[WhiteSeal]

TECAN Fluent programming

Hi All,

In my lab we have just installed the Tecan Fluent and I have started to find out how to start to program with the FluentControl software. There are a couple of questions that were not clearly addressed by Tecan when I asked them up front.

1. how can I create a subscript that can perform repeated segments in my normal script. I'd rather have to make one subscript that I can 'call' every time I need it.

2. Is there an easier way to get the information on all the variables in the script and the script setup besides using the Fluent software?

I have programming experience with R and I see some similarities, but the software is a bit slow when I compare it to R.

Dirk

[Lymanara]

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[JakobHedegaard]

I am working in a hospital lab and am the happy user of two Sciclone NGS, a NGS Express and a Zephyr NGS. We use the robots for DNA cleanup, NGS lib prep, purification and QC.
There are a number of ready-to-install APPs for the robots: https://perkinelmer-appliedgenomics....ds-for-NGS.pdf
You can either pay Perkin Elmer to do the install and validation (expensive - fair enough) or you can get training in installing and validating the APPs yourself. It takes of course some time, but it pays in the end as increased flexibility in modifying and troubleshooting the APPs.
We find the robots reliable and use them routinely for our certified workflows.

A new Sciclone has been released with a hotel for tips and an integrated thermocycler: https://perkinelmer-appliedgenomics....q-workstation/

PS I am not employed or otherwise payed by Perkin Elmer....

[cement_head]

Replacement for BiomekFX

Hello,

We currently have a BiomekFX, but it's getting to be near EOL for support/add-ons. We're wondering what people are using? I will NOT buy Perkin-Elmer; they're overpriced and super-unhelpful for technical support. I also will NOT consider anything from GE Healthcare.

Other than that, I'm open to suggestions.

[gabrieltw]

@pzucchel

My answer to your question only applies to our own situation. Depends on what a lab is doing, I think you will get different answers.

For us, we process 200+ samples a week from picking BACs to loading finished libraries to MiSeq. 96 is absolutely necessaries because we process samples in plate format.

Aside from long processing time of 96 vs 1 tip, there is also issue of cross-contamination issue with one tip dispensing into multiple wells. Most of the reagents we have are in plate format as well. 80% of our protocol is done in 96, only few steps are in single tube format.

As for beads clean up, the only thing matter to us is getting enough library to get on the sequencer. Sequencing is cheaper nowadays, therefore, even if some samples fail, it will be cheaper to just prep it again instead of trying to get something working 100%.

[pzucchel]

Hi,

first of all, i need to disclose i work for https://www.andrewalliance.com - but i have no commercial intention and my purpose is to understand better and/or inform.

I am very interested in knowing more precisely the 96 channel need. Most of the time, we see that the flexibility of a single channel (or 8 channels) in repetitive mode overcomes the need of multiple heads. The use case is quite different: take for example a single channel device, it can aspirate from a tube and go to a microplate directly. On-the-fly dispensing can take as little as 5 seconds per well, and then you just need one (or few) aspirations from your tube. The moment you move to 8 or more heads, you always need an intermediate step - e.g. you need to transfer the tube content with a large volume (single channel) pipette to a reservoir, and then act with the multi-channel head. Conclusion: can you please clarify WHY the 96 channel head is necessary/enabling for you? We are aware in multiple operations our single channel robot is often as fast/effective than a multi-channel epmotion 5070, that actually has a higher cost.

Concerning magnetic beads, please don't miss our beadtender solution https://www.andrewalliance.com/wp-co...D_20161005.pdf

The reason i mention it is the following: we tried to replicate the existing manual beads-separation methods in a robot, and we actually couldn't see any improvement. Only analyzing in details the behaviour of the beads and redesigning the process, we have obtained a reproducibility that is 5x better than any other manual method we could test.

[DRYTCYV]

Hi everyone,

For the RNA part, we use the truseq RNA stranded sample prep kit and protocol.
For the RAD part, we use the Baird protocol. We just replaced the column purification steps with AMPure XP purification steps.

Basically, somebody is watching after the machine:
- to refill tips: we use a lot of them (and I mean a lot!).
- to empty the trash (due to tips).
- to seal plates: we didn't buy an automatic sealing machine as the price seemed to high regarding the benefits. It's still possible to automatize that step.
- to put plate in the PCR machine for incubations and PCR. Same as previous point: it's a choice we made but it can be automatized as well.
- and of course to be ready when something goes wrong: because Murphy's law is powerfull.

So if you can afford it, the machine can be self-sufficient and a camera may be enough (except for the last point ^^).

Here are what convinced us to use a TECAN EVO as Liquid Handling Instrument:
- the machine was available for free from another facility in our institute: I can't deny it's a BIG BIG pro-point
- we already had a TECAN Genesis so we knew how to program/manipulate such a device.
- programs are "easy" to create: if you change your protocols, you don't need to wait until the robot manufacturer implements it on your machine. You can do it yourself.

But as I didn't had the opportunity to manipulate another instrument, I can't tell you if it's better or worst than something else.
All I can say is that it's satisfying for our use.

Thank you huguesparri for your input!

We had some meetings and my last informations are:

- Epmotion 5075 : The software looks easy to work, I thought it was more difficult. Huge error rate when pipetting 1ul (+- 15% in a special ISO).


- Perkin Elmer Sciclone: Still the most expensive equipment, and looks like we are always dependent from the tech support to design the protocols.

- Hamilton NGS Start : Still waiting for a reply to some questions: basic accessories, the price for the extra.

- TECAN Evo NGS:

• Very good impressions about everything.

• Completely customized and appear to be simple to design your own protocols.

• Like the Hamilton, it comes with the Dynamic Positioning System : All pipetting channels to move independently in the Y- and Z-axes.

