LightCycler® Software version 3.5 Users Manual
Contents
1. ATfRAaara For Idaho Technology s LightCycler 24 amp 32 Instruments Idaho Technology Inc 390 Wakara Way Salt Lake City Utah 84108 USA 1 800 735 6544 ph 801 736 6354 fax 801 588 0507 it idahotech com www idahotech com LightCycler Software v3 5 Users Manual 2 Idaho Technology Inc gt gt gt Customer and Technical Support Reach Us On the Web Idaho Technology s Web site is http Awww idahotech com We strongly encourage users to visit our Web site for answers to frequently asked questions Additional manuals parts and accessories are available on line Reach Us By E mail Contact Idaho Technology by e mail in the following areas support idahotech com Technical Support it idahotech com General Support Reach Us By Phone Technical support is available during the following times 8 a m to 5 30 p m Mountain Standard Time For technical support call 1 800 735 6544 United States and Canada 1 801 736 6354 Utah IDD Prefix 1 801 736 6354 International Reach Us By Fax To contact Idaho Technology by fax use the following numbers 1 801 588 0507 United States and Canada IDD Prefix 1 801 588 0507 International Copyright 2001 Idaho Technology Inc LightCycler Software v3 5 User s Manual March 2001 All rights reserved Printed in the United States of America The information contained in this document is subject to change without notice Idaho Technology makes no warran2. Gee Cre Tempe 9 Cyes Mode 34 0 20 0 o t None 2 50 30 20 0 0 0 None 3 94 Lo a2 a a 0 Step Printing a Quantification Analysis Summary Select Print Summary Report from the Report menu to print analysis reports The report is three pages long on letter sized paper it may differ if printing to other paper sizes The header of each page contains information about the current user the file name the LightCycler ID the Run software version the LCDA software version and when and by whom the experiment was run The report is arranged into the following categories Experimental Protocol Fluorescence Settings Quantification Settings Baseline Noise Band Analysis and Sample Information Note that results will only display currently selected samples Select Summary Report Preview from the Report menu to preview an analysis report illustrated below This displays how the report will appear as before printing Click the button with the printer icon at the top right to print Select a viewing option from the menu or click one of the four view buttons Click on the magnifying glass buttons to zoom in or out The button with the blue horizontal diamond icon selects full page width view while the button with the vertical dia mond icon selects full page view LightCycler Software v3 5 Users Manual 24 Idaho Technology Inc At the bottom of the window a field s
3. eae Sample 25 highlight it by clicking nt nt on the sample name Use Shift click to display multiple sample curves Use Control click to selective ly add or delete sample curves Once Baseline Adjustment is completed proceed to the next window by using the tab functions Only samples selected in this step will be displayed in subse quent graphs Analysis Notes Fields are located in both the quantification and Melting Curve analysis win dows that allow the user to enter remarks pertaining to the reaction run The software automatically saves these remarks in the ABT file generated for each run These remarks will not be erased when the user exits LCDA Exporting Data This function is found under the Quantification pull down option The user can save any data generated by the analysis software in a tab delimited spread sheet format Under the data menu use the Export function to create a spread sheet file from the Baseline Adjustment Noise Band or Standard curve graphs Idaho Technology Inc 21 LightCycler Software v3 5 Users Manual External Standard Curve This function is located under the Quantification pull down option Create External Standard allows the user to generate a standard curve file xsc from an open run file Import Standard File allows the user to load a standard curve file into a run file where no standard curve data exists One sample of a known concentration must be included in each run a
4. evant for analysis Data that fall below the noise band is excluded from analysis 20 Cycle Number Log Fluorescence vs Cycle Number Graph The bottom graph of the Noise Band tab option displays log fluores cence vs cycles shown below This graph only displays sample fluores cent data above the defined noise band No background fluorescence should be included on this graph If there is any back ground fluorescence readjust the noise band ho Technology Inc 29 LightCycler Software v3 5 Users Manu Step 3 Analysis Fit Points Only Introduction After background fluorescence is defined the fluorescence data is now ready for quantification analysis For Fit Points Analysis LCDA uses the intersection between the log linear phase of a fluorescence curve and an arbitrarily defined threshold band to generate a crossing point value In a Second Derivative Maximum analysis a crossing point is calculated from the second derivative maximum of each data curve For a set of standards the crossing point value represents a fractional cycle number This value is plotted against log concen tration to generate a standard curve where the unknown sample concentration is determined Number of Points Box Enter the number of points used to define the log linear phase of each curve in the Number of Points box shown below The default value is set at two and it is typically used Once the user sets t
5. 5 Although the dyes used with the three color LightCycler Instruments have differ ent emission maxima and the detection filters have been optimized to these maxima there is some bleed over of fluorescent signal from one detection channel into the next when multiplexing with color This bleed over is minimal between the F1 fluorescein channel and F2 Cy5 but more significant between the F2 channel and F3 Cy5 5 To overcome this problem IT has developed software that can be used to compensate for between channel cross talk The use of color compensation software is restricted to hybridization probes SYBR Green is analyzed in channel 1 with no requirement for color compen sation Color compensation is also not required for single color detection with either Fluorescein Cy5 or Fluorescein Cy5 5 probe pairs Color Compensation Software The IT color compensation software uses an experimentally generated calibra tion file as a reference against which all data are calibrated A calibration file only needs to be created once and should contain the dyes that are used to generate experimental data This calibration file is stored in the LightCycler Software data folder and can be used repeatedly to analyze multicolor applica tions Creating a calibration file Calibration files are designated as CCC files and are created by the user This requires running a calibration experiment as described below 1 Four LightCycler sample t
