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  • Nextera XT library size

    Hello everyone,

    I processed 2 C.elegans samples with Nextera XT protocol (Miseq), and before i continue to beads normalization i checked the non-normalized samples on Bioanalyzer (attached image). I didn't proceed to the normalization step, because i had some doubts about the sequencing performance in this kind of samples, since i have only peak after 400-500 bp.

    I checked the starting amount, I quantified (Qubit) and I didn't load more than 1ng.
    The tagmentation step was like that in the manual (55ºC > 5min).

    Any idea from what could influenced the tagmentation?
    If it was you, did you continued to sequence?

    Thanks in advance
    Attached Files

  • #2
    Hi!
    difficult to say. Did you check that the batch of transposase is not old? avoid repeated freeze/thaw cycles especially now that the temperature outside is higher.
    Are your samples resuspended in water? check for inhibitors, the transposase is sensitive to contaminants and salt in general.
    But most importantly, if you use the XT kit 1 ng is the upper limit! you don´t need to use that much, stay in the range 100-500 pg (and 11-13 cycles PCR) and you should get great libraries. Good luck!

    Comment


    • #3
      Originally posted by Simone78 View Post
      Did you check that the batch of transposase is not old? avoid repeated freeze/thaw cycles especially now that the temperature outside is higher.
      Are your samples resuspended in water? check for inhibitors, the transposase is sensitive to contaminants and salt in general.
      This part, everything ok, new kit; resuspended in water.


      Originally posted by Simone78 View Post
      if you use the XT kit 1 ng is the upper limit! you don´t need to use that much, stay in the range 100-500 pg (and 11-13 cycles PCR) and you should get great libraries. Good luck!
      Thanks for the tip!

      Today we did another test: 6 and 10 min of tagmentation.
      The non-normalized samples profile (HS DNA Bioanalyzer) are almost equal.

      The first conclusion, size really matters !!!
      C.elegans genome is ~110 Mb and looks like its out of Nextera XT limits.

      Anyone with more experience in this type of organisms (genome size) and Nextera XT kit?

      Comment


      • #4
        Hi,
        In my experienced your Bionalyzer looks great !!
        If you sequence that library you will obtain 400bp in most of your reads.
        Good luck

        Comment


        • #5
          Abarcelo, why do you think the library looks great? The peak centers around 1000 bp which is supposed to be the absolute max of what you should be loading onto a flow cell. Those large fragments are going to flop around everywhere leading to really low clusters passing filter.

          Comment


          • #6
            Originally posted by microgirl123 View Post
            Abarcelo, why do you think the library looks great? The peak centers around 1000 bp which is supposed to be the absolute max of what you should be loading onto a flow cell. Those large fragments are going to flop around everywhere leading to really low clusters passing filter.
            Exactly, that´s my doubt!
            The sample that I let 10 min at the tagmentation step, the bioanalyzer profile shifted a little to the left* (starting at >300bp), but i still have some concerns about the cluster density and the possible low coverage in some regions.
            Last edited by DRYTCYV; 08-01-2013, 12:56 AM. Reason: *left and not to the rigth

            Comment


            • #7
              if you want to get shorter libraries either you have to decrease the input or increase the enzyme. Incubating longer doesn´t help (or very little), because of the way how the transposase works. The enzyme works as a dimer and carries both the adaptors. Once it has ligated them to your DNA it becomes inactive. So, if all the molecules of enzyme have already "unloaded" their oligo, incubating longer at 55 degrees doesn´t solve the problem.

              Comment


              • #8
                Only a portion of the DNA that you see in the bionalyzer will go to the clustering process and for my experience the small fragments are the ones. I bet that if you sequence your library 90% of the pair-end will be around 400 bases, that means if you run a 2x250 most of your reads will be 250 long and at the end it will give a good assembly.
                We used to work with S. cerevisiae and with some microorganismes and that was we found.
                In the other hand if we had a nice bioanlyzer curve starting to 200bp until 1000bp most of the fragments had around 200 and only a few reads have actually 250 bases long.
                The point is,what you see in the bioanlyzer is not what you found inside the flow cell.
                In terms of overall coverage we obtained at least 95% in one run and 99% in some cases.
                It takes me a while and several experiments to realize that at least for the novo assembly the kind of bionalyzer that you get are actually the good one.
                I think that Illumina have now some illuminanotes that agree whit my argument.
                Hope it helps !!

