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Old 03-31-2009, 03:52 AM   #1
BeFuPe
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Default cDNA synthesis kits with titanium

I want to sequence ESTs from a RNA pool. I heard good things about SMART cDNA synthesis kits from Clotech and Evrogen. Also, I read different papers that have used this technology. My concern it is that I also read from "cDNA Frequently Asked Questions" from Roche, that SMART kits are not recommended with titanium kits because the size of the cDNA fragments obtained, ussually small in size and fail to nebulize properly. Any suggestions?
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Old 03-31-2009, 06:32 AM   #2
kmcarr
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Could you post a link to that FAQ. I searched the web site but could not find it. I'm interested in what they mean by the cDNA fragments are usually small in size.

We have run many, many cDNA projects on the 454 using GS 20, GS-FLX and now Titanium. The vast majority of these used SMART cDNA preparation, some with the Evrogen normalization, some without. We have had generally good results. Compared to genomic DNA sequencing cDNA sequencing generally produces fewer filter passed reads (more failures due to mixed reads) and the mean length is shorter. There is also more variability from library to library which probably is a DNA quality issue. I'd be happy to provide more information if you like.
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Old 03-31-2009, 07:08 AM   #3
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Thank you very much for your quick response. I would appreciate as much information as you can provide me since we are new in this business. The FAQ document it was provided from Roche Support Department by email, it is not public in the website but I can forward it to you if you wish.
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Old 04-16-2009, 06:24 AM   #4
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For the reasons already pointed out by kmcarr

"Compared to genomic DNA sequencing cDNA sequencing generally produces fewer filter passed reads (more failures due to mixed reads) and the mean length is shorter. There is also more variability from library to library which probably is a DNA quality issue."

I would advise doing a comprehensive titration for each cDNA sample processed. Also if you're going ahead with normalization you'll notice a deffinate shift in fragment sizes when run on a gel.
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Old 04-16-2009, 11:57 PM   #5
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Quote:
We have run many, many cDNA projects on the 454 using GS 20, GS-FLX and now Titanium. The vast majority of these used SMART cDNA preparation, some with the Evrogen normalization, some without. We have had generally good results. Compared to genomic DNA sequencing cDNA sequencing generally produces fewer filter passed reads (more failures due to mixed reads) and the mean length is shorter. There is also more variability from library to library which probably is a DNA quality issue. I'd be happy to provide more information if you like.
Hi,
we run only few project on our 454 (titanium only) and we were surprised about the amount of filtered reads and the length of the high quality reads. could you please tell me (if possible) the mean length and the percentage of passed filter?

For example, for our last run (4 regions, cDNA from chicken and duck), for 2 regions (chicken) we only have ~15% of passed filters (whereas Roche expects more than 50% for a titanium run - information from GSFLX Titanium metrics, but it may be for a genomic DNA project). Problems came from dots (cf attached spreadsheet).

Gerald
Attached Files
File Type: pdf 09_04_03_filters_TCAG_summary.pdf (81.9 KB, 138 views)
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Old 04-17-2009, 01:23 AM   #6
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Hi Gerald

Are you doing a size exclusion with the cDNA and a titration (concentrate the titration at the lower end)? Also, because fragment size is usually shorter for cDNA it may be worth loading the beads on the PTP at a lower density. As I've said before don't expect cDNA runs to be as successful as runs using gDNA.
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Old 04-17-2009, 05:43 AM   #7
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Quote:
Originally Posted by gerald2545 View Post
Hi,
we run only few project on our 454 (titanium only) and we were surprised about the amount of filtered reads and the length of the high quality reads. could you please tell me (if possible) the mean length and the percentage of passed filter?

For example, for our last run (4 regions, cDNA from chicken and duck), for 2 regions (chicken) we only have ~15% of passed filters (whereas Roche expects more than 50% for a titanium run - information from GSFLX Titanium metrics, but it may be for a genomic DNA project). Problems came from dots (cf attached spreadsheet).

Gerald
Gerald,

Your filtering output does not surprise me at all. We have seen very similar results with many of our cDNA runs on the 454. In the past, with the GS FLX Standard, most failed reads were rejected by the Mixed filter; now with the Titanium it seems that more are rejected by either the Dot or Short Quality filter. I don't know if this a difference in the sequence itself or in the filtering algorithms.

Our read lengths for cDNA samples often show a bimodal distribution, with one peak at the expected 450-500nt range and second peak in the 100-150nt range. This results in a lower mean and median read length but those statistics don't give the full picture in these cases.

Remember that all of the performance numbers which Roche publishes (e.g. 50% filter passed reads, 400-450nt mean read length) are based on sequencing very well behaved genomic DNA.

Our protocol for preparing cDNA for 454 (SMART kit with modified first strand primer) was originally developed when our instrument was a GS-20, then carried through to the GS FLX. It may be a time to develop a new strategy for the Titanium.
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Old 05-20-2009, 05:17 AM   #8
Emanuel Heitlinger
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Hi all,
the Evrogen MINT kit worked better than the SMART kit for me. The SMART kit produced strange concatenated primer/adaptor artefacts. Currently it is used without any polyT modifications producing ~75.000 good quality sequences of ~250nt from 1/4 plate GS FLX run.

