Post on 06-Feb-2018
Principle of Real-Time qPCR
AND
Applications
Han-Oh Park, Ph.D.
President & CEO
BIONEER CORPORATION
- -
Principle of Real-Time
qPCR AND Applications
Exicycler
Understanding Real Time PCR Data
(2007 6 )
(BIONEER CORPORATION) 49-3175 1992 8 28 5,67511,350,000 (1 500)www.bioneer.co.kr 116(06), 100(05), 92(04)
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Principle of Real-Time qPCR
Principle of PCR
Basics of PCR
Polymerase
Primer
Extension
72
Cycling
Cycling
1 Cycle
Principle of PCR
Cockerill FR III. Arch Pathol Lab Med. 2003;127:1112
Basics of PCR
Disadvantages of PCR
End-Point PCR analysis is not quantitative
Disadvantages of PCR
What is Wrong with Agarose Gels?
Poor precision
Low specificity
Size-based discrimination only
Low sensitivity/Resolution
Short dynamic range (< 2 logs)
Possibility of human errors
Cross-contamination
Results are not expressed as numbers
Ethidium bromide staining is not very quantitative
1. The problem of traditional quantification when using agarose gel
Real-Time PCR
Real-Time PCR
Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection
2. Optical
Components
3. Thermal
Cycler
1. Fluorescence
Dyes
Key components of Real-Time qPCR
Principle of Real-Time PCR
Real-
Time
qPCR
Nigel Walker, NIEHS
Principle of Real-Time PCR
Real-time PCR vs. Conventional PCR
Advantage of Real-Time PCR
Real-Time PCRPCRSensitivityHighLowSpecificityHigh-use specific probesLow-only size discriminationQuantitative resultsYes-Specific fluorescenceNo-EtBr stainingDetection methodProbe-specific FluorescenceAgarose gelElectrophoresisDetection rangeWide rangeShort range (
Amplification plot: The plot of cycle number vs. fluorescence signal which correlates with the initial amount of target nucleic acid during the exponential phase of PCR
Baseline: The initial cycles of PCR during which there is little change in fluorescence signal (usually cycles 3 to 15)
Glossary of Real-Time PCR
Baseline
Threshold
Ct
Amplification plot
cycle
Fluorescence
1. Terminology and definition used in real time PCR
Threshold: the fluorescence measurement at which product can be distinguished from background. Threshold should be set in the region associated with an exponential growth of PCR product
Ct (Threshold cycle): The cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the log of he initial copy number
Glossary of Real-Time PCR
Baseline
Threshold
Ct
Amplification plot
cycle
Fluorescence
1. Terminology and definition used in real time PCR
Detection Chemistry
1. Introduction of Exicycler
Primer & Probe
Primer
Short (Often < 50 nt) oligonucleotide sequence of DNA
Complementary to the beginning and the end of the target DNA sequence
Needed to initiate the synthesis of new DNA in a PCR reaction
Involved in AMPLIFICATION
Probe
A single-stranded DNA with a specific base sequence
Labeled with fluorescence dyes (TaqMan probe)
Used to detect the complementary base sequence of target DNA/RNA by hybridization
Involved in DETECTION
Reporter dye / Quencher dye
Primer
Probe
Detection Chemistry
DNA binding agents
- Intercalating method: SYBRGreen I
Fluorescent dyes
Hydrolysis Probe
- TaqMan probe, Molecular Beacon
Hybridization Probe
- Dual oligo FRET probes
Primer based Probe
- Scorpion
Intercalating dye fluoresces more brightly when bound to dsDNA.
DNA binding dyes are inexpensive compared to the other probes.
SYBR Green I, EtBr
Intercalating method
SYBR Green dye
Intercalating method
SYBR Green dye
1. Advantages:
- Cheap, easy to use
- Does not inhibit the reaction of amplification
- Does not require any fluorescent probe
- Does not require any particular expertise for the design of the probes
- Is not affected by mutations in the target DNA
2. Disadvantages:
- Impossible to make sure of specificity of amplicons
- Bad pairing can lead to positive forgeries or an over-estimate of the
quantification
- Still unspecified mutagen capacity
Fluorescent reporter dye at the 5 end is quenched by fluorescent quencher dye at the 3 end.
When amplification occurs the TaqMan probe is degraded due to the 5'-->3' exonuclease activity of Taq DNA polymerase, thereby separating the quencher from the reporter during extension.
The TaqMan assay accumulates a fluorescence signal.
TaqMan probe
TaqMan Probe
TaqMan probe
TaqMan Probe
1. Advantages:
- Increased specificity
- Better capacity of multiplexing
2. Disadvantages:
- Little expensive (dual-labeled probe)
- Less effective and less flexible compared to other techniques in the
real-time detection of specific mutation
- Require the design of probes
A molecular beacon begins as a stem-and-loop structure. The sequences at the ends of the probe match and bind, creating the stem
When the probe binds to a single-stranded DNA template, the structure unfolds, separating the quencher from the dye and allowing fluorescence.