                Comment


                • #9
                  Originally posted by Simone78 View Post
                  if you want to get shorter libraries either you have to decrease the input or increase the enzyme. Incubating longer doesn´t help (or very little), because of the way how the transposase works. The enzyme works as a dimer and carries both the adaptors. Once it has ligated them to your DNA it becomes inactive. So, if all the molecules of enzyme have already "unloaded" their oligo, incubating longer at 55 degrees doesn´t solve the problem.
                  Yes, looks like there's a limitation in the tagmentation step. But if the Nextera XT manual indicate to use a maximum of 1 ng, do you think that there's a genome size limitation?

                  Originally posted by abarcelo View Post
                  Only a portion of the DNA that you see in the bionalyzer will go to the clustering process and for my experience the small fragments are the ones. I bet that if you sequence your library 90% of the pair-end will be around 400 bases, that means if you run a 2x250 most of your reads will be 250 long and at the end it will give a good assembly.
                  We used to work with S. cerevisiae and with some microorganismes and that was we found.
                  In the other hand if we had a nice bioanlyzer curve starting to 200bp until 1000bp most of the fragments had around 200 and only a few reads have actually 250 bases long.
                  The point is,what you see in the bioanlyzer is not what you found inside the flow cell.
                  In terms of overall coverage we obtained at least 95% in one run and 99% in some cases.
                  It takes me a while and several experiments to realize that at least for the novo assembly the kind of bionalyzer that you get are actually the good one.
                  I think that Illumina have now some illuminanotes that agree whit my argument.
                  Hope it helps !!
                  Thank you abarcelo, i think you posted very important ideas.

                  Comment


                  • #10
                    [QUOTE=DRYTCYV;112489]Yes, looks like there's a limitation in the tagmentation step. But if the Nextera XT manual indicate to use a maximum of 1 ng, do you think that there's a genome size limitation?

                    I don´t think that genome size matters that much. Adey et al. in their original paper (Genome Biology, 2010), besides the standard library prep, also did some PCR-free library where they started from 100 and 200 ng of genomic DNA (E. coli and human) and that apparently didn´t matter. Most likely they used what is now sold as Nextera kit (not the XT). However, for the XT it should be the same, only the input is scaled down.
                    I haven´t tried it myself. I usually use the XT on full-length cDNAs with an average size of 1.5-2 Kb.

                    Comment


                    • #11
                      Hi everyone,

                      I'm going forward with this experiment, but now I have others doubts, in the dilutions.

                      In the manual, they say to take 5ul from each sample and pool, then take 24ul (from the pool) and dilute in 576ul of HT1.
                      Is the minimum 600ul?
                      Since we have only 2 samples what should we do? Use 12ul from each?
                      The non-normalized library bioanalyzer profile is like the one of "Nextera Library Validation and Cluster Density Optimization" pdf -Panel B High concentration library.

                      What's your opinion? Should i be concerned about load to much if load 12ul from each?

                      Thanks in advance

                      Comment


                      • #12
                        Possibility of tagmentation without fragmentation with Nextera XT kit

                        Hello everyone,

                        I would like to know if it's possible to do the fragmentation of my cDNA amplicon without any fragmentation using the Nextera XT kit. PCR amplicons have already sizes between 150 and 600 bp. these PCR products will be sequenced with Miseq.

                        Thanks in advance

                        Comment


                        • #13
                          Not possible with Nextera. You could either add the adapter sequences with PCR or ligate adapters to the ends of your cDNA using a standard Truseq Nano, a kit from NEB, or another supplier. In the future, you could add some adapter sequence to your targeting primers, then do another PCR to add the rest of the sequence required for flow cell binding.

                          Comment


                          • #14
                            Hi,
                            If you got fragments bigger than 300-400bp, i'll say to give a shot!
                            The transposase need a minimum of +-300bp to cut and add adaptores, so in some of your fragments will be possible.

                            Comment


                            • #15
                              Except that he's posted this somewhere else and forgot to mention here that there are barcodes ligated to the ends of these samples.

                              Comment

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