Only problem: High amount of rRNA contamination ~50% in the best preparations.
Does anybody else have this problem using SMART or MINT kits?

I'm looking forward to get my first titanium runs...
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Old 05-24-2009, 06:09 AM   #9
Emanuel Heitlinger
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Wowwww
I just found all this:
http://www.bio.utexas.edu/research/m...b/Methods.html
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Old 05-24-2009, 09:39 AM   #10
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Thanks Emanuel for the website!

The sequencing center told me they have previously experienced the same problem. Sequencing mainly SMART concatemers. They suggested I use the minimum number of PCR cycles. I'm also expecting my titanium run.
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Old 11-02-2009, 10:07 AM   #11
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Default Does a broken T primer help ?

Hello everybody
We had on our first cDNA sequencing try, using a MINT kit used on a 1/4 Titanium plate. No concatenates, but a bimodal length distribution with a first peak around 70 nt and a second around 500 nt; about 50000 reads are below 100 nt, and 100000 above. Reads with a 5' polyT make up 50 % of the short, bad class. We used a first strand primer with 30 T, which I suspect to bring difficulties with pyrosequencing. Does anybody has experience with broken T primers ?

Thanks ahead!
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Old 11-03-2009, 06:09 AM   #12
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My impression of Roche's policy towards cDNA library construction is that they were not satisfied with any of the methods available and therefore they refused to recommend any of them. In the fullness of time Roche would release a method or kit that would produce acceptable results. I find this maddening as both Applied Biosystems and Illumina have had expression analysis solutions available for many months. Roche/454, who have been in the "next gen" game a year longer, are still dragging their feet.

But I also get the impression there were a couple of methods that at least approached the Platonic ideal of a perfect 454 cDNA library. These were (1)ribosomal depletion (eg, polyA+ isolation) of total RNA, random primed cDNA synthesis. Nebulization, etc as for genomic libraries or (2) oligo dT 1st strand synthesis using an offset restriction enzyme site (eg Mme or EcoP) adaptor. The adaptor and most of the polyA/T tail then is removed from the cDNA via restriction digest. The latter would likely involve some tedious tricks (eg methylation) to avoid cutting Mme or EcoP sites in the cDNA proper.

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. It is of some significance, however; because SMRT cap adaptors, for instance, if they are sufficient to trigger high levels of bleed-over filtration, would be problematic. Especially if, as I believe is the case, the fragmentation broken ends are less often ligated than the extant capped ends.

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Old 11-03-2009, 11:08 AM   #13
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Quote:
Originally Posted by Emanuel Heitlinger View Post
Hi all,
The SMART kit produced strange concatenated primer/adaptor artefacts.
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.

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Old 11-04-2009, 08:16 AM   #14
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Quote:
Originally Posted by pmiguel View Post
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. It is of some significance, however; because SMRT cap adaptors, for instance, if they are sufficient to trigger high levels of bleed-over filtration, would be problematic. Especially if, as I believe is the case, the fragmentation broken ends are less often ligated than the extant capped ends.

--
Phillip
Thank you Phillip, this was a quick and informative answer. I do not fully follow your point about primers and bleed-over, though. If a primer sequence cause bleed over from well-to-well, many PCR products should be difficult to sequence in depth, and presumably not only SMART RT-PCR products. In our run with Evrogen RT_PCR kit, reads bearing in 5' the M1 cDNA primer are indeed in large excess (73%, for =<50% expected), so at least the M1 sequence seems safe for that matter. Could it be that what is bleeding is rather the too bright light of the polyT strech that follow every other 5'M1, making the neighbor beads difficult to read ?
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Old 11-04-2009, 10:58 AM   #15
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Quote:
Originally Posted by jmbon View Post
Thank you Phillip, this was a quick and informative answer. I do not fully follow your point about primers and bleed-over, though. If a primer sequence cause bleed over from well-to-well, many PCR products should be difficult to sequence in depth, and presumably not only SMART RT-PCR products. In our run with Evrogen RT_PCR kit, reads bearing in 5' the M1 cDNA primer are indeed in large excess (73%, for =<50% expected), so at least the M1 sequence seems safe for that matter. Could it be that what is bleeding is rather the too bright light of the polyT strech that follow every other 5'M1, making the neighbor beads difficult to read ?
I did not mean that the adaptor sequence caused bleed-over, only that it might look sufficiently like bleed-over to trigger the bleed-over filter.

Again, this is nearly pure speculation on my part, but the the reads that trigger the bleed-over filter will not be seen among your filter pass reads. They would have been filtered out.

I presume, the bleed-over filter would only be triggered if two beads in close physical proximity gave a similar sequence for some number of flows.

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Old 11-20-2009, 05:59 AM   #16
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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
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Old 11-20-2009, 08:11 AM   #17
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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. Upside:potentially 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...

--
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Last edited by pmiguel; 12-03-2010 at 05:12 AM. Reason: To disable smilies in text!
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Old 11-20-2009, 08:35 AM   #18
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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
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Old 11-20-2009, 08:49 AM   #19
Emanuel Heitlinger
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Does Roche make the new version of newbler available to users of 454 data?
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Old 11-20-2009, 08:51 AM   #20
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Quote:
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.

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