Molecular Beacon
Molecular Beacon Probe
Molecular Beacon
Molecular Beacon Probe
1. Advantages:
- Increased specificity
- High flexibility for probe design
- As the probes are not hydrolyzed, they are used at each cycle
2. Disadvantages:
- Little expensive (dual-labeled probe)
- Less effective and less flexible compared to other techniques in the
real-time detection of specific mutation
- Require the design of probes
FRET method designed two specifically probe. It labeled with different dyes, such as at the 5 end of donor probe and at the 3 end of acceptor probe.
At close proximity, the donor dye is excited by the light source and the energy is transferred the acceptor dye. Subsequently, fluorescent light is emitted at a different wavelength.
Hybridization Probe (FRET)
FRET Probe
Applications of Real-Time PCR
1. Introduction of Exicycler
Real-Time PCR Applications
Clinical Diagnostics
- Bacterial/ Viral pathogen detection
- Absolute pathogen quantification
Drug therapy efficacy / drug monitoring
Differential gene expression
RNAi
- siRNA validation
SNP Genotyping
Others
- Food pathogen testing
- Forensic studies
Real-Time PCR Applications
Structural AssayUses DNA, typically genomic extractionsSingle Nucleotide Polymorphism
Functional AssayUses RNA extractionsUses reverse transcriptase to generate cDNA templatesDifferential expressionDiagnostics involving gene expressionRNA interference
Clinical Diagnostic AssayUses DNA or RNA extracted from patients samplesViral/bacterial pathogens
Quantification Strategies
Absolute quantification
- Used to quantitate unknown samples by interpolating their
quantity from a standard curve.
- The standard is a known DNA sample whose concentration is
known absolutely.
- The accuracy of the absolute quantification assay is entirely
dependent on the accuracy of the standards.
Relative quantification
- Used to analyze changes in gene expression in a given sample
relative to another reference sample.
- A comparison within a sample (DNA or cDNA) is made with the
gene(s) of interest to that of an endogenous control gene.
- Quantification is done relative to the control gene.
Detection of specific genes in pathogens (virus, bacteria, fungi, etc) isolated from patients samples
Quantification: copy numbers of infected pathogens
Therapeutic Drug Monitoring/Screening
Varicella-Zoaster Virus Real-Time PCR
106 copies
Absolute Quantification
Pathogen Detection/Clinical Diagnostics
The Ct value correlates strongly with the starting copy number.
It is linear with the log of starting DNA concentration.
Ct value
Absolute Quantification
Sample 1
Ct:14
Conc: 2,500copy
Threshold
Absolute Quantification
Compares transcriptional levels of genes between control and experimental samples
- Tissue distribution of target gene
- Drug screening/Drug efficacy
- Gene expression profiling after drug treatment
Endogenous controls are used to normalize the data
Endogenous controls are genes common to both control and experimental samples that do not change their expression levels under the experimental conditions
- GAPDH
- -Actin
- 18s ribosomal subunit
Differential Gene Expression
Relative Quantification
Experiment Design (Traditional / Relative quantification)
PCR using same amount of mRNA after spectrophotometer quantification
Cell line A
mRNA
Cell line A + Drug
mRNA
Relative Quantification (PCR)
Experiment Design (Real-Time PCR /Relative quantification)
Cell line A
Cell line A + Drug
Relative Quantification (Real-Time PCR)
Actin
Target
Actin
Target
A > B (21 : 2 times much)
Sample A : 16 Ct
Sample B : 17 Ct
=> Delta Ct = 1 Ct
A > B (24 : 16 times much)
Sample A : 17 Ct
Sample B : 21 Ct
=> Delta Ct = 4 Ct
Relative Quantification (Drug-induced gene expression)
Control cell lines
Control cell lines
+ Drug A
Drug A treatment induced decreased target X gene expression
Relative Quantification
Relative Quantification (Tissue distribution / Delta Ct Methods)
Brain tissue
Liver tissue
Actin Target X
Actin Target X
Actin : 17 Ct
Target : 21 Ct
=> Delta Ct = 4 Ct
Actin : 22 Ct
Target : 26 Ct
=> Delta Ct = 4 Ct
Relative Quantification
Single Nucleotide Polymorphism (SNP)
DNA sequence variations that occur when a single nucleotide (A, T,
C, or G) in the genome sequence is altered
How many SNPs are there in humans today?
- Human mutation rate is ~ 2.5 x 108 mutation/site/generation
- ~150 mutations/diploid genome/generation
- 6.3 billion people in the world = 945,000,000,000 mutations in the
world today
The most common type of sequence difference between alleles
Provide a way to detect direct associations between
allelic forms of genes and phenotypes
SNP Genotyping
Allelic Discrimination Assays (Single Nucleotide Polymorphisms)
G
G
C
C
T
T
A
A
SNP Genotyping (Real-Time PCR)
SNP Genotyping (Real-Time PCR)
A
A
A
T
T
T
G
G
G
C
C
C
RNA interference:
double stranded RNA (siRNA) forms a complex (RISC) that binds to target mRNA and leads to its degradation
Target gene expression can be quantified by Real-Time PCR
RNAi: siRNA validation
Multiplex Analysis
Different dyes for each target (Example: FAM, TET, VIC and JOE)
Real-time detection of four different retroviral DNAs in a multiplex format.