6. Curves Pekaes m m I Enable Weighted Ft Load Defaults General Allows the user to display con i i i j ifi ks Initial Directory ExportData DataSelection centration data in either scientific nota Te Eene T tion or significant figures want jon deta in rE in C Significant Figures About Displays information regarding soft ware version software authors and copyright statements Load Defaults 17 Customize Graph Window In LCDA the user can customize each graph by selecting the corre sponding graph icon to open the Customize Graph window This graph ia icon is located near the top right corner of each graph For each axis the user can control the following parameters Automatic Fit Adjusts the max imum and mini mum axis values to fit the dis played data This option must be IF Automate Fe deselected to Wee manually set the maximum or minimum values Logarithmic Sets the axis to a log 5 A Meinun FF Aulematis Fit 7 Minor Tisks arithmic scale porr T Lagotto ARTETA TRE EEE EETA o ooia2 ooze oaos 00230 imo ree B wmo onse ooosas 00003 Ti Minor Ticks Places tick marks between the numbered ticks on the axis Maximum X or Y Enter the maximum value to be displayed on a given axis To use this feature Automatic Fit must be deselected Minimum X or Y Enter the minimum value to be displayed on this axis To use this feat
7. crossing point The results of the quantification analysis are displayed at the top left hand side of the page Note If some of Position Name Standard Calculated Concentration F Crossing Point the columns are 1e5 1 000E 05 1 057E 05 2153 hidden click and Replicate of 1e5 1 000E 05 1 001E 05 21 62 drag on th Replicate of le5 1 000E 05 9 602E 04 21 69 divider b a ae I Replicate of1e5 1 000E 05 9 676E 04 21 67 iviaer between the col Semple 5 1 037E 05 21 56 umn headers until the 1e4 1 000E 04 1 114E 04 25 06 Gata 1S completely dis Replceolted 1000E 0 1 0886 04 2506 eplicate of le played If more space is Replicate of1e4 1 000E 04 1 184E 04 24 97 needed to display data Sample 10 1 332E 04 2478 select the left hand 163 1 000E 03 6 825E 02 29 44 Replicate of1e3 1 000E 03 7 260E 02 29 34 boundary of the Replicate of1e3 1 000E 03 1 016E 03 28 82 Fluorescence vs Cycles Peplicateof1e3 1 000E 03 1 038E 03 28 78 graph and drag it towards Sample 15 8 438E 02 29 11 the right 1e2 1 000E 02 1 245E 02 3211 i Replicate of1e2 1 000E 02 1 418E 02 31 90 Replicate ofle2 1 000E 02 1 257E 02 32 09 Replicate of1e2 1 000E 02 4 501E 02 30 09 Sample Selection Sample 20 1 047E 02 32 38 To displ n amplifi tel 1 000E 01 lt 1 039E 01 gt 36 00 9 disp ayan amp Replicate of1e1 1 000E 01 lt 1 039E 01 gt 36 00 cation curve from an Replicate ofle1 1 000E 01 4 895E 01 33 57 individual sample Replicate oftel 1 000E 01 lt 1 039E 01 gt 36 00 Rate
8. features in LCDA in comparison to the old quantification data analysis software use the Transition Document included with the software package Bacolino Adjustnant Analysis Notos T None ouarificaion Anaye is window Anthmete Proportional Second Denvotve Maximum Normalized a M Sea eee EE 185 TOMES 1 057E 05 215 aie Eh res ae tives RES sy onoe 02 i404 2508 Pepieateofled TWlel 114 14 a Replesico to Mle 109 04 pan Bopirateoftod 100E 1184 04 a Serge 10 ized 2470 18 ooneso3 eaosE 22 aa Papiicere r193 1 000E a3 Ropicato of 103 zai opesteett03 1 a ane Sergio 15 st zant 1E 1245602 azn 2 100E LMEE 02 310 JMET 125760 20 TOE 450 aama Sopo 20 104 za Tel rmen an 2600 Fepleatect et TUE SI aa eplestee 1e1 1OQ0E t 08 Bg Fepiemearle 1EM lt 1 eas Serle 2s Grouped eons 1 000E 05 1 057E 05 Gouped SD GroupelMecns 000E 04 1114 04 Grouped S D 11121814 GroupodMeone 0006 03 E82E 02 Groupee 18171819 GroupesMeone L000E 02 12456 02 Groupee Fluaieeoance FI 21222324 Groupee Means 1 0006 01 Grouped S D Sample Table Descriptions of run samples are displayed in the Sample Table to the left of the Fluorescence vs Cycle Number graph The samples are displayed in the sequence they were loaded in the carousel Samples are identified by their graph color carousel position sample name standard concentration if any calculated concentration if calculated and
9. file from a color compensation experiment Please see Chapter 5 for instructions on how to set up a color compensation run This button is inactive if the current data file has more than four samples The Report menu allows the user to print out the graph of interest The Window menu allows the user to arrange the program windows and provides a list of active windows The Help menu allows the user to set directory preferences and access an electronic version of this manual LightCycler Manual Opens an electronic version of this manual Preferences Facilitates the setting of certain options to allow the user to customize the program for convenient data analysis 15 Initial Directory Allows the user to cus tomize the directory path upon opening of a run file Export Data Allows preferences for exported numerical data to follow either English conventions period for radix comma for thousands or Continental European comma for radix period for thousands Data Selection Offers options for user including choice of program to open flu orescence display and background points Meting Curves General j i Export Data Data Selection When the open file dialog appears the directory want to start in is The last directory from which chose a file LightCycler Data Directoy This one EAUighiCycler Usere Kennp Ei Load Defauis Cancel Guantiication MelingCuves Initial Directoy Expoit
