The Daily Dongle

Instant Movie of Your Data... Easy

You can view a FlowJo graph or layout in movie form. It's quick and easy, it won't hurt the data, and you may be surprised at what you see.

To make a movie, you add an additional parameter to the graph then view the data in relation to 30 or so slices of that parameter. If time is already a parameter in your data it will be the default choice.

But don't get bogged down in mere scientific details. The point here is to just try it once and see if you like the perspective.

• Open a graph and set the axes to forward scatter and side scatter.
• Choose psuedocolor as the graph type and put a check in the Smoothing box.
• From the Graph menu select Make Movie. You will see the dialog below.

• Select a parameter to be your "control."
• Select the Display accumulates..." button to show the data accumulating as more channels of the control parameter are displayed in the movie frames. The Show movie... option is selected by default. You DO have the QuickTime player installed, don't you?
• Click on Make Movie choose a name and directory and wait a moment for the frames of your movie to be created and displayed.

Other graph types make equally interesting movies. You need not choose psuedocolor. The first check box toggles between showing an equal number of the control parameter's channels per frame and and showing an equal number of events per frame. Try both...

You can scrub through the movie by moving the "play head" triangle back and forth along the time line. You can also scrub the movie by clicking or pressing and holding the arrow keys on the keyboard.

You can view the movie I made as I wrote this here.

For all the movie-making details, you can read the Web docs.

batching stats in layouts - with help of grids

Just like there are many ways to immunophenotype feline subcutaneous preps (skin the cat, as some call it) - there are many ways for the FlowJo V7 users to workaround on the missing FlowJo V8 (mac)  features by using existing good ol' tricks ;)

The V8 layout editor allows users to define little stat tables along side plots, which will iterate and batch like chart elements. V7 doesn't have that yet but you can achieve identical functionality by using the grid tool:

What's going on in this graph? I used our advanced tutorial data to create some mock-up gating. I overlayed the ungated FSCxCD45 plot with a CD14 FITC+ gate and a lymph/CD16+ gate.

The grid underneath the legend shows GM (Geometric Mean) values for the FL1/2/3 channels. I set up the upper row of the grid to automatically fetch the $P#S keywords for those channels. That way if the next file has CD3/4/8, the graph will be automatically updated (as will the stats)

So what did I put into the grid that it automatically renders the values? In the upper row, I inserted some text boxes. I made a textbox using the "A" tool in the layout. You can then select a keyword from the keyword list pulldown to choose one of the $P#S keywords:

performing this action inserts this text string:

<Key dataset="1" originalDataset="1" name="$P3S"/>

You can then clone the first textbox by alt-dragging the first one as many times as you need labels. Then double click each one and edit the number to be $P4S, $P5S.. etc..

So how about the bottom column of the grid?

For that I dragged pre-existing stat nodes from the workspace window to the layout. usually you end up with something like this:

(Cy5PE) CD45 : Geom. Mean: 115.39

Which is too much info. I know what channel and stat I chose. How do I change this to just show me the number? Double click the stat:

(Cy5PE) <Key dataset="1" originalDataset="1" name="$P5S"/> : Geom. Mean: <Statistic dataset="1" originalDataset="1" path="ForSc, OrthSc subset/Cy5PE+" name="Geom. Mean" id="Cy5PE"/>

So that you can see it, I highlighted the number-only part in BOLD. Get rid of the rest of it and it'll become just the number.

Then just drag the different textboxes into the grid. This is the worskpace I used to make this demo.. you can use it with the tutorial data linked above, in V7.1.3 and higher.

Download layout_stats.wsp

Graph Sets

FlowJo layout editor has an option to create multiple graph combinations from a right click menu:

For example selecting "histograms only" or "all parameters in order" would look like the attached graphics (click to view)
histograms_only.png

all_in_order.png

This is a pretty sweet timesaver when working on new templates. Just make all plots and delete ones you don't want.

Another useful trick is storing graph sets. Imagine you drag a plot into layout and with a few mouse clicks you can turn it into this (or any other pre-defined graph template):

here's how to do define a "graph set":

• drag some gate into the layout
• right-click, make multigraph overlay
• then for each plot you'd like to add, right-click, multigraph overlay, add plot.
• you can style each plots size, type, axis controls, fonts, foreground/background colors...
• once it's done, right-click, name and save this setup (will make an .xml file somewhere on your disk)

here's how to apply a graph set:

• drag some gate into the layout
• right-click, make multigraph overlay
• right-click, use named setup - find the xml file defined above, and voila.

controlling # of events displayed- is it necessary?

