[Kevin McCarthy]

New Thread opened

All: A new thread (http://seqanswers.com/forums/showthread.php?t=1670; "Updated Polonator ready for launch!") has been opened, since we are now accepting orders for the Polonator. My apologies to tlitman, who says he tried to contact me and didn't get a reply. I receive a number of email inquiries (my email is in the contact info on the web site www.polonator.org), and had been under the impression that I had been successful in seeing that everone got a reply. I do occasionally find an inquiry in my Junk folder, but usually scan this before deleting the contents. I did have an issue with this forum in which I no longer got an automatic notification when someone posted. We were busy with a number of design changes to the Polonator, and I assumed that the thread had simply gone silent. I would encourage interested parties to use the new thread, and I just received a notification email, so that system is definitely working. I will do my best to reply to all questions there, as well as continue to respond to email inquiries.
Kevin McCarthy

[tlitman]

The setup price is similar for both systems; today, SOLiD might even be less expensive due to an attempt to get into the HTS market. The estimated running cost per run is higher for SOLiD, unless you express it as cost per sequence, in which case, SOLiD appears as the more economical model, but again, taking the ease - end thus: the time and cost - of analysis into consideration, Illumina seems as the more preferred system.
BTW: No need for an extra particle size analyser.

[danachow]

I have asked the Polonator team for information on the system, but they never cared to reply

So, we have tested SOLiD against Illumina for microRNA quantitation
Here is my conclusion:

1. Both systems apparently measure the amount of miRNAs quantitatively
2. The SOLiD system outputs 30M sequences per run (high density)
3. The Illumina system outputs 6M sequences per run (standard density)
4. The SOLiD system outputs a number of "isomiRs", which in our analysis are spurious reads due to "overfitting" to the reference genome
5. The Illumina data are easier to analyse (no "color-space" issue)

Recommendation:
If ease of analysis is of importance, Illumina appears as the first choice.
If sequence output is of importance, SOLiD has the lead.

Comments?

I'd say the price difference between this both systems is quite significant too. Perhaps that should be taken into consideration. Was told a particle size analyzer is needed as well, anyone to clarify that?

[Chipper]

I have asked the Polonator team for information on the system, but they never cared to reply

So, we have tested SOLiD against Illumina for microRNA quantitation
Here is my conclusion:

1. Both systems apparently measure the amount of miRNAs quantitatively
2. The SOLiD system outputs 30M sequences per run (high density)
3. The Illumina system outputs 6M sequences per run (standard density)
4. The SOLiD system outputs a number of "isomiRs", which in our analysis are spurious reads due to "overfitting" to the reference genome
5. The Illumina data are easier to analyse (no "color-space" issue)

Recommendation:
If ease of analysis is of importance, Illumina appears as the first choice.
If sequence output is of importance, SOLiD has the lead.

Comments?

If ease of sample prep is of importance, Helicos is the first choice. If company size and on site support is of importance, the Invitrogen/ABI/SOLiD will be hard to beat.

[tlitman]

Polonator - SOLiD - Illumina

I have asked the Polonator team for information on the system, but they never cared to reply

So, we have tested SOLiD against Illumina for microRNA quantitation
Here is my conclusion:

1. Both systems apparently measure the amount of miRNAs quantitatively
2. The SOLiD system outputs 30M sequences per run (high density)
3. The Illumina system outputs 6M sequences per run (standard density)
4. The SOLiD system outputs a number of "isomiRs", which in our analysis are spurious reads due to "overfitting" to the reference genome
5. The Illumina data are easier to analyse (no "color-space" issue)

Recommendation:
If ease of analysis is of importance, Illumina appears as the first choice.
If sequence output is of importance, SOLiD has the lead.

Comments?

[horigen]

Another question

Hi Kevin, I have another question.

Current protocol uses dual biotin modification and SA beads to couple the primers to the beads. To my knowledge, this can also be done by covalently coupling using amino modification and carboxyl beads, simply following Dynal's protocol:

http://tools.invitrogen.com/content/sfs/manuals/650.111213%20Dynabeads%20MyOne%20Carboxylic%20Acid%20(rev003).pdf

If this approach is feasible, the cost can be greatly reduced. Additionally, covalently coupling also provides much more stable immobilization, which will eliminate many potential problems in following operations.

