Uncovering Pseudomonas aeruginosa interactions through comprehensive analysis of gene expression data

The bounty of
the commons.
Casey Greene
@GreeneScientist

Image: William R Shepherd

It ain’t what you don’t know
that gets you into trouble. It’s
what you know for sure that
just ain’t so.
- Attributed to Mark Twain

Everybody knows there
are 4 subtypes of HGSC.
… everybody but
Greg.

Tothill et al. Clinical Cancer Research. 2008
One hundred and seventy one tumors consistently
segregated into one of the six k-means clusters.
Most of the remaining tumors (80 of 114) could be
further assigned to one of the molecular subsets by
performing class prediction.
171 clustered cleanly
80 could be assigned
34 ???
12-40% unclear

The Cancer Genome Atlas, Nature. 2011
The silhouette width was computed to ﬁlter out expression proﬁles
that were included in a subclass, but that were not robust
representatives of the subclass. This resulted in the removal of 51
of 135 samples of the Differentiated subclass; 12 of 107 samples of
the Immunoreactive subclass; 0 of 109 samples of the Mesenchymal
subclass; and 13 of 138 samples of the Proliferative subclass..

Uniﬁed Bioinformatics Pipeline
curatedOvarianData
Remove
•  <130 tumors
•  Custom array
technology
Clustering
Analyses
SAM
Overrepresented
Pathways
Survival
Match Clusters
Dataset
Inclusion
Criteria
TCGA
Tothill
Yoshihara
Bonome
Mayo*
Keep
•  Histology
•  High Grade
Sample
Inclusion
Criteria
Gene
Selection
Criteria
Keep
•  1500 MAD
•  Union

Remove
•  <130 tumors
•  Custom array
technology
Clustering
Analyses
SAM
Overrepresented
Pathways
Survival
Match Clusters
Dataset
Inclusion
Criteria
TCGA
Tothill
Yoshihara
Bonome
Mayo*
Keep
•  Histology
•  High Grade
Sample
Inclusion
Criteria
Gene
Selection
Criteria
Keep
•  1500 MAD
•  Union
Uniﬁed Bioinformatics Pipeline
Are HGSC subtypes consistent?

k-Means and NMF are consistent.

Cross-population analysis does
not support four subtypes.

Why didn’t TCGA (2011) ﬁnd this?

Why didn’t Konecny (2014) ﬁnd
this?

What about TCGA’s re-analysis of
Tothill?

What if you re-analyze Tothill
without LMP samples?

What if you re-analyze Tothill
with LMP samples?

Comprehensive cross-population analysis of high-
grade serous ovarian cancer supports no more than
three subtypes
bioRxiv: http://dx.doi.org/10.1101/030239
github: http://github.com/greenelab/hgsc_subtypes

Research is to see what everybody
else has seen and to think what
nobody else has thought.
- Albert Szent-Györgyi
Image by J.W. McGuire/NIH

Image from You Don’t Know Jack. Vol 3.

If you showed 16,000 computers 10 million
images from youtube, what would they see?
Le et al. 2012

•  Pseudomonas aeruginosa compendium
•  > 100 different experiments
•  Many different labs

Analysis with Denoising Autoencoders of
Gene Expression (ADAGE)
Tan et al. Pac Sym Bio 2015; Tan et al. mSystems 2016.

The worst thing that can be said about your
grant is that it's a ﬁshing expedition.
- Tom Blumenthal (@ 2016 Gold Lab Symposium)

- Tom Blumenthal (@ 2016 Gold Lab Symposium)
If you engage in a ﬁshing expedition you can
actually catch ﬁsh.

Image by Peter (stockispicts)

The Transcription Factor Anr
Controls P.a. Response to Low O2
Low O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
O2
Anr
CF Lung Epithelium

Node42 reﬂects Anr Activity
E−GEOD−17179
}wt
}
}
Δanr
Δdnr
E−GEOD−17296
}
}
}
}
}
}
Δanr
ΔroxSR
Δanr
ΔroxSR
wt
wt
}
}
EXP
STAT
O2
E−GEOD−52445
O2
Node42 - Anr Activity
E−GEOD−33160
O2
A
−15 0 10
Value
Color Key Color Key
−10 0 10
Value
Color Key
Value
−10 0 10
−10 0 15
Color Key
Value

New Experiment Validates Node 42’s
Low-O2 Signature
CF lung epithelial cells
Jack Hammond
E−GEOD−17179 E−GEOD−17296
B
}
}
Δanr
wt
}
}Δanr
wt }
}Δan
wt
−5 0 5
Color Key
Value
Color Key
Value
−4 0 4
Color Key
Value
−2 0 2
Microarray RNAseq
PAO1
RNAseq
J215
CE−GEOD−17179
}
}
Δanr
Δdnr
E−GEOD−17296
}
}
}Δanr
ΔroxSR
wt
}STAT
D−52445
D−33160
B
}
}
Δanr
wt
}
}Δanr
wt }
}Δanr
wt
−5 0 5
Color Key
Value
Color Key
Value
−4 0 4
Color Key
Value
−2 0 2
Microarray RNAseq
PAO1
RNAseq
J215
C

Cross-platform normalization of microarray and
RNA-seq data for machine learning applications
Thompson, Tan, Greene. PeerJ.
Jeff Thompson

Cross-platform normalization of microarray and
RNA-seq data for machine learning applications
Thompson, Tan, Greene. PeerJ. 2016

