DNA sequencer recommendations?

[Bryan Bishop]

Hey all,

I am buying a used DNA sequencer. I'd like to spend somewhere between $500 to $5000 on it. I'd also prefer something local to or within roadtripping distance to Austin, Texas, but that's not as important to me. Does anyone have particular model recommendations?

My first idea was to buy the exact model as the socal-diybio group and get an ABI Prism 310. Alex was telling me the other day that their sequencer was a gift, and that's why it's in such rough shape. The advantage of me buying the same model is that I could conceivably help troubleshoot their problems and we'd be able to share whatever experience back and forth. It's the "neighborly" thing to do I guess. In this scenario, I'd have to wait for one to show up on eBay or at a liquidation auction somewhere.

Here's the ABI Prism 310 docs/specs:
marketing: https://products.appliedbiosystems.com/ab/en/US/adirect/ab;jsessionid=ys2cN8pMZ2kBjLv3nlWHZphplKHjkhtznKbp2vMQjQJkblgRws29!-946835675?cmd=catNavigate2&catID=600525&tab=DetailInfo
specs: https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=600525&tab=TechSpec
ANs: https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=600525&tab=Literature

My second idea was to go see if Raymond could get a DIYbio enthusiast deal with Illumina ;-). This will primarily be used either at the local hackerspace or my hangout spaces, for education / self-enrichment / global world domination and scheming thereof. So something like 50 to 150 bp read lengths would be more than acceptable for my purposes, especially if the chemistry isn't proprietary for running the machine.

I figure $5k is a nice upper bound; somewhere between $1k and $4k I hope to be able to buy a operational sequencer with documentation. I am not at all eager to spend three or four months fixing up a junker.

So anyway, does anyone have recommendations for a DNA sequencer in this budget range? Bonus points for anything that is easily moddable, i.e. with firmware I can flash/crack.

- Bryan
http://heybryan.org/
1 512 203 0507

[Mackenzie Cowell]

http://www.go-dove.com/event-15124/BioPharma-Exchange-291-Online-Auction-Hosted-by-Forest-Laboratories/lot-291631/Perkin-Elmer-ABI-Prism-377-DNA-Sequencer

http://www.go-dove.com/event-15121/BioPharma-Exchange-290-Online-Auction-North-America/lot-490222/Applied-Biosystems-Applied-Biosystems-377-DNA-Sequencer

good luck; cool idea.

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[Cory Tobin]

Refurbished, working ABI 310s go for around 15-30k. Ours has a busted
laser ($7k to replace) so right now we're looking into selling it.

Brand new Illumina HiSeq machines go for around $700k. Even if they
give you a deal it will probably be a little out of your price range
;) This will be the case for any sort of high throughput machine.

I'm not sure you'll be able to find a working capillary sequencer for
under 5k. My guess would be no. You can definitely find an old gel
sequencer for under 5k. I used to work with an ABI 377. Pouring gels
for those things is a pain in the ass but if you don't mind the extra
labor they are definitely in your price range. There is one on LabX
right now for 4k
http://www.labx.com/v2/spiderdealer2/vistaSearchDetails.cfm?LVid=8977344
not sure about the condition though.

If you're just interested in getting DNA sequenced and don't care
about hacking the hardware, just outsource it. Looks like the core
facility at UT Austin does service outsiders
http://www.icmb.utexas.edu/core/DNA/DNA_Sequencing/DNAsequencing.htm
You're only saving a few dollars by doing it yourself, not counting
the capital cost of the machine or your time.

-cory

[Nathan McCorkle]

automating things like silica/phenol+chloroform/EtoH extractions of
DNAs would really be where you could innovate. Some sort of microglass
handling system like CoryG has been talking about. One of those
extractions is pretty much all you need to do to DNA before sending it
to a core facility.

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[Jordan Miller]

50-150 bp you can read off a physical gel, why would you need to spend any significant amount of money on a black box that you're not going to be legally allowed to understand how it works?

if you especially want this capability for educational purposes, I can assure you nothing is more rewarding than easy assays that make sense, especially when the comparison is a black box vendor device.

otherwise, I agree just send it off for sequencing. you will still get the raw 4Peaks data back that you can use for education.

jordan

[J Adams]

Hi All;

To further Cory's discussion.

1) We sell refurbished ABI 310s in the 12-15K ranges, including installation
and 90 day warranty. The 310 is a single capillary system so you don't get
much throughput.

2) He also brings up the ABI 377. If you have a lot of samples to run and a
very little budget this is the cheapest method for both sequencing and
genotyping. You can run 96 samples in 2-6 hours depending on the
application. It is old technology, but we still have about 25 to 50
customers that still use theirs daily and LOVE THIS MACHINE. But, as he
points out, it is also the DIY people that like this machine because pouring
a gel can be a pain. The price for this machine is about the same as the
price for a refurbed ABI 310 (12-14K).

3) For genome sequencers the price for a full featured genome sequencer is
coming down! We recently launched our Max-Seq which has throughput
comparable to the Hi-Seq but the price is only 280K (much less than 700K!).
And the cost of running these systems is coming down as well. I suspect
this trend will only continue. You can now get the low throughput (100Mb)
machines for ~100K.

