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Old 09-15-2011, 03:31 PM   #1
Anthony.287
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Default Enrichment Numbers

I have a few new questions about enrichment beads that Im hoping you folks can help me with. In the SV protocols, the number of capture beads in the Lib-A manuals is different from those in the Lib-L manuals (2.0x10^6 vs. 2.4x10^6). Only the SV numbers are different, not MV or LV. Does anyone know if the numbers are actually different, or if this is a typo?
Also, during our training, we were taught to calculate bead enrichment values differently than the manuals describe it, and were given Excel spreadsheets with the calculations we were taught. Basically, the manuals use the enriched beads/total number of input beads; we were taught to use the enriched beads/actual beads recovered. I was wondering how other users are determining enrichment %s, and what percentage range tends to give good results with the calc we were taught?
Thanks!
Anthony
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Old 09-16-2011, 06:21 AM   #2
uhany
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Hi Anthony,

Yes capture beads in Lib A protocol is different and that is ok. I determine by enriched beads/total no. of beads and usually get good results from up to 30% recovered emulsions. thanks,
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Old 09-19-2011, 01:38 PM   #3
Nicole 454 Sequencing
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Hi Anthony,

The numbers in the protocols are accurate and the Small Volume Lib-A protocol does actually use fewer beads.

As far as enrichment percentage, either method can be utilized. The calculation in the manual is assuming there is 100% bead recovery. As the manual states, calculating the % breaking efficiency (Section 3.6.1, Step 8; GS FLX Titanium Series emPCR Method Manual – Lib-A SV) is an optional step, therefore the final calculations are based off the assumed recovery of 100% (2.0E6 million Capture Beads).

For a single Small Volume emulsion, you will want to be in the range of 100,000 – 400,000 enriched Capture Beads for best quality.

Feel free to contact your local Roche Representative with any further inquiries. http://my454.com/contact-us/technical-support.asp

Nicole
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Old 09-20-2011, 04:09 AM   #4
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What is the number of input beads for GS Jr Lib-L then? The manual gives an example of caclulation for 10e6 - is this the actual input number of capture beads?
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Old 09-22-2011, 05:55 AM   #5
Nicole 454 Sequencing
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Hi Yazimik,

The number of capture beads supplied within the GS Junior emPCR Reagents kit is 10 million. The enrichment calculations given in the manual are based off of this input and suggest between 500,000 and 2,000,000 enriched beads for best sequencing quality.

This information can be found in our Junior user manuals located at http://my454.com/my454/documentation...df-manuals.asp.

Regards,

Nicole
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Old 09-22-2011, 06:26 AM   #6
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We run many GS Junior emPCR Lib-L kits. When determining % initial bead recovery we use the 10e6 number. However, we adjust our %enrichment based on the recovered beads and not the original 10e6. This number will trend higher, but hopefully by only a couple of percentage points. For example our %enrichment based on initial bead recovery may be 20.2% (but if we had used the 10e6 number it would have been 18-19%).

PS... I would take any advise "Nicole 454 Sequencing" suggests. She rocks!
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Old 09-26-2011, 05:04 AM   #7
Dynamac
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Default Enrichment Too High

During a MV emPCR workup, I have washed the enriched beads up to 15 times to remove the white un-enriched beads, yet I still get enrichments up around 45%, which apparently is too high for good results. Anyone have thoughts of this?

Plus, I consistently underload the PTP regions even though I'm supposedly loading 2E+6 beads per region. Is this somehow related to the enrichment problem? I'm confident that the Coulter Counter is reading correctly; I checked it against concentration standards provided by Beckman.

Barry
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Old 09-26-2011, 05:09 AM   #8
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Quote:
Originally Posted by Dynamac View Post
During a MV emPCR workup, I have washed the enriched beads up to 15 times to remove the white un-enriched beads, yet I still get enrichments up around 45%, which apparently is too high for good results. Anyone have thoughts of this?

Plus, I consistently underload the PTP regions even though I'm supposedly loading 2E+6 beads per region. Is this somehow related to the enrichment problem? I'm confident that the Coulter Counter is reading correctly; I checked it against concentration standards provided by Beckman.

Barry
OK.. I know this is going to sound silly but it's true. Originally we were using a Matrix pipette (set at the slowest possible speed) to transfer our emPCRs to the plate and we got terrible %enrichment (30-50%). We did a side-by-side with a repeater pipette (recommended by 454) and what a difference! We went from 30% enrichment down to 8%. AND - since we made the switch - our SVs pretty much predict the MVs and LVs now within a couple of percent.

As for the loading of the PTP... you load 2million but typically you'll only see a little over 1million. I think that is standard - and if you look at Roche's benchmark, I think they even indicate that.

Good luck!
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Old 09-26-2011, 05:38 AM   #9
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Interesting. I used a 1 mL pipette and tried to estimate 100 L per well because I was doing a MV emPCR and the repeater tip wouldn't fit into the MV tube! I suppose I could transfer the emulsion into another tube for the MV runs.

As for the underloading, I'm getting about 750,000 wells per region. So I'm losing about half a million wells per plate.

