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

89.9090909091000810.363636363600000-19.3636363636-3.9090909091-4.54545454550039.90909090910025.7272727273-11.1818181818-3.0909090909000-14.0909090909000000000-9.90909090910-3.727272727300000000-310.27272727270-2.7272727273-14.72727272730000000000-24.090909090900000000-0.1818181818001040000000000052.909090909146.5454545455-29.81818181821.363636363671.636363636427.2727272727-23.3636363636-80-19.72727272730-8.4545454545

89.9090909091000768.363636363600000139.6363636364-3.909090909175.454545454500-0.09090909090069.7272727273-11.1818181818-3.090909090900013.909090909100000000052.0909090909015.272727272700000000-3-1.727272727301.2727272727-20.7272727273000000000013.909090909100000000-0.181818181800300000000000052.9090909091-58.4545454545-4.8181818182-6.6363636364106.636363636426.2727272727-23.3636363636-8030.2727272727068.5454545455

78.9090909091000844.363636363600000-7.3636363636112.0909090909-14.545454545500-0.09090909090012.7272727273-11.18181818182.90909090911400-9.090909090900000000043.0909090909021.272727272700000000-313.27272727270-2.727272727344.27272727270000000000-3.090909090900000000-0.1818181818200-360000000000088.909090909128.545454545532.1818181818-0.6363636364141.636363636461.2727272727-23.36363636364053.2727272727085.5454545455

169.9090909091000914.3636363636000027217.636363636489.090909090938.45454545450033.909090909100-14.2727272727-11.181818181811.909090909100076.909090909100000000062.09090909090108.272727272700000000-37.27272727270-2.7272727273-11.72727272730000000000-11.090909090900000000-0.1818181818003000000013000055.909090909141.545454545537.18181818182.3636363636101.636363636489.2727272727-14.363636363614021.27272727270102.5454545455

202.9090909091000954.36363636360000021.636363636494.090909090922.454545454500-0.090909090900-0.2727272727-8.1818181818-3.0909090909330041.90909090910000000001.0909090909073.272727272700000000-3-1.727272727370-2.7272727273-20.7272727273000000000016.909090909100000000-0.181818181800520000000000072.909090909162.5454545455-23.8181818182-16.6363636364100.6363636364118.27272727271.6363636364-4019.27272727270191.5454545455

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526.90909090910001068.3636363636900000-19.3636363636490.090909090974.454545454500-0.090909090900-14.2727272727-11.1818181818-3.0909090909000-20.090909090900000000019.09090909090474.2727272727000030927700-319.2727272727394-2.727272727317.2727272727000000030017.90909090910000014600-0.1818181818005900000000000424.9090909091-34.45454545458.18181818182.363636363647.6363636364473.2727272727-23.3636363636507.27272727270521.5454545455

976.90909090910001018.3636363636495000031.6363636364914.090909090943.454545454500-0.0909090909002.7272727273-11.1818181818-3.0909090909250038.90909090910000000002.09090909090886.27272727272300083276400-319.27272727279307.272727272711.2727272727000000000033.909090909100000000-0.1818181818001500000000000829.9090909091-35.4545454545-28.818181818211.363636363656.6363636364869.2727272727-23.3636363636-80-22.72727272730871.5454545455

1758.90909090910001198.36363636361255000019.63636363641691.090909090913.454545454500-0.09090909090013.7272727273-11.1818181818-3.090909090900060.909090909100000000020.090909090901689.272727272760001779173600-33.27272727271898-2.7272727273-20.72727272730000000000-8.090909090900000000-0.181818181800321000000600001642.9090909091-1.454545454535.1818181818-10.636363636453.63636363641722.2727272727-23.3636363636-8041.272727272701687.5454545454

3162.90909090910001038.36363636362479000031.63636363643118.090909090911.45454545450060.90909090910019.7272727273-11.181818181820.90909090910009.909090909100000000054.090909090903060.272727272700003356324900-34.27272727273429-2.72727272732.2727272727000000000029.909090909100000000-0.18181818180023000000000002812.9090909091-39.4545454545-22.818181818225.363636363635.63636363643138.2727272727-23.3636363636-10-22.727272727302985.5454545454

