The Daily Dongle

FlowJo v765 Mac Command Line

FlowJo v7.6.5 has a nifty command line feature extension for templates. (The FlowJo license allows Mac users to run both our C version (currently v9), and our Java version (currently v7.6.5); the feature sets of the 2 programs being slightly different). 

For repetitive analysis workspaces amenable to templates, the command line feature allows one to use a script to run FlowJo, open a template, and have it load/analyze data, and output reports (layouts and tables). Nice to get your computer to do some work while you are at the ballgame, or home asleep!

 

Here is an example:

1. first set up your FlowJo template.

Autobatch templates

(note: currently Layouts and Tables work in templates, group FCS Export won't execute from templates)

2. On your mac use a text editor to write your shell script to open FlowJo with command line arguments.

-Below is a simple example shell script on my mac to open a FlowJo template, load some data (the template outputs a Table, and a Layout), then flowjo quits.

Screengrab before running the script:

(double click image to make larger)

And, after double clicking the file "flowjo765mac autobatch.command"  

(terminal window opens and runs the script, causing flowjo to open, load the template workspace, load data, export a layout and table, and quit)

 

One can use Mac OSX built in Applescript language and/or Automator to make more complex automation scripts, including Folder actions.

OS X Folder Actions guide

FlowJo Table Options

For many FlowJo power users, FlowJo tables are integral to their flow data analysis. A few users have written into support lately asking how to modify the first column of the table (typically sample name).

 

The Issue:

The default in FlowJo tables is to use the same naming convention as the workspace, plus add numbers before the name. People ask how to get rid of the numbers shown in the picture below:

 

Here is how:

In the table editor, before creating the table, one needs to modify the table creation options (iteration). Instead of using the default (Group tubes by numerical order), one can choose to Group by keyword.

With the Table Editor window open, click the Tables Menu and choose Iteration-Options to open the Batch Iteration Options Dialog Window. (pict below). Set the options to Group tubes by keyword $Fil (or sampleID, or whatever keyword you prefer. $Fil is the standard fcs keyword for the original tube filename saved on the cytometer).

 

More info on Table options:

http://www.flowjo.com/v9/html/tables.html

http://flowjo.typepad.com/the_daily_dongle/2011/09/patient-day-tables-keyword-iteration-.html

 

Layout Batching with a control

Background

Many of our users know about the nice time saving FlowJo feature of batching Layouts with a control (the control doesn't change in each overlay graph). Someone recently asked us about making a batched layout with a control, but wanted to skip the graph that shows the Control tube plotted against the Control tube.

Solution

If you set up your Groups so that your controls are in one group, and your samples are in another (separate from the All-Samples group), then when you batch the layout, choose to iterate only over the tubes in the samples group. (double click the screengrab below for a larger picture)

 

Results:

Comp Matrix file tips

This post deals with compensation matrix file issues for our Mac version and covers:

• Comp matrix file format
• Loading a comp matrix file 
• Comp parameter default naming

 

File Format:

If you save a compensation matrix from flowjo and open the file In a text editor (say TextWrangler), you can see that the values are tab separated with end-of-line CR  (Mac classic). The Parameter names in the text file have to match the parameter names in your FCS data file. (click image below to enlarge)

 

Loading a comp matrix file

From the "Platform : Compensate : Load Matrix Form File" menu item in Flowjo v9, pick your matrix file.

(If someone sent you the matrix file and it is greyed out, the file type may not be TEXT. Some servers will change the HFS filetype. No worries. Open the file in TextWrangler and resave, or use the Terminal unix command SetFile. i.e. SetFile -t TEXT filename

If your matrix list gets long, one can remove an unused matrix from the matrix list in V9.

Comp parameter default naming

By default, FlowJo v9 will name compensated parameters by enclosing the original parameter name with < >. If you want to change the default for a matrix, edit the matrix file changing the " <  >" to what you like, maybe "Comp_"  like in our Java version v7.6.5. 

One can also load a Flowjo v9 matrix file into our Java version, v7.6.5, if it is named with the .mtx extension. 

Patient-Day Tables (Keyword Iteration)

Many Datasets can benefit from tables created using keyword iteration. A couple users have asked us about this lately. Setting up keyword iterated tables is not so intuitive the first time, so lets walk through it together using a dataset containing tubes from multiple patients, collected on multiple days. (Our Advanced Tutorial makes use of this dataset type).

The default in flowjo table batching creates a row for each tube. For a Patient-Day dataset this may look like the graph below. Admittedly, not the most desireable table, with summary stats not applicable.