New Experiment Validates Node 42’s
Low-O2 Signature
CF lung epithelial cells
Jack Hammond
E−GEOD−17179 E−GEOD−17296
B
}
}
Δanr
wt
}
}Δanr
wt }
}Δan
wt
−5 0 5
Color Key
Value
Color Key
Value
−4 0 4
Color Key
Value
−2 0 2
Microarray RNAseq
PAO1
RNAseq
J215
CE−GEOD−17179
}
}
Δanr
Δdnr
E−GEOD−17296
}
}
}Δanr
ΔroxSR
wt
}STAT
D−52445
D−33160
B
}
}
Δanr
wt
}
}Δanr
wt }
}Δanr
wt
−5 0 5
Color Key
Value
Color Key
Value
−4 0 4
Color Key
Value
−2 0 2
Microarray RNAseq
PAO1
RNAseq
J215
C
E−GEOD−17179
}
}
Δanr
Δdnr
E−GEOD−17296
}
}
}Δanr
ΔroxSR
wt
}STAT
D−52445
D−33160
B
}
}
Δanr
wt
}
}Δanr
wt }
}Δanr
wt
−5 0 5
Color Key
Value
Color Key
Value
−4 0 4
Color Key
Value
−2 0 2
Microarray RNAseq
PAO1
RNAseq
J215
C
E−GEOD−17179
}Δdnr
E−GEOD−17296
}Δanr
45
60
B
}
}
Δanr
wt
}
}Δanr
wt }
}Δanr
wt
−5 0 5
Color Key
Value
Color Key
Value
−4 0 4
Color Key
Value
−2 0 2
Microarray RNAseq
PAO1
RNAseq
J215
C
E−GEOD−17179
}wt
}
}
Δanr
Δdnr
E−GEOD−17296
}
}
}
}
}
Δanr
ΔroxSR
Δanr
ΔroxSR
wt
}
}
EXP
STAT
−GEOD−52445
−GEOD−33160
B
}
}
Δanr
wt
}
}Δanr
wt }
}Δanr
wt
−5 0 5
Color Key
Value
Color Key
Value
−4 0 4
Color Key
Value
−2 0 2
Microarray RNAseq
PAO1
RNAseq
J215
C

ADAGE analysis of publicly available
gene expression data collections
illuminates Pseudomonas aeruginosa-
host interactions
github: http://github.com/greenelab/adage
Tan, Hammond, Hogan, and Greene. mSystems. 2016

I didn’t want to just know the
names of things. I remember really
wanting to know how it all worked.
- Elizabeth Blackburn
Image: US Embassy Sweden

Activity volcano plot
Fold Change
-log(p)

Pareto activity selection
Fold Change
-log(p)

volcano + networks =
pathway-style analysis

We can measure gene-gene
similarity with ADAGE weights.
…
…

ADAGE similarity captures
functional similarity.
Tan et al. in prep.

ADAGE-based pathway analysis
reveals underlying mechanisms
Tan et al. in prep.

Where do we have (enough) data?
Greene et al. Pac Sym Bio. 2016

ADAGE webserver coming soon!
http://www.greenelab.com/webservers

Open science can be
hypercollaborative.

Open Science != Reproducibility

Computational biology should be
reproducible:
•  Accurately
•  Quickly
•  Without contacting original authors

Cloud computing allows
“Continuous Analysis” at scale.
Research
Cluster

Semi-Supervised Learning of the
Electronic Health Record for Phenotype
Stratiﬁcation
github: http://github.com/greenelab/DAPS
Reproducible computational workﬂows
with continuous analysis
github: http://github.com/greenelab/continuous_analysis

Research Parasite Awards
(The “Parasites”)
Selection criteria for the work in question:
•  The awardee must not have been involved the design of the experiments
that generated the data.
•  The awardee published independently of the original investigators, and the
original investigators are not authors of the secondary analyses but are
appropriately credited in the manuscripts.
•  The awardee may have extended, replicated or disproved what the original
investigators had posited.
•  The awardee has provided source code and intermediate or ﬁnal results in a
manner that enhances reproducibility.

Additional selection criteria for the Junior
Parasite award:
•  The awardee must have published the work at the training stage of their
career (postdoctoral, graduate, or undergraduate). If the awardee has
assumed a position as an independent investigator she or he should not have
been in that position for more than 2 years.
•  The award will be based on work described in a single manuscript
(submitted alongside the nomination letter).

Additional selection criteria for the
Sustained Parasitism award:
•  The awardee must be in an independent investigator position in academia,
industry or public sector.
•  The awardee must be a last or corresponding author on the three
manuscripts submitted alongside the nomination letter.
•  At least a five-year period must have elapsed between the publication of the
first manuscript and the final manuscript.

Details
•  Submit by October 14, 2016 @ 5PM HST
•  Additional Instructions at
http://greenelab.com/parasite-award
•  COI rules are strict! I can only talk about rules.
2017 Selection Committee:

Greene Lab:
Jaclyn Taroni (Postdoc)
Daniel Himmelstein (Postdoc)
Jie Tan (Grad Student)
Gregory Way (Grad Student)
Brett Beaulieu-Jones (Grad Student)
Amy Campbell (Postbacc)
René Zelaya (Programmer)
Matt Huyck (Programmer)
Dongbo Hu (Programmer)
Kathy Chen (Undergrad)
Mulin Xiong (Undergrad)
Tim Chang (Undergrad)
Roshan Ravishankar (Undergrad)

Collaborators:
Deb Hogan & Jack Hammond

Data:
All investigators who publicly release their gene
expression data.

Images:
Artists who release their work under a Creative
Commons license.

Funding:
Gordon and Betty Moore Foundation
National Science Foundation
Cystic Fibrosis Foundation
National Institutes of Health

Find us online:
http://www.greenelab.com
Twitter: @GreeneScientist
Calvin and Hobbes. Bill Watterson

Uncovering Pseudomonas aeruginosa interactions through comprehensive analysis of gene expression data

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