2223 Functional Genomics (KW3
V)
Radboud University
Functional Genomics
(02-02-2023)
Kevin Booij
,Inhoud
Functional Genomics (02-02-2023) College 1 – introduction.................................................................4
Functional Genomics (02-02-2023) College 1 – “Epigenomics 1”...........................................................7
Lecture 3 - Mhlanga_FG1_3D_Genome_1_Genome Architecture 1 (09-02-2023) Functional Genomics
(developmental genomics)...................................................................................................................22
Concept of Chromatin conformation capture (3C / 4C / 5C / , the conceptual steps of the protocol,
differences in applications) and interpretation of experimental results...............................................27
Models of enhancer function (looping) and interaction principles.......................................................29
How do boundaries and their effects on gene expression relate to chromatin topology (TADs)?.......30
Sequencing and Genomics....................................................................................................................34
3D Arcitecture of DNA packing.............................................................................................................38
Chromosome Conformation Techniques:.........................................................................................38
Chromosome conformation capture (3C).........................................................................................38
Hi-C:..................................................................................................................................................38
Chia PET:...........................................................................................................................................39
Arcitectural Proteins maintain chromatin loops...............................................................................39
CTCF:..........................................................................................................................................39
Boundaries / Insulators:.............................................................................................................40
Cancer Genomics..................................................................................................................................40
Point Mutation.................................................................................................................................40
How you do Exome Sequencing.................................................................................................40
Cancer Landscapes.....................................................................................................................41
Mutational Landscape of Glioblastoma......................................................................................41
Rearrangements...............................................................................................................................41
Small alterations.........................................................................................................................41
How mutations accumulate........................................................................................................42
Genome Engineering & Genome-wide screens....................................................................................42
How to disrupt gene function?.............................................................................................................44
CRISPR Cas (Can be used to read more about CRISPR Cas)...................................................................53
Explanation of CRISPR-Cas9 Genome editing Technology................................................................53
CRISPR/Cas9-based genome editing in eukaryotes..........................................................................54
CRISPR as experimental tool.............................................................................................................55
Modifications of CRISPR/Cas9 systems.............................................................................................55
,Somatic mutations in cancer................................................................................................................59
Rearrangements in cancer....................................................................................................................61
Considerations when designing a screening assay...............................................................................64
,Functional Genomics (02-02-2023) College 1 – introduction
Introduction:
Focused on reading literature.
Lectures and slides provided study information.
80 % exam
20 % practical on march 3rd
,Functional Genomics (02-02-
2023) LE 1 – “Sequencing
Genomics”
Sanger sequencing
o DNA chains elongates at -OH group on ribose
o Primer, dNTPs, polymerase, ddNTP with fluorescent label, Reading colours sequence
base sequence of DNA.
o Tumor research, multiple graphs at one place = mutation.
PCR
Next Generation Sequencing (NGS)
Illumina sequencing
See powerpoint PDF.!!!
Ion torrent sequencing (NGS)
Powerpoint, ken je al
Use short reads to compare it to the reference genome.
Mapping/alignment of the genome
,Single Molecule Real Time sequencing (PacBio)
SMRT sequencing uses mixed dNTPS attached to fluorophores. In contrast to Illumina, SOLiD and
Semiconductor methods, SMRT sequencing used a polymerase fixed to the bottom of the well which
pull the template DNA through. The retention time of the fluorescent dNTP at the bottom of the well
serves as the read-out.
Nanopore sequencing
Base sequence is determined using changes in current over the membrane. DNA can be sequenced
by threading it through a microscopic pore in a membrane. Bases are identified by the way they
affect ions flowing through the pore from one side of the membrane to the other. The Oxford
Nanopore MinION is a hand-held genome sequencer that directly connects with a laptop (via USB).
1. One protein unzips the DNA helix into two stands. A second protein creates a pore in the
membrane and hold an adapter molecule.
2. A flow of ions through the pore creates a current. Each base blocks the flow to a different degree,
altering the current.
