Genomikáróláltalában, főbb metodikák, összehasonlításuk 2 ...biotech.szbk.u-szeged.hu/biotechmsc/MG_MSC_2011_ora1.pdf · Functional genomics Related to transcriptomics and

1. Genomikáról általában, főbb metodikák, összehasonlításuk

2. Második generációs szekvenálás alkalmazási területei 1.

3. Második generációs szekvenálás alkalmazási területei 2.

4. Mutagenezis technikák/ Harmadik generációs szekvenálás

Genomics is the study of the genomes of organisms.

Milestones:- Full sequence of f-X174 bacteriophage (5368 bp) 1977, Frederick Sanger -The first free-living organism to be sequenced was that of Haemophilus influenzae (1.8 Mb) in 1995, Hamilton Smith - Shotgun technique 1998, Celera Genomics- Fruit fly (Drosophila melanogaster) in 2000- Human in 2001 (3.3 Gb)- Human in good quality in 2007 (less than one error in 10,000 bases and all chromosomes assembled)-Today: more than 1000 prokaryotic genomes, more than 2500 viruses and around 100 eukaryotic genomes (more than half are fungi) are fully sequenced

Genomics

Functional genomics

Personal genomics

Metagenomics

Nutrigenomics

Psychogenomics

Nitrogenomics Hydrogenomics etc.

Pharmacogenomics

Functional genomics

Related to transcriptomics and proteomics

Only 1.5 % of the human genome encodes proteins (ca. 20 000 genes)

Personal genomics

Pharmacogenomics

Nutrigenomics

Psychogenomics (Behavioural genetics)

The role of genes in behaviour.

Metagenomics

Nitrogenomics

Techniques applied in Genomics

General features:- High-troughput- Generate huge amount of data- Data evaluation is often the main challenge

1. Microarray techniques

2. Sequencing techniques

1. Microarray techniques

Arrayed series of thousands of spots of DNA oligonucleotides, called probes. Probes can beshort sections of genes that are used to hybridize a cDNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified bydetection of fluorophore- or chemiluminescence-labeled targets to determine relativeabundance of nucleic acid sequences in the target.

Microarray experiment

Microarray’s weaknesses

Ioannidis et al; Nat Genet 41 (2009)

- arrays prerequisite known sequences- examination of similar sequences is almost impossible by arrays (cross-hybridization, specificity issues)- array reproducibility shows huge fluctuations

2. Sequencing techniques

1. Traditional sequencing: non-high-throughput(not detailed here, see Gabor Rakhely’s lecture)1.1. Maxam-Gilbert method1.2. Chain termination method (Sanger method): key principle is the use of

dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators1.3. Shotgun sequencing (Sanger chemistry)

Chain termination method Shotgun seq

2. High-throughput sequencing NGS (next generation sequencing)

2.1. Second generation sequencing (pyrosequencing, sequencing byligation), this is the present454 Life Sciences: pyrosequencingIllumina: sequencing by synthesisABi SOLiD: sequencing by ligation

2.2. Third generation sequencing (nanopore sequencing, one moleculesequencing), this is the futurePacific BiosciencesHelicosComplete GenomicsEtc.

Need for library preparation in a host

• Labour and time - intensive, expensive

• Toxic regions are not represented

• Host genome contaminations

Low throughput

• strand synthesis and base determination are separated

• need for electrophoretic step

• high unit cost (cost/bp)

No need for library preparation in a host

• immobilized template fragments, PCR methods

• labour, time and cost effective

High throughput

• several millions of sequencing /run

• synthesis and sequencing are not separated

Sanger sequencing

NGS (Next Generation Sequencing)

No competition, but complementation

Long read, low coverage

Short read, huge coverage(especially SOLiD and Illumina)

Use in: De novo sequencing, validation

Use in: Resequencing, SNP analysis, RNA-Seq

Main characteristics of sequencing generations

Illumina platform

454 FLX

October 2007

SOLiD V 1.0

June 2008

SOLiD V 2.0

August 2010

SOLiD V 4

ABi SOLiD platform

Comparison of NGS technologies

Illumina

Mix DNA Library

& capture beads

(limited dilution)

454 FLX Technology

“Break micro-reactors”

Isolate DNA containing beads

• Generation of millions of clonally amplified sequencing templates on each bead

• No cloning and colony picking

Create

“Water-in-oil”

emulsion

+ PCR Reagents

+ Emulsion Oil

Perform emulsion PCR

Adapter carrying

library DNA

A

BMicro-reactors

Centrifuge Step

Load Enzyme

Beads

44 μm

Load beads into

PicoTiter™Plate

454 FLX Technology

Photons

Generated are

Captured by

Camera

Reagent Flow

PicoTiterPlate Wells

Sequencing

By Synthesis

(pyrosequencing)

Sequencing Image Created

454 FLX Technology

Enzymes needed:- DNA polymerase, ATP sulfurylase, luciferase, apyrase

Template: ssDNA

Addition of one of the four dNTPs in each step

Flow Order

• Count the photons generated for each “flow”

• Base call using signal thresholds

• Delivery of one nucleotide per flow ensures accurate base calling

TACG

1-mer

2-mer

3-mer

4-mer

KEY (TCAG)

Measures the presence

or absence of each

nucleotide at any given

position

454 FLX Technology: Basecalling

Summary of 454 FLX

• Read length: 350-450 bases

• Throughput: 400MB/slide/run

(average bacterial genome size: 5 MB)

• Homopolymer problem

(caused by proportionality of light intensity)

Illumina Technology

Step 1-6DNA Fragmentation

Adaptor ligationTemplate amplification

Illumina Technology

Step 7-12

Base determination

(sequencing by synthesis,

differently labeled nucletides,

laser excitation, fluorescence

detection)

Base imaging

Multiple cycles

Illumina Technology

Summary:

• Read length: 35-50 bases

• Throughput: 8 GB/flowcell/run

(1600x coverage on 5 MB bact. genome)

• High accuracy (no homopolymer issue)

Flow cell

SOLiD™ Chemistry