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Old 02-15-2012, 11:36 AM   #1
vlee2
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Default short read length on XL+

I have problem with XL+ machine. Since we've got an upgrade last fall, we have not been able to get a good run. Our first run of control beads gave us about 740 bp average read length, and since we are having problems, recently we did another control beads run which gave us about 650bp average length. Our samples consistently produce 450-470 average length. My initial thought was that 1600-1800 recommended length of library is too high. So I prepared two sets of libraries with 1100 and 1500 average length, did emPCR, collected supernatant after first denaturation step, concentrated it and ran on BioAnalyzer. The pictures are attached. The conclusion is that there is very small deference between 1100, 1500 and 1800 bp libraries. But it does not bring me closer to the solution of our problem.
Does anybody have a similar problem? What is the way to overcome?
Any thoughts?
Attached Images
File Type: jpg 344 L2S1 library.JPG (57.5 KB, 49 views)
File Type: jpg 344 L2S2 library.JPG (53.6 KB, 32 views)
File Type: jpg 344 L2S1 after emPCR.JPG (49.8 KB, 32 views)
File Type: jpg 344 L2S2 after emPCR.JPG (49.7 KB, 26 views)
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Old 02-15-2012, 11:05 PM   #2
flxlex
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Once our instrument was finally fixed (or is it?) we ran into the same problem. Our first run gave a peak around 600 bases, not bad relative to Titanium, but not near the promised 750-800 bp. After emailing Roche, we were told that the learning curve for FLX+ is longer than for Titanium, so they advised us to keep trying (nice...). Also, they specifiy the library requirements as follows: "peak size ranging from 1400bp to 1800bp with less than 10% of the fragments being smaller than 650 bp, and a peak as sharp as possible"; also, they would worry more about too short fragments, relative to too long ones.

So, that's what we will try, I guess...

That said, there seems to be very mixed results with FLX+, some labs get it going, some labs, like ours, struggle for a long time...
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Old 02-16-2012, 04:26 AM   #3
MissDNA
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We are yet to have our 1st FLX+ run. Our upgrade was in January and only this Monday the Tf run was performed due to lack of reagents. After the run was processed I sent the run report to Roche support, and only then they realized we didnīt have the sofware version 2.6 installed! When the upgrade was done Rocheīs engineer installed the new version on our server but apparently not all the parts.
Now we have to re-process and send the results again.

Problem is: since the run ended yesterday the sequencer did not stop. That Ok button that shows up when the run is finished, didnīt appear and the sequencer light is still green. It appears as some process is still running even though it says the run ended. I already reported the problem to Roche, and they havenīt given me an answer yet.
I thought about hiting abort but I donīt wanna do that without Rocheīs answer because I am afraid of messing up with the software. Any ideas?

As for FLX+ runs we already have 4 libraries made according to Rocheīs especifications: "peak size ranging from 1400bp to 1800bp with less than 10% of the fragments being smaller than 650 bp, and a peak as sharp as possible", and I am really looking forward to running then but we still are waiting on XL+ reagents.

We got parts of the kits starting in Dec., and the XL+ reagent supposedly arrives till tomorrow. 2 months to receive a complete kit. Making science in Brazil is really tough!

As soon as I get some data, I shall report.
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Old 02-16-2012, 06:21 AM   #4
vlee2
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MissDNA,

recently we had exactly the same software problem as yours
Quote:
Originally Posted by MissDNA View Post

Problem is: since the run ended yesterday the sequencer did not stop. That Ok button that shows up when the run is finished, didnīt appear and the sequencer light is still green. It appears as some process is still running even though it says the run ended. I already reported the problem to Roche, and they havenīt given me an answer yet.
I thought about hiting abort but I donīt wanna do that without Rocheīs answer because I am afraid of messing up with the software. Any ideas?
I checked the sequencing log and since it said the sequencing has completed, I aborted the run. After data analysis everything looks good. We seems did not mess anything by aborting the run after sequencing was done.
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Old 02-16-2012, 07:32 AM   #5
MissDNA
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Thanks, vlee2. I talked to Rocheīs tech support and he told me to abort the run. The software was still stuck, then we did a system stop and aftwards system stop. Now we are going to run a pre wash and if everything works well put a XLR70 run tomorrow, still with version 2.5.3. Next week we will upgrade our software to 2.6.

