Decoding & Understanding

Genomes
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What is a Genome?
• A complete set of an organisms DNA is called its genome

• Human diploid genome is 6.37/6.27 (female/male) Gigabase pairs

(Gbp)

• Weighs 6.41 picograms and end-to-end would be 205 cm long

• Full copy in the majority of cells in the body

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How is DNA packed into the cell nucleus?

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Aside - Polytene Chromosomes
Occur when many
chromosomes align with
each other without cell
division
Allowed the visualisation of
chromosome bands leading
to the idea that genes
resided on chromosomes.
Found in pea plants (aka
Mendel) and Drosophila (aka
Morgan).

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In Person Quiz 1
In Person Quiz 1
DNA Sequencing

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DNA Synthesis is also now cheap

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Sequencing technologies
Sanger Sequencing
• Sometimes called chain-termination
sequencing or dideoxy sequencing.
• Based upon in vitro DNA replication
• Utilises random incorporation of
modified, fluorescently tagged bases
onto the growing DNA strand.
• The 4 standard bases are tagged with
a different fluorophore so they can be
distinguished from one another.

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Sanger Sequencing

10 Adapted from - https://www.youtube.com/watch?v=gDdoGcGS014

DNA Sequencing technologies
Sanger Sequencing

• Used for routine sequencing

• Reads are 500 -1000bp

• Efficient and reliable

• Low throughput

• Requires known primers

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NGS DNA Sequencing technologies
Next Generation Sequencing technologies

• High-throughput, multi parallel

sequencing

• Up to 600 billion bases in one reaction

• Like Sanger sequencing, is based

upon in vitro DNA replication

• Does not require known primers

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NGS DNA Sequencing technologies
Next Generation Sequencing technologies
• Different sequencing chemistries (sequencing
by synthesis, sequencing by ligation,
pyrosequecing and ion semiconductor
sequencing )
• All require samples to be prepared into
libraries
• All require specialised machines with solid
surfaces to create ‘clusters’ of DNA
• Machines output raw data and increasingly
this is directly used for bioinformatic analysis

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NGS DNA Sequencing
sequencing by synthesis
• Genomic DNA is fragmented

• 5′and 3′adapters are ligated

• Adapter-ligated fragments are PCR

amplified.

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NGS DNA Sequencing

• Library is loaded into a flowcell

• Fragments are captured on a lawn of

surface-bound oligos complementary
to the library adapters

• Each fragment is then amplified into

distinct, clonal clusters.

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NGS DNA Sequencing

• All four terminator–bound dNTPs are

present during each sequencing cycle

• Unlike Sanger sequencing, these

modified bases can be converted back
to a ‘regular’ bases and do not halt the
reaction i.e. no normal bases required

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NGS Sequencing

17 Adapted from - https://www.youtube.com/watch?v=gDdoGcGS014

NGS Sequencing
• Used for high throughput analysis
• highly accurate sequencing that greatly
reduces context–specific sequencing
errors, even within repetitive sequence
regions
• Expensive and requires dedicated
machines
• Not suitable for routine sequencing
• Enables personal genomics

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Personal Genomics

• A new age of eugenics?

• Gattaca (1997) Written and

Directed by Andrew Niccol

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In Person Quiz 2
In Person Quiz 2
What does my genome tell about me?
Some genetic risk factors with strong effects can be identified (e.g.
BRCA alleles and breast cancer).

But most ‘gene’ associations are weak and effects are hard to
predict.

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Human Eye Colour

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Human Eye Colour

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What does my genome tell about me?
Some genetic risk factors with strong effects can be identified (e.g.
BRCA alleles and breast cancer).

But most ‘gene’ associations are weak and effects are hard to
predict.

Gene to phenotype associations are complex and penetrance can

vary.

