Ipa Software

IPA
October 2013
Academia Sinica
Gene ()
IPA Certified Analyst

I. Data Upload and How to Run a Core Analysis
IPA
II. Functional Interpretation in IPA IPA

Hands-on Exercises
III. Comparison Analyses
IV. Using IPA to Explore microRNA Impacts on Molecular Mechanisms of

Disease
microRNA
Hands-on Exercises
V. Data Analysis & Interpretation in IPA
- Case study for association of DNA copy number alterations and
ovarian cancer
VI. Q&A
Proprietary and Confidential
58
IPA
Ingenuity Pathways Analysis

Signaling PathwayMetabolic Pathway
Transcription regulator

:
IPA-Core Analysis mRNA, miRNAprotein
IPA-Tox Analysis:
IPA-Metabolomics Analysis: (Metabolomics)
59
Workflow for Dataset Analysis

IPA
Genomic, exon, miRNA,

SNP, protein arrays;
Any molecule lists;
Other proteomic &
metabolomic assays
Identify functions,
diseases, and canonical
pathways associated with
your data
60
Key Terminology
Observation:
An experimental condition such as a time point, disease subtype, or
compound concentration
Expression Value:
Numerical value indicating level of expression, significance, or other
assay result for a specific identifier (gene, RNA, protein, or chemical)
Reference Set:
The set of molecules used as the universe of molecules when
calculating the statistical relevance of biological functions and
pathways with respect to a dataset file. The set of molecules are the
user's dataset or molecules in Ingenuity's Knowledge Base (genes,
endogenous chemicals, or both).
Focus Molecule:
Molecules that are from uploaded list, pass filters are applied, and are
available for generating networks
61
IPADataset
ID
Replicates
Average
Other observations
62
IPADataset
p-vlauefold-change
IPA
Excel Sheet
Excel SheetID (Gene Symbol, Refseq
number, Uniprot number, HMDB)
Excel Sheet 20 observations (20
)
Observation3 (ex. p-Valuefold-change)
Head row ()
IPAcut-off
cut-offcut-off
IPAAnalysis-Ready Molecules
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Dataset
IPA Log Ratio
Observation :
Observation 1 : Smokers vs. NonSmokers

Observation 2 : Early COPD vs. NonSmokers
Observation 3 : COPD vs. NonSmokers
* observation
64
After Today, You Should Be Able To:

Define the following IPA terms
Reference Set
Observation
Expression Value
Network Eligible Molecules
Functions/Pathways/Lists Eligible Molecule
Focus Molecule
Upload a dataset
Run an analysis using IPA Best Practices
Access the Analysis Summary Page
65
Live Demo
66

IPA

Hands-on Exercises

Disease
microRNA
Hands-on Exercises
ovarian cancer
VI. Q&A
71
Interpret Downstream Biological Functions
Identify over-represented biological functions and predict how

those functions are increased or decreased in the experiment
72
Mechanistic Networks
How might the upstream molecule drive the observed expression changes?
Hypothesis generation and visualization

Each hypothesis generated indicates the molecules
predicted to be in the signaling cascade
73
IPA
Functions analysis:
Canonical Pathways : Signaling PathwayMetabolic Pathway

Upstream Analysis: Upstream molecules
Networks : Build Tool

Overlay Tool
74
What Does the p-Value Mean?

The smaller the p-value, the less likely an observed
proportion of genes mapping to a function or pathway is a
result of chance. We then infer that there is a biological
effect.
A p-value of 0.05 indicates that you should expect to observe the
gene proportion mapping to a function or pathway by chance in one
out of 20 (5 out of 100) repeated experiments of the same size
75
After Today, You Should Be Able To:

Define the terms: Focus Molecule, Functional Category,
Function, and Effect on Function
Access Functional Analysis for a dataset
Customize the bar charts according to your preferences
Describe Ingenuitys Functional Categorization
Describe the significance values calculated for Functions/
Pathways
Access Canonical Pathways and Network for a dataset
View the molecules involved in a Canonical Pathway and
Network
View the Canonical Pathway diagram with data overlaid
76
Live Demo
77
What Does the Z-Score Mean?