10. interval It only measures the width of the curve at 2 3 peak area Only one curve can be calculated and viewed at a time on the graph The graphical display changes when another sample is selected however all the calculated data will display The Clear All button erases all calculated data from the sam ple display the column headers until the data is completely displayed If more space is Note If some of the columns are hidden click and drag on the divider between required select the left hand boundary of the graph and drag it towards the right LightCycler Software v3 5 Users Manual 40 Idaho Technology Inc Fluorescence d F1 dT vs Temperature Graph This graph displays the melting curve profile for the selected sample The fitted melting peak curve is displayed as a thin solid orange line If two or more peaks are selected the individual Gaussian curves will display as orange dashed lines Weighted Fit The Weighted Fit option puts emphasis on points closer to the melting peak rather than outlying points This option minimizes the effect of melting peak shoulders and nonspecific peaks on the Gaussian fit This option is enabled by default Extra Manual Tm As in the old Melting Curve Analysis software product melting temperatures can be estimated by manually positioning a cursor under the desired melting peak The user can position any of four color coded cursor bars on the graph Drag th
11. 8 H i Log Concentration vs Cycle Number Graph The crossing point data for each sample designated as a standard is plotted against the known concentration generating a standard curve of Log Concentration vs Cycle Number shown below The linear regression for the curve is plotted as a dashed line Values for the slope of the line the Y intercept the standard error and the lin ear correlation coefficient r are displayed numerically to the left of the graph a E Linear Regression Crossing Points Cycle Number Slope 1 log E this value represents the overall reaction efficiency Y intercept Calculated using log CT log E 31 Error The Mean squared error may indicate sample volume variations The crossing Line is set to minimize standard curve data using the mean squared error Fit Points analysis features a Minimize error function and will automatically display the lowest mean squared error Clicking the Minimize Error button activates a software algorithm that shifts the crossing line back and forth until a minimal error value is achieved r value The correlation coefficient or r value varies from zero to nega tive one for a regression line with a negative slope The r value meas ures how well the linear regression line fits An r value of zero means that the plotted regression line explains nothing about the relationship between cycle number and l
12. C060298hpvmelt ABT 21 BEC060398hpvrr A BEC060298hpvmet2 ABT a BEC060498hpv 2 BEC060298hpvmelt3 ABT Sl BEC0E0498hpvrr a BEC060298hpv ABT BEC060398hpv ABT a BECO60598hpv a BECO60298hpv2 ABT a BECO60398hpv4 ABT a BECO60598melt 2 BECO60298hpv4 48T 8 BEC060398hpv5 ABT a BEC0E0II8hpvrr 1 m eee Files of type Lighteycter Files ABT z Cancel Data Selection Introduction This window displays the contents of the selected data file The top graph dis plays the temperature and fluorescence acquisition parameters while the bot tom graph displays the actual data acquisition FI BG seq 2 Quanificaton Meting Curve 1 1 i 1 00 00 00 00 0252 00 0545 00 08 38 woe 0014 24 Temperature Tracna Flurescence Acquistion In the top graph the user must define the run section for analysis by bracketing the area of interest Using the two green cursor bars bracket the selected area of interest The bottom graph also displays the bracketed fluorescence data by cycle time or temperature In each window enlarge or reduce the graph size by clicking the horizontal Y divider between the graphs and adjusting it up or down Temperature vs Time Graph In this window shown on following page a graph displays the temperature cycling conditions and fluorescent data acquisitions of the run Areas of fluores cence acquisitions are highlighted i
13. Data Data Selection When export data want numbers to appear in the following format General Engish Period for radix point comma for thousands Continental European vice versa Load Detauts Preferences Quantification Melting Curves Initial Directory Export Data D When open a fle the program that want LCDA to select is The frst program The fust quantification program The first melting curve program When open a file display fluorescence data by H When open a fle that contains color compensation data Activate color compensation Background points rangefrom 2 to 6 Load Detauts Cancel anu 16 jaho Techno Quantification The user can set default settings for the options in the 1 oe Quantification analysis window When pao quads anabc Iwarh i Sat wih Analysis Baseline G Fit Points C None Arthmetic C Second Derivative Proportional Thresh Ft Points Follow Noise Band Number of fit points 2 v C AutoPosion T Display Fit Points Noise Band Number of standard deviations above 3 B backaround noise Melting Curves The user can set the default settings for the options in the pote Melting Curve analysis window unification Meting Cuves General When perform a meting curve analysis want to start with Melting Curve Method Polynomial with Background Correction I7 Enable Digital Fiter for Melting
14. analysis method Select the standards known concentrations and samples to be included Select Quantification External Standard Curve Import External Standard from the Quantification window task bar Select the desired standard curve file xsc and open it The software will load the standard curve file and calculate the concentrations The use of an external standard curve is noted in a field above the standard curve graph The path and name of the imported external standard curve will appear in the Summary Text Report Printing Quantification Data The Report menu at the top of the window will print out any data from a quan tification analysis The user can print an analysis summary and or individual graphs 23 LightCycler Quantification Report User Kenny LightCycterID 0 Filename _LARAPIDAst RAPDlyeyitimaiOlotiStst ABT LC Run Version RAPID Run 1 24 LCDA Version 31 105 This experiment wes run on Apr 03 2000 by All Users Experimental Protocol rskm Cycles 40 Type Quantification Fluorescence Display Mode Fin Segment Temperature Held Time Slope 2 Taret Step Sie Sep Dehy Acquisition Number Target CC er Cerec Temp CC ce Cycles Mode L 94 o 20 o o a None 2 55 0 20 0 o a None 3 4 10 3 o 0 0 Single rskmmelt Cyes 1 Type Autorun Fluorescence Display Mode Fin Segment Temperature Hold Time Slope 2 Target Sep Size Sep Deby Acquisition Number Targececy
