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Old 08-04-2016, 09:50 AM   #1
thermophile
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Default cutting cost for library prep

We do a small number of nextera libraries. Not enough to justify really delving into the kit and doing lots of experimentation to reduce costs. But I'm always wanting to cut the easy costs. So was thinking of just doing 1/5 reactions (follow their protocol but 1/5 the volumes for everything).

I'd guess that this is fairly common way to cut costs. So am posting to request any tips or cautions from people doing this before we start?
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Old 08-04-2016, 01:55 PM   #2
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There are few papers on this subject. Generally reduce reaction volumes and input in proportion, strip transposase by adding SDS directly followed by PCR with mix of Illumina index primer and P5, P7 primers (flow cell binding motives). PCR reactions need to be done with a third paty polymerase at 50 ul for good yield.
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Old 08-05-2016, 04:44 AM   #3
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I've been looking at this paper to do the exact same thing... http://journals.plos.org/plosone/art...l.pone.0128036. My reasoning is small genomes/high throughput. I'd be interested to hear how it goes for you thermophile.
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Old 08-05-2016, 06:32 AM   #4
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We don't do enough to justify rewriting the protocol that much. Our last run with 1/2 reactions looked identical to full reactions and used the same procedural steps so I think I'll stick to that unless we suddenly are getting full plates of genomes to prep.

thanks for the replies
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Old 08-09-2016, 01:21 AM   #5
Simone78
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Quote:
Originally Posted by thermophile View Post
We don't do enough to justify rewriting the protocol that much. Our last run with 1/2 reactions looked identical to full reactions and used the same procedural steps so I think I'll stick to that unless we suddenly are getting full plates of genomes to prep.

thanks for the replies
I cut reaction volumes by a factor of 10 using a liquid handling robot and the Nextera kit (exactly the same protocol, no changes). Tagmentation is done in 2 ul, I add 0.5 ul NT (or 0.2% SDS) followed by 1 ul i5/i7 (1:5 dilution to avoid excess adaptors and save money) and 1.5 ul NPM. Input is 50-150 pg/rxn.
Even without a robot this is perfectly doable with a pipette.
I this way a 3000 USD kit will last for about 1000 samples instead of 96!

/Simone
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Old 08-09-2016, 06:24 AM   #6
thermophile
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Thanks for that! Our liquid handler doesn't go under 1ul, but good to know you were able to just reduce vol.
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Old 08-11-2016, 12:22 AM   #7
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Quote:
Originally Posted by Simone78 View Post
I cut reaction volumes by a factor of 10 using a liquid handling robot and the Nextera kit ....

/Simone
Dear Simone

Thanks for this info, which robot are you using, and how do you deal with magnetic beats separation and shaking, we have seen here some problems on our way for cutting cost

Does anybody have some tips on how to use a more time saving method than Qbit for DNA measurement? We would like to use a plate reader or similar to reduce hands-on time

Alexander
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Old 08-11-2016, 04:28 AM   #8
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Quant-iT PicoGreen dsDNA Assay Kit can do the plate format
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Old 08-11-2016, 07:29 AM   #9
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Further reduce costs for picogreen (which is already much cheaper than qubit). You can do 50 ul reactions of picogreen (25 dilute sample, 25 standard diluted reagent) in 384 well plates, rather than 200ul reactions that manufacturer suggests.
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Old 09-26-2016, 04:55 PM   #10
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Please see this publication showing nextera XT being prepared in 2, 4 and 8 uL and results for single cell RNAseq being compared and showing no loss of complexity.
http://jla.sagepub.com/content/early...216630741.full

And this one having samples prepared using same method but not mentioned in detail in methods.
http://www.cell.com/cell/pdfExtended...674(16)30740-1
libraries were normalized and prepared in 4 uL total volume.
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Old 09-26-2016, 10:59 PM   #11
Simone78
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Originally Posted by docsascha View Post
Dear Simone

Thanks for this info, which robot are you using, and how do you deal with magnetic beats separation and shaking, we have seen here some problems on our way for cutting cost

Does anybody have some tips on how to use a more time saving method than Qbit for DNA measurement? We would like to use a plate reader or similar to reduce hands-on time

Alexander
I use a Tecan Freedom Evo 200 with a 384 head. To save time and avoid (decrease) cDNA losses I don not perform any ethanol wash after binding. I just remove the supernatant and elute the cDNA from the beads. Since I am resuspending the cDNA in 15 ul (and I dilute it even more for the library prep) the leftover salt from the RT/preampl does not affect the efficiency of the downstream reactions.
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Old 05-22-2017, 01:10 AM   #12
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Quote:
Originally Posted by Simone78 View Post
I cut reaction volumes by a factor of 10 using a liquid handling robot and the Nextera kit (exactly the same protocol, no changes). Tagmentation is done in 2 ul, I add 0.5 ul NT (or 0.2% SDS) followed by 1 ul i5/i7 (1:5 dilution to avoid excess adaptors and save money) and 1.5 ul NPM. Input is 50-150 pg/rxn.
Even without a robot this is perfectly doable with a pipette.
I this way a 3000 USD kit will last for about 1000 samples instead of 96!

