Welcome to 3 part webinar series on Digital RNA sequencing

Digital RNA sequencing for accurate gene expression profiling

Part 1: What is digital RNAseq?

Speaker: Eric Lader, Ph.D.
Senior Director, Research & Development, QIAGEN

Date: Feb 17th, 1 pm EST, 10 am PST, 6 pm GMT

Part 2: Digital RNAseq for gene expression profiling

Speaker: Raed Samara, Ph.D.
Global Product Manager, NGS, QIAGEN

Date: Feb 24th, 1 pm EST, 10 am PST, 6 pm GMT

Part 3: Molecular Insight into gene expression profiling using digital

RNAseq: data analysis tutorial
Speaker: Melanie Hussong, Ph.D. Scientist, NGS
Jean-Noel Billaud, Ph.D. Principal Scientist. Bioinformatics

Date: March 1st, 1 pm EST, 10 am PST, 6 pm GMT

Sample to Insight
 Webinar on

Molecular Insight into Gene Expression using Digital RNAseq

Data Analysis Tutorial

Melanie Hussong Ph.D., Scientist, NGS

Jean-Noel Billaud, Ph.D., Principal Scientist, Bioinformatics

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 2
 Molecular Insight into Gene Expression using Digital RNAseq:
Data Analysis Tutorial
Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D.
Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 3
 Today’s Agenda

Why use Targeted Next Generation Sequencing (NGS) to study gene expression?

Targeted Enrichment
 QIAseq Targeted RNA Panels

QIAseq Primary Data Analysis

 Read Mapping
 Molecular Barcode Counting

QIAseq Expression Analysis

 Workflow
 Interface
 Data Interpretation: Ingenuity Pathway Analysis (IPA)

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 4
 Outline of a RNAseq Experiment

Want to Study Gene Expression?

For example:

■ Have 96 samples
■ Investigate 500 genes
■ Only have a limited amount of total RNA
■ Budget per sample is $100

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 5
 Gene Expression Profiling

Accuracy
PCR-based Limited sample & assay throughput
Requires a lot of RNA

Throughput power
Whole transcriptome sequencing (WTS) Expensive
Complex data
Easy data analysis
High background noise
Microarrays
Requires a lot of RNA
Limited dynamic range
Manageable data
Traditional targeted RNA sequencing Low per-sample cost
Amplification bias

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 6
 QIAseq Targeted RNA: High Throughput Digital NGS

Simple to use, complex behind the scenes

 Complete, Integrated System: Sample to Insight

□ Sensitive and highly specific
□ Extremely flexible in experimental design (n samples x n assays)
□ Simple for end user to address bioinformatically
□ Requires no rRNA depletion or blocking or dT selection
□ Makes best use of limited NGS read budget
□ Flexible content
• Leverages QIAGEN’s content know-how
• Disease and pathway focused panels
• Ready to use, easy to modify, and fully custom panel content all in one kit

 When? Who? Why?

 Scientists with known gene list or pathway

 Follow up on broader experiments, such as WTS or microarray

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 7
 QIAseq Targeted RNA Workflow: from Sample to Insight

Sample Insight

RNA Synthesize Library Library Sequencing Data Data

Extraction cDNA Construction Quantification Run Analysis Interpretation

Molecular Barcode Assignment MiSeq Primary Analysis for MT count Ingenuity Pathway
Purification NextSeq QIAseq RNA Quantification Portal Analysis (IPA)
Gene Specific PCR Ion Torrent Secondary Analysis for
Purification Platform Agnostic Normalization
Target Enrichment and Index Assignment
Purification

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 8
 QIAseq Targeted RNA Library Construction Schematic
MT: 12-base unique barcode
RNA Sample
GSP1, GSP2: Gene-specific primers
RS2, FS2: Universal PCR primers

cDNA Synthesis
Molecular Barcoded
forward gene-specific primer
Primer Extension
and Molecular Barcoding GSP1
MT
RS2
GSP1 and GSP2
never see each other,
Double QIAseq Bead Cleanup thereby minimizing primer dimers

FS2 GSP2 RS2

6 Hours 1st Stage PCR

Reverse gene-specific primer

QIAseq Bead Cleanup

Adaptor and Index

2nd Stage PCR
library construction
and sample indexing
Adaptor and Index

QIAseq Bead Cleanup

Library Quantification
Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 9
 QIAseq Targeted RNA Panels