TR: Transcription regulator
TR
+1
-1
+1
Literature-based effect TR has

downstream genes
-1
Predicted activation state of TR:

1: activated (correlated)
-1: inhibited (anti-correlated)
An absolute z-score of 2 is
considered significant. An upstream
regulator is predicted to be:
Activated if the z-score is 2
Inhibited if the z-score -2
84
Upstream Regulators and Mechanistic Networks

Upstream Regulator
Regulator
Dataset Molecules
Mechanistic Network
Algorithm chains
interacting regulators
together to create a
Mechanistic Network
Upstream
Regulator
Additional
Upstream Regulators
Dataset Molecules
85
Ratio vs. Significance

Ratio is calculated as the number of genes from your
dataset that overlap with the Canonical Pathway in
question divided by the total number of genes that are
represented in that Canonical pathway
It is meant to measure the amount of overlap
Significance is calculated in the same way as Functional

Analysis
It is meant to measure the confidence of overlap
Either value is acceptable are and are different ways to

look at which pathways are overlapping/represented by
your dataset
86
How Networks Are Generated

1. Focus molecules are seeds
2. Focus molecules with the most
interactions to other focus molecules
are then connected together to form
a network
3. Non-focus molecules from the

dataset are then added
4. Molecules from the Ingenuitys
Knowledge Base are added
5. Resulting Networks are scored and
then sorted based on the score
93
Hands-on Exercises I
1. Upload a dataset into IPA. You may use your own or we
can provide you with an example.
2. What is the top function associated with your dataset?
3. How can you find out what main functions a Canonical
Pathway (or group of Canonical Pathways) is involved in?
4. What are the functions of the top network in this analysis?
95

IPA

Hands-on Exercises

Disease
microRNA
Hands-on Exercises
ovarian cancer
VI. Q&A
96
Comparison Analysis allows you to analyze changes in

biological states across observations
1. First run a Core, Tox or Metabolomics Analysis on
your multiple datasets that represent timepoints or
dosage treatments
2. Then use Comparison Analysis to understand which

biological processes, clinical pathology endpoints,
diseases, and pathways are relevant to each timepoint
or dose.
97
IPA Compare Data Feature

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Unique to each list
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List 2
Common across all lists
Compare
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List 3
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Union across all lists
98
Live Demo
99

IPA

Hands-on Exercises

Disease
microRNA
Hands-on Exercises
ovarian cancer
VI. Q&A
113
Filter Datasets for Biomarkers or miRNA Targets
miRNA
Data
88 data
points
miRNA
Target Filter
13,690
targets
Molecule
Type
1,090
targets
Pathways
(Cancer/
Growth)
333
targets
mRNA
39
32
targets targets
Use Pathway tools to build hypothesis for microRNA to mRNA target association
114
Using Biological Context in miRNA Target ID

Goal: Utilize newly discovered microRNAs to better
understand biology around potential mRNA
targets/disease
Challenge: New and rapidly evolving field with different
measurement techniques and prediction algorithms
leading to variability in data
Need: Identify mRNA targets to microRNAs using
biological and experimental information, correlate
microRNA and mRNA target expression, specify easy to
use confidence levels of interaction predictions, annotate
mRNA targets with biological context, pathways, species,
etc., all within a single workflow
Outcome: Reduce time of identification of relevant mRNA
targets from months to minutes
115
Tagline
(1 Target Scan search) x (each microRNA in your data set) =

A LOT of targets
116
Live Demo
117
Hands-on Exercises II
Overall Exercises:
1. Use the COPD analytical results in exercises I.
2. What is the observed effect on the Xenobiotic
Metabolism Signaling Canonical Pathway in the Early
COPD group?
3. In the COPD group, focus on the function Cellular
Movement. Select these genes and add them to a new
My Pathway in your IPA account. How many of the
proteins in this pathway are enzymes?
4. In Early COPD vs NonSmokers observation Upstream
Regulators chapter, filter the Molecule Type to only
Transcription Factors, which molecule is predicted to be
Inhibited with the lowest z-score?
122
Hands-on Exercises II cont.