4) As for laser replacements, Azco offers a laser replacement "package" that
includes the laser, labor and travel to install it for $5,500 and we even
perform a PM on you system. Or, if you wish Cory, we will even purchase
your 310 if you are no longer using it!

5) As for using LabX, I always recommend "buyer be Ware", I have many
customers that tried saving a few thousand dollars buying an instrument on
Lab-X only to find it ended up costing more than they would've spent if they
just purchased a refurbished instrument from one of the instrument vendors
like Azco. For example, if the 377 for 4K needs a new laser, well that adds
5.5K to the price (now 9K), if the computer needs changed that adds 3.5K
(now 12.5K) to the price, and if the main board needs replaced that adds
7.5K to the price (now 20K!). So, as you can see, if you just spend 12K
then you get a system that has a new laser in it that will last 3-5 years,
you get installation and training, and you get a system that is warranted to
work!

I hope this helps!

Regards;

J Adams

-cory

--

[Ruediger Trojok]

lets do a DIY version of craig venter's Sorcerer II and go down the
amazone to sequence everything that comes across :P
Does anyone know a crazy billionare that would hand out that money to
me to buy a ship and the illumina sequencer?
It is for sure great fund and very educating.

> right now for 4khttp://www.labx.com/v2/spiderdealer2/vistaSearchDetails.cfm?LVid=8977344

>  not sure about the condition though.
>
> If you're just interested in getting DNA sequenced and don't care
> about hacking the hardware, just outsource it.  Looks like the core

> facility at UT Austin does service outsidershttp://www.icmb.utexas.edu/core/DNA/DNA_Sequencing/DNAsequencing.htm

[eriksonj]

Wishful thinking on getting an Illumina machine. They don't give them
away free or cheap, and you're certainly going to be spending a few
thousand per run on reagents alone. Quite honestly, Illumina doesn't
give a shit about their own paying customers, and if you don't have
money they don't want to talk to you. Also, the computing power
needed to handle the data is significantly more than your personal
computer.
In reality, there is more to the sequencing that just the chemistry,
you need to figure out what to do with the data. If you really think
you have something worthy of sequencing, just send it out to a
sequencing provider. You will save the money and learning curve.
That being said, if you want a cheap garage sequencer and don't want
to be pouring the acrylamide gels (which I've done alot and
passionately hate) look at a pyrosequencer, from Pyrosequencing AB
(now Biotage). 25bp reads, pretty bulletproof, and very user
friendly. It sucks for homopolymers, but other than that, I think you
will like it.

And crazy billionaires won't fund your ship...but they will fund your
sequencing start up if you're the right people with a crazy
imagination and a passion to change the world. Maybe you know the
guys I'm talking about...

[Mackenzie Cowell]

I'll join the amazon, uh, "Merlin" cruise.  Kickstarter. :)

I'm sure we could rig something DIY up.  http://www.fishingfury.com/wp-content/uploads/2009/03/fail-owned-best-failboat-ever-fail.jpg

Mac

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[Hugues]

Hi guys,

just bringing this thread back on top since it's from 2011, eons ago technologically speaking LOL.

Are there new opportunities to buy a second hand sequencer for personal lab usage ? anything available for a couple thousands bucks, or it's still too early ??

[Nathan McCorkle]

On Tue, Oct 25, 2016 at 11:43 AM, Hugues <lalibe...@gmail.com> wrote:
> Hi guys,
>
> just bringing this thread back on top since it's from 2011, eons ago
> technologically speaking LOL.
>
> Are there new opportunities to buy a second hand sequencer for personal lab
> usage ? anything available for a couple thousands bucks, or it's still too
> early ??

The pricing can be in the hundreds if you catch the right deal... I
think bioCurious even got one for nothing that they took apart (not
sure of the details on that).

[Bryan Jones]

We just got a MinION at our biohacker space. The starter package is $1000. Haven't had a chance to play with it yet though.
https://nanoporetech.com/products/minion

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[Hugues]

Thanks Bryan,

I just sent them a mail yesterday, their reply below:

It's still not all clear to me how long the cells last and how many times you can use them, still a newbie to this. Can I run say, a plant sample one day, then the next day a yeast sample, and so on... Or is it just for human DNA ? And how many days i can run alternating samples like this ? And is it for targeted sequencing ? then where do i get the primers ? Or the cell just reads everything we throw at it ? Replacement cells are not cheap based on the prices on their web site.

I would like to figure out how much it would cost me per year if say, i run 3-4 samples per month...

"How many samples you can run per for cell depends on your target region of interest/genome size and the depth of coverage you are looking to achieve per sample. On the newest R9.4 chemistry you can generate as much as 10 Gb of 1D data per flow cell, so you could use this as a starting point for your calculation. Although we ask that flow cells be returned after 8 weeks from receipt, bulk orders can be "drawn" upon as needed  - meaning that if you purchase 12 flow cells for example, you can schedule actual deliveries as needed for your experiments. This means that your remaining flow cells will automatically be updated to the latest chemistry as we continue to improve our pores, membranes and hardware."