■ Cancer Transcriptome (411)

■ Inflammation & Immunity Transcriptome (491)
■ Signal Transduction PathwayFinder (421)
■ Stem Cell & Differentiation Markers (309)
■ Molecular Toxicology Transcriptome (386)
■ Angiogenesis & Endothelial Cell Biology (356)
■ Apoptosis & Cell Death (280)
■ Extracellular Matrix & Cell Adhesion Molecules
(437)

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 10
 QIAseq Targeted RNA Data Primary Analysis Web Portal

■ FREE Complete and Easy to use Data Analysis with Web-based Software can be found at:
ngsdataanalysis.sabiosciences.com/NGSRNA/

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 11
 QIAseq Targeted RNA Data Analysis Upload Files Interface

■ File Upload Tab

• .fastq or fastq.gz files (Illumina Platforms)
• .bam files (Ion Torrent)

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 12
 QIAseq Targeted RNA Data Analysis File Management Interface

■ File Management Tab

• Delete/Manage Files

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 13
 QIAseq Targeted RNA Data Analysis RNA Quantification Interface

■ RNA Quantification Tab

• Submit Runs for Analysis

• View and download Output Files

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 14
 QIAseq Targeted RNA Data Analysis Quantification Interface

■ RNA Quantification Jobs Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 15
 QIAseq Targeted RNA Data Analysis Quantification Interface

■ RNA Quantification Jobs Tab

• Select the appropriate Catalog

Or Custom Catalog Number

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 16
 QIAseq Targeted RNA Data Analysis Quantification Interface

■ RNA Quantification Jobs Tab

• Select Sample Files to be analyzed

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 17
 QIAseq Targeted RNA Data Analysis Quantification Interface

■ RNA Quantification Jobs Tab

• File Lanes 1 for MiSeq/HiSeq and Ion Torrent

• File Lanes 4 for NextSeq

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 18
 QIAseq Targeted RNA Data Analysis Quantification Interface

■ RNA Quantification Jobs Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 19
 Data Analysis for QIAseq Targeted RNA Sequencing

QIAseq Targeted RNA Data Analysis workflow

Molecular
Barcode
Adaptor Read
Count
Trimming Mapping
(Primary
Analysis)

■ Adaptor Trimming
□ Remove the adaptor sequences from the reads

■ Read Mapping
□ Identify the possible position of the read within the reference
□ Align the read sequence to reference sequences

■ Molecular Barcode Counting

□ Merges unique barcodes with mapped reads

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 20
 Read Mapping

Adaptor Trimming and Alignment of Reads to the Reference Genome

 Trim universal PCR adaptor sequences off the 3’ end of reads.

 Reads that are less than 55 bp in length after adapter trimming are likely oligo dimers and
are dropped.

 The first 12 bp are removed from each read as they are the Molecular Tag (MT) sequence.
 Append the MT sequence to the read identifier.

 Using STAR RNA read mapper, reads are aligned to GRCh38 reference genome.
 STAR uses Gencode transcript models to aid in aligning reads that cross introns.
 Reads that do not map well are dropped.

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 21
 Process Read Alignments

Process Read Alignments

□ At least 60 bp of the reads must align otherwise the reads are dropped.
□ As most oligos are less than 30 bp, this ensures that at least 30 bp of the endogenous
RNA sequence is present in the read.

□ Drop reads that are generated by off-target or unintended priming by identifying the 5’
primer at the start of the read.

□ If the genome alignment at the primer start site is not very good then these reads are
dropped as the boundary between the MT region and the primer region of the read is
ambiguous. Hence dropping these reads improves MT counting accuracy.

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 22
 Count Molecular Tags

Count Molecular Tags

 For each amplicon, cluster together the 12 bp read sequences from the MT region that have
low edit distance from each other. Reads in a cluster putatively originated from the same
MT priming event.

Five replicates of one transcript MT = 1

 All of the above reads have the same MT ( ) although there are five replicates of one
Transcript they have a MT Count of one.

Five unique transcripts of a gene MT = 5

 All of the above reads have different MTs and are five unique MTs for one
Transcript they have a MT Count of five.