Here's a cost calculation:

Dual biotin approach:
$1199 10mL=100mg Dynabeads® MyOne™ Streptavidin C1 (Invitrogen Dynal 650-02)
$450 5umol scale Dual biotin modification (IDT /52-Bio/)
-
$1649 Total

Covalently coupling approach:
$353 10mL=100mg Dynabeads® MyOne™ Carboxylic Acid (Invitrogen Dynal 650-12)
$100 5umol scale Amino modification (IDT /5AmMC6/)
<$0.5 10mg Coupling reagent EDC (Sigma E1769)
-
$453 Total

These are for 100mg beads = 1e11 beads = 4e10 enriched beads = 20 runs.

If the calculation is correct, cost save is about ($1649-$453)/20 = $60/run = 15% of $400/run.

Have you ever tried this approach? Is there any problem preventing this approach from being used?

Thank you.

[Sungjoon Kim]

Flow cell geometry

Hi, Kevin

I'm in one of the groups who are waiting for the release of Polonator.

I have questions on Polonator.

First, what is the flow cell geometry of each lane? Each lane's imaging area is 224mm^2. Can we know the dimension? I couldn't find the flow cell dimensions at the polonator.org

Second. what makes the bead yield low? It seems that the bead usage of 1/6 very low..

Regards,

Sungjoon

[horigen]

questions about protocols

Hi Kevin, I have questions about the protocols.

In the sequencing protocol, the unextended forward primers will be digested by Exonuclease I, but why do you cap them using Bridge 2 before digestion?

Also, your protocol says that Cap 3 is added after the other two caps, but the protocol in the wiki adds the 3 caps together, which is right?

Thank you.

[zoegy]

My bad, I just read 1st page and did not know the above posts. But I am still waiting about the performance data.

[zoegy]

Hallo all,

This is my first post in this forum. I have been watching this instrument, since it looks like the "viable" solution for me which works in 3rd world country. I am waiting Kevin post about performance data, since he has promised that the data will be released this august.

Kevin, would you post the update ?

[Kevin McCarthy]

I've attached a PDF detailing the Polonator initial biochemistry from a graphical perspective. This is a paired end tag approach, and is of necessity more complex than sequencing single tags, or even doing gene expression studies. My innovative contribution was to notice that Courier New is a fixed pitch font. The first six protocols constitute sample prep, and only the last protocol takes place within the Polonator, whose task is essentially the automation of the seventh protocol. I have an animated PowerPoint that runs through the protocols in a much more dynamic fashion, but it needs a few tweaks before I upload it.

Enjoy,

Kevin

[ECO]

Hey Kevin, should be fixed now. I failed to turn on permissions properly...users had permission, the forums did not...learn something everyday.

And I've updated the user rank to make people with 10 posts a "member" and senior member is "100" posts. Any suggestions for better rank names would be greatly appreciated...

[Kevin McCarthy]

ECO: Being on the East Coast, I had to finally pack it in last night. I see the paper clip in your attachment, but trust me, it isn't there in my Post Reply screen. I would post a screenshot, but... Catch 22. I think that you really can block attachments by some posters, and for whatever reason, I've landed in that camp. I am and have been logged in, so that's not the issue. I do note that my status, under my name on the left, is "Junior Member". What's it take to get seniority around here?

Kevin

[ECO]

ECO: "You may not post attachments", at least according to the site. Then again, "You may not edit your posts", and I've been able to do that. I am using "Post Reply". Maybe I just can't find the icon. I do see the "Insert Image" and "Insert Link" icons.

Kevin

Hrm...it's a paper clip above the text box on the same row as the font choice, or scroll down and there is a "Manage Attachments" button in the "Additional Options" box.

If it's not there, you must not be logged in...maybe you're rejecting cookies and not staying logged in.

Anyone else reading this fascinating discussion able to attach files?

[Kevin McCarthy]

ECO: "You may not post attachments", at least according to the site. Then again, "You may not edit your posts", and I've been able to do that. I am using "Post Reply". Maybe I just can't find the icon. I do see the "Insert Image" and "Insert Link" icons.

Kevin