3. The adapter molecule keeps bases in place long enough for them to be identified electronically.
Compared to Sanger sequencing, advantages of the next-generation technologies mentioned thus
far, including 454/Roche, Illumina, and SOLiD, alleviate the need for in vivo cloning by clonal
amplification of spatially separated single molecules using either emulsion PCR (454 and SOLiD) or
bridge amplification on solid surface (Illumina). In addition to providing a means for cloning-free
amplification, these methods use single-molecule templates allowing for the detection of
heterogeneity in a DNA sample (e.g., identifying mutations present only in a subpopulation of cells),
which is a significant advantage over Sanger sequencing.
Whole and targeted genome sequencing
Goal: mutation discovery
Probable germline mutation Reference genome as control
Probably de novo mutation own cells as reference.
RNA-sequencing
Isolate RNA
Reverse transcriptase into cDNA
Produce second strand cDNA
Fragment double stranded cDNA
Ligate adapters
PCR amplify
Sequence
Map to genome
o Gaps in graph introns are removed since we look at mature mRNA!
Looking at expression of the genes
Chromatin immune-precipitation (ChIP)-sequencing
Used to identify where a protein is contacting to the chromatin (so where it is located in the
genome)
Can be applied to:
o DNA binding transcription factors
o Vasal transcription machinery (RNA pol II)
o Histone modification
Process
o Crosslink protein to DNA
o Shear DNA strands by sonicating
, o Add bead attached to antibodies to immunoprecipitated target protein
o Unlink protein and purify DNA
o Sequencing
o Map to genome
DNasel/ATAC sequencing
Where is the chromatin mainly closed and where are they open? Potential places for
transcription factors to bind? Sequence strand between chromatins are sequenced!
Transposons, loaded with a side of sequencing adapter. Both adapters sequence in
between can be measured since it is dsDNA.
Overlap with start sites/ promotor sites.
DNaseI-seq and ATAC-seq are used to determine which regions in the genome are most
accessible. For instance to determine where nucleosomes are displaced to allow
transcription factors to bind.
The concept of the approach: Tn5 is a transposase that inserts a piece of DNA into the
genome. However, it can only insert this DNA at positions where the chromatin is accessible
This has been used to directly add sequencing adaptors into the genome. Combined with PCR
this also directly provides fragments of the right size.
Functional Genomics (02-02-2023) College 1 – “Epigenomics 1”
Epigenomics
Is the study the complete set of epigenetic modifications on the genetic material of a cell,
known as epigenome.
Reversable modifications on a cell’s DNA or histones that affect gene expression without
altering the DNA sequence.
, ---
Nucleosomes consist of an octamer of histones wrapped with DNA.
Nucleosome is the repeating unit of chromatin.
Lots of variants of histones.
Histone tails:
Stick out physically of the structure.
o Writers
Chemical modifications generally take place at accessible
Lysine (acetylation, methylation and ubiquitylation) and Arginine
residues (methylation). Phosphorylation can take place at Serine and
Threonine residues.
H3K4, histone 3 – lysine K4 methylation
Acetylation: Acetylation removes the positive charge on the histones, thereby decreasing the
interaction of the N termini of histones with the negatively charged phosphate groups of
DNA. As a consequence, the condensed chromatin is transformed into a more relaxed
structure that is associated with greater levels of gene transcription.
Methylation: Methylation of histones can either increase or decrease transcription of genes,
depending on which amino acids in the histones are methylated, and how many methyl
groups are attached.
Ubiquitination: The addition of a large macromolecule, such as ubiquitin, to a histone tail would lead
to a modification of the high-order chromatin structure. Additionally, ubiquitination represents a
signal for successive histone modifications and/or a signal for recruitment of other proteins to the
chromatin.