15. ars will display on the Temperature vs Fluorescence graph Set the two left side cursors of the temperature transitions to define the beginning slope and set the two right side to define the ending slope It is best to set the cursors as close to the transition points as possible As the user adjusts the cursors the lower graph will automatically rescale 75 Temperature C Digital Filter The digital filter is used to reduce aberrations in the data due to mechanical effects The filter employs the Solvitzky Golay method to smooth melting curve data A third degree polynomial fits the data using a moving window of 10 C Temperature vs d F1 dT This graph shows the sample derivative melting curve data displayed as melt ing peaks The melting temperature of each peak is used as a method of prod uct identification Sample Selection Sample table is displayed to the left of the Fluorescence vs Temperature graph Each sample is displayed in the sequence it was loaded in the carousel Each sample is identified by its color in graph carousel position and sample 38 Idaho Techno name To display a data curve from an individual sample highlight it by clicking on the sample name Use Shift click to display multiple sample curves Use Control click to selectively add or delete sample curves Step 2 Peak Areas This tab function is especially useful for obtaining precise product melting tem peratures In addition thi
16. ature vs time graph bracket the melt segment with the vertical cursor bars It is possible to analyze only part of a melt by defining the area using these cursor bars The Melting Curve analysis window displays fluorescence curves for all sam ples from the selected run Three tab options display at the top of the window The user follows the steps in each option to define the analysis parameters The tab options are described below 1 Melting Peaks The user defines a calculation method for the genera tion of melting peaks Options include a Linear or a Polynomial data fit both options are described in more detail in the later sections The user also selects a method to correct background fluorescence This function compensates for nonspecific effects of temperature on fluorescence making data easier to evaluate e a e A A A h h Auh b e e aaa a 35 2 Peak Areas Once melting peaks are generated the analysis software is then used to calculate the area under each melting peak This type of analysis is necessary if the peak area is used as a measure of concen tration Additionally the software calculates the melting temperature for each selected sample w Extra Manual Tm The melting temperature Tm of any product is determined to a fraction of a degree The Tm is determined by manually moving any of four cursors in this window The product melting tempera ture is the highest p
17. dization probes Normalized All fluorescence curves are displayed as a percent of maxi mum fluorescence 100 fluorescence is represented by the value 100 This function brings all fluorescence curves to the same plateau value of 100 This option is recommended only for viewing purposes because the Fit Points Method may calculate data incorrectly Step 2 Noise Band Introduction The Noise Band is set to discriminate non informative fluorescence data Typically this is data acquired in the early part of amplification that is not distin guished from fluorescence noise If the Fit Points method is used the user tCycler Software v3 5 Users Manual 28 aho Technology Ir must define the noise band In the Second Derivative Maximum method the software automatically sets the noise band If the second derivative maximum method is used proceed to the Step 3 Analysis Fluorescence vs Cycle Number Graph When the Noise Band tab is selected two graphs will display The Fluorescence vs Cycle Number graph is the top graph shown below Unlike the graph shown in Baseline Adjustment this graph is used to optimize the noise band position The noise band displayed as a red horizontal line is initially set three standard deviations above the Gap Basan Slepa Nowa Sapa ana baseline The user can adjust the noise band up or down to define which data points to include for analysis Only data points above the noise band are considered rel
18. e cursor bars with the mouse or enter the desired value in the box next to the cursor color and hit enter The numerical value for the melting temper ature will display beneath the graph next to the cursor color
19. e user can increase or decrease C to Average by using the vertical slider Below is a comparison between the Linear and Polynomial methods Linear This method emulates the old software and does not generate a derivative estimate as accurate as the polynomial fit If the Linear method is used Set the Degrees C to Average to 2 when working with SYBR Green default setting Program generates the data points as a simple linear curve Requires fewer points to produce an acceptable calculation Polynomial This method of calculation is more robust than the linear fit and thus recommended for all cases of Melting Curve analysis If the Polynomial method is used Set the Degrees C to Average a value between eight and ten for hybridization probes Use the default setting of 4 when using SYBR Green I Program uses the data points to generate a fifth order polynomial curve Requires more points to produce a more accurate calculation Note Generally the polynomial fit generates a more accurate derivative estimate than the linear fit The user can also select Linear or Polynomial With Background options Background correction or subtraction is necessary and recommended to com pensate for nonspecific effects of temperature on fluorescence Background subtraction is strongly recommended for the calculation of peak areas Jaho Technology Inc 37 If a method with background subtraction is selected four vertical cursor b