Each reaction contained four sets of PCR primers specific for unique HIV-1, HIV-2, HTLV-I, and HTLV-II nucleotide sequences and four molecular beacons, each specific for one of the four amplicons and labelled with a differently coloured fluorophore.
HIV-1: Fluorescein
HIV-2: Tetrachlorofluorescein
HTLV-1: Tetramethylrhodamine
HTLV-II: Rhodamine
Vet JA et al. PNAS (1999)
Improving Reproducibility
Use clean bench (hood) Use aerosol resistant tips Use calibrated micropipettors Use large volumes (5L and up) Pipette into each reaction vessel once
Good Technique
Poor Technique
Same Reagents, Different Results
Special Features of ExicyclerTM
The ExicyclerTM is a real time PCR system developed by Bioneer. The ExicyclerTM is equipped with an optical system that fits above the thermal cycler. It readily utilizes most fluorescent dyes, providing the widest choice of excitation / emission wavelengths. It can also be used as a standard thermal cycler for general PCR reactions.
Real Time Quantitative Thermal Block
Exicycler
Superior optic module
Light pipe technology (patented)
Light source
Short Arc Lamp
Life: 3,000 hrs
High Power, high quality, low maintenance
Excitation filter
Six position (blank 1, filter 5)
Wavelength range: 490-640nm
5 multiplexing
4) Emission filter
Six position (blank 1, filter 5)
Wavelength range: 520-670 nm
5 multiplexing
Powerful 2D CCD detector
High resolution and great sensitivity
Resolution : 325K pixel
Thermal gradient
Characteristics of ExicyclerTM
Superiors of ExicyclerTM
Analysis can be viewed in real time.
Melting curve can also be viewed in real time.
Thermal gradient is compatible.
Range of fluorophore excitation and emission is 490~670 nm.
It can be used for multiplexing with 5 different fluorophores.
Reliable results without using reference dye.
Not necessary to decontaminate the sample block.
96 samples can be tracked simultaneously.
All data is accessible as Excel file and jpg image.
Result reports, everyone can open on internet explorer.
Software is friendly to beginner at qPCR.
USB communication interface.
Auto loading.
Patents of ExicyclerTM
Real time monitoring apparatus for amplified nucleic acid product
Appl. Con. : ROK
Appl. Date: Jun. 18, 2002
Appl. No. : 10-2002-0033965
Real time monitoring apparatus
Appl. Con. : ROK
Appl. Date : Apr. 3, 2003
Appl. No. : 10-2003-0021145
*
Optic module of ExicyclerTM
Light pipe
Camera lens
Lamp
Comparison of Optics
Exicycler
Other Company
More homogeneous light intensities among wells
=> Reliable results without using reference dye
Comparison of Optics
Exicycler
Other Company
Without reference dye
With reference dye
Without reference dye
Diversity of fluorescence dyes
Classified by light sources
Classified by light source
Classified by sensor and thermal cycling
Classified by sensor and thermal cycling
Market trend
Comparison
NameiCyclerSmartCyclerRotorGeneLightcycler2ABI7500Opticon 2ExicyclerCompanyBio-radCepheidCorbettRocheApplied biosystemMJ researchBioneerDetectionSimultaneousSimultaneousSequentialSequentialSimultaneousSequentialsimultaneousType96 well16 tube32, 72 tube32 capillary96 well96 well96 wellGradientOxOOLightsourceTungsten Halogen LampLEDLEDBlue LEDTungsten Halogen LampLEDShort Arc lampDetector10 bit CCDSiliconPhotodetectorPMTPhoto-hybrid10 bit CCDPMT16 bitCCDEx /Emrange400~700 450~650 470~660 470~710 488~650 470~700 490~670 Multiplex4 color 4 color4 color6 color5 color2 color5 color
Experimental Results in ExicyclerTM
SYBR Green I data
Without reference dye
Chart2
41.545454545550.5454545455-72.8181818182-52.7272727273-51.5454545455-35.5454545455-15.18181818180-7.181818181856.5454545455003.5454545455000-79.636363636436.8181818182-70.4545454545-24.636363636450.545454545555.363636363641.8181818182122-21.454545454525.363636363633.8181818182126.0909090909230.818181818218.0909090909
-35.454545454588.5454545455-9.81818181820.2727272727-61.5454545455-10.5454545455-11.181818181809.818181818265.54545454550070.5454545455000-9.6363636364-23.1818181818-25.4545454545-5.636363636430.545454545528.363636363612.818181818214-95.454545454532.3636363636-19.18181818187.0909090909126.8181818182-31.9090909091
-10.4545454545-18.454545454520.1818181818-9.72727272738.4545454545-20.54545454559.81818181820-7.181818181820.54545454550021.54545454550006.3636363636-86.181818181813.5454545455-4.636363636418.5454545455-44.6363636364-42.18181818180-5.4545454545-31.6363636364-19.1818181818-15.9090909091201.81818181821.0909090909
-30.454545454517.5454545455-16.81818181820.2727272727-42.545454545518.454545454528.8181818182014.818181818276.545454545500-6.4545454545000-90.6363636364-19.1818181818-14.4545454545-4.6363636364-34.454545454517.3636363636-12.181818181806.5454545455-12.63636363640.81818181828.0909090909172.8181818182-15.9090909091