On Wednesday, 26 October 2016 06:24:11 UTC+2, Bryan Jones wrote:

We just got a MinION at our biohacker space. The starter package is $1000. Haven't had a chance to play with it yet though.
https://nanoporetech.com/products/minion

...

[Hugues]

Ok. But which brand/model can we now find in this price range ?
Thanks

[Scott]

Think outside the box! Long before the advent of DNA Sequencers we use to run our own Sanger sequencing gels. Basic gel electrophoresis at it's best! The multi-lane gels were polyacrylamide and either 36 inches or 48 inches long for resolution. It sucked when the gel leaked before setting! Back then the reactions were labeled with P32 or S35 but I'm certain the modern fluorescent DNA dyes would work since you have one terminator per lane - yes, 4 lanes per sequence run! A single run would give us ~200-250bp of sequence (on a good day!) but the modern PCR-based reactions would improve that. 

Mind you the cost of a commercial sequence run would likely be be less but this would be a fun DIYbio project.

Cheers,

Scott

http://www.opensciencenet.org

[Hugues]

Thanks Scott,

What do you guys think about this one:

http://www.minipcr.com/product/minipcr-dna-discovery-system/

Anyone has tried it yet ?

thanks

On Sunday, 1 May 2011 02:37:09 UTC+2, Bryan Bishop wrote:

[SC]

That isn't a sequencer.  It's a PCR machine and an electrophoresis unit.  You won't be able to get sequence out of it.

[SC]

The error rate of the minion is so high it's pretty much useless.  Over 38% error, it's a complete waste of time and money:

http://www.sciencedirect.com/science/article/pii/S2214753515000224

[Xabier Vázquez-Campos]

Update your info. That paper talks about the R7 chemistry. They are now with the R9.4. They have change prep and pore proteins since then.
1D-reads go over 90% and 2D-reads over 99%

[Xabier Vázquez-Campos]

PS: I talk about accuracy of single reads

[Hugues]

But wait, i'm confused now. I thought we could read a DNA sequence with gel electrophoresis, no ?

Isn't what we can Sanger sequencing, with the help of dideoxynucleotides ?

example here:

https://www.youtube.com/watch?v=c2bAtGycqx4

but i'm new to this, so maybe i missed something ? or this method works but it's too long and nobody uses it anymore ?

[Abizar Lakdawalla]

Seriously, not worth doing a DIY sequencer. I worked at Applied Biosystems, the guys who commercialized the fluorescent gel based sequencer, then the fluorescent capillary sequencer. And at Illumina who commercialized next generation sequencing. It is a lot of work, mostly in optimizing.

Have attached a graphic which summarizes most of the sequencers out there.

For the gel based - yes you can DIY but you will need.

a) Long glass plates (at least 30-50 cm long) and spacers to create the parallel vertical plates to pour the gels in

b) a high voltage power supply - about 3000 V that is > 100W

c) acrylamide monomers to create the gel. These are neurotoxic.

d) Labeled sequencing primers (with fluorescent dye or with radioactivity)

e) Pure DNA templates (what you want sequenced) with adapters stuck on the ends (region where the sequencng primer will bind) - can be created by PCR

f) Sequencing mix - contains dideoxy terminated nucleotides with normal nucleotides. Enzymes, buffers, etc.

g) ability to dry the gel and expose to a large X-ray film of using radioacivity.

The bible for capillary sequencing is at this link

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[Abizar Lakdawalla]

I agree, the chemistry is substantially improved. They just assembled a small genome with ONT reads only. They do concern Illumina as being the most likely to disrupt with a bigger box (called the promethion).

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[Kermit Henson]

That`s half right. Is not as easy and fast , as one can guess.

The trick is that you need to label the last nucleotide of your "fragment" sequence in order to know "which nucleotide goes next".

In short, you cut your DNA in random length fragments (10, 100, 56, 2, 17bp, etc..). After this, you label each fragment at the end extreme (isotope, fluorescence, whatever). Then, you run the electrophoresis to separate the different fragments lengths. After this, you start reading in your gel nucleotide by nucleotide, so if you have a 1000bp fragment... well, enjoy the reading ;) 

Sorry if there is any mistake, but I think that you can get an idea of the whole process. Also, take in mind that to perform this process is not as easy as it may seem (specially for the gel part)

About the miniPCR kit. I have one of those PCR and it works like a professional one  (a side that it makes a LOT of noise due to the fan cooling system). So, yes: in theory you could used to sequence (in case you know and can label your fragments). BUT, to run the gels you should use an acrilamide gel, not agarose (which means you need "another" electrophoresis system).

My recommendation: purify your samples and send it to any company (ex: BGI, Macrogen...) or contact to any university's core facility.

It will be cheaper, faster and will save you from a lot of headaches. And, if there is any error, you will know and (most probably) will get a re-sequencing for free.

[Hugues]

ok thanks guys,

all clear.