Data uploaded

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Ross, David J. (Fed)

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Jan 20, 2017, 11:36:31 AM1/20/17
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The new data set for NIST Gaithersburg is uploaded.

We actually took the data a week ago, but we had a proposal deadline yesterday, so I didn’t get to uploading until today.

 

Let me know if you’ve got any comments or questions.

 

Thanks-

Jake Beal

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Jan 20, 2017, 11:39:12 AM1/20/17
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Thanks, David; I'll try to take a look before we meet on Monday.

Thanks,
-Jake


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Ross, David J. (Fed)

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Jan 20, 2017, 12:23:36 PM1/20/17
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Jake-

If you do get to it, but only have limited time, you might want to take a quick look at the number of cell counts and only analyze data for one of each strain.

We had two cultures that seemed to be at low densities. One of them was in plate 1 (EV, I think) and the other in plate 2 (1091, I think).


Thanks-



From: sbsc-flow...@googlegroups.com <sbsc-flow...@googlegroups.com> on behalf of Jake Beal <jake...@gmail.com>
Sent: Friday, January 20, 2017 11:39 AM
To: sbsc-flow...@googlegroups.com
Subject: Re: [sbsc-fc] Data uploaded
 
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Jake Beal

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Jan 23, 2017, 11:25:27 AM1/23/17
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Hi, David:

I've run the analyses, which we can talk about in detail at the meeting later today. For now, I will share with everybody my current summary of behavior across all three datasets that I have analyzed.

In short: the standard deviation for strong green cells is at our target level, while all else is worse.

The imprecision appears to come primarily from issues in the biological side of the protocol, as evidenced by the large intra-group differences between single-color and dual-color expression.  This, in turn, affects my ability to compare single-red as well, since I am using the dual-color controls for channel-to-channel conversion.

This further confirms one of our basic suppositions, that unit-scaling protocols that depend on culturing cells are highly unreliable, as well as further motivating the need to ship a prepared set of sample around for everybody to run through their cytometers.

Thanks,
-Jake


On Fri, Jan 20, 2017 at 11:23 AM, Ross, David J. (Fed) <david...@nist.gov> wrote:

Jake-

If you do get to it, but only have limited time, you might want to take a quick look at the number of cell counts and only analyze data for one of each strain.

We had two cultures that seemed to be at low densities. One of them was in plate 1 (EV, I think) and the other in plate 2 (1091, I think).


Thanks-




Sent: Friday, January 20, 2017 11:39 AM

Subject: Re: [sbsc-fc] Data uploaded
Thanks, David; I'll try to take a look before we meet on Monday.

Thanks,
-Jake

On Fri, Jan 20, 2017 at 10:36 AM, Ross, David J. (Fed) <david...@nist.gov> wrote:

The new data set for NIST Gaithersburg is uploaded.

We actually took the data a week ago, but we had a proposal deadline yesterday, so I didn’t get to uploading until today.

 

Let me know if you’ve got any comments or questions.

 

Thanks-

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Nicholas DeLateur

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Jan 23, 2017, 11:27:22 AM1/23/17
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Brian and I are at a conference 4-5; will write up a message tomorrow. 

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Jake Beal

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Jan 23, 2017, 11:29:41 AM1/23/17
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Can you give me a one-word summary status answer now, though: are you ready to send samples around?

Thanks,
-Jake


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Ross, David J. (Fed)

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Jan 23, 2017, 2:29:12 PM1/23/17
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Jake-

Questions I will have for follow-up discussion:

What exactly are the quantities that you are plotting the mean-fold-error and geometric mean of? A short formula and definitions of terms would be helpful.

How are you subtracting background?

Do you determine geometric mean by taking the average of the log of the quantity? Or by “fitting” the data to a log-normal distribution with something like a maximum likelihood or Bayesian approach?

 

Also, in general, I’d like to see more explicit written statements about the plans for the working group and related interlabs to facilitate better understand among the different labs regarding the goals and expected level of commitment. For someone like me, coming in to the group later than the rest of you, I feel like there is a lot of background that I am missing.

 

Thanks-

David

 

 

 

From: sbsc-flow...@googlegroups.com [mailto:sbsc-flow...@googlegroups.com] On Behalf Of Jake Beal
Sent: Monday, January 23, 2017 11:25 AM
To: sbsc-flow...@googlegroups.com
Subject: Re: [sbsc-fc] Data uploaded

 

Hi, David:

 

I've run the analyses, which we can talk about in detail at the meeting later today. For now, I will share with everybody my current summary of behavior across all three datasets that I have analyzed.

 

In short: the standard deviation for strong green cells is at our target level, while all else is worse.