4951.90909090910001208.36363636364399000031.63636363645220.09090909099.454545454500-0.09090909090027.7272727273-11.1818181818-3.090909090900038.909090909100000000056.090909090905072.272727272700005660554000-330.27272727275784-2.7272727273-9.72727272730000000000-37.090909090900000000-0.181818181800-9000000000004763.9090909091-61.45454545452.1818181818-68.636363636449.63636363645264.2727272727-23.3636363636-80-27.727272727304992.5454545455

7148.90909090910001212.36363636366841000041.63636363647708.09090909090.454545454500-0.09090909090065.7272727273-11.1818181818-3.090909090900085.909090909100000000044.090909090907470.272727272700008396830400-342.2727272727852418.272727272725.2727272727000000000015.909090909100000000-0.1818181818008000000000007102.9090909091-43.454545454523.1818181818-41.636363636426.63636363647756.2727272727-23.3636363636-8015.272727272707317.5454545455

9332.90909090910001416.363636363691790000110.636363636410149.090909090916.454545454500-0.09090909090033.7272727273-11.1818181818-3.0909090909330088.909090909100000000098.0909090909010080.2727272727700011122108230013034.272727272711064-2.727272727311.272727272700000000005.909090909100000000-0.18181818180032000000000009394.909090909133.545454545511.18181818185.363636363655.636363636410315.2727272727-23.3636363636-805.272727272709538.5454545455

11245.90909090910001266.3636363636111090000-3.363636363612004.090909090924.45454545450031.909090909100-14.2727272727-11.1818181818-3.090909090900074.909090909100000000026.0909090909011818.27272727270000131501282900-3-1.727272727313013-2.7272727273-20.7272727273000000000016.909090909100000000-0.181818181800-30000000000011302.909090909118.5454545455-13.8181818182-14.636363636439.636363636412097.2727272727-23.3636363636-80-24.7272727273011348.5454545455

12566.90909090910001344.363636363612422000055.636363636413232.09090909094.454545454500-0.090909090900-10.2727272727-11.1818181818-3.0909090909000107.909090909100000000067.0909090909013005.272727272727000144611416200-313.27272727271435232.272727272715.2727272727000000000051.909090909100000000-0.18181818180060000000000012428.9090909091-3.45454545452.1818181818-28.636363636436.636363636413358.2727272727-16.3636363636-80-39.7272727273012525.5454545455

13533.90909090910001354.363636363613259000031.636363636414087.0909090909-14.545454545500-0.090909090900-14.2727272727-11.1818181818-3.090909090900041.909090909100000000018.0909090909013745.272727272774000152941500300-344.2727272727152404.272727272725.27272727270000000000-21.090909090900000000-0.1818181818006727000000000013107.909090909110.545454545558.1818181818-1.636363636443.636363636414157.2727272727-0.363636363600-1.7272727273013247.5454545455

14524.90909090910001385.3636363636139260000-8.363636363614633.0909090909-3.545454545500-0.09090909090024.7272727273-11.1818181818-3.090909090900062.909090909100000000017.0909090909014162.272727272764000159081566300-356.27272727271574833.272727272714.2727272727000000000020.909090909100000000-0.181818181800440000000000013587.9090909091-4.45454545451.1818181818-70.636363636474.636363636414781.2727272727-23.3636363636-4025.2727272727013741.5454545455

15239.90909090910001336.363636363614191000019.636363636414814.090909090926.454545454500-0.09090909090043.7272727273-11.1818181818-3.090909090900056.909090909100000000020.0909090909014447.272727272772000160091589500-322.2727272727159198.272727272732.2727272727000000000034.909090909100000000-0.181818181800-10000000001013802.909090909118.545454545529.1818181818-10.636363636453.636363636414995.2727272727-17.36363636368014.2727272727013878.5454545455

15696.90909090910001400.363636363614433000063.636363636415126.0909090909-14.545454545500-0.090909090900-14.2727272727-11.1818181818-3.0909090909000111.909090909100000000064.0909090909014653.272727272711000162491610200-328.272727272716225-2.727272727312.2727272727000000080018.909090909160000000-0.18181818180033000000240790014113.9090909091-45.45454545458.1818181818-42.636363636496.636363636415145.2727272727-14.3636363636006.2727272727014077.5454545455

16150.90909090910001594.363636363614540000036.636363636415150.09090909099.454545454500-0.09090909090018.7272727273-11.1818181818-3.0909090909460037.909090909100000000043.0909090909014764.272727272761000162381610900-348.27272727271623819.27272727278.2727272727000000000024.909090909100000000-0.181818181800-320000000000014067.9090909091-19.454545454523.1818181818-26.6363636364-4.363636363615174.2727272727-23.3636363636-80-11.7272727273014067.5454545455

