moderne geräte zur dna-sequenzierung
TRANSCRIPT
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Moderne Geräte zurDNA-Sequenzierung
ABI Pyroseq 454 Solexa Solid
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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
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ABI 3730Probendurchsatz
1.0 Mb/d 1.5 Mb/d 2.1 Mb/d
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Pyro-SequenzierungPSQTM 96
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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
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PPi
ATP
PyrosequencingPyrosequencing
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PyrosequencingPyrosequencing
sequence to analyze:
A/GCTGCCT
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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�
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Locking template’s 3’ ends Locking template’s 3’ ends
Utting et al., Biotechniques 37:66-73 (2004)
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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.
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A Revolutionary Method for Rapid Whole Genome Sequencing and AssemblyAnd more……..
454Genome Sequencer GS20/FLX
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454Nature, July 31 2005
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• 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
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454Workflow
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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
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454emPCR
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454Sample loading
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454Sequencing
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454Single read
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454Read length & quality
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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)
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454Ultra deep sequencing
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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
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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
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• ~250 nt/read• 100 Mb/run• 7h
• paired end sequencing in one well
454GS FLX
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• 84 cycles of 4 nucleotides ~250 b• 168 cycles > 400 b occasionally
• paired end sequencing in one well
454Outook
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Solexa
Advanced genetic analysis
one billion bases at a time
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SolexaTechnology
• Clonal Singe Molecule Array TM
• Sequencing By Synthesis (SBS)
• reversible fluorescent labels
• reversible 3’OH blocking
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SolexaLinker ligation
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SolexaSolid-phase clonal single molecule PCR
“Bridge amplification”
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100μm
colony of ≈1000 single-stranded DNA templates
Single well of 454 Life Sciences
PicoTiterPlateTM (to same scale)
SolexaSolid-phase clonal single molecule PCR
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SolexaSequencing by synthesis (SBS)
removal of fluorescent labels & 3’OH blocking*
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100μm
colony of ≈1000 single-stranded DNA templates
SolexaSequencing by synthesis (SBS)
1 2
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SolexaAssembly
read length: 25-30 bp
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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
%
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SolexaRead length
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SolexaApplications
• Sequencing & re-sequencing
• expression profiling
• small RNA identification & quantification
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ABI AgencourtSOLID
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SOLIDHardware
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SOLIDLibraries
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SOLIDMate-pair library
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SOLIDMate-pair library
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SOLIDBead deposition
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SOLIDProbes
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SOLID4-color ligation
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SOLID4-color ligation
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SOLIDDetection
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SOLIDCleavage
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SOLID2nd cycle ligation
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SOLID2nd cycle detection
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SOLID2nd cycle cleavage
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SOLIDReset after 5th cycle
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SOLID6th cycle ligation
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SOLIDCycling scheme
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SOLIDPaired ends – two sequences
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SOLID2-base encoding
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SOLIDPerformance
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genome.fli-leibniz.deLectures