phenoDist Cell-based High-throughput Screening

Intensity(reporter) based screen Plate reader (intensity readout)

120 Cell seeding Mini. reaction

Image based screen

Microscope (image readout) Hit Identification

Negative control Sample Difference intensity screen 100 500 400

image screen ??

Image quantification

features features cell cell size shape int. ... size shape int. ...

1 50 0.3 650 ... 1 30 0.67 430 ... ?? 2 64 0.8 800 ... 2 40 0.4 788 ...

......

Dimension reduction Phenotypic distance

- 50 0.3 650 ... - 30 0.25 1200 ... 0.70

Distance Calculation Phenotypic Distance Methods

Computation steps

Tanaka 2005 Young 2008 Perlman 2004 Fuchs 2010 Loo 2007 1. Feature selection

Supervised SVM weight 2. Data Transformation PCA Factor analysis KS statistic classification vector

3. Distance calculation Phenotypic Distance via Classification

Negative control Sample

How similar/different are these two cell populations?

How easily can we separate the cell populations from each other, when they are mixed? Quality Control

Replicate correlation Replicate similarity Control separation 8 between replicates 25 RLUC 0.95 between non-replicates UBC CLSPN 20 0.90 6 0.85 15 4 0.80 Density Density 10 0.75

RLUC 2 5 0.70 Phenotypic strength (replicate 2) UBC CLSPN 0.65 0 Sample 0 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.6 0.7 0.8 0.9 Phenotypic strength (replicate 1) Phenotypic distance Phenotypic strength Hit Identification

0.95 PLK1 0.90 0.85 0.80 0.75 RLUC 0.70 Phenotypic strength (replicate 2) UBC

CLSPN 0.95 0.65 Sample COPB2 0.60 0.65 0.70 0.75 0.80 0.85 0.90 Phenotypic strength (replicate 1) 0.90 RLUC 0.85

UBC 0.80 0.80 0.85 0.90 AKAP7 PANK4 CLSPN Hit Identification with Phenotypic Similarity

RAC1 PINK1 MAP3K3 PIK3R4 PRPSAP2

GK MRC2 PRKY PRKDC MPZL1

MGC5601 PDGFRB PANK4 PRKCH MAPKAPK3

PAK6 PACE-1 NTRK1 PMVK EXOSC10

PLK1 PKM2 PKIB CDKL5 GUCY2D

GTF2H1 AKAP7 GCK ADRBK1 ADRB2

COPB2 ERBB4 ABI1 TESK2 STK39 Hit Identification with Phenotypic Similarity

RAC1 PINK1 MAP3K3 PIK3R4 PRPSAP2

GK MRC2 PRKY PRKDC MPZL1

MGC5601 PDGFRB PANK4 PRKCH MAPKAPK3

PAK6 PACE-1 NTRK1 PMVK EXOSC10

PLK1 PKM2 PKIB CDKL5 GUCY2D

GTF2H1 AKAP7 GCK ADRBK1 ADRB2

COPB2 ERBB4 ABI1 TESK2 STK39 Hit Identification with Phenotypic Similarity

RAC1 PINK1 MAP3K3 PIK3R4 PRPSAP2

GK MRC2 PRKY PRKDC MPZL1

MGC5601 PDGFRB PANK4 PRKCH MAPKAPK3

PAK6 PACE-1 NTRK1 PMVK EXOSC10

PLK1 PKM2 PKIB CDKL5 GUCY2D

GTF2H1 AKAP7 GCK ADRBK1 ADRB2

COPB2 ERBB4 ABI1 TESK2 STK39 Hit Identification with Phenotypic Similarity

CDKL5 ABI1

PKM2 RAC1 MAP3K3 GK COPB2 AKAP7 NTRK1

PMVK

MRC2 PINK1 MPZL1 ADRBK1 PLK1

PIK3R4 ACAD10 PDGFRB PRPSAP2 TESK2 GTF2H1 Hit Identification with Phenotypic Similarity

RAC1 MAP3K3 GK

CDKL5 ABI1

PKM2 RAC1 MAP3K3 GK COPB2 AKAP7 NTRK1

PMVK

MRC2 PINK1 MPZL1 ADRBK1 PLK1

PIK3R4 ACAD10 PDGFRB PRPSAP2 TESK2 GTF2H1

PLK1 PMVK MPZL1 MRC2 TESK2 ADRBK1

PIK3R4 PDGFRB PRPSAP2 ACAD10 PINK1 GTF2H1 Future Development

Expand to compound screening analysis e.g., dosage responsive curve, IC50 value

1.0 0.9 0.8 0.7

0.6 Phenotypic strength 0.5 Increasing concentration Developed by

Xian Zhang Greg Pau Michael Boutros Wolfgang Huber

Division Signaling and Functional Genomics Genome Biology Unit German Cancer Research Center EMBL