Resent-From: <djw@bu.edu>
From: Shmulik Motola <shmulikm@mit.edu>
Date: June 17, 2013 3:44:17 PM EDT
To: "Waxman, David J" <djw@bu.edu>
Subject: Re: question re. Bioanalyzer/Prosize tracings

Hi David,
 
The calculation of how many cycles we need to perform is based on the initial bioanalyzer concentration we perform here.
 
D13-2457 has a concentration of 9nM which is more than sufficient for sequencing (we can sequence staring 1nM).
D13-2459 has a concentration of 12nM
D13-2460 has a concentration of 6.8nM
D13-2461 has a concentration of 9nM.
 
Also, at what DNA concentration does the library no longer give optimal Seq results?  Are the above 4 samples OK to Sequence?
 
Yes, all 4 samples are good for sequencing, 1nM is our threshold.
 
One last question - the Bioanalyzer/Prosize report for D13-2453 gives a final ng/uL concentration of 0.644 that appears to be much lower than is indicated based on the tracing shown earlier in that same report, where a very high yield of fragments is observed.  Any explanations for that?
 
D13-2453 has a concentration of 3.1nM where I do not see an earlier report for that. Will you be able to point me to that?
 
To conclude – almost all the samples look good for sequencing and passed our advanced analytical and qPCR measurements. Only 2 needs further qPCR analysis (D13-2443 and 2444), once this is complete, they will go on the queue for sequencing.
 
Please let me know if more information is needed.
 
Best,
 
Shmulik

From: Waxman, David J
Sent: Monday, June 17, 2013 3:15 PM
To: Shmulik Motola
Cc: Pierrick Millet
Subject: question re. Bioanalyzer/Prosize tracings
 
Hi Shmulik,
 
Could you perhaps help us understand the Bioanalyzer (ProSize) results for 4 of our samples?
 
D13-2457
D13-2459
D13-2460
D13-2461
 
All four samples show very low yields (1-2 ng/uL from 224-586 bp), whereas many other samples from the same experiment, and with a very similar level of DNA, give up to 10-fold higher yields of DNA (e.g., D13-2463 through D13-2468, at 7-24 ng/uL).  Were the four sample above all processed on the same SPRI run, and was that run the same as for D13-2463 through D13-2468?
 
Also, at what DNA concentration does the library no longer give optimal Seq results?  Are the above 4 samples OK to Sequence?
 
One last question - the Bioanalyzer/Prosize report for D13-2453 gives a final ng/uL concentration of 0.644 that appears to be much lower than is indicated based on the tracing shown earlier in that same report, where a very high yield of fragments is observed.  Any explanations for that?
 
david
 
=============================
On Jun 17, 2013, at 2:14 PM, Pierrick Millet wrote:

Hello,

Please find attached to this email the traces of your samples.

To clarify:

D13-2443 = G90-M1
D13-2444 = G90-M2
D13-2445 = G90-M3
D13-2446 = G90-M4
D13-2447 = G91-M1
D13-2448 = G91-M2
D13-2449 = G91-M3
D13-2450 = G91-M4
D13-2451 = G92-M1
D13-2452 = G92-M2
D13-2453 = G92-M3
D13-2454 = G92-M4
D13-2455 = G92-M5
D13-2456 = G92-M6
D13-2457 = G92-M7
D13-2458 = G92-M8
D13-2459 = G92-M9
D13-2460 = G92-M10
D13-2461 = G92-M11
D13-2462 = G92-M12
D13-2463 = G92-M13
D13-2464 = G92-M14
D13-2465 = G92-M15
D13-2466 = G92-M16
D13-2467 = G92-M17
D13-2468 = G92-M18

We are now proceeding with sequencing.

Cordially,
Pierrick

Pierrick Millet
BioMicro Center
Massachusetts Institute of Technology
31 Ames Street, Building 68-316
Cambridge, MA 02139

Tel: 617-715-4533 
http://web.mit.edu/biomicro
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E.pdf>