20. experiment with the 4 tubes LightC ycler Software v3 5 Users Manual 12 Idaho Technology Inc Creating a CCC file ge ennpgennggenn In LCDA open the calibration data set Your data should look something like this Temperciure Tracing Fuorescencs Accuistien Compensation Lol on eanimerse 2 You will be required to IEA een re give the calibration file savei eaten l a name that will be saved as a CCC file Note Only files with four tubes will be acknowl edged as calibration files 3 The software will cali Filename FITC Cy5 Cy55 cCq sae brate each channel by Coc WE m Le colour Compensation Files hai subtracting fluores a cence bleed through from the other channels You have now created a reference calibration file that can be used for color compensating all experimental data sets that use the same fluors Performing Multicolor Experiments To use a calibration file to color compensate data follow the steps described below 1 Open the data file in LCDA 2 Bracket the segment to be analyzed 3 Select the fluorescent display that you are interested in i e F2 or F3 If you would like to see both the F2 and F3 data at the same time open the data file in LCDA again This will create another window of the same data set where you can look at the signal Load Colour Compensation Calibration Data Look it from the other chan nel Use the Window panta
21. from a melt is to be the pull down menu analyzed below the x axis Fluorescence vs Cycle Number The fluorescence data acquired within the bracketed section of the cycle dis plays in the bottom graph of the Data Selection window Buy asd d 08 074 zos zos 054 Flucrescence m 84 034 024 TEE The three options available for the X axis are Time Temperature or Cycles The Select a Program pull down option will automatically set the X axis to the appropriate setting Fluorescence Display Adjacent to the graph at the bottom of the Data Selection window is the fluores cence display pull down option It automatically defaults to the fluorescence specified in the run file The user can choose display options among single channels F1 1 F2 1 F3 1 and a ratio of data from two channels F1 F2 F1 F3 F2 F1 F2 F3 F3 F1 or F3 F2 Typical fluorescence displays and their applications are shown below Display Fluorescence Mode Display Application Ch 1 F1 FAM Fluorescein SYBR Green Ch 2 F2 Fluorescein Cy5 F2 F1 Ch 3 F3 LC Red 705 Hybridization Probe Pairs Cy5 5 Channel 3 F3 is only available with specially modified instruments If you are unsure about the type of dyes and filters in your instrument please consult Idaho Technology If you are unsure about the type of dyes and filters in your instrument please consult Idaho Technology Data Selection Options The available pu
22. function a Gaussian curve fitting function and precise cursor position ing in the analysis For a comparison of the new LCDA analysis with the old LC24 and LC32 melting curve analysis see the transition document included with your software package A melting curve is obtained by monitoring the denaturation profile of a PCR product using either SYBR Green or hybridization probes Melting curves are used to determine the melting temperature of the products formed in an amplifi cation reaction Since the melting temperature of a DNA product is characteris tic of its length and sequence melting temperature is used as a form of product identification replacing the traditional gel based methods The LightCycler analysis software automatically differentiates the melting curve data and dis plays it as melting peaks the area under a melting peak is also used to esti mate product concentration Additionally the melting profile of a sequence spe cific probe is used for rapid genotyping and mutation detection Overall melting curves provide valuable information about any DNA product 3 5 Users Manual 34 Idaho Technology Inc For a quick overview of the new features in LCDA in comparison to the old Melting Curves data analysis software use the Transition Document included with the software package To analyze melting curves the data must display graphically as fluorescence vs temperature in the LCDA Data Selection screen In the temper
23. g up this exponential curve are converted to a linear curve upon logarithmic plotting A user can also define a standard curve using this analysis In this curve the analysis soft ware calculates concentrations of different samples and standards and represents them as crossing points as fractional cycle number where sample fluorescence values are just above background Melting Curve A product melt can be performed after the amplification run This function displays fluorescence curve profiles acquired during the slow denaturation of PCR products As the melt progresses the tempera ture gradually increases to a point where the DNA product begins to melt If SYBR Green or hybridization probes are used the dye or probes sepa rate from the template resulting in decreased fluorescence Options for melting peak analysis include differentiation of melting peak displays inte gration of melting peak areas and determination of product melting tem perature LightCycler Software v3 5 Users Manual 4 Idaho Technology Inc Opening a Run File After LCDA is opened a menu listing the LightCycler run files will automatically display Use the pull down menu to select the computer directory or drive Choose the data for analysis by highlighting the file name in the menu To open a run file double click the run file or select the run file and click Open The Data Selection window for that run file will automatically display Look in 2 BE
24. he crossing line the number of points can be changed to improve the error and r value The Show Fit Points option will display the data points used and the threshold crossing point on the fluorescence vs cycle number graph Do not select more points than the number that fall within the log linear phase of the lowest sample concentration Log Fluorescence vs Cycle Number Graph The graph shown below is similar one displayed in Step 2 Noise Band On a logarithmic plot the linear segment of the fluorescence curve represents the product doubling after each cycle This typically comprises three to six cycles and contains the most informative data The selected points are highlighted by black crosshairs while the calculated crossing points for sample curves are highlighted as red crosshairs The calcu lated crossing points are also displayed along the threshold line 30 The cycle threshold Step 1 Baseine Step 2 Noise Band Step 3 Analysis band crossing line dis plays as a horizontal green line The threshold band also defaults to the same position as the noise band To define the start of the log linear phase move the thresh old band up or down Set the band so that it intercepts the log linear phase of each sample while noting the error and r value This is especially important with samples having low initial copy numbers Minimal error and an r value as close to negative one is desirable E
