immunological bioinforma cs automated analysis of facs data

Immunological  Bioinforma/cs  

Automated  analysis  of  FACS  data  

Ali  Al9ntaş  

(slides  from  Juliet  Frederiksen)  

Overview

1.  Introduc/on  to  Flow  Cytometry    

2.  Automated  analysis  of  Flow  Cytometry  data  

Introduc/on  to  Flow  Cytometry  

Quick  introduc/on  to  Flow  Cytometry  

First commercial flow cytometer: PDP 11 computer (1969)

Let’s start with a little walk down memory lane…

Introduc/on  to  Flow  Cytometry  

Schematic of a modern Flow Cytometer

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Figure  adapted  from  Introduc2on  to  Flow  Cytometry  Data  Analysis,  Invitrogen    

Introduc/on  to  Flow  Cytometry  

At the interrogation point…

Introduc/on  to  Flow  Cytometry  

Up close and personal…

Introduc/on  to  Flow  Cytometry  

Case story: You want to study the state of HIV infected individuals immune system. To do this you identify 8 cellular markers (antigens) that would give you a good representation of what you want to study. For each of these 8 markers you find fluorochrome-labelled mAbs. Then the “level” of each colour is measured on each cell as it passes by the laser.

Introduc/on  to  Flow  Cytometry  

The types of signal detected by the flow cytometer

Figure  adapted  from  Introduc2on  to  Flow  Cytometry  Data  Analysis,  Invitrogen    

Introduc/on  to  Flow  Cytometry  

Schematic of a modern Flow Cytometer

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-.&/*01(23$%*

Figure  adapted  from  Introduc2on  to  Flow  Cytometry  Data  Analysis,  Invitrogen    

Introduc/on  to  Flow  Cytometry  

Filters

Figure  adapted  from  Introduc2on  to  Flow  Cytometry,  Abcam    

BP  =  Band  pass  

SP  =  Short  pass  

LP  =  Long  pass  

Dichroic  =  45  degree  angle  

Figure  from  Flow  Cytometry  -­‐  A  Basic  Introduc2on,  Michael  G  Ormerod      

Introduc/on  to  Flow  Cytometry  

More details

Figure  adapted  from  Introduc2on  to  Flow  Cytometry  Data  Analysis,  Invitrogen    

Introduc/on  to  Flow  Cytometry  

Spectral Overlap

Introduc/on  to  Flow  Cytometry  

Compensation

Spillover Fluorescence

FL-1 FL-2

A - 17%

B 6% -

Introduc/on  to  Flow  Cytometry  

Now comes the interesting stuff!

Introduc/on  to  Flow  Cytometry  

What does the output from the flow cytometer look like?

Introduc/on  to  Flow  Cytometry  

What does the output from the flow cytometer look like?

Introduc/on  to  Flow  Cytometry  A

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What do you do with the output?

Quick  introduc/on  to  Mass  Cytometry  

A  deep  profiler's  guide  to  cytometry,  Trends  Immunol.,  Bendall  et  al.  

Automated  FCM  data  analysis  

Quick  introduc/on  to  Flow  Cytometry  

Let’s start with a little walk down memory lane…

A  deep  profiler's  guide  to  cytometry,  Trends  Immunol.,  Bendall  et  al.  

Automated  FCM  data  analysis  

Typical scenario with FCM data analysis…

Automated  FCM  data  analysis  

Tools available on internet

•  NetFCM •  SPADE •  SPICE •  FLOCK •  flowCore

Automated  FCM  data  analysis  

Let’s check out NetFCM

What is NetFCM? An online tool for the analysis of FCM data sets. What is the input to NetFCM? Compensated FCS files, marker information and gating strategy.

Automated  FCM  data  analysis  

Let’s check out NetFCM

What type of analysis does NetFCM perform? (1)   Principle component analysis

Automated  FCM  data  analysis  

Let’s check out NetFCM

What type of analysis does NetFCM perform? (2) K-means clustering

Automated  FCM  data  analysis  

Let’s check out NetFCM

What type of analysis does NetFCM perform? (3) Extreme Value Detection

Automated  FCM  data  analysis  

Let’s check out FLOCK

Automated  FCM  data  analysis  

Let’s check out FLOCK

FLOCK is an online tool that uses a density-based clustering approach to algorithmically identify biologically relevant cell populations.

Automated  FCM  data  analysis  

Let’s check out FLOCK

Automated  FCM  data  analysis  

http://imcportal.org/education/video/martin-zand-visualization-of-high-dimensional-data

Automated  FCM  data  analysis  

An overall pipeline for R/Bioconductor

Figure  adapted  from  Introduc2on  to  Flow  Cytometry  Analysis  in  R  by  Ryan  Brinkman  (17.06.13)  

Exercise  /me!  

Let’s  do  some  analysis!