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  • #16
    Hi everyone,

    If you've not heard, earlier this week we announced the availability of a new cDNA library preparation protocol. This is a straightforward, general utility transcriptome sequencing method, that takes advantage of an existing Roche ds cDNA synthesis kit and the newly released Rapid Library kit from 454. Importantly, we've also released an updated version of our assembler software that provides powerful de novo transcriptome assembly capabilities.

    For more information please contact your local Roche representative or see the following link on the 454 site:



    Additionally, customers can have a peek at all the new manuals at the my454 website:



    Best regards,
    Jason
    Technical Product Manager
    454 Life Sciences, A Roche Company

    Comment


    • #17
      Originally posted by 454Sequencing View Post
      Hi everyone,

      If you've not heard, earlier this week we announced the availability of a new cDNA library preparation protocol. This is a straightforward, general utility transcriptome sequencing method, that takes advantage of an existing Roche ds cDNA synthesis kit and the newly released Rapid Library kit from 454.
      [...]
      Best regards,
      Jason
      Hi Jason,
      Thanks! Long awaited.

      For everyone without access to the PDFs:

      Major Revelation
      The cDNA procedure uses a new "Rapid Library Prep Method" which
      (1) Asks for only 500ng of input DNA
      (2) Skips the ssDNA step altogether!?!?! Doesn't replace it with suppression PCR either. Pretty much the straight Roe lab methodology there.

      No nick-translation, either. Seems like they are counting on that to occur during emPCR?

      Anyway, about the cDNA method:

      So the issues with cDNA sequence on the 454 were mainly:

      (1) polyA tails in high numbers ruin a run
      (2) DNA in the 1-2kb range is not efficiently fragmented by nebulization.

      The protocol solves the first issue by using random primed 1st strand synthesis. (Downside: will have to get rid of the rRNA prior to 1st strand synthesis. Upsideotentially much less 3' sequence bias.)

      Second problem dealt with by fragmenting the RNA with heat+ZnCl2.

      Other features:
      Starting amount of RNA: 200ng. After fragmentation to mean length of 500 nt that would be about 800 billion molecules. Probably 100 billion are in the desired size range, if all goes well.

      No amplification. Minimum acceptable yield of "library" is 7.3 billion molecules. That is a fluorometric determination. So only double stranded molecules will be counted. Of course that includes:

      RNA-DNA hybrids. Probably low numbers of these due to the RNaseH activity of AMV Reverse Transcriptase.

      dsDNA that has PA on one end and PB on the other.
      dsDNA that has PA or PB on both ends
      dsDNA with PA or PB on one end and no adaptor on the other
      dsDNA with no adaptors

      Well, it is supposed to be "Rapid". Interesting...

      --
      Phillip
      Last edited by pmiguel; 12-03-2010, 05:12 AM. Reason: To disable smilies in text!

      Comment


      • #18
        Hi Phillip,

        One clarification - the Rapid Library protocol actually employs a single adaptor moiety, and no longer an A-B pair of adaptors. This is why there is no more ssDNA melt step and why there are no worries of the different adaptor-library combinations you mentioned. Additionally, the RL Adaptor is fluorescently labeled which enables direct quantitation following library synthesis. Your local Roche rep should have access to some material on this topic that would be of assistance.

        Best regards,
        Jason
        Technical Product Manager
        454 Life Sciences, A Roche Company

        Comment


        • #19
          Does Roche make the new version of newbler available to users of 454 data?

          Comment


          • #20
            Originally posted by 454Sequencing View Post
            Hi Phillip,

            One clarification - the Rapid Library protocol actually employs a single adaptor moiety, and no longer an A-B pair of adaptors. This is why there is no more ssDNA melt step and why there are no worries of the different adaptor-library combinations you mentioned. Additionally, the RL Adaptor is fluorescently labeled which enables direct quantitation following library synthesis. Your local Roche rep should have access to some material on this topic that would be of assistance.

            Best regards,
            Jason
            Wacky! Great, I'll contact him. Wonder if you guys went with the Solexa Y-adaptor approach or something else.