Not all FCS files are created equal. In the context of this entry - number of events can vary. Some folks acquire a set number of events total, some count things in a gate, some count seconds. In the end you have some uniform method for acquiring a "sample".

Let's leave the statistics of population comparison to the experts and focus on the visualization part. Few times a month some of you call and ask,

I'm comparing a sample with X events to a sample with Y events, but the graphs look weird side by side. How can I fix it?

The first suggestion is to use a count independent display type. I would suggest considering the contour plot (or zebra, or smoothed pseudocolor, or density). Many software packages include it, and it is popular because it creates form out of the density trend.

But those are just words. How about a picture. We acquired files with 10 fold increases from 10 to 1M events. Shown below as ungated histogram of CD3 and the CD3 gate displayed as CD4 vs CD8:

Except the first two, the pattern is almost indistinguishable from one plot to the other. Except the double-positive population. This little demonstration was used on the cover of Dr. H. Shapiro's Practical Cytometry 4th edition.

*update 9/13*
I've updated the graphic with the 1M (bottom)plot. I also saved the workspace and the FCS files in this zip file for anyone wanting to explore it. You will need FlowJo 8.2b8 or higher, http://treestar.com/beta.htm

Download counts.zip

 

Despite this simple and elegant solution, many users want to simply set the number of events displayed on the plot to be the same. In FlowJo this option is only viable via the Dot Plot graph, in which you can control number of events shown.

How - PC:
while in the graph window, expand "Options" and enter a number in the "Num. Dots:" filed. numbers between 1-100 will be interpreted as PERCENTAGE OF TOTAL EVENTS. Numbers 101+ will be interpreted as NUMBER OF EVENTS. Then click Edit, Save to Preferences.

How - Mac:
best to do thru preferences. Click FlowJo menu, Preferences,  Graphs tab-  and change the options to what you want in the "dot plots" area.

What about if you don't want to use dotplots or contours?  here are your options:

gate on event# or time parameter (histogram):
you can squeeze in a gate on top of your gate-of-interest which will control the number of events downstream by gating on time parameter or event# parameter. This will not give you exact number of events control. It also does not take a uniform sampling of all events in the file, just a cluster based on the order in which events were acquired (that assumes perfectly uniform sample distribution and sample flow)

export specific number of events (mac only, PC too in a few weeks)
you can export a file or a gate, by clicking on it, and choosing "export" from the workspace menu. The export dialog allows you to control number of events. I believe the sampling here is random.

boolean gates getting your mac down?

Exciting news this morning: boolean gates were optimized in 8.2b8 (to be posted today here). How much ?

the exact values were 2.83m vs. 0.33m.

what we are measuring here:

I created a template with a FSC/SSC gate, under which I created 6 histogram gates: CD3+/-, CD4+/-, CD8+/-. I selected all 6 gates and created 64 boolean combinations. I then attached this gate tree to the group and saved as template.

Then I opened the template in 8.1.1 vs. 8.2b8 and added 8 files with 200K events and measured the time required to calculate all gates.

If you are new too bolean gates, don't forget to look at my boolsheet :)

Running FlowJo HASP driver on AMD Opteron 64 bit computers

One of our users sent in this dongley tip for those of you who are lucky enough to work on AMD Opteron 64 bit workstations:

I am running FlowJo 7.1.2 on a dual AMD Opteron 64 bit system.  Unable to install HASP HL drivers out of the box.  This seems to be an issue with AMD Opteron based systems.  Work around (not for the faint of heart as it involves modifying the boot.ini file twice):

Do the following

1.  Locate the boot.ini file in the root of your boot drive, where the Operating System was installed (in most cases, this is C:\). You may need to go to the Tools menu in any Windows folder and enable the Show Hidden Files and Folders option and uncheck Hide Protected Operating System Files under Folder Options.

