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Old 11-20-2009, 10:14 AM   #21
kmcarr
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Jason,

Is it possible to do MID tagging with the RL protocol? Also, when is v2.3 of the software going to be released?
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Old 11-20-2009, 11:14 AM   #22
454Sequencing
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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.
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Old 11-26-2009, 07:50 AM   #23
blouro
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Quote:
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:

http://www.454.com/applications/tran...sequencing.asp

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

http://www.454.com/my454

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
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Old 11-30-2009, 04:44 AM   #24
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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

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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
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Old 07-23-2010, 05:43 AM   #25
sylvain
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Question SMART adaptor concatemers

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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 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
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Old 11-10-2010, 06:29 AM   #26
TonyBrooks
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Default 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
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Old 12-02-2010, 01:05 PM   #27
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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
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Old 12-03-2010, 04:19 AM   #28
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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.

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Old 12-03-2010, 12:46 PM   #29
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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
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Old 12-06-2010, 03:52 AM   #30
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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.
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Old 12-10-2010, 09:57 AM   #31
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Thanks again for your thoughts, Phillip. I've been doing some comparisons between different assemblies using different approaches and it seems that using the 454 kit may be preferable to the normalization approach. With all the sequencing issues associated with it, the increase handling, and longer protocol, I'm not sure the normalization is worth it. It might save us a little cash in the long run, but the incovenience and troubleshooting might just wash all that away. From comparing, assemblies (albeit from different species/tissues), it appears the quality of data that we get from the 454 kit is much better than the normalization approach, subsequent contigs appear longer, and there's less opportunity for stuff to go wrong. You do lose something in coverage, but it's not terrible if you input with something that's ribo-depleted or poly-A selected. And, since sequence length is so important in de novo assembly, it seems trying to maximize that might be our best approach to getting a good reference assembly. Anyway, that's my current two cents, if anyone is interested.
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Old 12-10-2010, 10:07 AM   #32
pmiguel
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Are you normalizing from random primed cDNA of total RNA?

I think that if you are normalizing oligo dT primed cDNA, then that would be the source of your shorter contigs, not necessarily the normalization itself.

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Old 01-04-2011, 12:31 PM   #33
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Originally Posted by pmiguel View Post
Anyway, we are dancing around something here and Roche is actually somewhat less mysterious than I think would normally be their wont. That is, they explicitly warn of the negative effects of polyA or polyT tracts. These tracts will blaze bright, obscuring nearby beads and possibly consuming all the dATP (or dTTP) in the flow before reaching the end of the tract. Hence the creation of the "broken oligo dT primer". This method apparently does not satisfy Roche, but their disapproval is somewhat muted.

But I think there is a second, related, issue in play. About this, Roche is more typically inscrutable, but one hears whispers. That is, one is led to believe that the image analysis software is sensitive to well-to-well bleed-over. Two beads sitting in adjacent wells of a PTP, if they emit too similar a sequence, are presumed to be "overlapping" and one or both are discarded. I'm not privy to what the threshold is for such a filter, if it does even exist, to kick in.
I think this is quite well documented in Roche manuals. I read the manuals describing signal processing tweaking possibilities and from what I remember at the very moment is that exactly for amplicon sequencing there is different processing because the sequences are synchronized between adjacent cells. Some filters are therefore turned off or increased to compensate for those previously turned off and also the width of the region from which the signal is summed up is different (wider). So, when you extract from your own machine xml templates and compare the files for shotgun vs. amplicon you may infer some knowledge and interpret it with the manual rather easily. I think some of these tricks should be probably applied to the cDNA cases when some templates contain very same sequence from their 5'- or 3'-end:


Here is a list of some differences in signal processing:
  • well-screening is disabled for Ti amplicon experiments
  • only EcTF group of control bead sequences is used to estimate error rates in the run instead of both groups
  • most likely error subtractor has a special value for amplicons
  • doValleyFilterTrimBack is disabled for amplicons
  • TrimBack scale factor is stricter for amplicons than for shotgun


Here is a list of some differences in basecaller settings:
  • PHRED-based quality trimming is turned off for amplicons
  • flowRadius parameters is increased to 32 (do not know what is the value for shotgun)
  • useCorrectionGlobalLimit is enabled for amplicon so that no n-mers can skew some algorithm calculation (maybe it is about CAFIE step?)


In summary, study the gsRunProcessor pipeline settings and play with some of the values. You can even disable dot or mixed filter, it is described in the manual as an example. Look what reads you yield with such filters disabled and then move on tuning the other parameters. I would be tempted to say that you can even to try process the cDNA runs with pre-defined amplicon settings and just see what happens.
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Old 01-05-2011, 03:55 AM   #34
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Hi Martin2,
Yeah, I suppose were I to spend a few weeks probing the muddy depths into which Roche has sunk their gsRunProcessor pipeline I might be able to divine their intent. But thanks for your outline, I do appreciate it. This is exactly the sort of discussion I think useful. (I dare say it might happen more frequently if Roche did not feel the need to hide their manuals away from the non-devout.)
I suspect most of the issues we were having when I posted (more than 1 year ago) what you quote had to do with the 454s particular susceptibility to homopolymers. Even now the only way I know to discover if a library is "poisoned" by long polyA tails is to fire up gsRunBrowser and iterate through the early images of a run.

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Old 01-28-2011, 06:09 AM   #35
heleneb
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Hi everybody.
Does anyone know this kit ? http://www.tebu-bio.com/Product/035SST10096
Is seems to be very quick to prepare a cDNA library with only 50 ng of poly(A) RNA so 5g of starting total RNA...
I am interested to know the opinion of people who have tried this kit...
Many thanks !
Helene
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