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Old 08-22-2008, 02:47 PM   #21
Samuel Kim
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Default Any updates

Hi, Kevin

Any updates on the performance of Polonator?

We are interested in evaluating one. What will be the lead time?

Regards,

Samuel.
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Old 08-22-2008, 05:24 PM   #22
Kevin McCarthy
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Samuel: Thank you for your interest in the Polonator. We have been busy over the last few months implementing some changes to the fluidics sub-system, the flow cells, and the software. It's been a bit intense, but I'm thrilled to report that the Polonator design is now frozen, and appears from everything we can see to be delivering on its promise. The Church Lab has been extremely rigorous in their testing of the Polonator, and commented recently that "We don't have to fiddle or tweak anything... We just hook them up, load flow cells, and run... they're perfect". Given the very high standards of the Lab, that's music to our ears.

Now that they have fully functioning Polonators, the Lab has begun to qualify the starting (26 bp paired end tag) biochemistry on the instruments. They are currently optimizing both the run biochemistry (which takes place within the instrument), as well as the upstream protocols. They are not yet ready to provide performance data, but we expect that this will be available in the near future. We plan an aggressive road map to drive performance upwards over time, but also recognize the need to put a stake in the ground soon with an initial set of performance data. This will be posted on the Polonator.org web site as soon as it is available.

We are shipping several more Polonators to our early adopters over the next couple of weeks, but we expect a bit of a pause before we enter wide release. The Church Lab is in one sense too competent; they and we agree that we need at least two of our independent early adopters to both confirm that the instrument and biochemistry are easy to use and reliable, and that they can readily replicate the Church Lab performance data, before we can begin to accept orders from the broader community. We have both inventory on hand and work in process to support initial demand, and are deploying the Danaher Business System's cellular manufacturing precepts to flexibly ramp capacity as required. Our lead time target is four weeks, but the backlog of potential users might skew this.

Stay tuned!

Kevin
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Old 08-22-2008, 05:51 PM   #23
ECO
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Kevin, glad to hear it's beginning to come out strong!

The launch chemistry is still 13bp x2, correct? Any comments on how many beads you're looking at per flow cell?
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Old 08-22-2008, 06:12 PM   #24
Kevin McCarthy
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ECO: Yes, the initial chemistry is 13 bp x 2. At a more granular level, we read 7 + 6 bases from each of two tags; the reads are not contiguous, since 4-5 bases of the 17 - 18 bp tags remain unread. I have written and been sitting on a character-based (PDF) run through the biochemistry; I've been meaning to post it to the Polonator.org site, and will do so shortly, but would post it here for the first time if I knew how. Do you support attachments? I don't see the icon to do this.

Kevin
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Old 08-22-2008, 06:15 PM   #25
Kevin McCarthy
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ECO: Ahh, I just saw that I am not yet cleared to post attachments. Resolve this, and I will post the base by base protocols PDF forthwith.

I also forgot to respond to your question regarding bead count. The flow cells were recently redesigned to better match the biochemistry time to the imaging time, among other things. The previous design had 18 circular wells of 15 mm diameter, fed in parallel from a fluidic perspective. The new flow cells have eight linear lanes (also fed in parallel) of 224 mm^2 imaging area each, with the ability (as had the previous 18 well design) to load independent libraries in each lane. We are still optimizing bead loading, but a fair starting estimate would be 7 to 10 e8 beads per flow cell (a run can consist of either one or two flow cells, but the run time is independent of the flow cell number).

Kevin

Last edited by Kevin McCarthy; 08-22-2008 at 06:47 PM.
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Old 08-22-2008, 07:19 PM   #26
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Quote:
Originally Posted by Kevin McCarthy View Post
ECO: Yes, the initial chemistry is 13 bp x 2. At a more granular level, we read 7 + 6 bases from each of two tags; the reads are not contiguous, since 4-5 bases of the 17 - 18 bp tags remain unread. I have written and been sitting on a character-based (PDF) run through the biochemistry; I've been meaning to post it to the Polonator.org site, and will do so shortly, but would post it here for the first time if I knew how. Do you support attachments? I don't see the icon to do this.

Kevin
Not sure why you can't post attachments, unless it's too big, or you're not going to "Post Reply" (the small "quick reply" box at the end of the thread does not support attachments).

