got a cluster density of 300K and the third was so high that the run was crap. Other people that tried XT on high-GC archaea had extreme overclustering, to the point that Illumina replaced not only their sequencing reagents but also the XT kit so they could make new libraries.

        Given that time is $$ and not wanting to constantly troubleshoot clustering, we decided to take the post-PCR cleaned libraries and treat them like any other library we make where we quantify with Qubit and find size distribution with the Bioanalyzer and then manually dilute and pool. It takes about an hour more to do all of that for 12 libraries compared to following the XT protocol, but in the long run it's worth it.

        If you're not doing a lot of sequencing though, and are willing to chance it on the cluster density being too low or too high, then give the normalization beads a shot. Worse that happens is the run fails and Illumina sends you replacement kits.

        Comment


        • #5
          Originally posted by mdalessio View Post
          I have a hard time blindly trusting the protocol
          ...That's sooooo true!!!!

          After the cleanup step (AMPure beads) of the amplification PCR you have your pit stop for QC. You can measure on Qubit or real-time pcr and check the library fragment size distribution on Bioanalyzer (HS DNA).

          My next experiment i'll by-pass the beads-normalization step and try just using Qubit or Real-Time.

          Cheers

          Comment


          • #6
            We are a core facility so we see DNA samples from very diverse sources. We ALWAYS check the post-PCR libraries by Bioanalyzer to ensure that there is sufficient material (this is just an estimate, not an accurate quantitation) and that the size distribution is suitable. We usually use bead normalisation and, in our hands, it works pretty well. Occasionally we see over- or under-clustering, but not often.

            Comment


            • #7
              You can also do qPCR post bead normalization. This, of course, is pretty much too late in regards to your question. However, it might spare you a wasted sequencing run.

              Comment


              • #8
                Thanks everyone.

                I am thinking that I will qubit and bioanalyze after the PCR clean up, but if things look good, I think I will attempt the bead normalization, and check again afterwards to see what happened. If all goes terribly, then I have proof that the bead normalization step messed up.

                Comment


                • #9
                  Does anybody know when they switched the XT protocol?

                  Comment


                  • #10
                    Originally posted by mdalessio View Post
                    Does anybody know when they switched the XT protocol?
                    As far as I know, the XT protocol has always incorporated the bead normalization and denaturing steps. The standard Nextera kit, which requires 50ng of input DNA does not include the bead normalization and denaturing, so are you sure you're not thinking of the standard Nextera kit and not the XT?

                    Comment


                    • #11
                      oh my goodness.

                      I just had to do the biggest face palm. I was trying to use the Nextera protocol, for the XT kit. I just want to slap myself. How could I be so stupid?!

                      Comment


                      • #12
                        Follow up due to my stupidity:

                        If I am out of the Amplicon Tagmentation Mix, do you think I could just use a regular DNAP instead? The transposome puts on the recognition sequence, and the PCR primers contain the sequence for the barcoding.

                        Comment


                        • #13
                          Originally posted by mdalessio View Post
                          Follow up due to my stupidity:

                          If I am out of the Amplicon Tagmentation Mix, do you think I could just use a regular DNAP instead? The transposome puts on the recognition sequence, and the PCR primers contain the sequence for the barcoding.
                          ATM is the mix that includes the transposomes while NPM is the polymerase master mix. If you're out of ATM, then you need to buy a new kit, but the NPM is the limiting reagent and you should have had a large excess of ATM. If you did start using the standard Nextera protocol with XT reagents, I'd call Illumina to see how best to proceed as the standard Nextera and XT adapters are incompatible.

                          Comment


                          • #14
                            Ah sorry, I meant NPM. I was flustered/upset about my obvious stupidity.

                            I did end up talking to Illumina, and all will be okay

                            Comment


                            • #15
                              We found the bead normalisation was OK but relying on it to achieve the correct cluster density was a wate of time. Most users want 1 good MiSeq run and the QT is too falky to guarantee this.
                              We real-time PCR QT teh final pool.
                              The most robust way is to use qPCR on all samplesand normalise before clustering.

                              Such a shame as the bead normalisation holds so much promise.

                              Comment

                              Latest Articles

                              Collapse

                              • seqadmin
                                Strategies for Sequencing Challenging Samples
                                by seqadmin


                                Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
                                03-22-2024, 06:39 AM
                              • seqadmin
                                Techniques and Challenges in Conservation Genomics
                                by seqadmin



                                The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

                                Avian Conservation
                                Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
                                03-08-2024, 10:41 AM

                              ad_right_rmr

                              Collapse

                              News

                              Collapse

                              Topics Statistics Last Post
                              Started by seqadmin, Yesterday, 06:37 PM
                              0 responses
                              11 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, Yesterday, 06:07 PM
                              0 responses
                              10 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, 03-22-2024, 10:03 AM
                              0 responses
                              51 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, 03-21-2024, 07:32 AM
                              0 responses
                              67 views
                              0 likes
                              Last Post seqadmin  
                              Working...
                              X