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Genome Analysis Genome Analysis: Mastercourse Biomolecular Sciences, May 2009 14-5-2009
Genome Analysis Genome Analysis: Mastercourse Biomolecular Sciences, May 2009 14-5-2009
Genome Analysis Genome Analysis: Mastercourse Biomolecular Sciences, May 2009 14-5-2009
1
Sequencing with fluorescent dyes
Used to determine
the reliability of the
data
Allows filtering
g
on quality
(required for building
contigs, sets of
overlapping DNA
segments)
2
Gene finding programs (predictions)
3
Use of tiling arrays transcriptome
4
Whole genome tiling array
• Full human genome
on 14 arrays with
each 6.000.000 probes
p
5
Example of wrong strand
6
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
7
Evolution of sequencing methods
• Sanger sequencing
Cloning and amplification of DNA in E. coli
Plasmid DNA isolation,, separate
p seq.
q reactions
8
Sequencing in picotiter plates
Pyrosequencing
Release
R l off pyrophosphate
h h
Is detected by a luciferase-
coupled detection system
9
Illumina method
1G
genome
analyzer
Data produced
In Tbytes
Sample preparation
10
Production of polonies (PCR colonies)
11
Multiple cycles allow sequencing
Short reads
of about 25 nt
Genome analysis
• Primary sequence of genomes
12
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
mRNA--Seq
mRNA
13
mRNA--Seq:
mRNA Seq: methods of DNA conversion
ChIP--Seq
ChIP
14
Genomic footprinting using NGS
15
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
16
Helicos
Nanopore sequencing
17
Single molecule real time sequencing
18