/Simone
Dear Simone and other Nextera users,

I was wondering how to go about the Tagmented DNA Clean-up in the Nextera protocol. It uses a Zymo spin column, which I guess will not be possible for a smaller volume (e.g. 2-10 ul). What do you do at this stage? Do you clean with beads? Skip the whole clean-up? Dilute the Tagmented DNA in buffer to make up for the original 50ul volume needed?

Thanks,

Arthur


Would it b
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Old 05-22-2017, 05:19 AM   #13
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Quote:
Originally Posted by Arthur45 View Post
Dear Simone and other Nextera users,

I was wondering how to go about the Tagmented DNA Clean-up in the Nextera protocol. It uses a Zymo spin column, which I guess will not be possible for a smaller volume (e.g. 2-10 ul). What do you do at this stage? Do you clean with beads? Skip the whole clean-up? Dilute the Tagmented DNA in buffer to make up for the original 50ul volume needed?

Thanks,

Arthur


Would it b

Hi Arthur,
no need to clean up the tagmented DNA before the final PCR. I do the tagmentation reaction in 2 ul, add 0.5 ul of NT buffer (or 0.2% SDS) and simply go on with the PCR mix (2.5 ul). 0.2% SDS will remove the Tn5 from the DNA and, after adding the PCR mix, will be diluted down and won´t interfere with the final PCR.
Only after PCR you will do a clean up and, if you want to multiplex tens or hundreds of cells, I would suggest to pool all of them and then do the cleanup, not the other way around.
Best,
Simone
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Old 05-23-2017, 08:25 AM   #14
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Quote:
Originally Posted by Simone78 View Post
Hi Arthur,
no need to clean up the tagmented DNA before the final PCR. I do the tagmentation reaction in 2 ul, add 0.5 ul of NT buffer (or 0.2% SDS) and simply go on with the PCR mix (2.5 ul). 0.2% SDS will remove the Tn5 from the DNA and, after adding the PCR mix, will be diluted down and won´t interfere with the final PCR.
Only after PCR you will do a clean up and, if you want to multiplex tens or hundreds of cells, I would suggest to pool all of them and then do the cleanup, not the other way around.
Best,
Simone
Hi Simone,

thank you for the message - this is so much easier than expected!

You mention after the PCR I should do a bead clean-up. The original protocol mentions to add twice the beads to the PCR product :

4 Add 30 μl AMPure XP beads to NAP2.
5 Add 30 μl AMPure XP beads to NAP2.
To me, this ratio of 20:30+30 looks like a mistake. Say I have 11ul of PCR products, would I add twice 16.5ul?

I hope someone can help me out there.

Cheers,
Arthur
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Old 08-04-2017, 08:49 AM   #15
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Hi Simone,

thank you for your answer. I experimented with scaling-down the Nextera protocol, with mixed results.

My main worry is that the transposome activity is not behaving as expected. See the TapeStation traces, before and after tagmentation. The 1kb peak has disappeared during tagmentation, which is good news (we are aiming for a 300bp library). But I fail to be convinced that there is any DNA left after tagmentation, looking at the TapeStation electropherogram.

What do you think about it?

Cheers,

arthur
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Old 08-04-2017, 09:52 AM   #16
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Hello Arthur45,

Make sure you have a very accurate quant of your input samples. Nextera is a mass driven setup so if you are off on your initial quant at all, can get results of too large/too small fragments very easily.
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Old 08-04-2017, 01:23 PM   #17
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hi Arthur 45,
sorry for the misunderstanding. You do need to clean up the cDNA after the first PCR (pre-amplification for the Smart-seq2 protocol). I am not really familiar with the latest version of the Nextera protocol but the 30 + 30 ul beads sounds a bit strange to me.
I know that Nextera is using the Zymo and Nextera XT is not. We decided to not use a column purifcation because it´s cheap and equally effective.
That said, I am not sure what you have there. A single fragment at 1 kb doesn´t look like a standard library (is it a ladder or similar?). Why don´t you just take some tot RNA , let´s say 1 ng, do some pre-ampl and try to tagment that? I agree that the Tape after tagmentation has no library. Don´t really know why, though
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Old 08-05-2017, 01:18 AM   #18
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Hi both,

thanks for your answers

About the 30+30 ul steps: I called Illumina since and the representative agreed that this *might* be a typo. He raised an issue for their protocol team. This team will change the protocol when the next version of the protocol will be out.

About my graphs: the graph with the 1kb peaks is my DNA before starting the library. The other graph is right after the tagmentation. It looks like there is no more DNA after tagmentation. I am starting to think that my input is not of the right concentration like ScienceGrrl said. And also, I read somewhere that DNA should be clean before starting a library, so I will try to clean my phenol-chloroform extractions with a spin column, just in case.

Cheers,
arthur
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