Pathway Analysis Jenny Wu Outline Introduction to NGS data - - PowerPoint PPT Presentation

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Pathway Analysis Jenny Wu Outline Introduction to NGS data - - PowerPoint PPT Presentation

Sep 25, 2023 8 likes •240 views

Introduction to Next Generation Sequencing (NGS) Data Analysis and Pathway Analysis Jenny Wu Outline Introduction to NGS data analysis in Cancer Genomics NGS applications in cancer research Typical NGS workflows and pipeline

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Introduction to Next Generation Sequencing (NGS) Data Analysis and Pathway Analysis Jenny Wu

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Outline

  • Introduction to NGS data analysis in Cancer

Genomics

– NGS applications in cancer research – Typical NGS workflows and pipeline – Open source software with GUI

  • Pathway Analysis and Software
  • Pathway Analysis goals and concepts
  • Commercial and open source pathway analysis software
  • Data analysis resources
  • Summary
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Next Generation Sequencing

Massively Parallel Sequencing: One can generate hundreds of millions of short sequences (up to 250bp) in a single run in a short period of time with low per base cost.

  • Illumina/Solexa GA II, HiSeq 2500, 3000,X
  • Roche/454 FLX, Titanium
  • Life Technologies/Applied Biosystems SOLiD

Reviews: Michael Metzker (2010) Nature Reviews Genetics 11:31 Quail et al (2012) BMC Genomics Jul 24;13:341.

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NGS in Cancer Genomics

Shyr et al.2013

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Data Analysis in the bottleneck

(wall.hms.harvard.edu)

Informatics

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Basic NGS Workflow

Olson et al.

QC and pipeline analysis Data interpretation Isolation of material PCR amplification End repair, size selection Library QC Cluster generation Instrument operation

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High Throughput Data Analysis Overview

Olson et al.

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http://www.broadinstitute.org/gsa/wiki/images/7/7a/Overall_flow.jpg http://www.broadinstitute.org/gatk/guide/topic?name=intro

Many Analysis Pipelines Start with Read Mapping

http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html

Genotyping (GATK) RNA-seq (Tuxedo)

Typical Data Analysis Pipelines

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Cancer NGS Data Analysis Pipeline-Software

Raw reads Analysis-ready reads

FASTQC, FASTX- toolkit, Trimmomatic

Mapped reads

Visualization (IGV, IGB, USCS GB……) BWA, STAR

……

Data Task Software

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Cancer NGS Application Specific Software

Cufflinks, MISO DESeq2,GATK MACS2, SISSRs Mapped reads Bismark, BS Seeker SomaticSniper, VarScan2, mutect freeBayes, Pindel, CNVnator

……

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Open Source Software with GUI

http://www.broadinstitute.org/cancer/software/GENE-E

Galaxy: Web based platform for analysis of large datasets

http://hpc-galaxy.oit.uci.edu/root https://main.g2.bx.psu.edu/ https://usegalaxy.org/

GENE-E: java based matrix visualization and analysis platform; includes heatmap, clustering, filtering etc.

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Commercial software for NGS analysis

  • Easy to use, no

command line skills required

  • Usually platform

independent

  • Little to no learning

curve

  • Limited flexibility
  • Harder to publish
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Outline

  • Introduction to NGS data analysis in Cancer

Genomics

– NGS applications in cancer research – Typical NGS workflows and pipeline – Open source software with GUI

  • Pathway Analysis and Software
  • Pathway Analysis goals and concepts
  • Commercial and open source pathway analysis software
  • Data analysis resources
  • Summary
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Why Pathway Analysis

  • Logical next step in any high

throughput experiments

  • Goal: to characterize biological

meaning of the joint changes in gene expression

  • Why? Often group of genes doing related

functions are changed

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Pathway and Network Analysis

Pathway Analysis Methods:

  • Functional category over representation:

discrete test for significance (BiNGO, David, IPA etc)

  • Continuous test (GSEA, PAGE)
  • Signaling Pathway Impact Analysis (iPathway

Guide)

Network Analysis: (WGCNA, Cytoscape etc)

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Functional Category Enrichment

  • Discrete tests: enrichment for groups in gene

lists

– Select gene list at some predefined cutoff – For each gene list and functional category cross-tabulate to get a 2X2 contingency table – Test for significance using Fisher’s exact test – FDR correction for multiple hypothesis testing

Differentially expressed Not differentially expressed total In the pathway a b a+b Not in the pathway c d c+d total a+c b+d n

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Functional Categories in Pathway Analysis

  • Gene Ontology

– Biological Process – Molecular Function – Cellular Localization

  • Pathway Databases

– KEGG – BioCarta – Broad Institute (MSigDB) – Commercial knowledge bases such as IPA

  • Other

– Transcription factor targets – Protein complexes – Self-Defined

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Commerical and Open Source Pathway Analysis Software

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Ingenuity Pathway Analysis Tool

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IPA Input file

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IPA results page

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Resources in NGS data analysis

Public forums: Computational resources available at UCI:

  • HPC: open source software
  • CLCbio, IPA, JMP Genomics…
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Summary

Thank you!

  • NGS technologies are transforming

cancer research.

  • Data analysis is a crucial part in NGS

applications

  • Pathway analysis concepts and software
  • Data analysis resources