25. he Fit Points method is selected follow this sequence 1 Baseline Adjustment 2 Noise Band 3 Analysis Step 1 Baseline Adjustment In the Baseline Adjustment tab option a single graph is displayed This graph shown below displays data according to fluorescence vs cycle number The same data is displayed from the previous Data Selection window As the base 27 line adjustment method is changed the software will automatically update graphical data The software will also recalculate any new changes in subse quent steps After an analysis method is chosen a baseline adjustment must be defined before data analysis proceeds Baseline fluorescence may vary among sam ples for a many reasons Some of these reasons include variations in sample preparation variations in DNA content and pipetting error In order to improve sample comparisons the baseline fluorescence for all samples should be set at zero The four available options for Baseline Adjustment include None No baseline adjustment Arithmetic A single value for initial fluorescence of each sample is sub tracted from each data point on the curve This function is useful when using SYBR Green I Proportional The sample data is displayed in terms of the proportional increase in fluorescence from baseline to plateau This function compen sates for sample to sample variations in reaction volume or template concentration This function is useful when working with hybri
26. hows which page is viewed and also the total number of pages To view a particular page enter a number in the Page Number field and hit return or tab through the pages using the arrow buttons Click on the Close button to exit the preview window and return to the quantifi cation window The user can print out the Baseline Adjustment Noise Band Analysis and Standard Curve graphs individually from the Report menu Select samples to be graphed from the sample key on the right The graph header displays the fol lowing information graph title data file name user name analysis program the date program was run person who ran program and date printed A sam ple key is printed beside the graph and some analysis parameters are listed below such as baseline adjustment noise band cursor position if present analysis method and color compensation if used Menus at the top of the window control graph printing The File menu contains several commands s 8 g 5 E Cycle Number Print Window This command sends the graph to the selected printer Printer Setup Allows user to choose printer paper size and paper orien tation Close Window Exits the graph window and returns to the quantification screen The Formatting menu allows the user to control some aspects of the graphs appearance Both the X Axis and Y Axis menu items contain the following options Precision Allows user to increa
27. ll down menus under the Data Selection window are File Setup Report Window and Help These menus are located in the task bar at the top of the window The File menu contains the following options Open Access another data file in a new window Print Window Generates a hard copy of the current window Exit Program Terminates the LCDA program The Setup menu contains the control of all settings in the Data Selection win dow These settings include the following Quantification Analysis Opens the quantification analysis window for the currently selected data Melting Curves Analysis Opens the melting curve analysis window for the currently selected window Analysis Data X Axis Facilitates the X axis setting to Cycles Time or Temperature Fluorescence Adjusts the fluorescence display Options include F1 1 F2 1 F3 1 F1 F2 F1 F3 F2 F1 F2 F3 F3 F1 or F3 F2 Export Facilitates graph data to a tab delimited test file which can be opened by other data analysis or graphing programs Color Compensation Only instruments that have been modified with a third channel can performed dual color reactions and require color compensation The simultaneous amplifi cation of two sequences in the same tube can be followed if two probes labeled with different dyes are used to detect the sequences Unless otherwise requested the IT three color LightCycler Instruments are designed to detect emissions from Fluorescein Cy5 and Cy5
28. log concentration vs cycle number graph is the bottom graph The crossing point data for each sample designated as a standard is plotted against the known concentration This generates a standard curve of Log Concentration vs Cycle Number shown below Values for the slope of the line the Y intercept the standard error and the lin ear correlation coefficient r are displayed numerically to the left of the graph Linear Regression 26 gt Slope 3957 Sad Inerespte 3838 BT pel Error 0 0490 The linear regression for the curve is plotted as a dashed line 33 Slope This value 1 log E represents the overall reaction efficiency Y intercept Calculated using log CT log E Error Mean squared error may indicate sample volume variations r value The correlation coefficient or r value varies from zero to nega tive one for a regression line with a negative slope The r value meas ures how well the linear regression line fits An r value of zero means that the plotted regression line explains nothing about the relationship between cycle number and log concentration while an r value of nega tive one means that the regression line explains the relationship perfect ly Melting Curve Analysis Introduction The Melting Curve analysis in LCDA offers several new options These include new options for background subtraction calculations for derivative data a digi tal filter
29. n pink while temperature tracings are illus trated in black Two green cursor bars are also displayed on the graph E g k n n n n n n n i n n n n 000000 00 0545 00 0838 001716 0020 03 Time fuminsec Temperature Tracing Fluorescence Acquistion Adjust the cursor bars to bracket the area of the cycle to be analyzed by using one of the following three methods 1 Choose the program containing the data of interest from the Select a Program pull down option This automatically moves the cursors to include the run portion where fluorescence was acquired and changes the X axis of the bottom graph to the appropriate setting ad Or manually adjust the cursor to bracket the area of interest Place the pointer on the cursor while holding down the left mouse button and drag the cursor to the desired position u w Or manually enter numerical values for the cursor Adjacent to the right top edge of the graph is a cursor button Click this button to open a dialog box and enter the cursor positions into the appropriate data fields Orientation Format Wh o 00 29 743 Cursor 1 Vertical Time Wh o19 25 215 Cursor 2 Vertical Time Note if the user manually sets cursors reset the x axis of the IF THEN SELECT bottom graph as follows Fluorescence data Cycles from the acquired once per pull down menu cycle is to be ana below the x axis lyzed Fluorescence data Temperature from