-21.4545454545-40.45454545453.1818181818-43.727272727336.454545454518.454545454592.8181818182011.8181818182-7.454545454500-6.454545454500061.3636363636-28.18181818184.5454545455-12.636363636436.5454545455-3.6363636364-19.1818181818012.5454545455-17.6363636364-11.181818181811.0909090909104.8181818182-32.9090909091
-19.45454545457.5454545455-23.818181818237.2727272727-9.5454545455-35.545454545515.81818181820-7.1818181818-27.45454545450048.5454545455000-39.636363636430.8181818182-26.4545454545-47.636363636415.545454545524.363636363622.8181818182043.54545454552.363636363643.8181818182-31.9090909091109.81818181822.0909090909
-14.4545454545-8.45454545452.1818181818-1.7272727273-27.545454545517.454545454524.81818181820-7.1818181818-7.454545454500-13.454545454500062.36363636363.8181818182-3.454545454545.3636363636-1.4545454545-21.6363636364-23.1818181818045.545454545538.363636363631.818181818226.090909090988.818181818240.0909090909
24.545454545518.545454545537.1818181818-73.7272727273-5.545454545572.454545454563.818181818201.8181818182-4.45454545450021.545454545500048.36363636368.818181818231.545454545518.36363636361.5454545455-3.63636363648.8181818182016.5454545455-0.63636363647.818181818244.090909090960.818181818224.0909090909
-19.454545454525.5454545455-11.818181818233.2727272727-34.545454545563.4545454545-39.18181818180-7.1818181818-21.45454545450054.5454545455000-41.63636363644.8181818182-43.4545454545-1.63636363642.5454545455-10.6363636364-32.1818181818022.5454545455-16.6363636364-34.1818181818-1.9090909091-8.181818181845.0909090909
44.545454545530.54545454554.1818181818-4.7272727273-1.5454545455-32.5454545455-28.18181818180-7.1818181818-20.454545454500-18.4545454545000-4.6363636364-45.181818181821.545454545546.3636363636-38.45454545456.363636363658.81818181820-42.454545454563.363636363638.8181818182-29.9090909091-208.1818181818-88.9090909091
-37.45454545455.5454545455-11.818181818221.27272727277.454545454514.4545454545-45.1818181818021.818181818254.545454545500-30.454545454500026.363636363645.8181818182-11.4545454545-9.63636363649.5454545455-5.636363636419.81818181820-46.4545454545-0.6363636364-17.181818181812.0909090909-205.181818181815.0909090909
38.5454545455-33.454545454510.181818181858.272727272779.4545454545-55.5454545455-71.18181818180-7.1818181818-35.454545454500-18.4545454545000-52.636363636442.81818181822.5454545455-2.6363636364-27.4545454545-26.636363636447.81818181820-6.454545454521.36363636364.8181818182-16.9090909091-170.181818181820.0909090909
45.5454545455-4.4545454545-12.8181818182-16.7272727273-10.5454545455-60.5454545455-52.18181818180-7.1818181818-27.454545454500-52.454545454500024.363636363641.818181818225.5454545455-26.636363636417.545454545568.3636363636-29.18181818180-46.4545454545-45.6363636364-46.1818181818-4.9090909091-147.1818181818-9.9090909091
16.5454545455-1.454545454518.181818181828.272727272730.4545454545-7.5454545455-17.18181818180-7.1818181818-49.454545454500-60.4545454545000-35.6363636364-24.1818181818-8.45454545455.363636363611.54545454556.363636363655.818181818220-52.4545454545-15.6363636364-26.1818181818-72.9090909091-110.181818181858.0909090909
62.545454545515.545454545528.181818181817.272727272746.454545454545.4545454545-18.18181818180-7.1818181818-35.454545454500-35.4545454545000-27.6363636364-53.181818181810.5454545455-51.636363636431.54545454556.3636363636134.8181818182020.545454545522.3636363636-10.1818181818-48.9090909091-112.181818181840.0909090909
57.5454545455-98.454545454554.1818181818-15.727272727338.454545454516.4545454545-2.18181818180-7.1818181818-31.454545454500-27.454545454500050.3636363636-44.181818181825.5454545455-42.6363636364-16.454545454540.363636363693.81818181820-75.454545454538.3636363636-34.1818181818-26.9090909091-100.181818181839.0909090909
79.545454545544.545454545582.1818181818-34.727272727385.4545454545-46.5454545455-25.18181818180-7.181818181813.5454545455005.545454545500056.3636363636-14.181818181893.5454545455-33.636363636447.545454545575.3636363636125.81818181820-67.454545454574.3636363636-42.1818181818-91.9090909091-103.181818181856.0909090909
119.5454545455-89.4545454545109.1818181818-27.7272727273114.4545454545-40.5454545455-69.18181818180-7.1818181818-24.454545454500-5.454545454500027.3636363636-67.1818181818130.5454545455-36.6363636364115.5454545455119.3636363636150.81818181820-26.4545454545150.363636363612.8181818182-50.9090909091-66.1818181818134.0909090909
271.5454545455-61.4545454545235.1818181818-9.7272727273268.4545454545-24.5454545455-49.18181818180-7.1818181818-54.454545454500-47.454545454500066.3636363636-88.1818181818290.5454545455-15.6363636364241.5454545455310.3636363636323.81818181820-18.4545454545218.3636363636-73.1818181818-17.909090909197.8181818182324.0909090909
