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1. 2o A tr t 1y A ye me 1 La tit Figure 8 Electropherogram showing two resolved alleles detected as a single peak Note For information on the tick marks displayed in the electropherogram see Examining Peak Definitions on page 9 Overview of the Analysis Parameters and Size Caller 11 ABI PRisM GeneScan Analysis Software for the Windows NT Operating System 12 Effects of Decreasing the Window Size Value Figure 9 shows that both alleles are detected after re analyzing with the polynomial degree set to 3 while decreasing the window size value to 15 from 19 data points EK 4140 4150 4100 4170 4180 4190 4200 4210 4220 4230 4240 4250 4200 4270 4280 4290 400 410 4920 490 440 450 400 47O 480 4 4400 40 4420 Figure 9 Electropherogram showing the alleles detected as two peaks after decreasing the window size value User Bulletin Overview of the Analysis Parameters and Size Caller Optimizing Peak Detection Sensitivity Example 2 Initial Figure 10 shows an analysis performed using a polynomial Electropherogram degree of 3 and a peak window size of 19 data points AJ J820 1890 1840 1850 1860 1870 1880 1890 1900 1010 1920 1930 1949 1980 1900 1970 1980 1990 2000 2010 2020 20390 2040 200 x Y Dye Sample Minutes Size Peak Height Peak Area Data Point Peak 3R 15 5 18 339 592. start point of a peak closer to its apex Slope Threshold for Peak Start value from zero to a positive number end point of a peak closer to its apex Slope Threshold for Peak End value to an increasingly negative number Note The size of a detected peak is the calculated apex between the start and end points of a peak and will not change based on your settings Overview of the Analysis Parameters and Size Caller 17 18 ABI PRiSM GeneScan Analysis Software for the Windows NT Operating System Slope Threshold Example Initial Electropherogram The initial analysis with a value of 0 for both the Slope Threshold for Peak Start and the Slope Threshold for Peak End value produced an asymmetrical peak with a noticeable tail on the right side EE 212 2w ao te aeo amo aio 24902200 2210 2720 2390 2290 2250 2300 270 22380 290 20 W0 VW Dye Sample Minutes Size Peak Height Peak Area Data Point 2Y 5 59 47 17357 1585 10752 2230 Figure 13 Electropherogram showing an asymmetrical peak User Bulletin Overview of the Analysis Parameters and Size Caller Electropherogram After re analyzing with a value of 35 0 for the Slope Threshold for After Adjustments Peak End the end point that defines the peak moves closer to its apex thereby removing the tailing feature Note that the only change to tabular data was the area peak size and height are unchanged BES 212 z
3. 1482 17703 1935 B96 3R 16 510 341 41 5354 1945 Figure 10 Electropherogram showing four resolved peaks detected as two peaks Overview of the Analysis Parameters and Size Caller 13 ABI PRiSM GeneScan Analysis Software for the Windows NT Operating System Effects of Figure 11 shows the data presented in Figure 10 re analyzed with a Reducing the window size value of 10 and polynomial degree value of 5 Window Size Value and Meee ee n n a e n a e e n e a e e t Increasing the w Polynomial Degree Value Figure 11 Electropherogram showing all four peaks detected after reducing the window size value and increasing the polynomial degree value 14 User Bulletin Overview of the Analysis Parameters and Size Caller Optimizing Peak Detection Sensitivity Example 3 Effects of Figure 12 shows the result of an analysis using a peak window size Extreme Settings value set to 10 and a polynomial degree set to 9 This extreme setting for peak detection led to several peaks being split and detected as two separate peaks EE 4010 4020 40390 4040 4050 4060 4070 4000 4090 4100 4110 4120 4130 4140 4150 4100 4170 4180 4190 4200 4210 4120 4230 Figure 12 Electropherogram showing the result of an analysis using extreme settings for peak detection Overview of the Analysis Parameters and Size Caller 15 ABI PrisM GeneScan Analysis Software for the Windows NT