Phosphorylation: Histone phosphorylation confers a negative charge to the histone, resulting in a
more open chromatin conformation. It is therefore associated with gene expression and is involved
in DNA damage repair and chromatin remodelling
Enzymes that can add chemical groups (writers)
o Kinases
o Histone/ lysine acetyl transferases (KATs/HATs)
V)
Radboud University
Functional Genomics
(02-02-2023)
Kevin Booij
,Inhoud
Functional Genomics (02-02-2023) College 1 – introduction.................................................................4
Functional Genomics (02-02-2023) College 1 – “Epigenomics 1”...........................................................7
Lecture 3 - Mhlanga_FG1_3D_Genome_1_Genome Architecture 1 (09-02-2023) Functional Genomics
(developmental genomics)...................................................................................................................22
Concept of Chromatin conformation capture (3C / 4C / 5C / , the conceptual steps of the protocol,
differences in applications) and interpretation of experimental results...............................................27
Models of enhancer function (looping) and interaction principles.......................................................29
How do boundaries and their effects on gene expression relate to chromatin topology (TADs)?.......30
Sequencing and Genomics....................................................................................................................34
3D Arcitecture of DNA packing.............................................................................................................38
Chromosome Conformation Techniques:.........................................................................................38
Chromosome conformation capture (3C).........................................................................................38
Hi-C:..................................................................................................................................................38
Chia PET:...........................................................................................................................................39
Arcitectural Proteins maintain chromatin loops...............................................................................39
CTCF:..........................................................................................................................................39
Boundaries / Insulators:.............................................................................................................40
Cancer Genomics..................................................................................................................................40
Point Mutation.................................................................................................................................40
How you do Exome Sequencing.................................................................................................40
Cancer Landscapes.....................................................................................................................41
Mutational Landscape of Glioblastoma......................................................................................41
Rearrangements...............................................................................................................................41
Small alterations.........................................................................................................................41
How mutations accumulate........................................................................................................42
Genome Engineering & Genome-wide screens....................................................................................42
How to disrupt gene function?.............................................................................................................44
CRISPR Cas (Can be used to read more about CRISPR Cas)...................................................................53
Explanation of CRISPR-Cas9 Genome editing Technology................................................................53
CRISPR/Cas9-based genome editing in eukaryotes..........................................................................54
CRISPR as experimental tool.............................................................................................................55
Modifications of CRISPR/Cas9 systems.............................................................................................55
,Somatic mutations in cancer................................................................................................................59
Rearrangements in cancer....................................................................................................................61
Considerations when designing a screening assay...............................................................................64
,Functional Genomics (02-02-2023) College 1 – introduction
Introduction:
Focused on reading literature.
Lectures and slides provided study information.
80 % exam
20 % practical on march 3rd
,Functional Genomics (02-02-
2023) LE 1 – “Sequencing
Genomics”
Sanger sequencing
o DNA chains elongates at -OH group on ribose
o Primer, dNTPs, polymerase, ddNTP with fluorescent label, Reading colours sequence
base sequence of DNA.
o Tumor research, multiple graphs at one place = mutation.
PCR
Next Generation Sequencing (NGS)
Illumina sequencing
See powerpoint PDF.!!!
Ion torrent sequencing (NGS)
Powerpoint, ken je al
Use short reads to compare it to the reference genome.
Mapping/alignment of the genome
,Single Molecule Real Time sequencing (PacBio)
SMRT sequencing uses mixed dNTPS attached to fluorophores. In contrast to Illumina, SOLiD and
Semiconductor methods, SMRT sequencing used a polymerase fixed to the bottom of the well which
pull the template DNA through. The retention time of the fluorescent dNTP at the bottom of the well
serves as the read-out.
Nanopore sequencing
Base sequence is determined using changes in current over the membrane. DNA can be sequenced
by threading it through a microscopic pore in a membrane. Bases are identified by the way they
affect ions flowing through the pore from one side of the membrane to the other. The Oxford
Nanopore MinION is a hand-held genome sequencer that directly connects with a laptop (via USB).
1. One protein unzips the DNA helix into two stands. A second protein creates a pore in the
membrane and hold an adapter molecule.
2. A flow of ions through the pore creates a current. Each base blocks the flow to a different degree,
altering the current.
