Genomics Gene expression Genome maping

Genomics

Gene expression • Genome maping • Genome sequencing • Genome annotations Structural genomics Nucleus DNA (Genome) pre-m. RNA Cytoplasm • DNA arrays and chips • RNA sequencing • (semi) q. RT-PCR • Northern blot + hybrid. • Transkriptional fusions on ati gul Re m. RNA (Transcriptome) Proteins (Proteome) Metabolites (Metabolome) • 2 D electrophoresis • Gel-free methods Mass spectrometry Protein sequencing • Translational fusional • Immunodetection • Enzyme activities • Chromatography • Mass spectrometry • NMR Functional genomics

History of genomes sequencing • 1977 bacteriophage øX 174 (5386 bp, 11 genes) • 1981 mitochondrial genome (16, 568 bp; 13 prots; 2 r. RNAs; 22 t. RNAs • 1986 chloroplast genome (120, 000 -200, 000 bp) • 1995 Haemophilus influenzae (1. 8 Mb) • 1996 Saccharomyces whole genome (12. 1 Mb; over 600 people 100 laboratories) • 1997 E. coli (4. 6 Mb; 4200 proteins) • 1998 Caenorhabditis elegans (97 Mb; 19, 000 genes) • 2000 Arabidopsis thaliana (115 Mb, 25 -30, 000 genes) • 2001 mouse (1 year!) • 2001 Homo sapiens (2 projects) • 2005 Pan, rice • 2006 Populus Technological improvements

DNA sequencing – principle (Sanger’s method) Polymeration from primer in the presence of low concentration of terminator (dideoxy) dd. NTP primer Random termination on all positions with occurance of the nucleotide

Original arrangement sequence - RI labelled primer - 4 separated reactions - with individual dd. NTP - dd. NTP: d. NTP (cca 1: 20 – (100)) - PAGE separation A T C G C T G G A T C T A G C Separation by size

Automated sequencing with fluorescence-labelled dd. NTP • Every dd. NTP labelled with different fluorescent dye – all • together in one reaction Separation by size in capillary – fluorescence detection

Next generation sequencing (NGS) - faster and cheaper!!! - parallel sequencing of high numbers of sequences! - no handling with individual sequences! Basic principles: Template - clone of identical DNA molecules (PCR) - single DNA molecule Reaction - synthesis of complementary strand - ligation of oligonucleotides (on the template) - DNA degradation by exonuclease - scanning of ss. DNA strand Detection - optical: substrate incorporation (fluorescent labelled substrates, linked luminiscent reaction) - electronic: products of degradation, NT in ss. DNA

NGS – comparison of basic parameters Method Single-molecule real-time sequencing (Pacific Bio) Ion Sequencing by semiconductor Pyrosequencing synthesis (Ion Torrent (454) (Illumina) sequencing) Read length 5. 000 -10. 000 (30. 000) bp up to 400 bp Reads per run 50. 000 Cost per 1 million bases (in US$) $0. 33 -$1. 00 700 bp Sequencing by ligation (SOLi. D sequencing) Chain termination (Sanger sequencing) 50 to 300 bp 50+50 bp up to 80 million 1 million up to 3 billion 1. 2 to 1. 4 billion N/A $1 $0. 05 to $0. 15 $0. 13 $2400 http: //en. wikipedia. org/wiki/DNA_sequencing $10 400 to 900 bp Examples of recently developed or developing technologies: 454 sequencing – pyrosequencing (Roche) - complementary strand synthesis Illumina – sequencing by synthesis - complementary strand synthesis SOLi. D - Sequencing by Oligonucleotide Ligation and Detection - ligation of labelled oligonucleotides Oxford nanopore technology - exonuclease degradation, el. current changes detection

Illumina – sequencing by synthesis (Solexa)

Illumina – seqencing by synthesis (Solexa)

Illumina – seqencing by synthesis (Solexa)

Illumina – seqencing by synthesis (Solexa)

Oxford nanopore technologies – direct sequencing http: //www. nanoporetech. com/sequences of one DNA strand - protein nanopore in membrane (alpha-hemolysin) - covalently bound exonuclease - monitoring specific decrease in current (met. C!)