594.5454545455-89.4545454545463.1818181818-52.7272727273541.4545454545-37.5454545455-22.18181818180-7.1818181818-29.454545454500-28.4545454545000-20.6363636364-33.1818181818611.545454545552.3636363636496.5454545455579.3636363636546.81818181820-12.4545454545490.3636363636-50.1818181818-119.9090909091313.8181818182546.0909090909
1055.5454545454-82.45454545451058.1818181818-19.72727272731075.4545454546-115.5454545455-77.18181818180-7.1818181818-55.454545454500-108.4545454545000-32.6363636364-71.18181818181111.5454545454-29.63636363641091.54545454541106.36363636361000.81818181820-114.4545454545953.3636363636-11.1818181818-56.9090909091879.81818181821134.0909090909
2001.5454545454-90.45454545451943.1818181818-31.72727272732030.4545454546-80.5454545455-133.18181818180-7.1818181818-72.454545454500-91.4545454545000-37.6363636364-154.18181818182118.5454545454-2.63636363641972.54545454542106.36363636362018.81818181820-21.45454545451943.3636363636-140.181818181844.09090909091826.81818181822083.0909090909
3634.5454545454-126.45454545453484.1818181818-30.72727272733732.4545454546-13.5454545455-29.18181818180-7.1818181818-39.454545454500-141.454545454500029.3636363636-67.18181818183873.54545454540.36363636363711.54545454543838.36363636363738.81818181820-91.45454545453483.363636363643.8181818182-63.90909090913526.81818181823938.0909090909
5979.5454545455-50.45454545455922.1818181818-61.72727272736224.4545454545-72.5454545455-52.18181818180-7.1818181818-56.454545454500-59.454545454500031.3636363636-115.18181818186348.5454545455-44.63636363646080.54545454556463.36363636366304.81818181820-79.45454545455860.3636363636-12.1818181818-51.90909090915684.81818181826424.0909090909
8928.5454545455-109.45454545458759.1818181818-30.72727272739337.4545454545-115.5454545455-86.18181818180-7.1818181818-82.454545454500-48.45454545450000.3636363636-46.18181818189380.5454545455-49.63636363648935.54545454559467.36363636369389.81818181820-48.45454545458721.3636363636-47.1818181818-1.90909090918543.81818181829707.0909090909
11506.5454545455-86.454545454511543.1818181818-133.727272727312128.454545454515.4545454545-48.18181818180-7.1818181818-11.454545454500-45.454545454500077.3636363636-102.181818181812076.5454545455-59.636363636411710.545454545512405.363636363612229.81818181820-37.454545454511149.3636363636-72.1818181818-87.909090909111210.818181818212625.0909090909
13616.5454545455-65.454545454513664.18181818183.272727272714686.4545454545-35.5454545455-23.18181818180-7.1818181818-36.454545454500-101.454545454500060.3636363636-52.181818181814345.5454545455-35.636363636413824.545454545514595.363636363614473.81818181820-63.454545454513376.3636363636-39.1818181818-116.909090909113557.818181818215222.0909090909
15277.5454545455-122.454545454515256.1818181818-11.727272727316169.4545454545-65.5454545455-74.18181818180-7.1818181818-60.454545454500-3.454545454500091.3636363636-47.181818181815864.5454545455-24.636363636415284.545454545516254.363636363615912.81818181820-18.454545454514885.3636363636-46.1818181818-110.909090909115140.818181818217011.0909090909
16287.5454545455-78.454545454516301.1818181818-67.727272727317044.4545454545-32.5454545455-31.1818181818028.8181818182-114.454545454500-65.454545454500057.3636363636-68.181818181816968.5454545455-36.636363636416244.545454545517201.363636363616961.8181818182015.545454545515913.3636363636-49.1818181818-84.909090909116303.818181818218030.0909090909
16558.5454545455-174.454545454516629.1818181818-72.727272727317455.4545454545-52.5454545455-125.18181818180-7.1818181818-80.454545454500-63.454545454500018.3636363636-65.181818181817393.5454545455-11.636363636416676.545454545517723.363636363617339.81818181820-34.454545454516504.3636363636-46.1818181818-102.909090909117174.818181818218573.0909090909
17071.5454545455-136.454545454517136.18181818187.272727272718009.4545454545-66.5454545455-107.18181818180-4.1818181818-99.454545454500-63.454545454500021.3636363636-72.181818181817868.5454545455-44.636363636417177.545454545518154.363636363617881.8181818182028.545454545517146.3636363636-60.1818181818-153.909090909117783.818181818219402.0909090909
17102.5454545455-179.454545454517031.1818181818-59.727272727318107.4545454545-58.5454545455-23.18181818180-7.1818181818-50.454545454500-62.454545454500082.3636363636-21.181818181818031.5454545455-47.636363636417145.545454545518100.363636363618103.81818181820-116.454545454517339.3636363636-42.1818181818-125.909090909117861.818181818219506.0909090909
17122.5454545455-127.454545454517227.1818181818-4.727272727318130.4545454545-135.5454545455-49.18181818180-4.1818181818-99.454545454500-49.4545454545000-33.6363636364-89.181818181817978.5454545455-81.636363636417229.545454545518226.363636363618058.81818181820-10.454545454517291.3636363636-87.1818181818-101.909090909118142.818181818219727.0909090909
