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Old 08-30-2013, 12:42 PM   #1
eab
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Default alternatives to Covaris

We are starting a sequencing core and considering whether to buy a Covaris machine. Do people think fragmentation of gDNA or full-length message cDNA (a la SMARTer ultra low for RNASeq) is going to be best done on a Covaris for the foreseeable future. Or is chemical/enzymatic fragmentation going to become a viable option?

Your carefully considered responses, gut instincts, etc. would be much appreciated.

Eli
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Old 08-30-2013, 12:50 PM   #2
mcnelson.phd
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Illumina/Epicentre have been making lots of progress on using transposons to fragment samples for genomes and transcriptomes, but the results are still not as good and unbiased as mechanical fragmentation methods. In some cases, like Nextera Mate-Pair, a Covaris is still needed for shearing the circularized fragments that were produced from the transposon at the very beginning.

Depending on what types of samples you'll be sequencing the most, a Covaris can definitely be a good investment. Of course, there are other instruments that could make life better as well for a small core, so it really depends on what you want to do and where you think you're better off allocating funds.
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Old 08-30-2013, 12:56 PM   #3
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Thanks very much for your reply. I had forgotten about Nextera! But my sense is that's not going to put Covaris out of business - do you agree? Even if bias improves?

If we're doing, say, lots of tiny-sample mRNA libraries using SMARTer Ultra Low, is there any way around a Covaris machine?

What other instruments would you recommend considering? Do you mean something that does what a Covaris does (I don't know of such an instrument)? Or do you mean spending money on a different aspect of the core, automation for library prep, high-throughput library QC, etc?

Basically, if we need to fragment dsDNA, is there any way around a Covaris for a respectable core facility?
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Old 08-30-2013, 03:56 PM   #4
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Since it appears that you're only going to be doing Illumina sequencing (MiSeq?), then the benefits of the Covaris for emulsion PCR based systems wouldn't be worth anything to you so really you're just looking at fragmentation.

Given that you state that you'll mostly be using the SMARTer library prep kit, it looks like a Covaris would be worth it.

We tested the M220 as a possible addition to our small facility, but we've decided that our $$'s are better spent on liquid handling for amplicon libraries. We have an S2 available to us in another building that we can use for things like TruSeq or the Nextera MP kits, but the majoriy of our genomes are done using Nextera(XT) and for transcriptomes we've used Nextera on cDNA or we use the ScriptSeq kits. It's possible do to Nextera XT on cDNA, but the results can be biased so I wouldn't recommend it for anything but very preliminary testing.

Bottom line, look like you're best bet is to get a Covaris, question would be which one...
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Old 09-01-2013, 04:21 AM   #5
eab
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OK, thanks, that is helpful. Yes, only Illumina, Miseq and Hiseq, now mostly TruSeq RNA and amplicon. SMARTer may be heavily used in the future, provided no alternative for single-cell libraries arrives on the market. How much better is it to shear the SMARTer product than to use Nextera?

Also, forgive my ignorance, but the use of Covaris in emulsion PCR is to reduce the fragment size for better emulsion generation?
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Old 09-01-2013, 05:39 AM   #6
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I have been using the SMARTer kit for RNA prep and my lab has a bioruptor. Having recently moved to this lab from a lab with a covaris I will say definitely go for the covaris.

At the moment I am am having problems with the Bioruptor and I am in doubt of its reproducability:

Aiming for a fragment size of 350bp, I was advised on 7 cycles 30s on/30s off (a total of 7 minute shearing). This gave me a fragment size over 1kb. 4 additional cycles improved to 500bp.

Another instance, I increased cycles to 14 cycles 30s on/30s off (14 min shearing), fragment size 800bp!

Compare this to the Covaris where I can get a reproducable fragment size of 350bp in 45s! It's a no brainer for me (shame it can't be said about my lab)!
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Old 09-01-2013, 12:39 PM   #7
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Thanks very much, that is helpful.

Does anyone think fragmentase or other enzymatic solution will in any way replace accoustic shearing?
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Old 09-02-2013, 04:23 AM   #8
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What liquid handling system(s) did you choose, by the way?

Quote:
Originally Posted by mcnelson.phd View Post
We tested the M220 as a possible addition to our small facility, but we've decided that our $$'s are better spent on liquid handling for amplicon libraries. We have an S2 available to us in another building that we can use for things like TruSeq or the Nextera MP kits, but the majoriy of our genomes are done using Nextera(XT) and for transcriptomes we've used Nextera on cDNA or we use the ScriptSeq kits. It's possible do to Nextera XT on cDNA, but the results can be biased so I wouldn't recommend it for anything but very preliminary testing.

Bottom line, look like you're best bet is to get a Covaris, question would be which one...
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Old 09-02-2013, 06:03 AM   #9
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As far as fragmentase or transposon mediated fragmentation go, I don't think that they will ever fully displace the Covaris for shearing. This is mostly because you get significantly better assemblies when you feed the assembler reads that have a tight insert size. However, we've been able to get 2Mbp contigs from Nextera data for some bacterial genomes, so that's one of the reasons that we didn't get a Covaris for ourselves.

As for liquid handling, we haven't made a purchase yet, still just demoing every instrument available. The QIAgility and EP5073/5075 are our top contenders so far, but neither has performed as well as we wanted during the demo. We're currently working with Qiagen and Eppendorf to resolve those issues so nothing has been decided yet.
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Old 09-02-2013, 06:31 AM   #10
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Hmm! Those two weren't even on my radar screen. Did you look at the Caliper machines or the IntegenX Apollo system at all?

What are your thoughts about the Qiagen and Eppendorf machines?
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Old 09-02-2013, 06:49 AM   #11
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Just from looking at the webpage for the Apollo, that system doesn't do liquid handling, so it wouldn't be on our radar. We tested a Kingfisher because I had talked with some people and read on this forum that it was better for things like AMPure cleanups than the liquid handlers, but we would only be using it for that step and thus it's not worth it for us when setting up 384 well PCR plates with unique indices is more critical of a step in our workflow.

Caliper I didn't think makes any liquid handling systems. Perkin-Elmer, their parent company, makes the Janus system I know, but that's designed for higher throughputs than we need.

The main reason we've been looking at the QIAgility and EPMotions are because of their flexibility compared to systems like the Kingfisher and because we're a small in-house facility that won't (hopefully) be accepting outside work then we don't need and can't justify something like the Janus.
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Old 09-05-2013, 06:27 AM   #12
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Default liquid handlers

Other liquid handlers you might want to check out:

Caliper Sciclone NGS
Hamilton
Agilent Bravo
Beckman FxP

- Genohub
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Old 09-09-2013, 02:29 AM   #13
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Genohub kindly mentioned the Biomek FXp, which is perfect for high throughput library construction.

Just thought I would butt in and mention the Biomek 4000. If you are considering liquid handlers in the EpMotion budget range then the B4K is the closest Beckman Coulter liquid handler. We already have prewritten methods for our amplicon cleanup and size selection chemistries (AMPureXP and SPRISelect). You can see more here

http://www.bclifesciences.com/automa...ish/index.html
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Old 01-27-2014, 11:00 AM   #14
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The Q800R system is a new alternative to a Covaris device. It offers higher throughput and costs much less. In addition, its compatible with all sequencing platforms including Illumina, Life Tech, Raindance, etc...
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