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  • #16
    Originally posted by whw View Post
    I originally thought that long adaptors would hybridize and lead to degradation of all species but a colleague recently tried digesting a TruSeq library and found no reduction in abundant species. Is it possible that the forked adaptors are actually preventing DSN from attacking any transcripts?
    Seems more likely that the DSN was swamped with duplexes derived from adapter-adapter annealing (just as you initially posit).

    Might work to perform normalization on the cDNA pre-ligation. Then re-synthesizing 2nd strands. Then blunting/ligation.

    The problem there would be that reverse transcription in the TruSeq is designed to work on a much lower amount of RNA (after removal of rRNA). So there might not be enough reagent to convert all that additional RNA.

    I guess you could generate cDNA using some other kit. Normalize, then enter TruSeq construction. At that point you might as well use the cheaper DNA TruSeq kit, though.

    --
    Phillip

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    • #17
      I tired DSN-Library from total RNA with TruSeq RNA Sample Prep Kit ver.2.
      Analysis of Agilent Bioanalyzer showed that DSN-Library contained abnormal peaks like ladders.
      I've never seen this abnormal ladder-like peaks in DSN-Library prepared with older mRNA-Seq Kit.
      Have you seen abnormal ladder-like peaks in DSN-Lib prep with TruSeq Kit?

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      • #18
        The ladder peaks may be from the control DNAs that are an optional part of the TruSeq kits. Did you use the control DNAs during library construction?

        --
        Phillip

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        • #19
          Hi, Phillip

          I considered the possibility of TruSeq's in-line-control contamination,
          but obtained reads were not aligned on sequence of in-line control DNA.

          I think that TruSeq adaptor could cause the ladder peaks artificially.

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          • #20
            Maybe I'm missing something obvious, but if the issues are with the adapters, then why not DSN treat the cDNA before end-repair, etc? -nevermind, I missed the bit about the cDNA synth being optimized for low-input poly-a, etc.
            Last edited by led_o_zep; 04-12-2012, 06:35 AM. Reason: reread earlier messages

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            • #21
              I have never done DSN on any TruSeq libraries but have been successful with older version of illumina RNA seq. I made libraries (purified PCR product) and performed DSN and amplified at the end. Never failed. My confusion is why it does not work on TruSeq library? Although TruSeq adaptors are much longer than illumina PE old adaptor, however the final libraries are the same construct(except with additional barcodes). Therefore DSN should work on TRuSeq libraries as well.

              Originally posted by led_o_zep View Post
              Maybe I'm missing something obvious, but if the issues are with the adapters, then why not DSN treat the cDNA before end-repair, etc? -nevermind, I missed the bit about the cDNA synth being optimized for low-input poly-a, etc.

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              • #22
                I am confused by this information. DSN is supposed to take place on either ligated cDNA or amplified libraries. How does it have anything to do with polyA selection?

                Originally posted by whw View Post
                I've heard that from Illumina as well but I'm not really satisfied with the answer. The issue during EPF is probably due to the use of oligo-dT beads to bind and clean up the reaction. I would guess that DSN treatment might degrade the poly-A tails, preventing the use of beads. However if you use a different isolation method, say a column based system, there's no reason it shouldn't work. As for the primer efficiencies I don't see any reason why you couldn't adjust the concentration for a smaller amount of sample.

                However! Maybe you tried all those things and they didn't work, what was your experience with DSN?

                We are interested in low abundance transcripts in lymph node tissue. Coverage is very important since we are essentially looking for needles in an immune cell haystack. I am hoping that DSN will improve our chances.

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                • #23
                  The sequencing primers in Truseq sequencing kit are a cocktail of all kinds of sequencing primers: those for ILMN pair end libraries, for truseq libraries and even for v1.5 sRNA libraries.

                  Originally posted by QTLdum View Post
                  As far as I can see from the TruSeq application note, it should be possible to enter the protocol at the "Incubate RFP" step after adding the Elute, Prime, Fragment mix. The mix should perform the fragmentation step.

                  My question for the DSN procedure with TruSeq is on the primers used for the final amplification steps after the DSN treatment. The Illumina application note for DSN indicates using the PCR Primer PE 1.0 and PE 2.0 from the old RNA sample prep kits. However, I assume these will not work with the subsequent TruSeq cluster generation and sequencing steps. Does any one have an idea on the sequence of the primers required for DSN in combination with the TruSeq kits?

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                  • #24
                    Originally posted by CC_seqanswers View Post
                    I am confused by this information. DSN is supposed to take place on either ligated cDNA or amplified libraries. How does it have anything to do with polyA selection?
                    The title of this thread is "DSN normalization with TruSeq kits". TruSeq RNA prep kits starts with fairly low levels of total RNA (0.1-4 ug). First step is poly A+ mRNA extraction followed immediately by fragmentation, cDNA synthesis and adapter ligation.

                    DNA normalization is problematic prior to adapter ligation, because the concentration of cDNA is very low -- pM. It is problematic after ligation/PCR, because the long adapters (60 bp) would be degraded by the DSN as they are, in effect, a low complexity component of the library.

                    Another issue is: if you are doing DSN normalization, you probably don't want to do polyA+ at all. But one presumes that the cDNA synthesis reagents in the TruSeq kit are provisioned for doing cDNA synthesis after rRNA has been removed. So one would expect to be at least 10x low on reagents for cDNA synthesis if one skips the polyA+ step.

                    Obviously, you could use some other kit or your own home brew batch of reagents to do fragmentation and cDNA synthesis, subject that cDNA to DSN normalization and then just use a TruSeq DNA kit to make your libraries. You might even be able to use dUTP for 2nd strand synthesis. That would allow you to create strand-specific libraries.

                    The TruSeq RNA prep kit blows away every other method we have undertaken for cDNA library construction on price, robustness, scaling for large number of samples. But it does not seem to be compatible with DSN normalization.

                    --
                    Phillip

                    Comment

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