 

 

More desireable is a table with different rows for each Day, different columns for each Patient, and useful table summary statistics in the last 2 rows. The table below is created by batching with Keyword Iteration.

If your tube files do not have appropriate keyword values, first create a keyword value series in flowjo. With appropriate Day and Patient keyword values for the tubes, open the Table Editor, choose Tables menu > Iteration Options, then set the Iterator to the Day keyword, and the Descriminator to the Patient keyword.

Now, set up the table by dragging nodes from the Workspace into the Table Editor from tubes that have the same Iterator Keyword Value (same Day in this case). Each dragged in node will create a column in a batched table, each change in the Iterator will create a new row in a batched table. (screengrab below, click on to open a larger image)

 

 

Note:

-The table columns use conditionally formatted numbers, in this case set to be red if the number is more than 2 standard deviations from the mean of the numbers in the column. (Conditional formatting gets set up before batching by selecting a row in the Table Editor and clicking the "i" button).

-Layout Editor batching using keyword iteration works in an analogous manner.

-The screengrabs are from our Mac version 9.3.3, but everything works similar in our Java version 7.6.x that runs on both Windows and MacOSX. 

FlowJo high resolution graphics with Powerpoint 2011

For FlowJo v9:

-copy from the layout, then in ppt2011 choose paste-special (ctrl-command-v).

If you are missing the y-axis label on the graph, this means you pasted instead of doing the paste-special thing. I guess ppt2011 thinks FlowJo graphs are special. If you still have ppt2004, normal paste will do just fine, and one can even right click on the pasted graph and ungroup. Later versions of ppt removed this nifty feature.

If you have dotted (or otherwise non-solid line) histogram plots, make sure to uncheck the FlowJo Output Preference for "Copy histograms as polygons". Otherwise the pasted image will only have solid lines.

 

For FlowJo v7.6.4 (our current Java version):

-save the FlowJo layout with a filename that ends with ".emf" (layout1.emf), then in ppt choose "Insert Picture from File" to get a nice high resolution graphic from FlowJo into ppt. The default file format can be set in the File Format Preference.

 

Patient-Time data

Ever want to look at how a statistic varies Day-to-Day from Patient-to-Patient on a single plot as shown in the graph below? One strategy is to use FlowJo v9, combining the use of keyword formulas and concatenation.

The dataset below contains a T-cell panel for two patients with tubes run on different days. The tube files contain keywords for Patient and Day (Our Advanced Tutorial Dataset). Gates were made in the workspace to get the frequency of CD4+CD8+ T-cells.

To make the graph:

- Create a keyword from the Freq of CD4+CD8+ statistic.
(see http://flowjo.typepad.com/the_daily_dongle/2011/07/focus-group.html)

-Concatenate the files, creating additional parameters for the Day, Patient, and CD4+CD8+ keywords.
(see http://flowjo.typepad.com/the_daily_dongle/2008/06/how-the-new-concatenation-works.html)

-Load the concatenated file back into the workspace, and display CD4+CD8+ vs Day.

-Switch the default pseudo-color plot to Polychromatic, assigning a color to the Patient parameter.

Or, another way to visualize and gate on the data:

Focus Group

A user wrote in to support the other day, wanting to select a group of samples (out of hundreds in the workspace) based on a Statistic. Many savvy FlowJo users know they can create Groups of samples based on Keyword criteria or staining protocol. What about creating a Group based on a Statistic (or formula)? 

One can do this in Flowjo v9 in the Workspace using Keywords and FJML formulas, and/or in the Table Editor.

Creating a Focus Group with a Keyword defined by a statistic

-Edit a newly created Keyword by double clicking the first cell in the Keyword column. Define the Keyword with an FJML statistical formula (begin the keyword with FJML=).

For example, One may want to create a Keyword based on the Statistic: percent CD4 CD8 double positive T cells. Once the gates and stat nodes are created, one easy way to get the FJML formula is to drag the stat for CD4+CD8+ into the Layout Editor, double click the stat in the LE to see the FJML. Copy/paste the formula into the Workspace Keyword:

 
 

For an overview of Keyword formulas, please see these previous posts: 

http://flowjo.typepad.com/the_daily_dongle/2011/01/fjml-keyword-examples.html

http://flowjo.typepad.com/the_daily_dongle/2010/05/fjml-in-fj-mac.html

 

Once the Keyword value is created for the first sample, Copy it to the other samples using Workspace menu > Copy Value to Group

Now, one can create a new (Focus) Group based on the desired criteria:

 

 

Creating a Focus Group from within a Flowjo Table

Create the Table, double click the stat column title to sort the column, select the samples of interest, click Create Group from Selected Samples: