Identification of genes by function interaction

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Identification of genes by function (interaction)
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Yeast two-hybrid screen for protein interactors
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Genomics
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Gene expression

• Genome maping
• Genome sequencing
• Genome annotations

Structural genomics
Nucleus
DNA (Genome)
pre-mRNA
Cytoplasm
mRNA
Functional genomics
mRNA (Transcriptome)
Proteins (Proteome)
Metabolites (Metabolome)
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History of genomes sequencing

• 1977 bacteriophage øX174 (5386bp, 11 genes)
• 1981 mitochondrial genome (16,568bp 13 prots 2
  rRNAs 22 tRNAs
• 1986 chloroplast genome (120,000-200,000bp)
• 1992 Saccharomyces chromosome III (315kb 182
  ORFs)
• 1995 Haemophilus influenzae (1.8Mb
• 1996 Saccharomyces whole genome (12.1Mb over 600
  people 100 laboratories)
• 1997 E. coli (4.6Mb 4200 proteins)
• 1998 Caenorhabditis elegans (97 Mb 19,000 genu)
• 2000 Arabidopsis thaliana (115Mb, 25-30,000 genu)
• 2001 mouse (1 year!)
• 2001 Homo sapiens (2 projekty)
• 2005 Pan, rice
• 2006 Populus

Technological improvements
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DNA sequencing principle(Sangers method)
Polymeration from primer in the presence of low
concentration of terminator (dideoxy) ddNTP
primer
Random termination on all positions with
occurance of the nucleotide
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A T C G

• Original arrangement
• sequence
• - RA labelled primer
• 4 separated reactions
• - with individual ddNTP
• - ddNTPdNTP (cca 120 (100))
• - PAGE separation

C
T
G
G
A
T
C
T
A
G
C
Separation by size
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Automated sequencing with fluorescence-labelled
ddNTP

• Every ddNTP labelled with different fluorescent
  dye all together in one reaction
• Separation by size in capillary fluorescence
  detection

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Genom sequencing is more than sequencing of DNA

• 1 sequencing reaction 300 800 bp
• Typical genom hunderts of millions to billions bp
  
• How to manage?

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Strategies of genome sequencing

• Classical strategy (Map-Based Assembly)
• - minimal quantity of DNA sequencing
• sorting of big DNA fragments, successive
  reading
• (human genome sequencing original strategy)
• - scaffold for genome sequence assemble
• - time consuming
• Whole genome shotgun (WGS)
• random (7-9x redundant) sequencing
• sorting of sequence data (Haemophilus)
• - problems with repetitive DNA
• Combination hierarchical shotgun, chromosome
  shotgun

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Hierarchical shotgun sequencing
Whole-genome shotgun sequencing
Production of over-lapping clones (e.g. BACs,
YACs) and construction of physical map
Shearing of DNA and sequencing of subclones
Assembly
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(No Transcript)
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Hierarchical shotgun sequencing

• First step library of big DNA inserts
• ( genome fragments)
• phage (l) vectors 30 kb
• cosmids 50 kb
• BACs (bacterial artificial chromosomes)
• 100-300 kb
• YACs (yeast artificial chromosomes)
• cca 0.5-1Mb

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Number of 100 kb BACs to cover the whole genome
(with 99.995 probability)
(Cullins, 2004)
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Physical BAC map of genome

• Arrangement (position, orientation) of individual
  BAC in the genome
• Fundamental for classical sequencing
• Very usefull for assembly of shotgun sequences
• How to make the map from BACs with unknown
  sequence?

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Map construction - BAC fingerprinting
Sequencing of DNA ends
Restriction sites

• 10-20x more bp in BACs than in the genome for
  map construction (Arabidopsis 20 000, rice -
  70 000)

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MTP, minimum tiling paththe lowest possible
number of BACs to cover the sequence
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Walking method of genome sequencing

• Redundant BAC library (tens of thousands)
  sequencing of BAC ends
• Sequencing of few (hunderts) seed clones
• Walking by BAC end sequence

seeds
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Teoretically one seed per chromosome is enough,
but many steps

• Better to use more seed clones

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Filling of gaps shorter clones are better

• - optimal libraries with different insert sizes
  (2, 10, a 50 kbp)

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Whole Genome Shotgun (WGS) and variations
genome
Sequencing of clone ends (known distance between)
plasmids (2 10 Kbp)
cosmids (40 Kbp)
500 bp
500 bp
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Genome (chromosome, BAC...) assembly

• Looking for overlaps in sequences
• Assembly to contigs
• Assebly to supercontigs using the information of
  sequence pairs (ends distance)
• 4. Complete consensus sequence

..ACGATTACAATAGGTT..
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Repetitive sequences and contig assembly
Repetition are problem, if they are longer than
sequencing run
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Use of physical map for genome assembly(STS
sequence tagged sites short sequences with
known position on chromosoms)
Supecontigs with scaffold (BAC-end sequences with
known distance)
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What to do with the genome sequence? To annotate!

• Searching for genes
• Automatic prediction of coding seq.
• Prediction of introns/exons
• Prediction according to related seq.
• Confirmation by cDNA and EST
• Prediction of function
• from experimentally characterized homologues

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Comparison of genetic a physical map
physical (bp)
genetic (cM)
Arabidopsis chromosome IV
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Large genomes alternative strategies of
sequencing- isolation of individual
chromosomes (wheat)- shotgun sequencing of
non-methylated DNA(maize)- sequencing of ESTs
(potato)
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• Expressed Sequence Tags (ESTs)
• short sequenced regions of cDNA (300-600 nt)
• usually gene fragments (primarilly originate from
  mRNA)
• highly redundant, but also incomplete!
• problems - no regulatory sequences (promotors,
  introns,...)
• only transcripts of certain genes

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Expressed Sequence Tags (ESTs)
Preparation of EST library

• - mRNA
• - RT with oligoT primer ? cDNA
• cleavage of RNA from heteroduplex
• RNAseH
• - 2nd strand cDNA synthesis
• - cleavage with restriction endonuclease
• - adaptor ligation cloning

sequencing
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Assembly of EST contigs - Unigenes
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Group Organism EST Genomic Asterids
Lycopersicon (tomato) Nicotiana
(tobacco) Solanum (potato) Rosids
Arabidopsis Brassica (oilseed
rape) Gossypium (cotton) Glycine
(soybean) Lotus Medicago
Populus (poplar) Monocots Hordeum
(barley) Oryza (rice)
Sorghum Triticum (wheat)
Zea (maize) Conifers Pinus (pine)

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New technology for DNA sequencing
20 Mbp for 4 hours Bacterial genome for 4 days
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454 technology (nanodrop sequencing)
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454 technology
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454 technology
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454 technology
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http//mammoth.psu.edu/rico.d/index.html