Bioinfo Tools for Functional Genomics

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Identification of Genes of Interest 
 
 
 
Prof. Gabriel Rodrigues Alves Margarido 
gramarga@usp.br 
 
ESALQ/USP 
Piracicaba 
Bioinformatics Tools for 
Functional Genomics 
 
 
 
Genetic Variability 
Functional Genomics 
Introduction 
 
 
 
Source: www.agencia.cnptia.embrapa.br Source: www.ruralcentro.uol.com.br 
Central Dogma of Biology 
Functional Genomics 
Introduction 
 
 
 
Organisms are Dynamic 
Functional Genomics 
Introduction 
 
 
 
Source: www.pioneer.com 
Organisms are Dynamic 
Functional Genomics 
Introduction 
 
 
 
Organisms are Dynamic 
• Time 
• Space 
• Condition 
• Intrinsic and extrinsic stimuli 
 
• Genes are dynamic! 
• Regulated Expression 
Functional Genomics 
Introduction 
 
 
 
Protein Pathways 
 
 
 
 
Functional Genomics 
Introduction 
Protein Function and Variability 
Functional Genomics 
Introduction 
 
 
 
Genetic Variability 
 
 
 
Functional Genomics 
Introduction 
Genome Sizes & Complexity 
Functional Genomics 
Introduction 
 
 
 
Functional Genomics 
Introduction 
 
 
 
Genes are Dynamic 
Functional Genomics 
Introduction 
 
 
 Genes 
C
o
m
p
le
x
ity
 
Regulation 
Genes are Dynamic 
Functional Genomics 
Introduction 
 
 
 
Changes in Genes (Alleles) 
Functional Genomics 
Introduction 
 
 
 
Changes in Gene Expression 
Functional Genomics 
Introduction 
 
 
 
Goals 
 
• How do we find the important genes? 
 
• How are they regulated? 
 
• How do they interact? 
Functional Genomics 
Introduction 
 
 
 
Goals 
Functional Genomics 
Introduction 
 
 
 
Definition 
 
• Aims to describe the functions of genes and 
proteins 
 
• Dynamic view 
• Transcription & Translation 
• Interactions 
Functional Genomics 
 
 
 
Definition 
 
• All molecular levels 
 
• Leverage and understand natural variability 
 
• Study experimental perturbations 
Functional Genomics 
 
 
 
Sequencing Costs 
 
 
 
Standard Experimental Layout 
Functional Genomics 
 
 
 
Sample 1 
Condition A 
Sample 2 
Condition B 
Isolate 
molecule 
Isolate 
molecule 
High-throughput 
Assay* 
Data analysis 
mRNA 
 
Transcriptomics 
 
 
 
mRNA 
 
 
• Single strand molecule 
• Complementary to DNA 
 
 
• Carries information from nucleus to cytoplasm 
Transcriptomics 
 
 
 
Gene Expression 
 
• Context-sensitive 
• Organs and tissues 
• Developmental program 
• Environmental stimuli 
• Diseases 
• Genic activity 
Transcriptomics 
 
 
 
Definition 
 
• Study of the characteristics and regulation of 
the population of functional RNA transcripts in a 
cell or organism, at a given moment in time 
 
• Population 
• Functional 
• Regulation 
Transcriptomics 
 
 
 
High-throughput 
 
• Closed system profiles 
• Microarrays 
 
 
• Open system 
• RNA-Seq 
Transcriptomics 
 
 
 
Transcriptomics 
RNA-Seq 
Transcriptomics 
 
 
 
RNA-Seq – Quantification 
Transcriptomics 
 
 
 
Differential Expression 
Transcriptomics 
 
 
 
Adapted from Hu et al. (2013) Nucl Acids Res 41:e39 
Photo: PicsFive/Shutterstock 
Differential Expression 
Transcriptomics 
 
 
 
Statistical Analyses 
• High dimensionality 
• 𝒑 >> 𝒏 problem 
 
• Correlated observations 
 
• Complex patterns 
 
• Statistical vs biological significance 
Transcriptomics 
 
 
 
Expression Patterns 
Transcriptomics 
 
 
 
Expression Patterns 
Transcriptomics 
 
 
 
 
 
 
• Expression 
patterns 
Classification 
• Do transcripts predict pre-defined groups? 
 
 
• Diagnostics 
Transcriptomics 
 
 
 
RNA – Data Analysis 
 
• Large number of observations 
• Experimental and biological noise 
 
• Goals 
• Transcripts: expression patterns 
• Samples: similarity and/or differences 
Transcriptomics 
 
 
 
Proteins 
 
Proteomics 
 
 
 
Proteome 
 
• Collection of all proteins expressed by a 
genome, cell or organism, at a given moment in 
time, under certain conditions 
 
• Collection 
• Expressed 
• Conditions 
Proteomics 
 
 
 
Definition 
 
• Large-scale analysis of proteins 
 
• Study of the collection of all proteins coded for 
by a genome 
 
• Study of all the proteins in a cell and/or in an 
organism 
Proteomics 
 
 
 
Techniques 
 
• Two-dimensional electrophoresis (2D-PAGE) 
 
• Mass spectrometry 
 
• Yeast two-hybrid system 
Proteomics 
 
 
 
Division 
• Quantitative proteomics 
• “Expression proteomics” 
• Protein levels at different conditions or 
points in time 
 
• Qualitative proteomics 
• “Identification” proteomics 
• Protein-protein interactions 
Proteomics 
 
 
 
Two-dimensional electrophoresis 
Proteomics 
 
 
 
Mass Spectrometry 
 
•Different 
 sequences 
 
•Different 
 spectra 
 
Proteomics 
 
 
 
Source: www.promega.com 
Protein-Protein Interaction 
 
Proteomics 
 
 
 
Yeast Two-Hybrid System 
 
Proteomics 
 
 
 
 
 
 
 
Finding Associations 
Functional Genomics 
Correlation 
Causal 
 
 
 
 
Identification of 
Genes of Interest 
Crosses and Linkage Mapping 
Identification of 
Genes of Interest 
 
 
 
 
From DNA to RNA 
Identification of 
Genes of Interest 
 
 
 
Identification of 
Genes of Interest 
 
 
 
Identification of 
Genes of Interest 
Correlation Networks 
 
 
 
Network Reconstruction 
Impact on Other Fields 
Cultivar Management 
 
Impact on Other Fields 
Pleiotropy 
Source: www.naturezabrasileira.com.br Source: www.ecodesenvolvimento.org 
Impact on Other Fields 
Crop Management - Harvest Planning 
Summary 
 
Overview 
 
Bioinformatics 
 
Functional Genomics 
Bioinformatics 
Identification of Genes of 
Interest 
 
 
 
Differentially Expressed Genes 
 
Bioinformatics 
Identification of Genes of 
Interest 
 
 
 
Networks 
 
Bioinformatics 
Identification of Genes of 
Interest 
 
 
 
Changes in Gene Expression 
Bioinformatics 
Identification of Genes of 
Interest 
 
 
 
Changes in Gene Expression 
Bioinformatics 
Identification of Genes of 
Interest 
 
 
 
Bioinformatics 
 
Bioinformatics 
Identification of Genes of 
Interest 
 
 
 
Bioinformatics 
 
• High-throughput 
 
• Hypotheses generating 
 
• Follow-up and validation 
Bioinformatics 
Identification of Genes of 
Interest