Tweet
I'm curious to know if anyone is using any automation to do TruSeq library preps. If so, what instrument do you use and why?
-
-
I'm bumping my own message, since I last inquired about it back in Aug. I recently looked at the Eppendorf automation for library prep, but it's more "semi-automated." The plates require manual sealing and the heat block does not have a heated lid. So really, it preps the plates and the user incubates them. I'm just curious if anyone has looked into this recently. (We already have a SPRIworks. I'm looking for automation to do the "manual" TruSeq protocol.) -
'fully automated' NGS library prep
It is possible with more flexible robots, such as the JANUS form PerkinElmer, to have a more fully automated yet flexible platform for NGS library prep. For example you can integrate a plate sealer for automated sealing. We use JANUS for Illumina library prep and Agencourt sureSelect hybridization setup & cleanup. In our lab we seal the plates automatically and incubate at up to 37 just on a temp control tile on the deck. Then we unseal them manually. It is also possible to automate the unsealing and even integrate a PCR machine, it only depends on your budget. The JANUS can also do normalization based on pico green or rtPCR.Comment
-
We do have some customers automating TruSeq in the UK using Biomeks. It's all a matter of how much you want automated but as a general rule sealing and unsealing plates gets very expensive. Get in touch with your local Beckman rep and they can show you our options with regards heated positions etc. Depending on your throughput, something like a Biomek NXp would do the job nicely.Comment
-
There is no good solution for automated sealing for PCR anywhere really. The effort drives systems that become complex beyond the workflow requirements, and result in a kind of HTS screening kind of zombie system that could process 100 plates a day, but is used for two or three plates a week. And then, even HTS systems rarely incorporate seals good for PCR successfully.
Also be aware that if you wish to automate sealing for PCR then you will also need an automated centrifuge. There are well designed options here that fit the bill, but they add from $50K to $75K to your solution, depending on the table space and robotic bump up needed to integrate it.
So you need to ask: How many times can I manually seal and unseal a plate in the process of running two or three hundred exomes (ie 2 or 3 plates worth) a week before I would rather pay ~>$100K to avoid it? 5 times? 10 times? 50 times?
CheersComment
-
I don't agree that there is "no good solution" for this. Again it depends on the degree of automation that you want and are willing to pay for. For complete automation yes you would need to integrate a sealer, PCR machine, centrifuge, and unsealer and this would get very pricey. A reasonable compromise is to add the sealer and do moderate temperature incubations on the deck of the robot, then prompt the user to centrifuge and unseal the plate. This cuts the number of user interactions with the semi-automated system from about 6 per day to about 3 per day for a library prep and will reduce lag times that can make for a very long day. Reducing time and attention required for library preps may indeed be worth it!Comment
-
Yes. Prompting, manual sealing and placing in a PCR machine is a "good solution." Automating that step has no good solution. (is that what I said?)
But "this" == Having robots hand off to a sealer and then pick from a sealer and try to have a machine remove seals (the hard part); this is wobbly at any price in my experience. You will see many efforts to innovate around this problem if you ever attend automation trade shows, for this reason, and the effort to innovate continues because it remains a problem for robotics. It has been a wished for (and passed over) capability on dozens of High Throughput Screening projects (hundreds or thousands of plates a day) with budgets running to millions that I have seen.
Makes no sense to think about automating sealing at current NGS throughput requirements.
But all the issues of the HTS world that really funded the maturation of so much good technology pressed to serve NGS, will become the issues of NGS as the complexity of the molecular biology sample prep work-flows are reduced, the level of multiplexing goes up, and the cost of sequencing all the way through to final informatics drops. Then, when sample prep becomes a bottle neck, the lack of a good solution for **automated** plate sealing and peeling will become an issue again. And, having waited 10+ years for one, I don't suspect that there will be one then either.
Vendors: There is still time!Comment
-
Actually, now that I read more carefully, I do see that eliminating an interaction for PCR setup is valuable. It creates the potential for starting a run late and resuming after the PCR machine is done and is keeping the plate securely at 4C.
Un-selaing is where the automation falls....Comment
-
There are a few good options for sealing multiple times during incubations and/or PCR. We use a Beckman Biomek deck with MJ thermocycler on deck. There is a reusable lid with a soft rubber gasket that seals PCR plates very well. Not sure who makes it but we got it from Beckman.
We use that for sealing on library prep PCR as well as the polyA step of the TruSeq method. All with no problems. Cleaning with 1M NaOH, DNAZap or RNAZap is all effective for cleaning. We tested contamination and evaporative loss pretty extensively.HudsonAlpha Institute for Biotechnology
http://www.hudsonalpha.org/gslComment
-
I agree it is un-sealing that is the problem. We have used the Plate-Loc from Velocity (now part of Agilent) for quite a while and it works very well for sealing the plates. Unsealing - most systems just use a piercing apparatus which can be a problem as you start to load multiple seals up onto the plate.Originally posted by mikebenway View PostComment
-
Has anyone looked at the Caliper system yet for NGS library prep?Originally posted by jlove View Post
Cheers,
NeilComment
-
Cancer survival rates have significantly increased in the last few decades in the United States, reaching a combined 70% 5-year survival rate by 2021. Behind this number, there are years of research to find new therapies, drug targets, and early detection methods. But there is one core challenge that keeps slowing down these advances, and it’s about drug resistance.
There is no single reason why many patients don’t respond to treatment as expected. Cancer is...Yesterday, 05:17 AM -
Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
-
I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
Comment