 

The imprecision appears to come primarily from issues in the biological side of the protocol, as evidenced by the large intra-group differences between single-color and dual-color expression.  This, in turn, affects my ability to compare single-red as well, since I am using the dual-color controls for channel-to-channel conversion.

 

This further confirms one of our basic suppositions, that unit-scaling protocols that depend on culturing cells are highly unreliable, as well as further motivating the need to ship a prepared set of sample around for everybody to run through their cytometers.

 

Thanks,

-Jake

 

On Fri, Jan 20, 2017 at 11:23 AM, Ross, David J. (Fed) <david...@nist.gov> wrote:

Jake-

If you do get to it, but only have limited time, you might want to take a quick look at the number of cell counts and only analyze data for one of each strain.

We had two cultures that seemed to be at low densities. One of them was in plate 1 (EV, I think) and the other in plate 2 (1091, I think).

 

Thanks-

 

From: sbsc-flow...@googlegroups.com <sbsc-flow...@googlegroups.com> on behalf of Jake Beal <jake...@gmail.com>
Sent: Friday, January 20, 2017 11:39 AM
To: sbsc-flow...@googlegroups.com
Subject: Re: [sbsc-fc] Data uploaded

 

Thanks, David; I'll try to take a look before we meet on Monday.

 

Thanks,

-Jake

 

On Fri, Jan 20, 2017 at 10:36 AM, Ross, David J. (Fed) <david...@nist.gov> wrote:

The new data set for NIST Gaithersburg is uploaded.

We actually took the data a week ago, but we had a proposal deadline yesterday, so I didn’t get to uploading until today.

 

Let me know if you’ve got any comments or questions.

 

Thanks-

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Ariel Haim Hecht

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Jan 23, 2017, 2:36:24 PM1/23/17
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I think those are great questions Dave. A few more that popped into my mind while looking at the data:

What is the meaning of the y-axis units “log 10 MEFL”? What’s meaning of plotting RFP values in MEFL? And I thought we were going to move away from using MEFL units since there is no fluorescein in these beads?
What do you mean by channel-to-channel conversion?
What do you mean by “dual-color controls”?

And I’ll second Dave’s last comment.

Thanks,
Ariel


John Sexton

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Jan 23, 2017, 2:46:07 PM1/23/17
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I'm going to take this opportunity to add my 2nd round of data analysis to the discussion (see attached). I'd appreciate some time to go over it during our meeting today.

This analysis only looks at GFP as a fluorescence source (i.e. strains harboring pL2f1091). I attempted to gate samples as best as I could and then plot RFI values ("gfp_rfi.png") and, when possible, MEF values ("gfp_mef.png"). No spectral compensation has been attempted yet.

As a reminder, the following light paths should have matching excitation and emission characteristics: LP06, LP35, LP48. Additionally, the following light paths have different emission characteristics, but may be candidates for spectral compensation: LP17 (LP12), LP20, LP36, LP49.

Code for this anaylsis currently exists at "2016_mini_interlab/data_analysis/20170104_JS_delateur_sebastian_nist".
  • I used arithmetic mean as the summary statistic for a sample population (I will argue that the geometric mean and the arithmetic mean should be mathematically related if I did a good job of gating and isolating log-normal cell distributions, though).
  • Bars represent the arithmetic mean of multiple replicates, and error bars represent the standard deviation of the population arithmetic means if multiple samples were available.
  • I also generated a bunch of plots to visualize gating and MEF calibration function calculations for beads and samples in the "plot_beads" and "plot_samples" folders for anyone who's interested in those details. File names will typically reference the sample or bead UID from the master spreadsheet: "20170104_JS_delateur_sebastian_nist.xlsx".
Some initial conclusions:
  • data corroborates claims made in the FlowCal paper that calibrating samples against beads accounts for changes in instrument detector voltage
    • Nicholas's green samples from 2 different voltages collapse nicely collapse to same MEF value.
    • The NIST Gaithersburg and NIST Stanford data sets at different voltages generally collapse down to common MEF values. A lot of variability exists between the NIST Gaithersburg samples, though.
  • the "trustworthy" (see gating) NIST Gaithursburg samples actually seem to collapse to MEF values which are pretty similar to the NIST Stanford MEF values!
  • the MIT samples ("DeLateur") and the NIST samples collapse to MEF values which are in the same ballpark (~3x off I think?), but not perfect
-John

joint_fluorescence_analysis.png
gfp_rfi.png
gfp_mef.png

Nicholas DeLateur

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Jan 23, 2017, 3:27:32 PM1/23/17
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Yes

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Jake Beal

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Jan 23, 2017, 4:00:21 PM1/23/17
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Thank you, Nicholas; I will look forward to the full report tomorrow!

Thanks,
-Jake


Yes

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