immunological bioinforma cs automated analysis of facs data
TRANSCRIPT
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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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!