4. Size standard curve is constructed using the method selected by the user e g local southern Size standard curve used to size all peaks in sample Fail All peaks present in size standard are detected 4 New standard for failed sample is defined regardless of migration or quality Information in the size standard definition is used to select the peaks of the size standard ratio matching is used based on the list of sizes defined in the Size Standard file Best fit curve of the detected sized standard fragments is constructed J Figure 27 Peak sizing flowcharts For each peak in the size standard the matched size of the peak is compared to the calculated size using the best fit curve previously constructed If the sizes differ significantly or the peaks cannot be found the sizing fails Jie Size standard curve is constructed using the method selected by the user e g local southern J Size standard curve used to size all peaks in sample Fail Sample is not sized J The user should 1 Make sure all fragments listed in the size standard definition are reflected in the analysis range 2 Make sure the primer peak is not interfering with smaller fragments If it is exclude the primer peak from the analysis 3 Make sure the higher fragments are resolved
5. e Size Call Range e Size Calling Method e Peak Amplitude Thresholds ABI Prism GeneScan Analysis Software Version 3 7 User Guide Changed from Macintosh versions e Smooth Options e Min Peak Half Width this user bulletin and the ABI PRISM GeneScan Analysis Software Version 3 7 NT and 3 1 Macintosh User Guides e Polynomial Degree e Peak Window Size e Slope Threshold this user bulletin and the ABI PRISM GeneScan Analysis Added for the Software Version 3 7 Windows NT version for Peak Start User Guide e Slope Threshold for Peak End e Window Size Baseline ABI PRISM Removed options from the Windows NT version Multicomponent GeneScan Analysis Software version 3 1 User s Manual Overview of the Analysis Parameters and Size Caller ABI PrRisM GeneScan Analysis Software for the Windows NT Operating System Analysis Parameters Dialog Box About the Analysis Parameters Dialog Box Example Use the Analysis Parameters dialog box to set analysis parameter values for data processing The default analysis parameter values are analysis guidelines This bulletin should serve as a guide for modifying these values as appropriate for each laboratory Figure 2 shows the Analysis Parameters dialog box with default values for GeneScan analysis software v3 7 1 on the Windows NT operating system Analysis Parameters g b il Figure
6. 2 Analysis Parameters dialog box displaying default values User Bulletin Overview of the Analysis Parameters and Size Caller Data Processing Smooth Options Parameter About the The Smooth Options parameter sets the degree of smoothing applied Parameter to the display of the analyzed electropherogram Smoothing may aid in data interpretation How the The Smooth Options parameter is applied after peak detection and Parameter Works affects only the display of analyzed electropherograms The peak heights and areas are calculated and displayed in the tabular data display based on the none smoothing option Selecting light or heavy smoothing will not affect the calculation of these values Smoothing Figure 3 shows the peaks from the same sample file after analysis Example using no smoothing black light smoothing green and heavy smoothing red All tabular data including peak height and area remain unchanged CA cc 4000 4026 4060 4075 4100 4125 4160 4175 zo No smoothing black mall Light smoothing green m Heavy smoothing red Dyelsampie Minutes Size Peak Height Peak Area Data Point Peak 3Y 3 1476 148 51 2968 16671 4024 4 3 71476 148 51 2068 16671 a024 BY 3 14 78 145 51 72968 16671 4024 Figure 3 Electropherogram showing the effects of smoothing on peaks from the same sample file Overview of the Analysis Parameters and Size Caller 5 AB
7. If they are not reduce the analysis range and change the size standard definition to reflect the missing peaks Attempt to get a better ratio match by changing the size standard definition and analysis range to analyze a smaller range containing only peaks of interest if plausible Attempt to guess values for any split peaks so that every peak displayed has a value oa 34 User Bulletin Overview of the Analysis Parameters and Size Caller Overview of the Analysis Parameters and Size Caller 35 Copyright 2002 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document Applied Biosystems ABI PRISM and its design and GeneScan are registered trademarks and AB Design and Applera are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation Macintosh is a registered trademarks of Apple Computer Inc All other trademarks are t