2   Right click on boot.ini, select Properties and remove the check mark from the Read Only box (if it is checked).

3.   Double click on boot.ini, it should open with wordpad

4.  Locate the line containing:
      multi(0)disk(0)rdisk(0)partition(1)\Windows="Microsoft Windows XP Professional" /fastdetect /noExecute = OptIn
      and change the text to:
multi(0)disk(0)rdisk(0)partition(1)\Windows="Microsoft Windows XP Professional" /fastdetect /Execute

5.  Save and close the file.

6.  Reboot your computer and install FlowJo and the dongle drivers.  If you already have installed Flowjo, you can just uninstall and then re-install the HASP HL driver.  You can confirm that this worked by opening up FlowJo after the installation.

7.  Edit boot.ini to revert back the original as shown in the first line in Step 4. Save the changes and close the file.

8.  Right-click on boot.ini, select Properties and check the Read Only box.

9.  Re-boot your computer.  FlowJo should find the dongle and it should work now.  The LED on the dongle will flash when FlowJo starts up.

These instructions are from the web site:  http://www.guidancesoftware.com/support/articles/installingdongledrivers.asp

FlowJo 7 on OS X vs. parallels vs. native - performance

FlowJo 7 can be executed in numerous ways:

• natively via .exe/virtual machine (diva friendly mode) on windows XP, 2K
• natively via webstart (not diva friendly, java conflict) on windows XP, 2K, OS X
• emulation via parallels on intel macs

so which of these will perform the fastest?

Results:

time is reported in decimal minutes (30s = 0.5m). for a simple analysis, the MacPro webstart performs best, iMac parallels worst.

The webstart version on the iMac performed similarly despite a big difference in CPU speed (3.4GHz dell, 2.0GHz iMac). This may be due to architectural changes. iMac's Core Duo has a 32KB L1 cahce and a 2MB shared L2 cache. Dell's Prescott P4 has a 16KB L1 cache and a 1MB L2 cache. The Mac Pro has a 4MB shared L2 cache per CPU, which may account for the fast performance.

V8 performed about 500% faster when executed natively on the MacPro (compared to Dell WS)

time is reported in decimal minutes (30s = 0.5m). for a complex analysis, the Dell webstart performs best, iMac webstart worst. I cannot explain why the webstart lags behind on the Mac Pro here.

interestingly, the iMac parallels almost matches the Dell..

V8 performed about 750% faster when executed natively on the Mac Pro (compared to Dell WS)


Test Details:

4x4 - A template was created with a set of quad gates on FSCxSSC. each of those quads had a set of quads on FL1 vs FL2.
4x4x4 - a template was create like above, but each of the FL1xFL2 quads had a set of quads on FL3 vs 4.

in both tests, 1GB (282 files, ~250K events each, 4Color, 7 parameters) of Calibur data was dumped into the template.

Mac Pro V7 WS - this was a standard Mac Pro 2.66 GHz with 3GB RAM total. FlowJo was executed via webstart.

Dell  V7 WS - Dell optiplex 280 workstation with a 3.4GHz P4 550 (prescott) CPU, 1GB RAM

Imac V7 WS - standard iMac 20 inch config, 2.0GHz intel core duo, 2GB RAM. FlowJo was executed via webstart.

Imac V7 P - same as above. we installed parallels and windows XP, with a 1.5GB RAM allocation. Data/templates were copied into this emulation and FlowJo was executed via .exe installer.

V8 crash on intel macs related to gating path length

If you use the macintosh version 8.1.1 thru 8.2b4, on an itel mac, you may sometimes expereince a crash in the layout editor when a certain chart is double-clicked. This has to do with the number of characters in the full gating path.

If you experience this problem, try to rename some of the gates using shorter names.

To rename gates: click on a gate, cmd+r. Dialog comes up, type in the new name.
this also works for group-owned gates - but for those, click on the GROUP instance of the gate (upper workspace pane)

FlowJo 7 on OS X via webstart

FlowJo for Windows version 7 can be executed on macintosh computers running OSX. The reason some folks are doing this is because of the lack of common workspace format. For now this only works with a serial number.

1. make sure you're running Tiger OSX. This might work in Panther, but I have not tested it. Run software update to get java 5 (1.5 b 6)
2. go to the windows downloads page. Click on the webstart link. You'll be given the normal Webstart certificate notification and some questions about desktop shortcuts etc.
3. Start the program. You'll be asked to enter a serial number or run in demo mode. If you already have a serial number for V8 on your mac, it'll work. However a SN from another computer will not work - unless it's a site license number and your mac is registered.

what to expect: it may run a little slower then V7 on windows. I'll be capturing this performance in the next few days, updates to follow..