Overall Exercises:
5. In studies of nicotine metabolism in smokers, it has been
estimated that 70% of a nicotine dose is metabolized to
cotinine. Which group express the highest effect on the
Nicotine Degradation pathway?
6. In observation Upstream Regulators chapter. Which
molecule is predicted to be activated in Both of early and
late COPD groups?
123

IPA

Hands-on Exercises

Disease
microRNA
Hands-on Exercises
ovarian cancer
VI. Q&A
124
Prioritization of Candidate Ovarian Cancer Genes

with IPA
Survey copy number alterations (CNAs) in ovarian tumors using Affymetrix

500K SNP Chip
Profile the expression patterns of these tumors and whole ovary normal
samples with Affymetrix U133A and B chips
Focus on drivers not passengers eliminate gene expression changes
that are not CNA-specific
125
Goals of study initial study, extended analysis in

IPA
DNA copy number variations are frequently observed in ovarian
cancer.
What are the most relevant alterations (recurrent CNAs)?
Causal genes in those regions?
Which copy number variations are functionally relevant to oncogenesis?
Identification of causal genes provides candidate therapeutic targets

and biomarkers
Help find genes with tumor-driving roles in ovarian cancer
What functions are these genes associated with?
Can we infer that modulation of those functions may be a major driving factor in the
selection of CNAs?
Prioritize those candidates based on biomarker, drug target, cellular and disease
knowledge
126
Prioritize Candidate Genes from CNA Study

Which are implicated in ovarian cancer? (search Ingenuity Knowledge Base)
What molecular interactions exist and do those interactions represent a collective
biological function? Potential driver for carcinogenesis? (IPA Core Analysis)
Does an assay exist to measure key, carcinogenesis-relevant gene products in a clinical
setting?
Identify exploratory clinical biomarkers (Overlay Biomarkers)
Narrow in on key genes, relationships that are relevant in multiple contexts, datasets
MECOM/SMARCA2/CCNE1
127
Additional Evidence Linking CNA Genes to Ovarian

Cancer
Which of them are implicated in ovarian cancer through other studies?
What is the evidence?
Search for all ovarian cancer genes in the Ingenuity Knowledge Base
Visualize those ovarian cancer genes in a pathway

Overlay CNA gene expression dataset
Focus on ovarian cancer genes whose gene expression is altered
based on the CNA study
128
Validation, Therapeutic Relevance in Ovarian

Cancer
13 of the genes
identified in this study
have been implicated
in previous ovarian
cancer gene
expression studies
with similar up/down
regulation patterns
Several are targets of
ovarian cancer drugs:
SPINT2
EPCAM
PTGER1
PRKC1
CKD4
HDAC10
129

setting?
MECOM/SMARCA2/CCNE1
130
Analysis Summary
Cell Death, Cell Cycle

networks and
Functional groups
Carcinogenesis and
Apoptosis pathways
132
Top scoring network for CNA-specific gene

expression changes
Points to key biological process that may be a driver of

carcinogenesis in ovarian tissue. Provides a pool of candidate
genes for further prioritization, validation (mRNA, protein
levels, functional validation).
133
Summary Report for Apoptosis Network
Highlights therapeutic relevance, expands biological understanding

of network.
134
Initial Conclusions from Pathway Analysis

Collection of expression changes that are specific to ovarian cancer
tumors and regions of high copy number alteration
Strong association with apoptosis
Biological process and genes may be potential drivers of carcinogenesis
Contains known anti-neoplastic drug targets: TNFRSF10B, IGF1R, VEGF,
MAPK11/12
135


setting?
MECOM/SMARCA2/CCNE1
136
What methods, assays exist? Identify exploratory

clinical biomarkers
137
Understand method, application of exploratory

biomarker
ELISA exists to measure IGFR1 levels and activity in clinical samples.
CASP8 is being used as a secondary outcome marker (impact on

apoptosis and cell cycle arrest) for CDDO anti-tumor therapy.
138


setting?
MECOM/SMARCA2/CCNE1
139
Integration of multiple lines of evidence highlights

MECOM
Upregulated in CNA-specific gene expression analysis of ovarian
tumors
Upregulated in other ovarian cancer studies (Findings in Ingenuity