30. nd designated as a standard The sample of known concentration should lie within the range of the external standard The fluorescence signal channel of the standard curve and selected run file should match Creating an External Standard Curve This option is used to create a separate standard curve file from an existing run file ty LightCycler Data Analysis Kenny Quantification alison 2 F2 E Ee Bepott Window Help Analysis fine Adiustmer lone jithmetic i ropartional ect Calculat Cre 7 0 000E 00 13 No Extemal Std Curve Load the desired run file to generate an external standard curve Select the standards known concentrations and samples to be included Select Quantification External Standard Curve Create External Curve from the Quantification window task bar In the pop up window enter a file name and designate a path where it will be saved This file will be labeled with a xse extension ycler Software v3 5 Users Manual 22 Idaho Technology In Importing an External Standard Curve This option allows the user to load a standard curve file into a run file without standard curve data Thus unknown sample concentrations can be calculated USING EXTERNAL STANDARD F1 FithW 2 18 99neupk63 ABT Linear Regression 357 Crossing Points Open the desired run file to be analyzed with an external standard curve Confirm the correct fluorescence channel and
31. no briotoday cce menu to move calb 1512 ccc between windows calib 1512 2 ccc calib7148 ccc cascade or tile are calib 1512 3 ccc 3 Monica s cee good options for dis play 4 From the color com f Fie neme pensation menu select Files oftype Colour Compensation Files z cancel 3 calib1512 1028 cce 3 newer cce 14 load calibration data P accoste Colo Compensation Quantilication Meling Curve and open the calibra impot CC Fie tion file that you would f 3 1 calib 1512 2 ccc like to use A i 2 Monica s cec 5 Alternatively use the B calb 1512 coe Select CC Data button 4 calib 1512 2 cce on the task bar at the 5 Colour compensation calibration was done on 28 Oct 1998 19 25 28 with data top of the screen and 6 calib 1512 ccc select Import CC file 7 calib51710 ccc You will notice that the Monicas ccc J 9 calib1512 1028 cce names of any calibra tion files used are dis played with the current file indicated with at check mark Once you have selected a calibration file the color compensated data will be immediately redisplayed on the screen To turn color compensation off click on the color compensation button on the task bar at the top of the screen or select disable from the color compensation pull down menu Click on the color compensation button again to redisplay the compensated data V 10 calib1512 1028 cce D a Click this button to create a cco
32. og concentration while an r value of nega tive one means that the regression line explains the relationship perfect ly Method Il Second Derivative Maximum Analysis The Second Derivative Maximum is completely automated and eliminates user input In this method the fractional cycle number is generated by the second derivative maximum value of each curve The rate of fluorescence changes the fastest during the second derivative maximum It is also the point where the sample fluorescent data is initially differentiated from the background fluores cence The second derivative maximum is often used to accurately predict sample concentration The fractional cycle number of the second derivative maximum for each sample is used to estimate sample concentration This method does not depend on defining the log linear portion of the data curves nor does it require a manual noise band setting If the Second Derivative Maximum method is selected follow this sequence 1 Baseline Adjustment 2 Analysis Note Noise Band is automatically done LightC ycler Software v3 5 Users Manual 32 Idaho Technology Inc Log Fluorescence Vs Cycle Number Graph In this analysis window two graphs are displayed The top graph shown below is the same Log Fluorescence versus Cycle Number graph from Step 1 Baseline Step 1 Baseine Step 2 Analyse Bas i Log Concentration vs Cycle Number Graph In this graph the
33. oint of the melting peak Step 1 Melting Peaks The Melting Peaks window is composed of two graphs a top graph displaying Temperature vs Fluorescence data and a lower curve displaying Temperature vs d F1 dT data The top graph is carried over from the Data Selection win dow and the negative first derivative of this data is used to convert melting curve data to melting peak data The melting peak data is displayed in the bot tom graph In this step the user has several options that will influence the manner the data is presented The options are listed and described below Calculation Method C to Average Digital Filter Calculation Method The user has two options to calculate the negative first derivative data presented in the Temperature vs dF dT graph Linear and Polynomial refer to the curve fit methods used to calculate the first derivative for the melting curves For a polynomial fit the approach that is used to estimate the derivative of a set of x y points involves examining each point in turn fitting a polynomial to the points surrounding it and using the negative first derivative of the polynomial fit A linear fit is simply a polynomial fit using a first degree polynomial 36 Degrees C to Average This value represents the sliding interval used to calculate the derivative data for the melting peaks The optimum num ber of degrees C to average varies depending on the selected calcula tion method Th
