1. Sequencing
tutorial
Peter HANTZ
EMBL Heidelberg

2. Dideoxy (Sanger) sequencing
Uni Osnabruck
M. Waterman
Dideoxy (Sanger) sequencing
Principle:
Gel electrophoresis: discrimination of 1 bp below ~1000 bp
Synthesis: starts with a DNA oligo, stops after incorporating a (marked) ddNTP
First ~ 60 bp uncertain (high relative mass of the fluo. dye)
Radiolabeling:
4 reactions
Csak kis resze dideoxy, gel erzekenysege 1 bp, kell oligonukleotid az inditashoz
Dye-termination:
4 fluorescent dyes,
one reaction

3. Pyrosequencing (Roche / 454)ds
Bead I.
Streptavidin coated
Odaragadthoz kepest komplementer lejon es kesobb felhasznaljuk, golyok kidobodnak
Library construction
A,B: short DNA oligos fused with
genomic DNA segments
B is biotinilated
Selection of dsDNA: streptavidin-coated magnetic beads
denaturation: AB strands collected
wiki

4. Pyrosequencing (Roche / 454)
Bead II.
Simple agarose beads coated with B oligos
Single sstDNA (singles-stranded template DNA)
with cA and cB oligo immobilized one on a bead
Bead-bound library emulsified (water-in-oil)
Lanyok a golyon, fiuk raragadnak, VIZCSEPP|!!!! Egyelore ketszalu dna, egyik szal kovalensen kapcs, a masik csak hibridizacio reven
PCR reaction:
One strand will be covalently bound to the bead
wiki

5. Pyrosequencing (Roche / 454)
denaturation, one strand is released
Following the selection of DNA-positive beads (enrichment),
Beads+reactants in wells having a diameter of cca 40um
Denaturalas: egy szal maradjon, seq by synthesis, ez csak egy szallal mukodik
wiki

6. Pyrosequencing (Roche / 454)
The reaction:
-addition of dNTP-s:
incorporation releases pyrophosphate
(only one phosphate is needed for the backbone)
-ATP sulfurylase converts PPi to ATP
-luciferase: acts in the presence of ATP
-Unincorporated nucleotides
and ATP are degraded by the apyrase
Leszakad pirofoszfat (2P), ebbol lesz ATP, feny detektalasa, egyszinu
-400,000 reads in parallel
-multiple consensus incorporations:
>higher signal intensity
>problematic...
wiki

7. Illumina (Solexa) sequencing
-making DNA library (~300bp fragments)
-ligation of adapters A and B to the fragments
Denaturalas, a szalak csak hibridizalnak
-binding the ssDNA randomly to the flow cell surface
-complementary primers are ligated to the surface
Illumina-Fasteris

8. Illumina (Solexa) sequencing
Bridge amplification:
initiation
Itt meg nem szekvenalasi cellal szintetizalunk. Az uj szal kovalensen kapcsolodik az uveghez, az eredeti elvesz
On the surface: complementary oligos
GeneCore

9. Illumina (Solexa) sequencing
Itt meg mindig nem syekvenalunk, csak soxorositunk, linearisan
EMBL Gene Core

10. Illumina (Solexa) sequencing
Data aquisition:
sequencing by synthesis:
“reverible terminator” nucleotides
blocked + fluorescently labeled
Itt a szekvenalas szintezis altal!! Fityego resz levagasaval inditom ujra
de-blocking to enable the synthesis
dye cleavage+elimination
wash step+repeat
TGCA
illumina.com

11. Illumina (Solexa) sequencing Mate-pair sequencing
Biotinnal fogjuk meg az ertekes reszt, TRUKKOS MODSZER MINDKET VEGEN VALO SZEKVENALASRA

12. Single Molecule Real Time Sequencing
Principle:
fluorescent label on the terminal phosphate of NTP-s
DNA polymerase:
cleaves this
incorporation lasts ~ mS
Detection:
"Zero-Mode Waveguide" holes:
near-field standing waves
(~Total Internal Reflection )
Pirofoszfat kilepes! Lenyeg: egyetlen molekula eleg, video
Present performance:
1,500 bp in read lengths
Wiki
Pacific Biosciences