Knowledge Base)
Target of miRNA upregulated in ovarian cancer (Dahiya et al, Johns
Hopkins )
In a complex with HDAC, which is a target of CML therapeutic
intervention
Role in CML - repress the TGF-mediated growth inhibitory signal
Binds SMARCA2
downregulated in CNA-specific gene expression analysis
target of miRNA downregulated in OC
binds ovarian cancer markers
140
MECOM/SMARCA2 hypothesis
CCNE1, SMARCA2, MECOM (EVI1), MBD3

relationship plays an important role in cell
proliferation, growth arrest checkpoints.
Deregulation of transcript levels and miRNA in
ovarian tumors suggest important area for
validation studies
Validate mRNA, protein levels & role as drivers of
carcinogenic processes
141
Conclusions
Evaluation of Copy Number Alteration (CNA) specific gene
expression changes provide valuable insight into genes that are
potential drivers of carcinogenesis
Analysis of CNA-specific gene expression changes in IPA identified
key processes, pathways that may be driven by these genes
Apoptosis networks
Molecular Mechanisms of Cancer, Death Receptor Signaling pathways
Provides an initial pool of candidate genes that may be useful as

markers of carcinogenesis in ovarian tissue
Examination of candidate genes in the context of multiple lines of
evidence narrows in on subset of genes for validation studies:
validation of protein levels, functional validation
Highlighting exploratory clinical biomarkers, therapeutic targets
Overlaying additional mRNA, miRNA datasets
142
Thank you
Office: +886-2-2795-1777#1169
Fax: +886-2-2793-8009
My E-mail: Genechen@gga.asia
MSC Support: msc-support@gga.asia
143

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IPA

IPA Certified Analyst

II. Functional Interpretation in IPA IPA

IV. Using IPA to Explore microRNA Impacts on Molecular Mechanisms of

Signaling PathwayMetabolic Pathway

Proprietary and Confidential

Workflow for Dataset Analysis

Genomic, exon, miRNA,

Proprietary and Confidential

Proprietary and Confidential

Proprietary and Confidential

Observation 1 : Smokers vs. NonSmokers

Proprietary and Confidential

After Today, You Should Be Able To:

Proprietary and Confidential

II. Functional Interpretation in IPA IPA

IV. Using IPA to Explore microRNA Impacts on Molecular Mechanisms of

Interpret Downstream Biological Functions

Identify over-represented biological functions and predict how

Hypothesis generation and visualization

Proprietary and Confidential

Canonical Pathways : Signaling PathwayMetabolic Pathway

Networks : Build Tool

Proprietary and Confidential

What Does the p-Value Mean?

Proprietary and Confidential

After Today, You Should Be Able To:

Proprietary and Confidential

What Does the Z-Score Mean?

Literature-based effect TR has

Predicted activation state of TR:

Upstream Regulators and Mechanistic Networks

Proprietary and Confidential

Ratio vs. Significance

Significance is calculated in the same way as Functional

Either value is acceptable are and are different ways to

How Networks Are Generated

3. Non-focus molecules from the

Proprietary and Confidential

II. Functional Interpretation in IPA IPA

IV. Using IPA to Explore microRNA Impacts on Molecular Mechanisms of

Comparison Analysis allows you to analyze changes in

2. Then use Comparison Analysis to understand which

Proprietary and Confidential

IPA Compare Data Feature

Unique to each list

Common across all lists

Proprietary and Confidential

Union across all lists

II. Functional Interpretation in IPA IPA

IV. Using IPA to Explore microRNA Impacts on Molecular Mechanisms of

Filter Datasets for Biomarkers or miRNA Targets

Using Biological Context in miRNA Target ID

(1 Target Scan search) x (each microRNA in your data set) =

Proprietary and Confidential

Hands-on Exercises II cont.

Proprietary and Confidential

II. Functional Interpretation in IPA IPA

IV. Using IPA to Explore microRNA Impacts on Molecular Mechanisms of

Prioritization of Candidate Ovarian Cancer Genes

Survey copy number alterations (CNAs) in ovarian tumors using Affymetrix

Goals of study initial study, extended analysis in

Which copy number variations are functionally relevant to oncogenesis?

Identification of causal genes provides candidate therapeutic targets