Föreläsning 7: Comparative -omics

What are omics

two definitions:

• “Omics aims at the collective characterization and quantification of pools of biological molecules that translate into the structure, dynamics and function, of an organism”

• “Analysis of large amounts of data representing an entire set of some kind,
  especially the entire set of molecules, such as proteins, lipids, or metabolites, in a cell, organ, or organism.”

Name the omics fields

• Genomic

• Transcriptomics

• Proteomics

• Metabolomics

• Epigenomics

• Phenomics

Each omics field provides one layer of insight — combined, they give a full picture of biological function. The flow reflects the central dogma: DNA → RNA → Protein → Metabolism

What is genomics?

• = the study of the full genetic complement of an organism

• Uses tools like DNA sequencing, bioinformatics and recombinant DNA technologies

• It helps understand gene structure, function, and variation across individuals and populations.

What are the research areas in genomics

• Functional genomics: investigate gene function and structure

• Structural genomics: determine a 3D structure for every protein

• Metagenomics: study of all
  microorganism in an environmental sample by DNA sequencing

Metagenomics is a research field in genomics. What is it?

• Used to study microorganisms, especially those that cannot be easily cultured.

• Sequences DNA directly from environmental samples (soil, water, gut).

• Commonly targets 16S rRNA gene:

  • Present in all microbes

  • Conserved and variable regions → allows classification

  • Helps discover new species never cultured before

What is transcriptomics

• Studies the transcriptome = all RNA molecules in a cell under certain conditions.

• Answers: which genes are active, at what levels and in which cells.

• Methods: RNA-seq or microarrays.

What are the challenges for studying RNA vs DNA

• RNA is easily degraded (by RNase):

  • unstable

  • RNase common

• Harder to examine RNA because:

  • relative RNA abundance may be low

  • may contain interfering molecules

  • may be difficult to isolate your sample

• Many types of RNA:

  • RNA isolation method affects your results

What are included in the RNA quality controls

• Purity: DNA and interfering molecules must be removed

• Quality: good quality needed = not degraded

• Quantity: need enough materials to work with

Typically investigated spectrophotometrically and on gelelectrophoresis/similar

What is the purpose of microarray analysis

To measure how much RNA is present by hybridizing labeled RNA to known DNA sequences on a chip to determine how much a gene is transcribed.

What are the steps in microarray analysis

1. A chip (microarray) contains thousands of short DNA probes.

2. RNA from a sample is converted to cDNA, labeled with fluorescent dyes.

3. The labeled cDNA hybridizes to matching DNA on the chip.

4. Fluorescence is measured to determine expression levels.

What are features and limitations with microarray analysis

Features:

• DNA probes are synthesized with photolithography, allowing high density chips (up to 300 000 oligonucleotides/cm²)

• Can analyze many genes at once, but only for known sequences

Limitations:

• Only works for known sequences

• Reproducibility may vary due to labeling/hybridization differences.

• Requires reference genes (e.g., actin) for normalization.

What is RNA sequencing

To capture and sequence the full set of RNA transcripts in a sample. Unlike microarrays, RNA-seq does not require prior knowledge of the sequences.

1. Isolate RNA (especially mRNA, which is only ~5% of total RNA).

2. Convert RNA to cDNA using unspecific primers.

3. Degrade RNA strand, synthesize second DNA strand.

4. Sequence the cDNA using high-throughput methods.

5. Assembly: Align reads to the genome to quantify expression and find splice variants.

What are the advantages of RNA sequencing

• Untargeted analysis

• Can detect splice variants

What is alternative splicing

One gene can produce multiple mRNA by including or excluding several exons

Why is alternative splicing important to consider

• 95% of human multi-exon genes undergo alternative splicing.

• Increases protein diversity.

• Involved in cell-specific functions (e.g., neurons vs thyroid).

• ~15% of genetic diseases are linked to splicing errors.

What is the goal of RNA isolation and what methods are used

The goal is to purify RNA, often only mRNA (5% of total mRNA), from tissues

Methods:

• Magnetic beads: coated with oligo(dT) to bind the poly-A tail of mRNA

• rRNA depletion: depleting rRNA (major part) from the total RNA

• cDNA capture: a method to capture cDNA and analyze the expression of genes

• Total RNA isolation: includes all RNA-types. Typically the first step of RNA isolation

Why is RNA isolation important

• The method of use affects which RNAs you detect

• mRNA isolation is critical for transcriptomics

What is the goal of RNA-seq assembly

To identify which parts of the genome are transcribed and at what levels.

What are the two main strategies for RNA-seq assembly

• Map RNA sequence reads directly onto the genome sequence

• Construct contigs and map these onto the genome sequence

What is Cap Analysis of Gene Expression (CAGE)

Purpose: identify transcription start sites by sequencing only the 5’ ends of transcripts.

1. Capture mRNAs via their 5’ cap using biotin and avidin beads.

2. Add special oligonucleotides with restriction enzyme sites. First strand of cDNa is synthesised. RNA is degraded. Second strand of cDNA is synthesized. cDNA is restricted into short fragments (downstream of the recognition site).

3. Sequence short restricted fragments starting from the 5’ end.

Transcriptomics is a study of all RNA transcripts in a cell at a given time and condition. What is the challenge with this and what are the new strategies to tackle this challenge.

Tissues contain many cell types. If analyzed as a whole, signals from small subpopulations may be masked.

New Advances:

• Single-cell transcriptomics: study individual cells. High cell-type resolution, no location info.