            --
            Phillip

            Comment


            • #21
              Jason,

              Is it possible to do MID tagging with the RL protocol? Also, when is v2.3 of the software going to be released?

              Comment


              • #22
                Emanuel - Yes. The easiest path is to follow up with your local Roche representative to get access to our software. Be sure to send me a PM if you don't meet with success.

                kmcarr - Yes, we have also released a kit of 12 Rapid Library MID adaptors that is presently available. The v2.3 software is also available at this point via your local Roche rep.
                Technical Product Manager
                454 Life Sciences, A Roche Company

                Comment


                • #23
                  Originally posted by 454Sequencing View Post
                  Hi everyone,

                  If you've not heard, earlier this week we announced the availability of a new cDNA library preparation protocol. This is a straightforward, general utility transcriptome sequencing method, that takes advantage of an existing Roche ds cDNA synthesis kit and the newly released Rapid Library kit from 454. Importantly, we've also released an updated version of our assembler software that provides powerful de novo transcriptome assembly capabilities.

                  For more information please contact your local Roche representative or see the following link on the 454 site:



                  Additionally, customers can have a peek at all the new manuals at the my454 website:



                  Best regards,
                  Jason
                  Hi Jason,
                  thanks for the info!

                  The normalization (Trimmer cDNA normalization kit) step is still possible between those "ds cDNA synthesis" and "Rapid Library" Roche kits?

                  Cheers,
                  Bruno

                  Comment


                  • #24
                    Dear Bruno,

                    You're welcome!

                    As we've only recently released the new cDNA Rapid Library protocol I'm unaware of any studies concerning the use of the Evrogen products in combination with this method. That said, I know that others have successfully used the Trimmer products with their own homebrew cDNA sequencing approaches using our earlier General Library preparation kit.

                    Best regards,
                    Jason

                    Originally posted by blouro View Post
                    Hi Jason,
                    thanks for the info!

                    The normalization (Trimmer cDNA normalization kit) step is still possible between those "ds cDNA synthesis" and "Rapid Library" Roche kits?

                    Cheers,
                    Bruno
                    Technical Product Manager
                    454 Life Sciences, A Roche Company

                    Comment


                    • #25
                      SMART adaptor concatemers

                      Originally posted by pmiguel View Post
                      I finally figured out what must be the source of adaptor concatamers that plague some SMART libraries.

                      If you are using the reverse transcriptase to add the 5' adaptor via MMLVs natural TdT activity (one to five C's are said to be added), then 5' concatamers would be the expected result. This is because MMLV will reverse transcribe RNA until it hits the end of the template, then it adds some C's and waits around for something to anneal at those C's. The 5' SMART oligo can anneal there. If it does, MMLV continues until it reaches the end of the 5' SMART oligo. (In essence MMLV has "switched" templates -- sometimes this is called "strand switching" or "template switching".)

                      After reaching the end of the new template (the SMART oligo) MMLV would again add non-templated C's.

                      This allows another 5' SMART oligo to anneal to the nascent strand, and the process can repeat over and over again, creating a string of concatamers.

                      The way to avoid this is to not add the 5' SMART adaptor to the reverse transcription. Instead just let the 5' SMART adaptor get added during 2nd strand synthesis/ amplification PCR. Basically a step-out from the non-templated C's that would terminate all the 1st strand cDNAs produced by MMLV. Taq polymerase doesn't add non-templated C's to the end of nascent strands, so no concatamers will result.

                      --
                      Phillip
                      Hello everybody

                      A this time, we have done six ½ Titanium run of cDNA sequencing. We always use SMART (SMARTER kit from Clontech) cDNA preparation without normalization. In the last run, we obtain at least 50% of SMART adaptor concatemer sequence.

                      Few mouths ago, Phillip has proposed a very elegant hypothesis to explain how this concatemers are created and suggested to modify the SMART original protocol in order to avoid this problem.

                      - Is anybody have tried this new protocol ?
                      - Do you think that the best solution is to prepare cDNA with the Evrogen MINT kit instead of SMART, even that Evrogen kit use the same technology of template switching ?