16149.90909090910001552.363636363614494000073.636363636415171.0909090909-5.5454545455007.90909090910018.7272727273-11.1818181818-3.0909090909140050.90909090910000000005.0909090909014720.272727272733000162391607400-319.272727272716204-2.7272727273-11.7272727273000000000012.909090909100000000-0.1818181818005619000000000013954.909090909119.545454545522.1818181818-28.636363636444.636363636415188.2727272727-20.3636363636-80-3.7272727273014110.5454545455

16459.90909090910001545.363636363614473000055.636363636414921.090909090915.454545454500-0.090909090900-2.2727272727-11.1818181818-3.090909090900031.909090909100000000077.0909090909014773.27272727270000162501608200-364.27272727271615914.272727272738.2727272727000000000033.9090909091110000000-0.181818181800420000000000014041.9090909091-4.454545454547.1818181818-31.636363636455.636363636415173.2727272727-23.3636363636-60-19.7272727273014129.5454545455

16584.90909090910001521.363636363614390000068.636363636414929.0909090909-1.545454545500-0.090909090900-14.2727272727-11.1818181818-3.090909090970050.909090909100000000028.0909090909014695.27272727270000161851597900-363.27272727271612565.2727272727-2.72727272730000000000-23.090909090900000000-0.181818181800-100000000000013981.9090909091-20.454545454516.18181818181.363636363633.636363636415115.2727272727-23.3636363636-80-40.7272727273014040.5454545455

16571.90909090910001363.363636363614529000064.636363636414990.0909090909-4.545454545500-0.09090909090018.7272727273-11.1818181818-3.090909090900091.909090909100000000054.0909090909014808.27272727274000162041611200-3-1.727272727316125-2.7272727273-20.72727272730000000000-33.090909090900000000-0.181818181800100000007000013962.9090909091-43.454545454534.1818181818-27.636363636443.636363636415016.2727272727-23.3636363636-80-18.7272727273014053.5454545455

16546.90909090910001598.363636363614417000020.636363636414962.0909090909-0.545454545500-0.090909090900-7.2727272727-11.1818181818-3.09090909090004.909090909100000000010.0909090909014668.27272727270000162921623700-318.272727272716115-2.72727272737.27272727270000000150031.909090909100000000-0.181818181800-170000000000013936.9090909091-6.4545454545-29.8181818182-11.636363636424.636363636415047.2727272727-23.3636363636-80-19.7272727273014033.5454545455

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

16616.90909090910001666.3636363636145250000-3.363636363614883.0909090909-14.545454545500-0.090909090900-13.2727272727-11.1818181818-3.090909090900044.909090909100000000047.0909090909014734.27272727270000162621619100-326.2727272727160160.272727272748.2727272727000000000016.909090909100000000-0.181818181800330000000000013903.9090909091-28.4545454545-13.8181818182-0.636363636450.636363636415108.2727272727-23.3636363636-80-32.7272727273013963.5454545455

test1

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Test1

Test3

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761731700079040000245731335127127630620901932130168387435248171180131157196110208179191235465770820888520743279751202515634677802121516313115916901722991552613172751460171119153162031126813729014484292011662171822502373202982676915812110222791216900008652

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13620291000144650000358131713192202151912400194165632334273762661901961511202271731201521762674601374019513585013050140549725562322137211657412680183725914028816326933623117052195164187203412563072892481991498821114521426527725526135934212446167220028613964155000015096

145812690001553300003581408739019821020030108519748165343428226114182951061961051611451562813921465417411596013984149108024173357145111847411337195174332522461833593003171131514312721124102972301931622271587819015922221921427228139141413350142205023814913207000016114

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15994318000166280000285155423192402092152700141171018845739023610919317911021314023825622134341915689232112102015068159601312801293931574826785130724318344123633618526541832413832125106204175019519221524023416512915313522625524726833538639014549180219023916250228000017543

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1631734500016768000027915462304251247331219020017716241432478272161196159126118185132233249227408159251631537201510915959752113145315751260100176922916314013333024824437627317932183180196232562333303073352431989919413030227532533530836337214430171167029216092212000017502

Ave

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