3. The adapter molecule keeps bases in place long enough for them to be identified electronically.
Compared to Sanger sequencing, advantages of the next-generation technologies mentioned thus
far, including 454/Roche, Illumina, and SOLiD, alleviate the need for in vivo cloning by clonal
amplification of spatially separated single molecules using either emulsion PCR (454 and SOLiD) or
bridge amplification on solid surface (Illumina). In addition to providing a means for cloning-free
amplification, these methods use single-molecule templates allowing for the detection of
heterogeneity in a DNA sample (e.g., identifying mutations present only in a subpopulation of cells),
which is a significant advantage over Sanger sequencing.
Whole and targeted genome sequencing
Goal: mutation discovery
Probable germline mutation Reference genome as control
Probably de novo mutation own cells as reference.
RNA-sequencing
Isolate RNA
Reverse transcriptase into cDNA
Produce second strand cDNA
Fragment double stranded cDNA
Ligate adapters
PCR amplify
Sequence
Map to genome
o Gaps in graph introns are removed since we look at mature mRNA!
Looking at expression of the genes
Chromatin immune-precipitation (ChIP)-sequencing
Used to identify where a protein is contacting to the chromatin (so where it is located in the
genome)
Can be applied to:
o DNA binding transcription factors
o Vasal transcription machinery (RNA pol II)
o Histone modification
Process
o Crosslink protein to DNA
o Shear DNA strands by sonicating
, o Add bead attached to antibodies to immunoprecipitated target protein
o Unlink protein and purify DNA
o Sequencing
o Map to genome
DNasel/ATAC sequencing
Where is the chromatin mainly closed and where are they open? Potential places for
transcription factors to bind? Sequence strand between chromatins are sequenced!
Transposons, loaded with a side of sequencing adapter. Both adapters sequence in
between can be measured since it is dsDNA.
Overlap with start sites/ promotor sites.
DNaseI-seq and ATAC-seq are used to determine which regions in the genome are most
accessible. For instance to determine where nucleosomes are displaced to allow
transcription factors to bind.
The concept of the approach: Tn5 is a transposase that inserts a piece of DNA into the
genome. However, it can only insert this DNA at positions where the chromatin is accessible
This has been used to directly add sequencing adaptors into the genome. Combined with PCR
this also directly provides fragments of the right size.
Functional Genomics (02-02-2023) College 1 – “Epigenomics 1”
Epigenomics
Is the study the complete set of epigenetic modifications on the genetic material of a cell,
known as epigenome.
Reversable modifications on a cell’s DNA or histones that affect gene expression without
altering the DNA sequence.
, ---
Nucleosomes consist of an octamer of histones wrapped with DNA.
Nucleosome is the repeating unit of chromatin.
Lots of variants of histones.
Histone tails:
Stick out physically of the structure.
o Writers
Chemical modifications generally take place at accessible
Lysine (acetylation, methylation and ubiquitylation) and Arginine
residues (methylation). Phosphorylation can take place at Serine and
Threonine residues.
H3K4, histone 3 – lysine K4 methylation
Acetylation: Acetylation removes the positive charge on the histones, thereby decreasing the
interaction of the N termini of histones with the negatively charged phosphate groups of
DNA. As a consequence, the condensed chromatin is transformed into a more relaxed
structure that is associated with greater levels of gene transcription.
Methylation: Methylation of histones can either increase or decrease transcription of genes,
depending on which amino acids in the histones are methylated, and how many methyl
groups are attached.
Ubiquitination: The addition of a large macromolecule, such as ubiquitin, to a histone tail would lead
to a modification of the high-order chromatin structure. Additionally, ubiquitination represents a
signal for successive histone modifications and/or a signal for recruitment of other proteins to the
chromatin.
Phosphorylation: Histone phosphorylation confers a negative charge to the histone, resulting in a
more open chromatin conformation. It is therefore associated with gene expression and is involved
in DNA damage repair and chromatin remodelling
Enzymes that can add chemical groups (writers)
o Kinases
o Histone/ lysine acetyl transferases (KATs/HATs)
