This document discusses functional genomics and its approaches. It defines functional genomics as the worldwide experimental approach to access the function of genes by using information from structural genomics. The key functional genomics approaches discussed are transcriptomics, proteomics, metabolomics, interactomics, epigenetics, and nutrigenomics. Modern techniques discussed include expressed sequence tags (ESTs), serial analysis of gene expression (SAGE), and microarray analysis.

1. FUNCTIONAL GENOMICS
DEEPALI DASH
02/PP/Ph. D/17
Dept. Of Plant Physiology

2. Genomics is the field of genetics that attempt to understand the content,
organisation, function and evolution of genetic information contained in whole
genome.
GENOMICS
Types of genomics:
•Structural genomics: Structural Genomics is a worldwide effort aimed at determining the
three-dimensional structures of gene products in an efficient and high-throughput mode.
•Comparative genomics : Comparative genomics is a field of biological research in which
the genomic features of different organisms are compared. The genomic features may
include the DNA sequence, genes, gene order, regulatory sequences, and
other genomic structural landmarks
•Functional genomics : Functional genomics is the worldwide experimental
approach to access the function of gene by making use of information and reagent provided
by structural genomics. - Hieter & Boguski 1997

5. Functional Genomics Approaches
Transcriptomics
Proteomics
Metabolomics
Interactomics
Epigenetics
Nutrigenomics

6. Functional Genomics Approaches
1. Transcriptomics: Transcriptomics studies measure gene
expression at the transcript or RNA level.
2. Proteomics: Proteomics approaches focus on which proteins
are expressed in a biological system but may also include
studies of protein structure.
3. Metabolomics: Metabolomics is the study of all metabolites
in a biological system
4. Interactomics: Together, transcriptomics, proteomics,
metabolomics describe the transcripts, proteins and
metabolites of a biological system and expected to provide a
complete model of the biological systems being studied.

7. Interactomics, is of specific relevance to
agriculture systems, particularly in understanding
disease. Interactomics is the study of the molecular
interactions and encompasses host– pathogen
interactions
5.Epigenetics: Epigenetics studies non-heritable
changes in the genome (e.g., DNA methylation and
histone modification) that affects observed phenotypes.
6. Nutrigenomics:. Nutrigenomics (or ‘nutritional
genomics’) focuses on understanding how diet affects
gene expression.
Conti…

8. •Transcription profiling
This is one of the most popular study types, also known as
'expression profiling'. It involves the quantification of gene
expression of many genes in cells or tissue samples at the
transcription (RNA) level.
The quantification can be done by collecting biological
samples and extracting RNA (in most cases, total RNA) following
a treatment or at fixed time-points in a time-series, thereby
creating 'snap-shots' of expression patterns.
Branches of Functional Genomics

9. Fig. schematic transcription profiling process

10. Genotyping
Genotyping studies are those which identify differences
in the DNA sequence (i.e genotype) of a sample. with the aim of
identifying differences in the genotype which may explain the
difference in phenotype.
Genotyping studies can be designed to identify DNA
sequence differences at three levels:
•Single nucleotide polymorphisms (SNPs, pronounced ‘snips’):
SNP analysis focuses on differences in the DNA sequence at the
single nucleotide level.
Example: SNP-profiling of plasma DNA in breast cancer patients

11. •Copy number variations (CNVs): CNVs refer to an increase or
decrease in the number of copies of a segment of DNA (e.g. a
gene, or a locus-specific DNA repeat element). Each 'copy' can be
as short as 50 bases or up to 100 kilo bases.
Example: A genome-wide copy number variant study of suicidal
behavior
•Structural variations: they are an order of magnitude larger
than CNVs and often cover megabases of DNA.
Example: Transcription profiling by high throughput sequencing
of different maize lines to discover and characterise 'presence-
absence variation' in the maize genome
Conti…

12. Epigenetic profiling
Epigenetics is the study of how biochemical
modifications or physical interaction of DNA/chromatin
affect gene regulation in a cell.
At the DNA level, methylation of CpG
dinucleotides (often located near gene promoters) can be
detected by first converting unmethylated cytosines
into uracil using bisulfite, which allows methylated and
unmethylated cytosines to be distinguished.
.
Example: Quantitative sequencing of 5-methylcytosine
and 5-hydroxymethylcytosine

13. DNA/RNA-protein interactions
Transcription factors, ribosomes and other
DNA/RNA-binding proteins can bind to nucleic acid
sequences and influence the transcription
and translation of genes. The immunoprecipitation
technique has also been applied to study protein
binding sites on RNA.

14. Meta-analysis
Meta-analysis is a branch of functional genomics in
which data from pre-existing experiments is combined to
create statistically more powerful models of a biological
process. This type of analyses has become popular as it
allows the identification of subtle events that could not be
detected in smaller studies. Functional genomics databases
such as ArrayExpress and Expression Atlas play an
important role in these studies as reliable, well annotated
sources of functional genomics data.
Example: Genome-wide analysis of over 106,000
individuals identifies 9 neuroticism-associated loci

15. Techniques in Functional Genome Analysis
1.Classical techniques and tools
• Inserstional mutagenesis : Insertional mutagenesis is mutagenesis of
DNA by the insertion of one or more bases.
e.g. Transposons and T- DNA tagging
• Sequence based mutagenesis : It is a molecular biology method that is
used to make specific and intentional changes to the DNA sequence of a
gene.
e.g. Physical and chemical mutagenesis
•Tarrget gene mutagenesis: Homologous recombination can be used to
produce specific mutation in an organism. Vector containing DNA sequence
similar to the gene to be modified is introduced to the cell, and by a process
of recombination replaces the target gene in the chromosome.
e.g. RNAi, Sense and antisense expression

16. 2. Mordern techniques and tools
a. sequence based approaches
EST
SAGE
b. microarray based approaches

17. Expressed sequence tags
•ESTs are short sequences of cDNA typically 200-400 nucleotides in length.
•Obtained from either 5’ end or 3’ end of cDNA inserts of cDNA library.
DRAWBACKS OF USING E.S.T
• Automatically generated without
verification thus contain high error rates.
• There is often contamination by vector
sequence , introns, ribosomal RNA,
mitochondrial RNA.
• ESTs represent only partial sequences
of genes.
ADVANTAGES OF E.S.T
• Provide a rough estimate of genes that
are actively expressed in a genome under
a particular physiological condition.
• Help in discovering new genes, due to
random sequencing of cDNA clones.
• EST libraries can be easily generated

18. EST INDEX CONSTRUCTION
“A collection of nonredundant and annotated EST sequence is known as gene
index construction
STEPS INVOLVED
• Remove vector contaminants
• masks repeats using Vecscreen
• Clustering (associates EST sequences with unique
genes)
• Derive consensus sequences results in longer contigs
• Coding region is defined by excluding introns and 3’-
untranslated sequences.
• coding sequence translated into protein sequence
• Alignment of these complied ESTs with genomic
sequence

21. Microarraybasedapproaches
A microarray is a pattern of ssDNA probes which
areimmobilized on a surface called a chip or a slide.
• Microarrays use hybridization to detect a specificDNA
or RNA in a sample.
• DNA microarray uses a million different probes, fixed
on a solid surface.
• Microarray technology evolved from Southern
blotting.
• To analyze the expression of thousands ofgenes in
single reaction, very quickly and in anefficient manner.

23. Application of Functional Genomics