16994.5454545455-130.454545454517069.1818181818-12.727272727318205.4545454545-72.5454545455-86.18181818180-7.1818181818-46.454545454500-20.4545454545000-45.6363636364-67.181818181817990.5454545455-20.636363636417216.545454545518234.363636363618119.8181818182015.545454545517453.3636363636-63.1818181818-108.909090909118030.818181818219656.0909090909
17176.5454545455-86.454545454517238.1818181818-12.727272727318269.4545454545-89.5454545455-41.18181818180-7.1818181818-73.454545454500-69.454545454500034.3636363636-76.181818181818115.5454545455-17.636363636417200.545454545518182.363636363618042.81818181820-33.454545454517429.3636363636-56.1818181818-165.909090909118099.818181818219567.0909090909
17208.5454545455-107.454545454517191.1818181818-58.727272727318201.4545454545-128.5454545455-100.1818181818010.8181818182-50.4545454545007.545454545500072.36363636361.818181818218074.5454545455-22.636363636417256.545454545518200.363636363617935.81818181820-6.454545454517579.3636363636-27.1818181818-70.909090909118119.818181818219560.0909090909
17387.5454545455-124.454545454517155.1818181818-60.727272727317955.4545454545-111.5454545455-46.18181818180-3.1818181818-8.454545454500-6.4545454545000-21.6363636364-49.181818181818275.5454545455-20.636363636417348.545454545518148.363636363617964.8181818182025.545454545517731.3636363636-35.1818181818-106.909090909118217.818181818219378.0909090909
17258.5454545455-179.454545454517241.1818181818-22.727272727318296.4545454545-30.5454545455-77.18181818180-7.1818181818-96.454545454500-77.4545454545000-26.6363636364-162.181818181818308.545454545556.363636363617184.545454545518179.363636363618249.81818181820-8.454545454517745.3636363636-44.1818181818-47.909090909118116.818181818219626.0909090909
17450.5454545455-71.454545454517015.1818181818-30.727272727318132.4545454545-63.5454545455-100.18181818180-5.1818181818-102.454545454500-25.4545454545000-39.636363636412.818181818218405.5454545455-20.636363636417229.545454545518315.363636363618102.81818181820-39.454545454517834.363636363632.81818181825.090909090918113.818181818219697.0909090909
17258.5454545455-34.454545454517087.1818181818-69.727272727318153.4545454545-6.5454545455-133.18181818180-7.1818181818-66.454545454500-19.4545454545000-72.6363636364-44.181818181818328.5454545455-8.636363636417218.545454545518263.363636363618085.81818181820-13.454545454517787.3636363636-54.1818181818-84.909090909118086.818181818219684.0909090909
test1
304000000000000000000000000000000000000050000000101218000000000030000000000021000000000000992148360000000
3010005040000000000000010000000000000089054000000000000000000000002017000000000056000000000000614260290000000
32300082700000003000000000000000000000056000000000000000000000000022000000000005600000000000099087350000300
31200092000000000000000000004600000000053014000000000000140000000000120000000000030000000000000840357704004300
3910001048000000010000000040000000000000007100000000000035000000000029000000000001270000000000030136499487000017011
3960001285000000030000000000002900000000086070000000000000400000000004900000000000910000000000024103082440012029018
37700015200000000390000000000018000000000180590000000000000000000000040000000000051000000000000100228757053403900
32100016180000027060000020000000000000007805000000000000050000000000044000000000008100000000000611340999408010052032
37700017280000010820000005803000011000000000960410000000001001760000000000073000000000007800000000000459921438807420056042
41400018930000022000010095000005300000000046063000000000800000000000005900000000000120000000000004282817115004624072036
367000191900000000000000123000012000000000520690000000033001330000000000036000000000009500000000000441209779400008031
3910002190000000414500000200000600000000069056000000000100000000000006400000000000910000000000043169255557000075046
35800021860000056000000000000046000000000280210000000000003500000000003800000000200600000000000075121819113441018054053
45600023680000000100040004000000600000000043043000000000120060000000000150000000000018400000000000811580761553100021016
4560002326000001590900000084000003400000000010506200000000000400000000000530000000000011000000000000815325681903000071093
4450002402000001211600000027061400110000000009606800000000015006500000000003600000000020044000000000001171406274225650120940110
536000247200002723793530034000015000970000000001150155000000000900900000000002800000000000110000000130000841536777185939220620127
5690002512000004198370000014303300620000000005401200000000000700000000000005600000000000132000000000001011746581841222540600216
735000260900000231689000005002526004200000000056027300008410000143000000000000280000000000015200000000000269108295217923739290240293
89300026269000000494890000000000000000000007205210000309277000213940380000000300570000014600000139000000000004537738771314770130480546
13430002576495000051918580000017002500590000000005509332300083276400021930103200000000007300000000000950000000000085876186140873000180896
