Moderne Geräte zurDNA-Sequenzierung

ABI Pyroseq 454 Solexa Solid

Kapillar-SequenziererABI 3730

                                                                 

Highest-quality sequencing •Capillary temperatures up to 700C to melt secondary structure •New separation matrix (POP-7™ Polymer) provides up to 1,100 bp reads •High pass rate and long reads reduce non-electrophoretic project costs •New basecaller with quality values improves raw and consensus accuracies

Maximum optical sensitivity •Simultaneous illumination of all capillaries •In-capillary detection •Backside-thinned CCD •High sensitivity supports maximum range of input DNA concentration •Dual-side capillary illumination provides very uniform optical detection

Maximum throughput •New high-speed separation matrix •Simultaneous injection of 96 samples •Automated polymer replenishment •Choice of capillary lengths supports 48 runs per day

Enhanced Automated Operation •Integrated autosampler and sample plate stacker •Internal (and optional external) bar code reader •On-board reagents for up to 100 runs

Lowest-cost DNA sequencing •High optical sensitivity reduces DNA and reagent consumption •Separation matrix consumption 30% lower than earlier models •Automation reduces labor and costs from human error •Very long reads reduce non-electrophoretic project costs •High throughput lowers depreciation, overhead, and service costs per result

ABI 3730Probendurchsatz

1.0 Mb/d 1.5 Mb/d 2.1 Mb/d

Pyro-SequenzierungPSQTM 96

PyrosequencingPyrosequencing

• Real time sequencing (Sequence by synthesis)

Detect incorporation of nucleotides Monitor run as it is proceeding

• Four-enzyme mixture

• Detection of pyrophosphate release upon nucleotide incorporation

• Fast, accurate and simple to use

PPi

ATP

PyrosequencingPyrosequencing

PyrosequencingPyrosequencing

sequence to analyze:

A/GCTGCCT

Primer designPrimer design

• Secondary structure:

Crucial to check for self-annealing

5’ overhangs may result in

background

Avoid unspecific annealing to template

5� ---GGCGGATCGATGACGAT TAGCAGTAGCTAGGCGG---5�

Locking template’s 3’ ends Locking template’s 3’ ends

Utting et al., Biotechniques 37:66-73 (2004)

Locking template’s 3’ ends Locking template’s 3’ ends

Utting et al., Biotechniques 37:66-73 (2004)

B

C

E

D

A

C/C

T/T

T/C

A Theoretical histograms for the genotypes determined by SNP rs226379. Positions of bases informative for the SNP are shown on yellow background.

B Pyrogram of a blank control experiment of single-stranded template without sequencing primer.

C Pyrogram obtained using the standard protocol.

D Pyrogram obtained using blOligo and E Pyrogram using ddCMP modified template.

Red arrows point to allele specific base.

The blue insets give the allele frequencies calculated from the peak heights.

A Revolutionary Method for Rapid Whole Genome Sequencing and AssemblyAnd more……..

454Genome Sequencer GS20/FLX

454Nature, July 31 2005

• 25 Mb• 99% accuracy• 4h

• de novo assembly Mycoplasma genitalium (0.58 Mb): 96% coverage, 99,96% accuracy

• 100x currant Sanger technology

454Nature, July 31 2005

454Workflow

dsDNA fragments

PA BLigation

Fill in

Capture on SA-Beads & WashAlkaline Elution

A B

P Bio

Bio

Bio

Bio

Bio

454Preparation of A/B fragments for sequencing

454emPCR

454Sample loading

454Sequencing

454Single read

454Read length & quality

GC normal, high-cloned coverage

% GC

% GC

green - 454 readsred - Sanger readsblack - GC content

454AT-rich sequences

GC poor, low-cloned coverage(discontinuous without 454 reads)

454Ultra deep sequencing

Background subtracted

Primer sequence

C to T ratio: 1/500

T only

Primer sequence

0.16%

Mut

atio

n F

req.

Mut

atio

n F

req.

Cov

erag

eC

over

age

454Ultra deep sequencing: rare mutations

Sanger

GAAATGGNTTTGCC

Unresolvable region

Mu

tati

on

Fre

q.

Co

vera

ge

GACATGAATTTG|| |||| ||||GAAATGAGTTTG

34% of readsGAAATG-GCTTTGCC|||||| | ||||||GAAATGAG-TTTGCC

39% of reads

GAAATGCAGTT-GCCAGG|||||| |||| ||||||GAAATG-AGTTTGCCAGG

21% of reads

454Ultra deep sequencing: subspecies, haplotypes

• ~250 nt/read• 100 Mb/run• 7h

• paired end sequencing in one well

454GS FLX

• 84 cycles of 4 nucleotides ~250 b• 168 cycles > 400 b occasionally

• paired end sequencing in one well

454Outook

Solexa

Advanced genetic analysis

one billion bases at a time

SolexaTechnology

• Clonal Singe Molecule Array TM

• Sequencing By Synthesis (SBS)

• reversible fluorescent labels

• reversible 3’OH blocking

SolexaLinker ligation

SolexaSolid-phase clonal single molecule PCR

“Bridge amplification”

100μm

colony of ≈1000 single-stranded DNA templates

Single well of   454 Life Sciences 

PicoTiterPlateTM (to same scale)

SolexaSolid-phase clonal single molecule PCR

SolexaSequencing by synthesis (SBS)

removal of fluorescent labels & 3’OH blocking*

100μm

colony of ≈1000 single-stranded DNA templates

SolexaSequencing by synthesis (SBS)

1 2

SolexaAssembly

read length: 25-30 bp

SolexaRead length

0

20

40

60

80

100

5 10 15 20 25 30 35 40 45 50

3 errors

2 errors

1 error

Perfect

%

SolexaRead length

SolexaApplications

• Sequencing & re-sequencing

• expression profiling

• small RNA identification & quantification

ABI AgencourtSOLID

SOLIDHardware

SOLIDLibraries

SOLIDMate-pair library

SOLIDMate-pair library

SOLIDBead deposition

SOLIDProbes

SOLID4-color ligation

SOLID4-color ligation

SOLIDDetection

SOLIDCleavage

SOLID2nd cycle ligation

SOLID2nd cycle detection

SOLID2nd cycle cleavage

SOLIDReset after 5th cycle

SOLID6th cycle ligation

SOLIDCycling scheme

SOLIDPaired ends – two sequences

SOLID2-base encoding

SOLIDPerformance

genome.fli-leibniz.deLectures