Genome analysis

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Dideoxy sequencing (Sanger)

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 Sequencing with fluorescent dyes

Phred scores the

peak quality
score 20 (P =0.01)
score 30 (P =0.001)
=0 001)

Used to determine
the reliability of the
data

Allows filtering
g
on quality
(required for building
contigs, sets of
overlapping DNA
segments)

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Example of genome sequencing

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 Gene finding programs (predictions)

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Experimental support predicted genes

• Identity to previously annotated sequence

• Similarity to sequences from other organisms

• Presence of conserved protein domains

• Sequencing of mRNA (cDNA, ESTs)

• RT PCR off selected

RT-PCR l t d genes

• DNA microarrays to monitor expression

• Sequencing all transcripts (RNA-Seq)

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 Use of tiling arrays  transcriptome

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Affymetrix Genechips: array

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 Whole genome tiling array
• Full human genome
on 14 arrays with
each 6.000.000 probes
p

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Examples of new transcript

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 Example of wrong strand

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Example of novel 5’ exon

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 Dark regions of genomes
• Approx. >10 fold more transcription takes place
than is expected based on known genes

• Transcripts corresponding to
 Introns
 Unknown 5’ and 3’ exons
 Intergenic regions

• Transcript of Unknown Function (TUFs)

 What do they regulate?
 What is their function?

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Next Generation Sequencing

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 Evolution of sequencing methods
• Sanger sequencing
 Cloning and amplification of DNA in E. coli
 Plasmid DNA isolation,, separate
p seq.
q reactions

• Next Generation Sequencing

 No cloning step
 PCR amplification (on beads or colonies)
 Massive pparallel sequencing
q g approaches
pp

• Next Next Generation Sequencing

 No PCR amplification step
 Single molecule sequencing approaches
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Amplification in microreactors (454)

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 Sequencing in picotiter plates

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Pyrosequencing

Release
R l off pyrophosphate
h h
Is detected by a luciferase-
coupled detection system

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 Illumina method
1G
genome
analyzer

Data produced
In Tbytes

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Sample preparation

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 Production of polonies (PCR colonies)

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Sequencing using reversible terminators

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 Multiple cycles allow sequencing

Short reads
of about 25 nt

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Genome analysis
• Primary sequence of genomes

• Which parts are transcribed into RNA?

 mRNA, small RNAs (miRNA, siRNA), other

• Where do DNA binding proteins act on the DNA?

• What is the epigenetic status of the DNA

 Chromatin
Ch ti structure
t t
 3D-organisation
 DNA-methylation status

• All dependant on tissue and condition

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 Powerful applications of NGS
• mRNA-Seq
 Method to quantify mRNA levels (as microarrays)

• S ll RNA
Small RNA-Seq
S
 Mine and quantify miRNAs, siRNAs, etc.

• ChIP-Seq
 Chromatin IP to select bound DNA sequences

• Genomic footprinting (nucleosomes and TFs)

• Methyl-Seq (or BS-Seq)

 Conversion of cytosines to uracil by bisulfite
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mRNA--Seq
mRNA

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 mRNA--Seq:
mRNA Seq: methods of DNA conversion

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ChIP--Seq
ChIP

Chromatin Immune Precipitation

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 Genomic footprinting using NGS

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Methyl--Seq (or bisulfite sequencing)

Methyl
• Bisulfite-conversion of cytosine
to thymidine, but not of methyl-C

• Compare reads to reference sequence

(including C to T conversions)

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 Next--Next Generation Sequencing
Next
• Single molecule approaches
 Sequencing by synthesis
• Visigen, using modified and immobilized polymerase
• Helicos, using immobilized single DNA molecules

 Direct sequencing
• Nanopore sequencing
• Single polymerase
imaging

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Helicos single molecule sequencing

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 Helicos

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Nanopore sequencing

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 Single molecule real time sequencing

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