2125000275612550000391695280000028000008100000000073017366000177917360005189800000000000031000000000001121000000600001671110656413717260008201712
352900025962479000051312226006100340240003000000000010703107000033563249000634290230000000000690000000000010300000000000284172710011931420701803010
53180002766439900005152242400000420000059000000000109051190000566055400003257840110000000000200000000000710000000000047925032613352680001305017
75150002770684100006177121500000800000010600000000097075170000839683040004485242146000000000055000000000008800000000000713168533311077600005607342
96990002974917900001301015331000004800330010900000000015101012770001112210823001333611064032000000000045000000000001120000000000094231454180139103190004609563
116120002824111090000161200839003200000000950000000007901186500001315012829000013013000000000000560000000000077000000000001133113016601231210100016011373
1293300029021242200007513236190000040000012800000000012001305227000144611416200015143523536000000000091000000000008600000000000124571083246120133627001012550
1390000029121325900005114091000000000000620000000007101379274000152941500300046152407460000000000180000000000014727000000000013136122887312714161238039013272
1489100029431392600001114637110000039000008300000000070014209640001590815663000581574836350000000000600000000000012400000000000136161073141581478504066013766
15606000289414191000039148184100000580000077000000000730144947200016009158950002415919115300000000007400000000000790000000001013831130596413714999616055013903
16063000295814433000083151300000000000001320000000001170147001100016249161020003016225033000000080058600000000001130000002407900141426638321801514998047014102
165170003152145400000561515424000003300460058000000000960148116100016238161090005016238222900000000006400000000000480000000000014096925348791517800029014092
1651600031101449400009315175900800330014007100000000058014767330001623916074000211620409000000000052000000000001361900000000001398313152461281519230037014135
168260003103144730000751492530000001200000520000000001300148200000162501608200066161591759000000000073110000000000122000000000001407010777431391517702021014154
169510003079143900000881493313000000007007100000000081014742000016185159790006516125681800000000001600000000000700000000000014010914676117151190000014065
169380002921145290000841499410000003300000112000000000107014855400016204161120000161250000000000006000000000009000000070000139916864471271502000022014078
1691300031561441700004014966140000070000025000000000630147150000162921623700020161150280000000150071000000000006300000000000139651050631081505100021014058
16983000309914468000045147940000000004900820000000009101477733000161371610300019160513110000000000031000000000001230000000000013990923391133149984000013940
169830003224145250000161488700000010000065000000000100014781000016262161910002816016369000000000056000000000001130000000000013932831674134151120008013988
Ave
366.09090909090001557.63636363640000019.36363636363.909090909114.5454545455000.09090909090014.272727272711.18181818183.090909090900020.090909090900000000052.9090909091046.72727272730000000031.727272727302.727272727320.7272727273000000000039.0909090909000000000.181818181800800000000000028.0909090909111.454545454529.818181818274.636363636483.36363636363.727272727323.36363636368040.7272727273024.4545454545
-62.0909090909000-1557.636363636400000-19.3636363636-3.9090909091-14.545454545500-0.090909090900-14.2727272727-11.1818181818-3.0909090909000-20.0909090909000000000-52.90909090910-46.727272727305000000-3-0.727272727309.2727272727-2.72727272730000000000-36.090909090900000000-0.181818181800-5900000000000-28.0909090909-12.4545454545-8.8181818182-26.6363636364-47.3636363636-3.7272727273-23.3636363636-80-40.72727272730-24.4545454545
-65.0909090909000-1053.636363636400000-19.3636363636-3.9090909091-14.545454545500-0.090909090900-14.2727272727-10.1818181818-3.0909090909000-20.090909090900000000036.090909090907.272727272700000000-3-1.72727272730-2.7272727273-20.72727272730000000000-19.0909090909170000000-0.181818181800-2400000000000-28.0909090909-50.454545454512.1818181818-14.6363636364-54.3636363636-3.7272727273-23.3636363636-80-40.72727272730-24.4545454545
-43.0909090909000-730.636363636400000-19.3636363636-3.909090909115.454545454500-0.090909090900-14.2727272727-11.1818181818-3.0909090909000-20.09090909090000000003.09090909090-46.727272727300000000-3-1.72727272730-2.7272727273-20.72727272730000000000-17.090909090900000000-0.181818181800-2400000000000-28.0909090909-12.4545454545-29.818181818212.3636363636-48.3636363636-3.7272727273-23.3636363636-80-37.72727272730-24.4545454545