                      Thanks a lot

                      Sylvain

                      Comment


                      • #26
                        Total RNA sequencing

                        Hi all
                        Has any one tried a random primed approach on total RNA with a DSN normalisation?
                        Hopefully we'll be able to remove most rRNA from the samples, but keep the non polyA RNAs that could still be interesting. Alternatively, has anyone validated the Roche rapid cDNA kit from mRNA with a DSN normalisation after end repair?
                        Thanks in advance
                        Tony

                        Comment


                        • #27
                          Has anyone tried the method that Phillip suggests for avoiding the concatamer issue with the SMART kit? I was wondering if people thought is was successful and worth doing. I was unsure about how common a problem this was. Is this due to the enzyme specifically? Can it actually add the C's to a DNA template? I was a little surprised to see some many people with a similar issue as we use the Clontech kit for cDNA synthesis in the our lab regularly for other applications and haven't noticed these type of problems. Is this issue just due to long RT reaction times? Also, did downstream amplification of first-strand products work fine? Wouldn't this contribute to some 3' bias in sequencing? Just curious what peoples thoughts were.

                          Thanks.

                          Cheers,
                          Nate

                          Comment


                          • #28
                            Just to be clear: we do not modify the SMART procedure in the way I describe above. I just mentioned it as a possible solution to a problem we do not seem to have but does plague others.

                            About the addition of an oligo C tail to the reverse transcribed strand: that is the basis of second strand synthesis in the SMART kit. SMART uses an oligo G tailed second strand synthesis primer.

                            --
                            Phillip

                            Comment


                            • #29
                              Thanks for your thoughts, Phillip. Glad to hear you haven't had these issues with your own data. Are you still using a normalization approach similar to what's been discussed or have you switched over to the Rapid Library 454 kit one? The 454 kit seems to avoid some of the sequence issues we are discussing here, but the lack of a normalization protocol still makes me less attracted to this approach.

                              As for the SMART oligo issue, I could be wrong, but I thought that the C tail was added during the first strand synthesis to allow the SMART oligo to bind to it and then the RT enzyme would finish off sequencing the complement on the first strand being constructed. One of the reasons I thought that SMART made it this way was to reduce non-specific binding along the transcript itself, because basically everything else would be double-stranded at this point. I was worried if I tried this that the non-specific binding of the SMART oligo would be problematic. Also, in the second strand synthesis, you use typically use a primer that is nested within the that SMART oligo that doesn't have that GGG tail, so I assumed you would only use the SMART oligo in this reaction and not the PCR primer they have. I was still a little concerned about how that SMART primer and the "cap" primer (3' primer) would interact given the regions of similar sequence between the two. That's why I was curious to see if anyone had tried this out. Seemed like an interesting possible solution. Thanks again.

                              Cheers,
                              Nate

                              Comment


                              • #30
                                Hi Nate,
                                Currently we don't normalize when we construct libraries.

                                I take your point as to the utility of allowing the double-stranded RNA-DNA 1st strand to block internal priming of the GGG tail of the 2nd strand synthesis primer. (My guess would be that if the adapter is at a much higher molar concentration than the cDNA strands, that adapter concatamers would be more likely to form.) And, as I mentioned previously, we don't attempt to decouple 1st strand and 2nd strand synthesis to avoid adapter concatamers.

                                If you were keen to try decoupling out, but did not want to risk internal 2nd strand synthesis, you could remove the MMLV RT used by the SMART kit after 1st strand synthesis (eg, using phenol, etc.) Then add the 2nd strand primer + some other polymerase capable of displacing the RNA strand. I could even be a RT -- just one that doesn't have MMLV's tendency to add oligo C tails.

                                If we get enough total RNA and our polyA yield is high enough, we use the rapid cDNA method. If not, we use the SMART kit in the Metz/Colbourne/Mokaitis/Buchanan-Carter method. The latter has lots of tricks embedded in it to allow construction of libraries (including normalized libraries, if you are so inclined) optimized for 454 sequence generation with limited amounts of total RNA.

                                In principle, I see no reason the Rapid cDNA method could not be modified to normalize. Just use PCR (with the pA/pB primers) to generate enough cDNA to treat with a double-stranded nuclease.
                                --
                                Phillip

                                Comment

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