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 23
 QIAseq Targeted RNA Data Analysis Quantification Interface

■ RNA Quantification Jobs Tab

Job Status

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 24
 QIAseq Primary Data Analysis Output

■ Features of the Primary Analysis Molecular Tag Report

Summary Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 25
 QIAseq Primary Data Analysis Output

■ Features of the Primary Analysis Molecular Tag Report

Summary Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 26
 QIAseq Primary Data Analysis Output

■ Features of the Primary Analysis Molecular Tag Report

Summary Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 27
 QIAseq Primary Data Analysis Output

■ Features of the Primary Analysis Molecular Tag Report

Summary Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 28
 QIAseq Primary Data Analysis Output

■ Features of the Primary Analysis Molecular Tag Report

Summary Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 29
 Read Details: Unique Captures per Target Gene Count

Sample by Sample, Gene by Gene Unique Barcode Counts (and Total)

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 30
 Read Details: Unique Captures per Target Gene Count

Sample by Sample, Gene by Gene Unique Barcode Counts (and Total)

Differential gene expression can be seen inter- and intra- samples

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 31
 Read Details: Unique Captures per Target Gene Count

gDNA Control Amplicons

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 32
 Read Details: Unique Captures per Target Gene Count

Housekeeping Reference Genes Amplicons

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 33
 QIAseq Targeted RNA Secondary Data Analysis Setup
■ Found in the GeneGlobe Data Analysis Center
■ Upload Data

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 34
 QIAseq Targeted RNA Secondary Data Analysis Setup

■ Analysis Setup

Uploaded Data

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 35
 QIAseq Targeted RNA Secondary Data Analysis Setup

■ Analysis Setup

Sample Manager

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 36
 QIAseq Targeted RNA Secondary Data Analysis Setup

■ Analysis Setup

Data QC

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 37
 Secondary Data Analysis: Normalization Against

■ Unique barcodes/total unique barcodes per sample

This method simply uses the sum of the molecular tag counts in each sample as the
normalization factor. However, it assumes that equal amounts of RNA were used in
the analysis and that a majority of the genes are expressed at a relatively stable level
across the Samples.

■ Housekeeping genes (one, some, all)

Housekeeping genes are ranked as most stable to least stably expressed.
Housekeeping genes stability ranking is based on GeNorm calculations.
Genes that have a stability factor of less than 1.5 are considered to be stably
expressed.

■ Genes of your choice

Calculate - fold change, p-values, generate heat maps, volcano plots

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 38
 QIAseq Targeted RNA Secondary Data Analysis Setup

■ Analysis Setup

Select Normalization Method – Total Molecular Tags

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 39
 QIAseq Targeted RNA Secondary Data Analysis Setup

■ Analysis Setup

Select Normalization Method – Housekeeping Genes

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 40
 QIAseq Targeted RNA Secondary Data Analysis

■ Analysis

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 41
 QIAseq Targeted RNA Secondary Data Analysis Plots and Charts

■ Plots and Charts

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 42
 QIAseq Targeted RNA Secondary Data Analysis Scatter Plot

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 43
 QIAseq Targeted RNA Secondary Data Analysis Volcano Plot

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 44
 QIAseq Targeted RNA Secondary Data Analysis Clustergram

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 45
 QIAseq Targeted RNA Secondary Data Analysis Export Data

■ Export Data

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 46
 QIAseq Targeted RNA Secondary Data Analysis Export Data

■ Gene Table Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 47
 QIAseq Targeted RNA Secondary Data Analysis Export Data

■ Fold Changes and p-value Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 48
 QIAseq Targeted RNA Secondary Data Analysis Export Data

■ Fold Regulation and p-value Tab

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 49
 QIAseq Targeted RNA Secondary Data Analysis Export Data

■ IPA Upload Tab

■ Biological Interpretation Using IPA

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 50
 QIASeq Targeted RNA Panel: Sample to Insight workflow

 Integrated library preparation

 Works with any RNA sample type
 Compatible with most sequencers
 Complementary data analysis tool for fold change analysis

Sample to Insight
Title, Location, Date 51
 The QIAGEN Knowledge Base: Content & Content-aware Analytics