-54.0909090909000-637.636363636400000-19.3636363636-3.9090909091-14.545454545500-0.090909090900-14.2727272727-11.1818181818-3.090909090900025.90909090910000000000.09090909090-32.727272727300000000-3-1.72727272730-2.7272727273-6.72727272730000000000-27.090909090900000000-0.181818181800-5000000000000-28.0909090909-27.4545454545-29.8181818182-39.6363636364-6.3636363636-3.7272727273-19.3636363636-802.27272727270-24.4545454545
24.9090909091000-509.636363636400000-19.3636363636-3.9090909091-4.545454545500-0.090909090900-14.2727272727-11.18181818180.9090909091000-20.0909090909000000000-52.9090909091024.272727272700000000-3-1.72727272730-2.727272727314.27272727270000000000-10.090909090900000000-0.18181818180047000000000001.909090909124.545454545519.181818181819.36363636363.6363636364-3.7272727273-23.3636363636-80-23.72727272730-13.4545454545
29.9090909091000-272.636363636400000-19.3636363636-3.909090909115.454545454500-0.090909090900-14.2727272727-11.1818181818-3.09090909090008.909090909100000000033.0909090909023.272727272700000000-3-1.72727272730-2.7272727273-16.727272727300000000009.909090909100000000-0.1818181818001100000000000-4.0909090909-8.4545454545-29.81818181827.3636363636-39.3636363636-3.7272727273-23.363636363640-11.72727272730-6.4545454545
10.9090909091000-37.636363636400000-19.3636363636-3.909090909124.454545454500-0.090909090900-14.2727272727-11.1818181818-3.0909090909000-2.0909090909000000000-34.9090909091012.272727272700000000-3-1.72727272730-2.7272727273-20.72727272730000000000-35.090909090900000000-0.181818181800-2900000000000-28.0909090909-11.4545454545-7.818181818212.3636363636-26.3636363636-3.727272727329.6363636364-40-1.72727272730-24.4545454545
-45.090909090900060.3636363636000007.6363636364-3.9090909091-8.545454545500-0.090909090900-12.2727272727-11.1818181818-3.0909090909000-20.090909090900000000025.090909090903.272727272700000000-3-1.72727272730-2.727272727329.272727272700000000004.909090909100000000-0.181818181800100000000000-22.09090909091.545454545510.181818181824.363636363610.6363636364-3.727272727356.63636363642011.272727272707.5454545455
10.9090909091000170.36363636360000088.6363636364-1.9090909091-14.545454545500-0.09090909090043.7272727273-11.181818181826.9090909091000-9.090909090900000000043.09090909090-5.727272727300000000-38.2727272727014.272727272739.2727272727000000000033.909090909100000000-0.181818181800-20000000000016.9090909091-12.4545454545-8.8181818182-31.63636363644.6363636364-3.727272727350.636363636412015.2727272727017.5454545455
47.9090909091000335.3636363636000002.6363636364-3.9090909091-14.5454545455000.90909090910080.7272727273-11.1818181818-3.090909090900032.9090909091000000000-6.9090909091016.272727272700000000-36.27272727270-2.7272727273-20.7272727273000000000019.909090909100000000-0.181818181800400000000000013.9090909091-29.454545454551.1818181818-3.636363636466.6363636364-3.727272727322.636363636416031.2727272727011.5454545455
0.9090909091000361.363636363600000-19.3636363636-3.9090909091-14.545454545500-0.090909090900-14.2727272727111.8181818182-3.0909090909000-8.0909090909000000000-0.9090909091022.27272727270000000030-1.7272727273010.27272727279.27272727270000000000-3.090909090900000000-0.181818181800150000000000015.90909090918.5454545455-20.81818181822.363636363610.6363636364-3.7272727273-23.3636363636-80-32.727272727306.5454545455
24.9090909091000632.363636363600000-19.363636363637.090909090930.454545454500-0.090909090900-12.2727272727-11.1818181818-3.0909090909000-14.090909090900000000016.090909090909.272727272700000000-3-0.72727272730-2.7272727273-20.7272727273000000000024.909090909100000000-0.181818181800110000000000014.909090909157.5454545455-4.8181818182-19.6363636364-26.3636363636-3.7272727273-23.3636363636-8034.2727272727021.5454545455
-8.0909090909000628.36363636360000036.6363636364-3.9090909091-14.545454545500-0.090909090900-14.2727272727-11.1818181818-3.090909090900025.9090909091000000000-24.90909090910-25.727272727300000000-3-1.72727272730-2.727272727314.27272727270000000000-1.0909090909000000001.818181818200-200000000000046.90909090919.545454545551.181818181816.363636363650.636363636437.2727272727-23.363636363610013.2727272727028.5454545455
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16616.90909090910001541.363636363614468000025.636363636414790.0909090909-14.545454545500-0.090909090900-14.2727272727-11.1818181818-3.0909090909490061.909090909100000000038.0909090909014730.272727272733000161371610300-317.27272727271605128.2727272727-10.72727272730000000000-8.090909090900000000-0.181818181800430000000000013961.9090909091-19.45454545453.181818181816.363636363649.636363636414994.2727272727-19.3636363636-80-40.7272727273013915.5454545455
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test1
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Test1
Test3
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Ave
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