13. The genome is fragmented randomly (sonication)
Assembling
Shotgun sequencing
The genome is fragmented randomly (sonication)
No positional and orientatin information is available
The fragments are sequenced
The results have to be assembled
A ket veguk van szekvenalva, itt a tartalom nem erdekes, nincs transzlacio, szetvagott kep analogia
Merging reads into contigs

14. Bridges of Königsberg Leonhard Euler, 1735 Graphs
Graphs
set of edges that connect pairs of nodes
used to model pairwise relations between certain objects
Bridges of Königsberg
Leonhard Euler, 1735
Find a path that visits each bridge (=edge) once!
Eulerian path problem: visit each edge once and only once:
linear-time algorithm

15. Hamiltonian Path Problem
Find a route that visits
each node (=each airport)
exactly once
This is an NP (Non-Polynomial) -problem
Nem-polinomialis, iszonyatos szamitasi kapacitast er
Nem polinomialis, iszonyatos szamitasi kapacitast igenyel
the amount of computation necessary,
using the most efficient algorithms known at present,
grows exponentially with the size of the route map

16. Traveling Salesman Problem
Traveling Salesman Problem
Find the shortest path which visits every vertex exactly once.
That is: the shortest Hamiltonian pathway
This is also an NP-hard problem...

17. The Shortest Superstring Problem
Given a set of strings,
find a shortest string that contains all of them
Input: Strings s1, s2,…., sn
Output: A string s that contains all strings s1, s2,…., sn as substrings,
such that the length of s is minimized
Equivalent of:
-finding the shortest Hamiltonian pathway
-TSP

18. Graph Theory helps DNA assembly
University of Maryland
"Translation" of the problem: a model
Nodes: reads
Edges: connects nodes if the corresponding reads overlap
Example: assembling a bacterial genome
Red lines - wrong assembly
Bold Black lines - good assembly
Assembling the reads = finding the shortest Hamiltonian pathway = TSP = SSP
NP...impossible...?

19. The Way Out: Constructing and analyzing de Bruijn Graphs
Finding Eulerian paths in the de Bruijn graph can lead to sequence reconstruction
Linear problem!
J. Kaptcianos

20. Thank You for Your attention!

21. Second-generation DNA sequencing "Sequencing by synthesis" methods
(Solexa)
300bp [normal] - 10kb [mate-pair]
(454)
1-10 kb, and 20 kb in expt. stage
DNA Colonies amplified by PCR: “Polonies”
(Solexa)
isothermal extension "bridge PCR"
note: even PCR-free!
(454)
emulsion PCR
Gelelfo: adott merettertomanyu dns-ek kivalasztasa; SEQ BY SYNTHESIS-mi ez, egyszalu dns, bazispar beepulese, fenykibocsatas
fluorescent imaging of the entire array
Reads:
(Solexa): ~50-80
(454): ~
Nature Biotech, vol. 26

22. Illumina (Solexa) sequencing flow cell: Paired-end sequencing
EMBL GeneCore

23. ABI: capillary electrophoresis sequencing and SoLID

24. Directed graphs
We assign a certain direction with the edges
The Eulerian Path Problem can be re-formulated accordingly:
Visit each edge 1! while passing along the edges in their direction
Note: Eulerian path might not exist!

25. (also known as Overlap-Layout-Consensus method)
M. Waterman
Examples:
tenyleg legrovidebb-e
kezdet
Red: repeats
(also known as Overlap-Layout-Consensus method)

26. directed graph representing overlaps between sequences of symbols
The Way Out: Constructing and analyzing de Bruijn Graphs
directed graph representing overlaps between sequences of symbols
Given sequences of symbols (~reads): ATG, TGG, TGC, GTG, GGC, GCA, GCG, CGT
"k-length fragments" (k=3)
Nodes: fragments of k-1 (k-1=2)
Edges: k-length fragments connecting overlapping vertices
Finding Eulerian paths in the de Bruijn graph can lead to sequence reconstruction
(Superpath problem, Merging transformation, etc.)
Linear problem!
J. Kaptcianos