• Spatial transcriptomics: keep track of location in tissue. Includes location info, may sacrifice single-cell precision depending on method.

What is single cell transcriptomics

• Measures gene expression in individual cells.

• RNA sequencing is done per cell

• Great for discovering unknown cell types or subpopulations

There are two types of spatial transcriptomics. Name and describe them

Imaging based: In situ hybridization of probes - detected by high resolution microscopy.

Sequencing based: In situ capturing of sequences - RNA coupled to adaptors

Imaging based spatial transcriptomics is a targeted analysis. What are the pros and cons with this

• Pro: high spatial resolution

• Con: limited to number of parallell genes

Sequencing based spatial transcriptomics is an untargeted analysis. What are the pros and cons with this

• Pro: more genes studied in parallel

• Con: lower resolution (not single cell)

What is proteomics

Proteomics refers to the systematic identification and quantification of the
complete set of proteins (the proteome) of a biological system (cell, tissue,
organ, biological fluid, opganism etc) at a specific point of time.

What are the two common techniques used in proteomics

• Mass spectrometry: Measures how fast peptides fly through a vacuum tube → gives m/z.

• 2D gel electrophoresis: proteins separated in 2 dimensions

Describe 2D gel electrophoresis

• Combines two rounds of gel electrophoresis (in different dimensions) or combines isoelectric focusing+gel electrophoresis

• Difficult to get 100% reproducible:

  • need to compare to reference proteins

  • can compare 2 proteomes in one round if proteins are differently labeled (different fluorescent markers)

• Proteins that differ can be cut out and studied further

Describe using mass spectrometry in proteomics

1. Proteins are separated (often using HPLC).

2. Proteins are digested (e.g. with trypsin) into peptides.

3. Peptides are ionized.

4. Mass/charge (m/z) ratio is measured using MALDI-TOF.

MALDI-TOF: Matrix-Assisted Laser Desorption/Ionization - Time Of Flight.

• Measures how fast peptides fly through a vacuum tube → gives m/z.

• Advantages: Very accurate. Can detect small differences (e.g. with isotope labeling like ICAT).

What is single cell proteomics

Similar to transcriptomics, proteomics can benefit from sampling single cells (instead of mixtures of several cell types) to avoid mixing signals from different cell types.

What is protein-protein interaction and why is it important

• Proteins often work together in complexes.

• Interactions reveal function and signaling pathways.

How can protein interactions be identified

• Phage display:

• Yeast two-hybrid:

• Functional protein array

Describe phage display

Create a library of phages that display different proteins. Screen them to see which ones bind to a specific target.

Describe yeast two-hybrid

Detect protein-protein interaction using transcription-based reporter system

1. System setup: Yeast cells are genetically modified to include a reporter gene (e.g. lacZ) that responds only to a functioning transcription factor (TF).

2. Splitting the TF:

   • TF is split into:

     • DB (DNA-binding domain) – binds DNA but can't activate transcription alone.

     • AD (Activation domain) – activates transcription but cannot bind DNA alone.

3. Fusion proteins:

   • The protein of interest ("bait") is fused to DB.

   • The potential interaction partner ("prey") is fused to AD.

4. Interaction = transcription:

   • If bait and prey interact, the DB and AD are brought together, reconstituting a functional TF and activating the reporter gene (visible as a blue color on X-gal plates).

What is metabolomics

Metabolomics refers to the systematici dentification and quantification of the metabolic products (the metabolome, substrates, intermediates and products of cell metabolism) of a biological system (cell, tissue, organ, biological fluid or organism) at a specific point in time.

What are the two common techniques used in metabolomics

• Mass spectrometry

• NMR spectrometry

Lipidomics is a branch of metabolomics. Describe it

• Study of lipid metabolic pathways and networks.

• Importance: Helps understand disease mechanisms (e.g. diabetes, obesity) and identify biomarkers for metabolic disorders.

What is epigenomics

• Epigenetics is the study of molecular processes that influence the flow of
  information between a constant DNA sequence and variable gene expression patterns, without changing the DNA sequence.

• This includes investigation of nuclear organization, DNA methylation, histone
  modification and RNA transcription

• Epigenetic processes can result in intergenerational (heritable) effects as well as clonal propagation (fortplantning) of cell identity without any mutational change in DNA sequence

What are the three main types of epigentic modifications

1. DNA Methylation:

   • Methyl groups added to CpG sites.

   • Silences genes (transcription OFF).

   • 5-methylcytosine is the common mark.

2. Histone Modifications:

   • Affects how tightly DNA is wrapped:

     • Acetylation = loose (euchromatin) → gene ON.

     • Methylation = tight (heterochromatin) → gene OFF.

   • Also includes phosphorylation, ubiquitination.

3. Non-coding RNAs (miRNA):

   • Can bind to mRNA and block translation or promote degradation

How do you study epigenetics (has the modification occured)

För att studera metylering: DNA behandlas med natriumbisulfat

• Kemisk behandling som påverkar cytosiner.

• Om cytosinen inte är metylerad → Den omvandlas till uracil.

• Vid sekvensering
  → Uracil läses som tymin → alltså sker en C → T-ändring i sekvensen.

• Om cytosinen är metylerad (5-mC)
  → Den skyddas och förblir cytosin (C).

• Resultat
  → Genom att jämföra med obehandlat DNA kan man se vilka cytosiner som var metylerade (de som inte ändrats till T).

För att studera histonmodifieringar:

• ChIP-seq

• Mass spectrometry

För att studera noncoding RNAs:

• RNA-seq