• Cancer Scoring
• Hereditary Disease Scoring
• Causal Network Analysis
• Druggable Pathways
• Disease Model-based Analysis

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 52
 Introduction to Ingenuity Pathway Analysis (IPA), Interpretation
Gene View, Chem View, and
Disease/Function View

Human and Mouse

Isoform Views

Canonical Pathways/Molecule
Activity Predictor
Upstream Analysis
Upstream Regulators/Mechanistic
Network/Causal Networks

Diseases & Functions

Downstream Effects Analysis

Regulator Effects

microRNA Target Filter

BioProfiler

IsoProfiler

Interaction Networks,
Build and Overlay tools

My Findings

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 53
 Biological Interpretation of Breast Cancer (using 2 breast cancer cell lines)

BT-549 and MDA-MD-231 Breast cancer cell lines

• Understanding the transcriptome in these cell lines when TOB1 is knockdown by siRNA

 What are the signaling or metabolic pathways involved, are they activated/inhibited? (Canonical
Pathways)
 What are the underlying transcriptional programs? (Upstream Analysis)
 What biological processes are involved and in what way? (Diseases & Functions)
 Identify Isoforms differentially expressed connected to cancer (IsoProfiler)
 What hypotheses can be drawn further? (Mechanistic Network, Causal Network, Regulator Effects)

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 54
 QIAseq RNA Panels: 2 Breast cancer cell lines

Upload the 254 QIAseq RNA panel dataset

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 55
 IPA: Analysis of QIAseq RNA panel of 2 Breast Cancer cell lines

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 56
 Canonical Pathways (CP) in BT-549 cell line

Understanding which Canonical Pathways (overlap) are involved and Determining the predicted
activity of this CP (Pathway Activity Analysis)

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 57
 Pathway Activity Analysis in BT-549 cell line

Cell Cycle G1/S Checkpoint Regulation is predicted to be inhibited (Z-score negative)

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 58
 Upstream Analysis in MDA-MB-231 cell line

Determining which transcriptional program is involved and in what way

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 59
 TP53-driven network in MDA-MD-231 cell line

TP53 is predicted to be activated when TOB1 siRNA is added

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 60
 TP53-driven network in MDA-MD-231 cell line

Overlay statistically significant diseases and functions

Apoptosis

Proliferation
of cells

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 61
 CTNNB1-driven Mechanistic Network in MDA-MB-231

CTNNB1 is predicted to be activated and drives a Mechanistic Network along with 18 other
regulators. This Network may explain the pattern of expression of 38 targets downstream (Not
shown here).

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 62
 Downstream Effect Analysis of MDA-MD-231 cell line

Apoptosis of tumor cell lines is

predicted to be activated

Apoptosis
of tumor
cell lines

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 63
 TCEB3 driven Causal Network in MDA-MB-231

TCEB3 Causal Network is predicted to be activated and to increase “Invasion of Cells”

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 64
 Regulator Effects in MDA-MB-231

Connecting the transcriptional program and the biological processes involved together via the
targets in the dataset.

Upstream
Regulators

Targets
Downstream

Diseases and
Functions

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 65
 IsoProfiler in IPA to discover isoforms that may drive tumor progression

Some VEGF isoforms are identified in the datasets that are specifically connected to EEC.

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 66
 Using QIAGEN Bioinformatics Approach

• Using IPA, we have been able to:

 Visualize the differentially expressed genes in the 2 breast cancer cell lines with TOB1 siRNA

 Understand which signaling pathways are involved in tumor progression in these cell lines

 Discover potential transcriptional program(s) that are induced or repressed in these BrCa cell lines treated with TOB1
siRNA

 Discover specific biological processes that participate in the tumor progression

 Identify Isoforms differentially expressed connected to EEC

 Highlight new hypotheses (ready to be tested and validated) that could explain cell invasion

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 67
Melanie Hussong, Ph.D. Jean-Noel Billaud, Ph.D.
Scientist, NGS Principal Scientist.
Bioinformatics

Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 68
Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 69
Sample to Insight
QIAseq RNA Part 3: Molecular Insight into Gene Expression using Digital RNAseq: Data Analysis Tutorial, 03/01/2016, Melanie Hussong, Ph.D. and Jean-Noel Billaud, Ph.D. 70