I think the problem was our GS sequencer software was already version 2.6 but gsRunProcessor still 2.5.3. Once we upload the new version we are gonna re-analyze the runs performed since the upgrade to see if it gets better. For XLR70 runs our machine seems to be performing better after the upgrade. Go figure.
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Old 03-23-2012, 11:42 AM   #6
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So now we had our 1st XL+ using a "normal" sample. Before sequencing I sent the bionalyzer data to Roche support and they said the library looked according to specified.

The output looks like an ok Titanium run. I already sent the reports to Roche and will wait on their feedback. I am disappointed but honestly after reading so many complains here, I didnīt have my expectations set too high.

I asked several times their specialist about the peak size around 1400-1800 bp being too long, he did not answer me. What I donīt understand is why they didnīt change cDNA library prep. leaving the peak around 900-1200 bp and bumped RL+ sizes that high. Does anyone know?
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Old 03-25-2012, 07:07 PM   #7
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Here is some output of some of the XL+ runs I've done. The latest run had much shorter fragments, but was apparently just above the minimum spec. The earlier runs were all pretty good and well within or above their specs. I wonder how much sample type affects read length? The recent run with shorter lengths was of metagenomic samples, whereas the longer lengths were more from bacteria, plasmids, viral samples.
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File Type: png readlength_GACT_chart5.png (7.4 KB, 51 views)
File Type: png readlength_GACT_chart8.png (9.8 KB, 52 views)
File Type: png readlength_GACT_chart.png (6.7 KB, 44 views)
File Type: png readlength_GACT_chart_cDNA.png (6.7 KB, 43 views)
File Type: png readlength_GACT_chart2.png (9.9 KB, 48 views)
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Old 03-26-2012, 02:31 AM   #8
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RCJK, it is nice to see XL+ can work. Our sample was a metagenome and we have another 2 runs on that project to perform. Have you had a good metagenomic run? For XLR70 some of our best runs were from metagenomic samples. I am still waiting on some Roche feedback about my case.
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Old 03-27-2012, 10:41 AM   #9
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RCJK - it IS nice seeing some XL+ results. We are getting ready to do our first XL+ run on bacterial genome rapid libraries. What did you find worked as cpb ratio on your libraries?
Again, thanks for sharing info as we seem to manage somewhat in the dark, too.
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Old 03-27-2012, 03:51 PM   #10
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MissDNA-I've only done 1 XL+ run with metagenomic samples (the 3rd image I posted) so maybe it was a fluke that it was shorter? I don't recall having any specifically bad XLR70 metagenomic runs in the past.

LAH-I have started using the Kapa kits to quantify my libraries (with an increased extension time). Based on those quantifications cpb inputs have ranged from ~0.1-0.4, but with most around 0.3cpb. For my first XL+ run, the libraries were quantified w/ the Roche adaptors as per the RL protocol, for those libraries a cpb of 4 seemed to work fairly well.
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Old 03-28-2012, 08:01 AM   #11
pmiguel
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Quote:
Originally Posted by vlee2 View Post
I have problem with XL+ machine. Since we've got an upgrade last fall, we have not been able to get a good run. Our first run of control beads gave us about 740 bp average read length, and since we are having problems, recently we did another control beads run which gave us about 650bp average length. Our samples consistently produce 450-470 average length. My initial thought was that 1600-1800 recommended length of library is too high. So I prepared two sets of libraries with 1100 and 1500 average length, did emPCR, collected supernatant after first denaturation step, concentrated it and ran on BioAnalyzer. The pictures are attached. The conclusion is that there is very small deference between 1100, 1500 and 1800 bp libraries. But it does not bring me closer to the solution of our problem.
Does anybody have a similar problem? What is the way to overcome?
Any thoughts?
So could you give more details about these pictures? The first 2 look very different from the second two. But which is what sample? Also what type of bioanalyzer chip did you run? What was your concentration method? What percent of your recovered supernatant did you run?

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Old 03-29-2012, 03:37 AM   #12
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@RCJK: Some of those runs you show are really good, and we can as of today only dream of them. This again comes back to the apparent incredible site-to-site variation in the implementation of GS FLX+. We are actually getting another, new(!) GS FLX+ instrument on loan for testing soon...

Last edited by flxlex; 03-29-2012 at 03:37 AM. Reason: Clarified
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Old 04-02-2012, 12:07 PM   #13
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Quote:
Originally Posted by MissDNA View Post
I asked several times their specialist about the peak size around 1400-1800 bp being too long, he did not answer me. What I donīt understand is why they didnīt change cDNA library prep. leaving the peak around 900-1200 bp and bumped RL+ sizes that high. Does anyone know?
I am not sure what you mean with "RL+" but if I got your question right, the cDNA templates are more prone to form secondary structures, impairing the polymerase movement. Therefore, would you use 1400-1800bp inserts most likely the polymerase would fall apart the template somewhere along the way. In contrast, if you insert inserts in the range 900-1200bp hopefully less 2D structures and also weaker would form, giving you some hope the polymerase can reach the 3'-end. I think this is quite a good advice from Roche, really.

