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  • Library quantification

    Hi,
    I am using qPCR and Bioanalyzer to quantify my libraries before sequencing in a GAII. Even so I still can not get it right. Each run is a surprise (not always good). Does anyone have a really good method?
    Thanks

  • #2
    if you still cant make it from qPCR conc to get the right density of flowcell. i prefer you to sequence for 6 cyc on GA and make the flowcell with the right conc.

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    • #3
      Do you mean spend a Flow Cell to determine the exact density?

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      • #4
        yes, deblock FC after 6 cyc and store in fridge. You can reuse the flowcell if you want to validate your GA.
        Last edited by sbsuser; 09-08-2010, 02:14 AM.

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        • #5
          If you're running a control lane on your flowcells you can also mix (a best guess) 10% of another library in with PhiX to get a pretty accurate quantitation and also check on the quality of the library.

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          • #6
            How can you mix another library in to phix to get accurate quantitation? Can you explain.

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            • #7
              Originally posted by sbsuser View Post
              How can you mix another library in to phix to get accurate quantitation? Can you explain.
              Yes, just mix in the other library prior to putting it on the flowcell. After the run extract any reads from that lane which didn't map to PhiX and those will mostly come from your other library. The PhiX stats don't get messed up (other than %mapping) and you still have enough diversity to do the phasing calculations.

              If you get around 10% of the other library from your lane we find that around 90% of the unmapped sequences come from this library. You can filter out most of the other PhiX reads by remapping against PhiX with relaxed constraints if you want, and you can get a pretty good idea of what's going on in your other library.

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              • #8
                is this abt the in lane Phix experiment?

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                • #9
                  Originally posted by sbsuser View Post
                  is this abt the in lane Phix experiment?
                  It's more the other way. Illumina now offer an option to spike small amounts of PhiX in with each lane for QC purposes, but these don't help if you need a control lane for calibration purposes. If you're still running a full PhiX lane you can spike in another library to that to get a low level of coverage which won't affect the normal function of the control lane.

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                  • #10
                    This is how we did it:

                    1. We wasted a FC
                    2. Amplified (~14 cycles) gel purified/size selected library (in the size range that you use to sequence)
                    3. Purified the PCR products
                    4. Nanodrop quantified the PCR products
                    5. Made dilution series to hyb to a FC
                    6. Pick the "best" dilution that gives desired cluster #
                    7. From this info, create a qPCR STD which you will use to quantify every subsiquent sample that is going on to sequencing. Make tons of this stuff (alliquoted in strip tubes and left in the -20C), and do not run out without using a set to quantify your next batch of STD!!
                    Our qPCR STD dilution series is this:
                    100pM, 30pM, 10pM, 3pM, 1pM, 0.3pM, 0.1pM, 0.03pM

                    As long as you always cluster the amount that you found in step #6, you should be great.

                    Let me know if this doesn't make sense.

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                    • #11
                      Am I correct to guess that the titration run on the flowcell would have to be repeated with each software upgrade and/or formulation change since that would affect cluster #?

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                      • #12
                        I wouldn't think so. The most difficult part here is aligning your qPCR results to your cluster #. After that, your cluster # should not change dramatically enough to have to redo the titration. If you see that your cluster # is going down/up all you have to do is change your concentration.
                        Example: If you are generally clustering at 8pM and you see that your cluster density has dropped across the board (even PhiX control lane), then you might want to start clustering at 10pM or 14pM (and change PhiX if necessary).....

                        It is possible to make a mistake when clustering the flowcell, so be careful. I occasionally get flowcells that will fail the first base report with low cluster density, but it was the process or the flowcell or the clustering that was the issue...not the concentration.

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                        • #13
                          Library qualification by qPCR

                          Hello everyone,
                          I am pretty new to this forum and stumbled on this post as even I am facing problem with qPCR quantification method
                          1.Has anyone encountered with, a higher qPCR concentration than the estimated concentration by pico green??
                          2. Also if assumed that qPCR is more accurate(w.r.t pico green) as it would give conc. of ssDNA ligated with adapters,then the cluster generation should be accurate with qPCR conc. but it doesn't happen so...the flow cell (V3,Hiseq 2000) remains under clustered.Can any one think of possible reasons??
                          3. This trend seems to change with every library.Is absolute quantification the right approach for Sybr green qPCR method?Also I am using the PhiX control provided by Illumina as my standard.
                          4.Do people take the "exact concentration of libraries" as determined by qPCR for cluster generation or they compare it with pico green to see the relative trend of concentration?

                          Would appreciate if any of the queries are resolved!!
                          Thanks in advance

                          Comment


                          • #14
                            There are factors that might confound qPCR. They include:

                            (1) Ethidium Bromide in your sample. I did not do a careful test of this. But someone in the lab tried spiking EtBr into a sample at a low level. It shifted the Ct by a cycle or two. Ethidium Bromide will also confound fluorimetry, so purifying it away after some size selection methodologies is important.

                            (2) Your phiX may not be the concentration and/or length that you think it is. Most current (v3) phiX libraries from Illumina are supposed to be about 500 bp. So far every one that we tested has been around that size. I think v2 (including v2 multiplex) phiX is supposed to be 350 bp. The v2 multiplex phiX has always tested out to 350 bp or so for us. But we saw one case where the non-multiplex v2 was 500 bp, rather than 350 bp. Further, I have seen a 500 bp library that had a very weak, but visible, 350 bp peak. Also one library had a ~70 bp peak. I don't know what the adapter length is for these library types, so I don't know if this is an adapter dimer. If it was, then it was 10% of the molar total. But would produce little signal during SYBR Green qPCR.

                            Since the phiX standards are likely calibrated primarily to produce a given number of clusters, the actual size of the library molecules may not be a great concern for Illumina. However, SYBR green assays do not directly determine molarity -- instead they rely on you to supply the correct average size of libraries. So:

                            (3) You must have an accurate determination of both your library's average length and your concentration standard's length.

                            (4) One more: TruSeq libraries, if they are run no-amp, cluster at least an order of magnitude lower than qPCR would suggest they should. Illumina tells you, that you must do at least one cycle of enrichment PCR to "break the fork" of the adapters. Taken literally, this would suggest that the adapters are covalently linked strand-to-strand by a chemistry that breaks during extended heating but not by NaOH. Alternatively the adapters might lack some critical "magic" present in the PPC -- PCR primer cocktail -- of the TruSeq DNA/RNA prep kits. So, these factors could result in issues, if true.
                            --
                            Phillip
                            Last edited by pmiguel; 03-09-2012, 05:02 AM. Reason: Added point (4).

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                            • #15
                              There is no magic in the PPC. Adapters are not a complete "library element"...which is the beauty behind the Y-adapter. You do not have complimentary ends...they are "sticky", and only contain half of what you need to cluster (only half of your library elements have what it takes to hybridize to the flowcell). They should however qPCR with more accuracy than an order of magnitude...as only the first cycle of qPCR should be affected...but maybe thats enough....I haven't done the math.

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