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Old 05-06-2015, 02:14 AM   #1
Gorbenzer
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Lightbulb MiSeq alternative/cheaper regents

Hi guys,
we own a MiSeq that we use quite a lot and we are now looking for alternative reagents to reduce cots.
There are non-illumina flowcells on sale?
Anyone use alternative reagents or dilute illumina reagents for library preparation?

Any suggestion on sample prep and reagent utilisation that could reduce running costs would be really appreciated!

Thanks to everyone that will share his knowledge
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Old 05-06-2015, 02:24 AM   #2
GenoMax
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For the run reagents/flowcells the answer is no. This is proprietary illumina technology. You may be able to save on library prep by using alternate methods but I will leave that for someone more knowledgeable in that aspect.

You could consider selling spare sequencing capacity you have to "make" some money to help defray your costs, if institutional policy allows for it. There are registered vendors on SeqAnswers.com that will help you to list your spare sequencing capacity.
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Old 05-06-2015, 12:04 PM   #3
Bukowski
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In my experience, vendors are generally trying to make profits - i.e. providing things to you with tolerances which are already guaranteed to maximise their returns with a little wiggle room. Diluting reagents rarely improves their performance...
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Old 05-06-2015, 01:32 PM   #4
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I recollect people using some kind of dilution of ABI reagents to get more sequencing out of kits, but back then the reagents were not packed in a disposable cartridge like MiSeq (I may be wrong about this).

Here is a product listing I found that makes a similar claim: http://www.moleculardevices.com/reag...ution-reagents
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Old 05-06-2015, 02:44 PM   #5
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Diluting the MiSeq reagents is virtually guaranteed to yield poor results. The two areas for cost saving are 1) qPCR for library quantification (to maximize the amount of data/number of pooled samples per run), and 2) DIY library prep (which uses routine molecular biology reagents/primers that can be purchased in bulk).
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Old 05-07-2015, 02:10 AM   #6
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Quote:
Originally Posted by HESmith View Post
Diluting the MiSeq reagents is virtually guaranteed to yield poor results. The two areas for cost saving are 1) qPCR for library quantification (to maximize the amount of data/number of pooled samples per run), and 2) DIY library prep (which uses routine molecular biology reagents/primers that can be purchased in bulk).
In fact, we would like to use alternative reagents for library preparation (bacterial genomes/metagenomes, 16S rRNA profiling and RnaSeq). Do you know any MiSeq-compatible kit?
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Old 05-07-2015, 05:02 AM   #7
HESmith
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The following (non-comprehensive) list of vendors offers Illumina-compatible kits: New England Biolabs (NEBNext), Kapa Biosystems (Hyper Prep), Diagenode (MicroPlex), Nugen (Ovation/Encore), Bioo Scientific (NEXTflex), Swift Biosciences (Accel-NGS), Rubicon Genomics (ThruPlex), Life Sciences (ClaSeek).
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Old 05-08-2015, 08:29 AM   #8
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Quote:
Originally Posted by GenoMax View Post
I recollect people using some kind of dilution of ABI reagents to get more sequencing out of kits, but back then the reagents were not packed in a disposable cartridge like MiSeq (I may be wrong about this).

Here is a product listing I found that makes a similar claim: http://www.moleculardevices.com/reag...ution-reagents
Genomax, you have revealed yourself to be a whippersnapper! "some kind of dilution", eh? I'm going to interpret your uncertainty here as being due to the fact that you were likely still in diapers when Bruce Roe's lab discovered you could do 1/20th scale ABI's Big Dye reagent reactions. Well, I'm not sure they discovered this, but they were the only ones who put it all on front street and published a protocol on their web site for running sequencing reactions using much smaller amounts of the reagent.

Still there:

http://www.genome.ou.edu/big_dyes_plasmid.html


This was using much less sensitive instruments than the final apex predator instrument of its kind, the ABI 3730XL. With this you can easily do 1/32nd reactions through a combination of smaller volume reactions and fairly high dilutions of big dye.

BTW, don't try to use Roe's dilution buffer for Big Dye 3.1. There was some change in the formulation of Big Dye between 3.0 and 3.1 that made it necessary to use ABI's dilution buffer. Never found out what the change was...

Oh wait, the OP wasn't looking for a trip down memory lane. Okay, I guess they just want an inexpensive library prep method. But I find this principle interesting. Basically expensive sequencing machine companies tend to generate most of their profits from reagent sales, not the original instrument sales. By far your single greatest cost for sequencing was the Big Dye reagent. So it was an attractive focus for saving money. But systems just didn't change as rapidly back then as they do now. There doesn't seem to be enough time these days to come up with a reasonable hack of a sequencing company's chemistry. By the time you had it up a running, there would likely be a new latest and greatest instrument system that used slightly different chemistry and made all your work a waste of time.

So, yeah, making cheaper libraries would be a good space to optimize.

--
Phillip
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Old 05-08-2015, 08:44 AM   #9
GenoMax
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Quote:
Originally Posted by pmiguel View Post
I'm going to interpret your uncertainty here as being due to the fact that you were likely still in diapers when Bruce Roe's lab discovered you could do 1/20th scale ABI's Big Dye reagent reactions.
I wish

More like I had stepped away from bench (having done my share of months of radioactive sequencing to get a few measly kb of (perfect) sequence). My recollection was based on second hand information, so my hesitation.
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Old 05-11-2015, 01:17 AM   #10
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Quote:
Originally Posted by HESmith View Post
The following (non-comprehensive) list of vendors offers Illumina-compatible kits: New England Biolabs (NEBNext), Kapa Biosystems (Hyper Prep), Diagenode (MicroPlex), Nugen (Ovation/Encore), Bioo Scientific (NEXTflex), Swift Biosciences (Accel-NGS), Rubicon Genomics (ThruPlex), Life Sciences (ClaSeek).
Thanks, i'll give a look at it!
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Old 05-11-2015, 04:36 PM   #11
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Quote:
Originally Posted by Gorbenzer View Post
In fact, we would like to use alternative reagents for library preparation (bacterial genomes/metagenomes, 16S rRNA profiling and RnaSeq). Do you know any MiSeq-compatible kit?
If you read papers carefully enough, you can always make your own homebrew master mixes for library prep. It will always be more expensive to buy from a vendor.

That being said, you could spend forever trying to optimize your own enzyme mixes, and your data or samples will end up suffering because of it. I have previously used half volumes of the Hyper Prep kit with no problems but I ended up just switching back to the full amounts because it ended up being such a tiny cost of the entire process anyway.

If you are doing bacterial genomics, the costs are actually very low. What you need is judicious experimental design and pooling of experiments to maximize the amount of data you can get per run. For example, in 16S community profiling depending on your experiment and input amount you can pool a ton of samples in a single MiSeq run. Same for bacterial genomes.
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