• It's possible to upload files with all the details about how many ul to pipette, from where to where and you can give the concentration that you want to normalize your samples and the software/robot can do the rest
  
  

[huguesparri]

For the RNA part, we use the truseq RNA stranded sample prep kit and protocol.
For the RAD part, we use the Baird protocol. We just replaced the column purification steps with AMPure XP purification steps.

Basically, somebody is watching after the machine:
- to refill tips: we use a lot of them (and I mean a lot!).
- to empty the trash (due to tips).
- to seal plates: we didn't buy an automatic sealing machine as the price seemed to high regarding the benefits. It's still possible to automatize that step.
- to put plate in the PCR machine for incubations and PCR. Same as previous point: it's a choice we made but it can be automatized as well.
- and of course to be ready when something goes wrong: because Murphy's law is powerfull.

So if you can afford it, the machine can be self-sufficient and a camera may be enough (except for the last point ^^).

Here are what convinced us to use a TECAN EVO as Liquid Handling Instrument:
- the machine was available for free from another facility in our institute: I can't deny it's a BIG BIG pro-point
- we already had a TECAN Genesis so we knew how to program/manipulate such a device.
- programs are "easy" to create: if you change your protocols, you don't need to wait until the robot manufacturer implements it on your machine. You can do it yourself.

But as I didn't had the opportunity to manipulate another instrument, I can't tell you if it's better or worst than something else.
All I can say is that it's satisfying for our use.

[DRYTCYV]

We use a Tecan evo to build RNAseq libraries and RADseq libraries and we are happy with it.
We often process 96 samples at the same time.

For the RAseq part, we ends up with less material than when we do it handmade so we need to start with more material. But sequencing results are similar.

I don't know for other liquid handling instruments and maybe it's due to our machine workbench configuration but having 1 person near the machine during nearly all the library construction process is mandatory.

Thank you huguesparri for your reply, it was very useful. Do you use official protocols or you design your owns?

For RNAseq we are thinking to use this one : http://www.nature.com/nprot/journal/....2014.006.html

We want to put the robot in a room, more or less a clean room, just to prepare libraries, so no one will be there...should we install a camera ?

Cheers

[huguesparri]

We use a Tecan evo to build RNAseq libraries and RADseq libraries and we are happy with it.
We often process 96 samples at the same time.

For the RAseq part, we ends up with less material than when we do it handmade so we need to start with more material. But sequencing results are similar.

I don't know for other liquid handling instruments and maybe it's due to our machine workbench configuration but having 1 person near the machine during nearly all the library construction process is mandatory.

[DRYTCYV]

Hi,

Our list just increase with 1 more brand, but we still have some questions/concerns that we're trying to get in contact with the tech support.

- Epmotion 5075 : In the beginning, they said that the software was very user friendly and the possibilities to customize your protocols. Then, was not so easy to do that.

- Perkin Elmer Sciclone: The most expensive equipment, and looks like we are always dependent from the tech support to design the protocols.
- Hamilton NGS Start : Still waiting for a reply to some questions: basic accessories, the price for the extra.

- TECAN Evo NGS: We received a good proposal for this equipment, we'll have a meeting with them next week
Any one already design their own protocol in any of this equipments? More information?

Cheers

[santorini]

I'll update with some info we've since learned about some of these instruments. We haven't decided on one yet either, so more feedback is appreciated!

- Epmotion 5075 - Agree with everything thermophile said. Easy enough to program with drag and drop, but lots of work needed to optimize your protocols, especially with beads/magnets. Also only has single and 8 channel pipettes, does not have the capability for 96 channel head.

- Sciclone - Only is capable of pipetting 200uL at a time, so if you need large volumes this may not be your instrument. The Agilent Bravo is similar, where you might need to pipette something multiple times to get the right volume. We ruled this one out for that reason.

So far we're pretty much between the Hamilton STAR and the BioMek FxP. We also looked into the Lynx platform from Dynamic Devices, but we're not sure about their pipetting accuracy.

[jteeee2]

We have both an Eppendorf 5075 TMX and a Perkin Elmer Sciclone. In terms of NGS capability, the Sciclone is far superior to the Eppendorf 5075. As thermophile mentioned, the real headache is fine tuning all of the plasticware. Even when you get that worked out, the instrument is not as good as the Sciclone in when doing the steps required for library prep (e.g. bead cleanups). We use the Sciclone to run several different library prep workflows (up to 96 libraries generated at one time). The Eppendorf we use for simple dilution and pooling of libraries. One downside to the Sciclone is that unless you know programming language, it's much more difficult for the user to make small changes without having an engineer from Perkin Elmer come out and do it. With the Eppendorf, making tweaks to protocols is very straightforward for the average user.

[thermophile]

I have the epmotion 5075 and the qiagility. They are obviously very different instruments. Our epmotion has the tmx and vac-but the vac hardly ever works, some clients have used it but for our lib prep we are all bead based. The software for the epmotion is frustrating, all point and click, but you can control everything that it does once you figure out how the software thinks. Expect to fine tune every plasticware that you use with the beads (adjust height, asp. speed, etc). The labware from eppendorf is just the starting point if you want to maximize your yields.

The qiagility is made to be a pcr/qpcr robot. If you can figure out how to translate your task into a pcr like task it will do it, if not too bad. I wouldn't recommend that instrument for anything but PCR (we use it for library normalization and pooling)

[DRYTCYV]

Hello everyone,

Does anyone have more information to update this post?

We're in the middle of the same process to choose a Liquid Handling Robot, and our list is:

- Hamilton NGS

- Eppendorf 5075

- Perkin Elmer Sciclone

Pro and Cons ? Any other options that we should check?

Thanks