Immunological Bioinforma cs Automated analysis of FACS data
Transcript of Immunological Bioinforma cs Automated analysis of FACS data
![Page 1: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/1.jpg)
Immunological Bioinforma/cs
Automated analysis of FACS data
Ali Al9ntaş
(slides from Juliet Frederiksen)
![Page 2: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/2.jpg)
Overview
1. Introduc/on to Flow Cytometry
2. Automated analysis of Flow Cytometry data
![Page 3: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/3.jpg)
Introduc/on to Flow Cytometry
![Page 4: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/4.jpg)
Quick introduc/on to Flow Cytometry
First commercial flow cytometer: PDP 11 computer (1969)
Let’s start with a little walk down memory lane…
![Page 5: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/5.jpg)
Introduc/on to Flow Cytometry
Schematic of a modern Flow Cytometer
!"#$%%&'()&"*+&,"#*
-.&/*01(23$%*
Figure adapted from Introduc2on to Flow Cytometry Data Analysis, Invitrogen
![Page 6: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/6.jpg)
Introduc/on to Flow Cytometry
At the interrogation point…
![Page 7: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/7.jpg)
Introduc/on to Flow Cytometry
Up close and personal…
![Page 8: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/8.jpg)
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.
![Page 9: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/9.jpg)
Introduc/on to Flow Cytometry
The types of signal detected by the flow cytometer
Figure adapted from Introduc2on to Flow Cytometry Data Analysis, Invitrogen
![Page 10: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/10.jpg)
Introduc/on to Flow Cytometry
Schematic of a modern Flow Cytometer
!"#$%%&'()&"*+&,"#*
-.&/*01(23$%*
Figure adapted from Introduc2on to Flow Cytometry Data Analysis, Invitrogen
![Page 11: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/11.jpg)
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
![Page 12: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/12.jpg)
Introduc/on to Flow Cytometry
More details
Figure adapted from Introduc2on to Flow Cytometry Data Analysis, Invitrogen
![Page 13: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/13.jpg)
Introduc/on to Flow Cytometry
Spectral Overlap
![Page 14: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/14.jpg)
Introduc/on to Flow Cytometry
Compensation
Spillover Fluorescence
FL-1 FL-2
A - 17%
B 6% -
![Page 15: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/15.jpg)
Introduc/on to Flow Cytometry
Now comes the interesting stuff!
![Page 16: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/16.jpg)
Introduc/on to Flow Cytometry
What does the output from the flow cytometer look like?
![Page 17: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/17.jpg)
Introduc/on to Flow Cytometry
What does the output from the flow cytometer look like?
![Page 18: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/18.jpg)
Introduc/on to Flow Cytometry A
n e
xam
ple
of a
m
anu
al g
atin
g s
tra
teg
y
What do you do with the output?
![Page 19: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/19.jpg)
Quick introduc/on to Mass Cytometry
A deep profiler's guide to cytometry, Trends Immunol., Bendall et al.
![Page 20: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/20.jpg)
Automated FCM data analysis
![Page 21: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/21.jpg)
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.
![Page 22: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/22.jpg)
Automated FCM data analysis
Typical scenario with FCM data analysis…
![Page 23: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/23.jpg)
Automated FCM data analysis
Tools available on internet
• NetFCM • SPADE • SPICE • FLOCK • flowCore
![Page 24: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/24.jpg)
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.
![Page 25: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/25.jpg)
Automated FCM data analysis
Let’s check out NetFCM
What type of analysis does NetFCM perform? (1) Principle component analysis
![Page 26: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/26.jpg)
Automated FCM data analysis
Let’s check out NetFCM
What type of analysis does NetFCM perform? (2) K-means clustering
![Page 27: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/27.jpg)
Automated FCM data analysis
Let’s check out NetFCM
What type of analysis does NetFCM perform? (3) Extreme Value Detection
![Page 28: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/28.jpg)
Automated FCM data analysis
Let’s check out FLOCK
![Page 29: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/29.jpg)
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.
![Page 30: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/30.jpg)
Automated FCM data analysis
Let’s check out FLOCK
![Page 31: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/31.jpg)
Automated FCM data analysis
http://imcportal.org/education/video/martin-zand-visualization-of-high-dimensional-data
![Page 32: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/32.jpg)
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)
![Page 33: Immunological Bioinforma cs Automated analysis of FACS data](https://reader034.fdocuments.net/reader034/viewer/2022051713/586e1d111a28ab633c8beda9/html5/thumbnails/33.jpg)
Exercise /me!
Let’s do some analysis!