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What is a gene and how many are there?
A gene is a section of DNA that encodes a function
Only a small percentage of genomes are translated into functional
protein products (<2% in case of mouse genome, FANTOM Consortium,
2005)
In the human genome it has been estimated that 86,245 different
protein products could be produced from 19,969 protein coding genes
by alternative splicing (Nurk et al. 2022). There may be twice the number
non-coding genes

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What is a gene and how many are there?
A gene is a section of DNA that encodes a function
Only a small percentage of genomes are translated into functional
protein products (<2% in case of mouse genome, FANTOM Consortium,
2005)
In the human genome it has been estimated that 86,245 different
protein products could be produced from 19,969 protein coding genes
by alternative splicing (Nurk et al. 2022). There may be twice the number
non-coding genes
Due to introns and other regulatory non-coding sequences, ~40% of
human genome may be required to produce proteins.

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Controlling gene expression

Enhancer + Promoter Open Reading Frame Termination sequence

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Transcription Factors
Transcription factors are proteins involved in transcribing DNA into
RNA.

Many have DNA-binding domains that allow them to bind to specific

sequences of DNA in gene promoters or enhancers. Transcription
factors can also interact with each other or additional protein co-factors
to change their DNA binding properties.

~1500 estimated transcription factors in the human genome

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Transcription Factors

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Salzer & Kumar Plos One 2010
Controlling gene expression

Epigenetic modifications

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Controlling gene expression

Epigenetic modifications

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Transcription Factors
Transcription factors are proteins involved in transcribing DNA into
RNA.

Many have DNA-binding domains that allow them to bind to specific

sequences of DNA in gene promoters or enhancers. Transcription
factors can also interact with each other or additional protein co-factors
to change their DNA binding properties.

~1500 estimated transcription factors in the human genome

Predicting functional enhancer sequences is difficult.

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Controlling gene expression

Enhancer + Promoter Open Reading Frame Termination sequence

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Protein Coding Genes

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 mRNA Splicing

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Pre-mRNA Splicing
RNA Splicing: catalysed by a large ribonucleoprotein (RNA & Protein)
complex called the spliceosome

The spliceosome is a highly dynamic assembled by sequential binding

and release of the small nuclear RNAs (snRNAs) and protein factors
that can influence splicing.

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Pre-mRNA Splicing

U2 (Major) U12 (Minor)

U1 U2 U11 U12
U5 U5
U4 U6
U4 U6 atac atac

>99% of introns <1% of introns

U2 U12
type type

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mRNA Splicing
Include Polypeptide A Include Polypeptide A

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mRNA Splicing
Include Polypeptide A Include Polypeptide A

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mRNA Splicing
Exclude Polypeptide B Include Polypeptide B

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mRNA Splicing
Inlcude Polypeptide C Exclude Polypeptide C

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Protein Coding

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Pre-mRNA Splicing

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Pre-mRNA Splicing
RNA Splicing: catalysed by a large ribonucleoprotein (RNA & Protein)
complex called the spliceosome

The spliceosome is a highly dynamic assembled by sequential binding

and release of the small nuclear RNAs (snRNAs) and protein factors
that can influence splicing.

Alternative splicing factors can produce a large diversity of protein

products from one gene

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Pre-mRNA Splicing

Drosophila Dscam: 38,016 different peptides from one gene

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Post Transcription regulation of RNA

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Post Transcription regulation of mRNA
Transcription control

RNA processing control

RNA transport control

Translation control

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Cloning

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Cloning

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Cloning
Inefficient: Multiple surrogates and many cloned embryos to achieve a single
successfully cloned animal.

Can be useful for valuable agricultural animals and rare species. Used
extensively for pets (~40K CHF to clone your dog)

Seems safe – clones and their progeny have normal lifespan and no seemingly no
adverse health issues

Proposals to clone extinct animals (e.g. Wolly Mammoth) rarely talk about the
number of surrogates required, in addition to other ethical issues.

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In Person Quiz 3
In Person Quiz 3
Outcome not guaranteed
Developmental differences

Environmental differences

Epigenetics

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 Thak You & Questions
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