In genomic DNA, there are are not many reasons to await a 2D structure formed solely by its single chain. That is why for shotgun DNA you may be willing to get longer inserts.
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Old 04-02-2012, 12:20 PM   #14
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Quote:
Originally Posted by martin2 View Post
I am not sure what you mean with "RL+" but if I got your question right, the cDNA templates are more prone to form secondary structures, impairing the polymerase movement. Therefore, would you use 1400-1800bp inserts most likely the polymerase would fall apart the template somewhere along the way. In contrast, if you insert inserts in the range 900-1200bp hopefully less 2D structures and also weaker would form, giving you some hope the polymerase can reach the 3'-end. I think this is quite a good advice from Roche, really.

In genomic DNA, there are are not many reasons to await a 2D structure formed solely by its single chain. That is why for shotgun DNA you may be willing to get longer inserts.
I meant Rapid Library construction for XL+ chemistry. Thanks for you explanation, noboby in Roche has ever explained to me that way.

However I still think 1400-1800 bp range is too large, and perhaps one of the sources of the problems we have with XL+ runs. It seems to me that when we have a library with those very large fragments, the slightest contamination with shorter fragments, which cannot be seen using the bionalyzer could the cause of the bellow specs runs we have been observing, since small fragments will amplify preferably than the very large one during emPCR.
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Old 04-02-2012, 01:23 PM   #15
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I donīt know why Roche decided to leave the cDNA library peak at 900-1200 bp, but one of the reasons might be that higher than that we can start loosing the smallest cDNAs encoding for small proteins of 10 kDa and above...
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Old 04-03-2012, 05:28 AM   #16
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Quote:
Originally Posted by madseq View Post
I donīt know why Roche decided to leave the cDNA library peak at 900-1200 bp, but one of the reasons might be that higher than that we can start loosing the smallest cDNAs encoding for small proteins of 10 kDa and above...
Guys, you are getting this backwards. The new FLX+ cDNA protocol uses 1400 bp+ templates. But since the read lengths are only 800 or so, maximum, there seems little point in making them so large. Especially since, as pointed out above, smaller messages will be lost entirely under such circumstances.


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Old 04-03-2012, 05:54 AM   #17
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Phillip, the new XL+ cDNA protocol is exactly the same as the "old" cDNA library prep, they just added GS FLX+ Series — XL+ to the name and changed dates. The avg lenght size remais between 600-1200 bp and the majority of the fragments should be around 900-1200 bp. The new RL+ protocol calls for peaks around 1400-1800 bp whereas the standard RL was around 600-900 bp.
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Old 04-03-2012, 06:36 AM   #18
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MissDNA,
Okay, I stand corrected. That makes the RL+ protocol even more mysterious.
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Old 04-03-2012, 07:13 AM   #19
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Exactly! I have been asking this question to our Roche specialists and they were no able to tell me why yet. One limited to say it was because R&D has probably determined as the ideal condition.

I asked Support about which emPCR conditions should be used for cDNA. Since cDNA protocol has not changed why not keep using the emPCR conditions for XLR70 and not XL+? I was told I should use XL+ emPCR when sequencing with XL+ kit. Do not understand why.

We are doing bead recovery today from 2 RL+ libraries and we are going to do the procedure to evaluate for the presence of small fragments before proceed with sequencing. If there is no small fragments, we are going to sequence tomorrow and hope for long reads. We are no longer using those bad bead recovery lots nor expired emPCR reagents.
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Old 04-03-2012, 07:11 PM   #20
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Default I dream of 500bp

Hi,
we have had two sample runs since the FLX+ upgrade with the libraries shown below.

Beads prepared once by us and then by Roche.

The first run averaged 250bp length, this last one 350 bp length.

Note all our FLXplus customers are sold sequence at above 500bp………so anything below that is non-delivery.

Apart from length the run looks pretty good.

Control run Type I control beads 713bp, our first run 300 bp, this run 428bp
Control run Type II control beads 470bp, our first run 220 bp, this run 305bp

Any ideas? Can we source a control library that will give 1-1.5 kb reads if machine is working well?

FGP
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