Pac. Bio: SMRT (Single-Molecule Real-Time) sequencing - direct sequencing of circular ss. DNA - sequencing by synthesis (polymerase and fluorescent d. NTPs) - modified bases – slowing down the reaction - low accuracy for single reads (repeated reading - circularity!) - molecules 7 kbp-15 kbp (- 20 kbp)! zero-mode waveguides (ZMWs) - fluorescent signal only from the well bottom (no light going through the well (70 x 100 nm), label cleaved upon incorporation (PPi)

Genome sequencing is not only sequencing of DNA • single sequencing read • 300 – 800 bp (Sanger, 454, Illumina) • 10 s of kbp (Pac. Bio, Oxford nanopore) • Typical genom hunderts of millions to billions bp How to manage?

Genome (chromosome, BAC. . . ) assembly 1. Looking for overlaps in primary sequences 2. Assembly to contigs to get short consensus sequences 3. Assebly to supercontigs using the information of sequence pairs (ends + distance) 4. Complete consensus sequence . . ACGATTACAATAGGTT. .

Repetitive sequences complicate contig assembly Repetitive seq. ? ? Repetitions are serious problem in assembly, if they are conserved and longer than sequence reads

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 -9 x redundant) sequencing – sorting of sequence data (Haemophilus) - problems with repetitive DNA • Combination – „hierarchical shotgun“, „chromosome shotgun“

Hierarchical shotgun sequencing Whole-genome shotgun sequencing Production of overlapping clones (e. g. BACs, YACs) and construction of physical map Shearing of DNA and sequencing of subclones Assembly Green (2001) Nature Reviews Genetics 2: 573 -583

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 -1 Mb

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?

Map construction - BAC fingerprinting = restriction analysis Sequencing of DNA ends Restriction sites - 10 -20 x more bp in BACs than in the genome for map construction (Arabidopsis – 20 000, rice - 70 000)

BAC fingerprinting ANIMATION of HIERARCHICAL SHOTGUN: http: //www. weedtowonder. org/sequencing. html

Minimum tiling path = the lowest possible set of BACs covering the whole sequence physical map arrangement and mapping and clone selection - by restriction fragment analysis - using terminal sequences and hybridization - by hybridization with markers with known position in genetic map

Filling of gaps clones to be sequenced X - sequencing of longer clone ends (clone end tracking) - optimal – libraries with different insert sizes (2, 10, a 50 kbp) - sequencing the linker sequence = filling the gap

Shotgun sequencing BAC/chromosome/whole genome random cleavage + direct sequencing (NGS) Alternatively (today) - long sequence reads (Pac. Bio, Oxford nano) Cosmids (40 Kbp): sequencing of clone ends (known distance between) ~500 bp

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 c. DNA and EST • Prediction of function – from experimentally characterized homologues

Fragment of Gen. Bank BAC clone annotation

Graphical interface of BAC annotation

Large polyploid genomes alternative strategies of sequencing: - isolation of individual chromosomes e. g. wheat – allows assembly of homeologous chromosomes (allohexaploid) - shotgun sequencing of non-methylated DNA (maize) - sequencing of ESTs (potato)

Expressed Sequence Tags (ESTs) -short sequenced regions of c. DNA (300 -600 nt) -usually gene fragments (primarilly originate from m. RNA) -highly redundant, but also incomplete! -problems: - no regulatory sequences (promotors, introns, . . . ) - only transcripts of certain genes

Expressed Sequence Tags (ESTs) Preparation of EST library - m. RNA - RT with oligo. T primer c. DNA -cleavage of RNA from heteroduplex RNAse. H - 2 nd strand c. DNA synthesis - cleavage with restriction endonuclease - adaptor ligation cloning sequencing

Assembly of EST contigs - Unigenes

454 technology - pyrosequencing up to 1 mil reads (lenght 700 - 1000 bp) one day (23 hour procedure) = 500 -800 Mbp

454 technology - pyrosequencing

454 technology

454 technology

SOLi. D™ System (Applied Biosystems) 2 Base Encoding Sequencing by Oligonucleotide Ligation and Detection - reads up to 75 b - 20 -30 Gb for a day! - high accuracy up to 99, 99 % - initial step – clonal multiplication (similar to 454) http: //appliedbiosystems. cnpg. com/Video/flat. Files/699/index. aspx

SOLi. D™ System Mix of 1024 octamers (number of variations NNN = 64) x 16 known dinucleotides Z = nucleotides universally pairing with any nucleotide (prolongation) – cleaved out after ligation labelling: 4 fluorescent dyes – each for 256 octamers (with just 4 known middle dinucleotides) -

5 independent reactions = each 10 – 15 times repeated ligations of labelled octamers starting from a primer with shifted end

Knowledge of the first nucleotide allows translation of color sequence to nucleotide sequence AATGCA GGCATG CCGTAC } alternative translation with different 1 st nucleotide