First Pass at Data Analysis

[John Sexton]

Hello all,

I took a first pass at doing a joint analysis on data from the DeLateur experiment, the NIST Gaithersburg experiment, and the NIST Stanford experiment. I only looked at pL2f1091 (GFP) strains for now, and I did not attempt to calibrate any of the data yet (so everything is currently in Relative Fluorescence Intensity (RFI) units). Even at this preliminary stage, there are some topics I'd like to highlight and discuss. Details are included below and stored in a file at "2016_mini_interlab/data_analysis/20170104_JS_delateur_sebastian_nist/notes.md". If we have time, I'd like to go over it during the meeting today. See y'all soon.

-John

Notes on RFI results via FlowCal Excel UI

=========================================

* NIST Gaithersburg and NIST Stanford need to employ better SSC thresholds

  * event lists are dominated by non-cell events

* center beads in FSCvSSC

  * FSC and SSC channels are independent of fluorescence channels; we can

    change the voltage as needed to make beads and cells appear cleanly in FSC

    and SSC while not affecting fluorescence channels (which need to be

    consistently measured at the same voltages between beads and samples)

  * NIST Stanford beads appear to be saturated or near saturated in SSC

  * DeLateur beads appear to be saturated or near saturated in FSC

* area ("-A") vs. height ("-H") for event quantification

  * "-A" works "better" for DeLateur beads

  * -H works "better" for NIST Gaithersburg and NIST Stanford beads

    * harder to say for samples. Comparing Arithmetic Mean of samples measured

      via "-A" and "-H" resulted in *very* similar values, though.

* looks like NIST Stanford doesn't have BL2 (LP0049) as originally thought

* cautiously optimistic / excited because I think there's signal in all the

  places where we wanted signal. Going to take some gating magic to clean

  everything up, but I think it can be done.

  * DeLateur pL2f1091 (green) FlowCal Excel UI Sample IDs:

    * DL007 - DL009

    * DL019 - DL021

    * DL031 - DL033

    * DL043 - DL045

    * DL055 - DL057

    * DL067 - DL069

    * DL079 - DL081

    * ^^^ all show decent, clean, HIGH signal

  * NIST Gaithersburg pL2f1091 (green) FlowCal Excel UI Sample IDs:

    * -A

      * plate 1

        * NG012 - don't see anything

        * NG046 - don't see anything

        * NG080 - don't see anything

      * plate 2

        * NG029 - minor secondary HIGH population on BL1-A

        * NG063 - *very* minor secondary HIGH population on BL1-A. Can't tell

                  about BL2-A.

        * NG097 - can't tell on BL1-A.  Minor secondary peark on BL2-A.

    * -H

      * plate 1

        * NG114 - see *very* minor secondary HIGH population in BL1-H

        * NG148 - see *very* minor secondary HIGH population in BL1-H and BL2-H.

                  BL2 is lower than BL1.

        * NG182 - see *very minor secondary HIGH population on BL2-H.

      * plate 2

        * NG131 - see clear minor secondary HIGH population in BL1-H.

        * NG165 - see clear minor secondary HIGH populations in BL1-H and BL2-H.

        * NG199 - see clear minor secondary HIGH populations in BL1-H and BL2-H.

  * NIST Stanford pL2f1091 (green) FlowCal Excel UI Sample IDs:

    * -A

      * NS_013, NS_017, NS_021

      * NS_037, NS_041, NS_045

      * NS_061, NS_065, NS_069

    * -H

      * NS_085, NS_089, NS_093

      * NS_109, NS_113, NS_117

      * NS_133, NS_137, NS_141

    * ^^^ all show minor secondary LOW peak which is likely non-cell events

      and primary HIGH peak which is expected GFP signal

    * density gating works better on -H FSCvSSC    

[Jake Beal]

Adding to John's work, my own TASBE-based analysis shows we've got pretty good agreement between NIST Stanford and MIT.

Thanks,

-Jake

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