Thanks for the help.

Barry
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Old 09-26-2011, 05:45 AM   #10
suefo
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Quote:
Originally Posted by Dynamac View Post
Interesting. I used a 1 mL pipette and tried to estimate 100 L per well because I was doing a MV emPCR and the repeater tip wouldn't fit into the MV tube! I suppose I could transfer the emulsion into another tube for the MV runs.

As for the underloading, I'm getting about 750,000 wells per region. So I'm losing about half a million wells per plate.

Thanks for the help.

Barry
We're using 1 mL combi-tips for SVs, 2.5 mL combi's for MVs and 5 mL combi's for LVs. And so far the diameter of the barrel seems to fit.
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Old 09-26-2011, 08:44 AM   #11
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Quote:
AND - since we made the switch - our SVs pretty much predict the MVs and LVs now within a couple of percent.
I've been having terrible trouble with the c.p.b values suggested by the SV titration not scaling up to MV - I keep getting far too high enrichment. I'm now off to buy some different tips for my multipipette so it fits in the MV tubes!

Thanks for posting your explanation!
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Old 09-26-2011, 09:22 AM   #12
Dynamac
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Let us know how it works out.
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Old 11-18-2011, 11:18 AM   #13
Anthony.287
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Thanks for all of the great help and info!
I have some questions about the comparison between the Matrix pipette and the repeater...was the Matrix set to pick up more volume than there actually was? I've noticed with our Matrix, which was programmed to fill 1250ul, that once it drew up all of the emulsion in the tube, it would continue to fill, since there was less than 1250ul of emulsion. This would cause the emulsion to bubble at the top of the tip, and I wonder if this could be breaking the micro-reactors. I too have not had any consistency between emPCR's, even from one SV to another, with the exact same library dilutions that have not gone through any freeze-thaw cycles between emPCRs. It would be wonderful if I could use the data from a titration to perform a bulk run, but that doesn't seem to be the case.
Also, doesn't the repeater dispense a lot faster than the Matrix? We have one that I just started using for wash steps, and it would be quite tricky to dispense slowly, with ours at least.
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Old 11-18-2011, 11:25 AM   #14
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Anthony,
The Matrix was VERY slow and yes, on the final pickup, we overshot the volume on the draw. The repeator is much much faster - we don't try to slow it down. We just shoot into the wells and move on.
I just did an SV titration this week, selected a cpb and then made a new dilution series for 2 LVs. I'm attaching the linear graph of that. We were pretty happy with the reproducibility of the repeator pipettor. The triangles are the LVs and the blue diamonds are the SVs.
Attached Images
File Type: jpg SV vs LV titration.jpg (58.6 KB, 18 views)
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Old 03-21-2012, 10:29 AM   #15
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@Anthony --> I am determining enrichment % based on the actual beads receovered, because I think that assuming 100% bead recovery is not the best way to proceed.

@Dynamac --> In my SV workup, I have stopped washing the enriched beads at the 6th wash (because I was following the protocol that recommended 3-6 times) even though there were still white un-enriched beads. In the end, the enrichments were very high (40%-70%). Do you think I should have kept washing?

@suefo --> Do you think that the decrease in the enrichment was due to the repeater pipette and it wasn't anything else? I am not questioning you, I am asking because I am very stressed in finding out what went wrong with my emulsions.

I have posted all the details of my emPCR on "Calculation of % Bead enrichment (454 FLX Titanium)": http://seqanswers.com/forums/showthread.php?t=18588

Thank you!!
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Old 03-21-2012, 10:53 AM   #16
Dynamac
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Christina - Those enrichment numbers seem a little high to me, so I would keep washing. As for my problem, it seemed to work itself out when I went from 5 copies/bead down to 2 copies/bead during amplicon sequencing (did not do a titration).
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Old 03-21-2012, 11:11 AM   #17
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Christina85... I am ABSOLUTELY sure it was the pipettor. I have photos and data to support this is a side-by-side experiment we did. We checked everything before hand as well... pcr plate, thermal cyclers, caps for the pcr plates, etc and we had Roche is 3 times. It wasn't until 454 came in with Roche and made this recommendation - now everything works like a dream. And there is correlations between our SVs and LVs (almost perfect). I can send you data / photos if you want to see them. Email me at sfoltin@umich.edu.
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Old 03-21-2012, 01:11 PM   #18
Anthony.287
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One thing I've been doing to increase accuracy (I hope) is perform 2 volume measurements and 2 bead counts for each tube, and then use the averages. Measuring ~1200ul with a pipette is pretty tricky, and I've found that 2 back-to-back measurements, with the same pipette, tip, and user can give differences up to 50ul, and two back-to-back bead counts (using different Accuvettes and different 3ul aliquots) can give differences up to 500,000 beads. Little deviations like these can certainly add up and make troubleshooting difficult, so I'm doing it all in duplicate.
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Old 03-22-2012, 04:30 AM   #19
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What do you mean exactly by
Quote:
Originally Posted by Dynamac View Post
I went from 5 copies/bead down to 2 copies/bead during amplicon sequencing (did not do a titration).
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