I have the PDF limit at 2MB now...let me know if that's not big enough. If you want to make it a new thread that will probably be best and easiest for people to find.
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Old 08-22-2008, 08:15 PM   #27
Kevin McCarthy
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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

Last edited by Kevin McCarthy; 08-22-2008 at 08:21 PM.
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Old 08-22-2008, 08:56 PM   #28
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Quote:
Originally Posted by Kevin McCarthy View Post
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?
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Old 08-23-2008, 02:24 AM   #29
Kevin McCarthy
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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

Last edited by Kevin McCarthy; 08-23-2008 at 02:27 AM.
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Old 08-23-2008, 07:15 AM   #30
ECO
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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...
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Old 08-25-2008, 06:54 AM   #31
Kevin McCarthy
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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
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Last edited by Kevin McCarthy; 08-25-2008 at 06:54 PM.
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Old 08-25-2008, 08:15 PM   #32
zoegy
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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 ?

Last edited by zoegy; 08-25-2008 at 08:17 PM. Reason: just incorrect word
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Old 08-25-2008, 08:52 PM   #33
zoegy
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My bad, I just read 1st page and did not know the above posts. But I am still waiting about the performance data.
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Old 08-27-2008, 07:54 AM   #34
horigen
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Default 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.
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Old 08-27-2008, 02:21 PM   #35
Sungjoon Kim
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Default 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

Last edited by Sungjoon Kim; 08-27-2008 at 02:24 PM.
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Old 08-27-2008, 08:54 PM   #36
horigen
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Default 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 DynabeadsŪ MyOne™ Streptavidin C1 (Invitrogen Dynal 650-02)
$450 5umol scale Dual biotin modification (IDT /52-Bio/)
-
$1649 Total

Covalently coupling approach:
$353 10mL=100mg DynabeadsŪ 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.

Last edited by horigen; 08-27-2008 at 09:08 PM.
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Old 09-07-2008, 12:07 AM   #37
genseq
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Quote:
Originally Posted by Kevin McCarthy View Post
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
Hi Kevin,

your "sample prep" is very sofisticated:

Library Construction Protocol (first stage, from Kevin McCarthy - Chief Technology Officer Dover Motion Systems):

1. DNA cleaning.
2. Shotgun shearin.
3. Fragments purification.
4. Blunting.
5. A-tailing.
6. “Circularization” (ligation).
7. Elimination of residual noncircularized material by exonucleosis with exonuclease I and III.
8. DNA purification.
9. Hyper-branched rolling-circle amplification.
10. Digestion with the restriction endonuclease Mme I.
11. Gel separation (separation the population of 66 to 68 bp oligo s).
12. Blunting.
13. The forward and reverse primers are then ligated to their respective ends of the 66 to 68 bp component, producing a 134 – 136 bp long oligo which consists of the 43 bp forward primer, the first 17 to 18 bp genomic tag, the synthetic 32-mer, the second genomic tag, and finally the 25 bp reverse primer.
14. Since each primer’s 5’ ends aren’t phosphorylated on the end that ligates to the genomic tags, this leaves a nick (a missing phosphor-diester bond) at these two points. E.coli DNA polymerase I and a dNTP mix are then used to translate this nick off each end of the oligo.
15. The template DNA is then amplified using PCR (12 cycles).



That is simplified protocol (see attach):

RNA-ligase Library Construction Protocol (for Danaher from genseq - Junior Member of Seqanswers):

1. DNA cleaning.
2. Shotgun shearin.
3. DNA denaturation.
4. Ligation OH_OH and P_P adaptors with ssDNA (RNA-ligase).
5. Fragments purification (gel separation).
6. PCR (12 cycles) with 5’-P primers (LD PCR for blunt ending).
7. “Circularization” (DNA-ligase T4).
8. Digestion with the restriction endonuclease Mme I.
9. DNA denaturation.
10. Ligation OH_OH and P_P adaptors with ssDNA (RNA-ligase).
11. Fragments purification (gel separation).
12. Real-time PCR.
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Last edited by genseq; 09-07-2008 at 09:11 PM.
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Old 09-13-2008, 10:54 AM   #38
genseq
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Default George M. Church about Polonator (June)

High-Speed Imaging for DNA Sequencing.

http://www.photonics.com/content/bio...res/92286.aspx
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Old 11-06-2008, 06:39 AM   #39
tlitman
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Exclamation 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?

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Old 11-06-2008, 07:01 AM   #40
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Quote:
Originally Posted by tlitman View Post
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.
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