34. s feature is a great tool for comparing product Tm s melting temperature of product and melting peak areas The peak areas of an unknown samples and known standard can be compared in the same amplifi cation tube In order to compare melting temperatures and peak areas among samples the nonspecific fluorescence must be minimized In Step 1 Melting Peaks select ing a calculation method With Background subtraction allows the user to reduce background fluorescence levels to a baseline value This allows valid comparison of product Tm s and melting peak areas The melting peak area window fits up to three Gaussian curves to the deriva tive melting curve data defined from the Step 1 Melting Peaks window Analyze each sample individually since the number of peaks may differ among samples The fit curves will display as a thin solid orange line on the graph 39 ma ma as 2 e os Ea 127 as vase 1280 OSD eg nes asn Ee 170 a2 Sample Display As each sample is analyzed the user must define the peak number that is fit ted to the data in Number of Peaks Box Peak area calculations are displayed in columns to the left of the graph according to sample color sample position sample name standard concentration empty for unknown samples area melting temperature and standard deviation For each sample the software can calculate up to three melting peaks Note that standard deviation is not a confi dence
35. se or decrease the number of decimal places for numerals on the axes Number Format Allows the axes to display in either decimal scientific or engineering notation Restore Defaults Recovers the default graph settings Cursors Off Hides cursors in the graphs where cursors are present Help Menu Features The LightCycler Manual can be accessed in the Data Selection Quantification and Melting Curve Analysis Screens For help with other features please call our product support line 800 735 6544 or you can reach us via e mail at support idahotech com Analysis Methods LCDA offers two options for data analysis Fit Points and the Second Derivative Maximum Initially the user must determine which Analysis Method to use Method I Fit Points After the user selects the Baseline Adjustment the Noise Band must be deter mined The Noise Band is a threshold line that is set above the non informative fluorescence data The user then selects a set of data points from the log linear region of the fluorescence curves This set of data points is used to generate a best fit regression line Crossing Line thus determining the fractional cycle number of the crossing point The Crossing Line may be manually adjusted to intersect the log linear phase of as many sample fluorescence curves as possi ble The Fit Points analysis method requires greater user input where the Second Derivative Maximum method does not automated by software If t
36. ty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Idaho Technology shall not be liable for errors contained herein or for incidental consequential damages in connection with the furnish ing performance or use of this material The new LightCycler Data Analysis LCDA software program allows the user to evaluate quantification or melting curve data acquired during a LightCycler Instrument run To open the LCDA software select the LCDA button in the Front Screen The LCDA software also opens immediately after a LightCycler Instrument run is completed Note that the previous Front Screen options Run Profile Quantification and Melting Peaks are still available and remain unchanged The L C D A software displays and analyzes data in the following three formats Data Selection When the user opens a data file the Data Selection win dow will automatically display This window displays the raw fluorescence and temperature data acquired during the run In this window the user must first define the data of interest before the analysis software can pro ceed This window gives the same information obtained in the Run Profile button of the old software Quantification The quantification analysis only uses values measured in the exponentially growing phase of the amplification This phase is termed the log linear phase because the points makin
37. ubes must be prepared Tube 1 should contain a reagent blank buffer no fluors Tube 2 should contain buffer and the Fluorescein probe at 0 2 uM Tube 3 should contain buffer and the Cy5 probe at 2 uM Tube 4 should contain buffer and the Cy5 5 probe at 2 uM nh After the tubes are prepared go into the Real Time Fluorimeter Set the temperature to 40 C wo Seek Tube 2 monitor the signal in F1 and set the gains so that the sig nal is between 60 80 Record the gain setting for F1 A Seek Tube 3 monitor the signal in F2 and set the gains so that the sig nal is between 60 80 Record the gain setting for F2 a Seek Tube 4 monitor the signal in F3 and set the gains so that the sig nal is between 60 80 Record the gain setting for F3 Roche LightCycler Real Time Fluorimeter Fle Hep TIC and Fluorescence 1000 950 s004 a504 s004 7504 ED EE oi 5 Loone J e eee Calibrated 6 Once you have recorded the gains for each channel move into the LightCycler software programming screen Set up the following cycling conditions 94 C 0 sec 20 C sec None 40 C 30 sec 20 C sec None 95 C 0 sec 0 1 C sec Cont This program is designed to record the fluorescence signal from each dye over a temperature range of 40 95 C 7 Run the
38. ure Automatic Fit must be deselected After the user has customized a graph select OK to save changes and return to LCDA Select Cancel to return to LCDA without saving graph changes Quantification The Quantification analysis of LCDA has several new features Most notable are the crossing point values calculated in faction cycle numbers These val ues are displayed in the analysis screens and allow the user to compare cross ing points among samples Additionally the software can calculate unknown concentration values In order to accomplish this the user should define at least two standards of known concentration prior to running the reaction LC32 user can define samples as Standards in the Load Screen portion of the Run function before running a reaction LC24 users need only to check the con centration column in the loading screen for the desired standard sample LCDA offers two methods to analyze fluorescence curves In one method Fit Points a noise band is used to define the beginning of the log linear phase of the curve which contains the most relevant data points The intersection of a second crossing line Threshold band is used to calculate product concentra tion In the second method Second Derivative Maximum the maximum sec ond derivative is used to define fractional cycle number and product concentra tion This function is completely automated and eliminates user input For a quick overview of the new
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