8. Operating System Peak Detection Slope Threshold for Peak Start and Slope Threshold for Peak End Parameters About These Parameters How These Parameters Work Guidelines for Using These Parameters 16 Use the Slope Threshold for Peak Start and Slope Threshold for Peak End parameters to adjust the start and end points of a peak This parameter can be used to better position the start and end points of an asymmetrical peak or a poorly resolved shouldering peak to more accurately reflect the peak position and area In general from left to right the slope of a peak increases from the baseline up to the apex From the apex down to the baseline the slope becomes decreasingly negative until it returns to zero at the baseline Apex Increasingly Increasingly positive negative slope A Baseline Q If either of the slope values you have entered exceeds the slope of the peak being detected the software overrides your value and reverts to Zero As a guideline use a value of zero for typical or symmetrical peaks Select values other than zero to better reflect the beginning and end points of asymmetrical peaks A value of zero will not affect the sizing accuracy or precision for an asymmetrical peak User Bulletin Overview of the Analysis Parameters and Size Caller Using These Use the table below to move the start or end point of a peak Parameters IF you want to move the THEN change the
9. window sizes The electropherogram shows the default Baseline Window Size value of 51 that appears in Figure 15 as the blue trace Note that all peaks in this cluster have been baselined FER _275 o0 2025 seso sors s700 gras s750 3775 seo 825 3850 3875 3900 3925 3950 3975 4000 4025 4060 4075 4100 4125 4160 4175 4200 4225 4250 4275 4000 4325 4350 4375 P an V L Figure 16 Electropherogram showing an allelic ladder with a cluster of peaks User Bulletin Overview of the Analysis Parameters and Size Caller Effects of Figure 17 shows an extreme Baseline Window Size value of 2801 that Extreme Increase appears in Figure 15 as the red trace 2801 is approximately the of the Baseline width in data points of all the peaks shown This increase resulted in Window Size an Overall raised baseline and many elevated peaks within the cluster e 2575 3600 3625 3060 3675 3700 3725 3760 3775 3800 3826 3850 3675 3000 3025 3960 3975 4000 4025 4050 4075 4100 4125 4160 4175 4200 4225 4250 4275 400 4025 4360 4075 BeeEEERESE EERE RE 216 107 Figure 17 Electropherogram showing a raised baseline caused by an increase in the baseline window size value Overview of the Analysis Parameters and Size Caller 23 ABI PRisM GeneScan Analysis Software for the Windows NT Operating System Effects of Extreme Decrease of the Baseline Window 24 Size Figure 18 shows an
10. I PRisM GeneScan Analysis Software for the Windows NT Operating System 4050 4075 4100 4125 4160 as awo al x v d T mN le Minutes Size Peak Height Peak Area Data Point BA ue me EE w 1463 14405 1347 200 3986 SY 1 1463 14405 134 s00 3986 Figure 4 Electropherogram showing the effects of smoothing on the smaller peak and baseline by changing the y scale from Figure 3 User Bulletin Overview of the Analysis Parameters and Size Caller Peak Detection Min Peak Half Width Parameter About This Use the Min Peak Half Width parameter to specify the smallest full Parameter width at half maximum height for peak detection This parameter can be used to ignore noise spikes How This The Min Peak Half Width parameter defines what constitutes a peak Parameter Works The software ignores peak half widths smaller than the specified value The way in which this version of the software defines the minimum peak half width is different than in previous versions Old Versions Current Version Half width of the peak measured Full width of the peak measured at from peak start half its height Figure 5 Defining the Min Peak Half Width Overview of the Analysis Parameters and Size Caller ABI PRiSM GeneScan Analysis Software for the Windows NT Operating System Peak Detection Polynomial Degree and Peak Window Size Parameters About These Para
11. User Bulletin ABI PRISM GeneScan Analysis Software for the Windows NT Operating System SUBJECT Introduction In This User Bulletin Purpose Intended Audience June 2002 Overview of the Analysis Parameters and Size Caller This user bulletin includes the following topics GeneScan Analysis Software Process 0 000 cee cence 2 Analysis Parameters 2 0 0 0 e eee eee eee eee 3 Analysis Parameters Dialog Box 0 00 0 e eee eee 4 Data Processing Smooth Options Parameter 5 Peak Detection Min Peak Half Width Parameter 7 Peak Detection Polynomial Degree and Peak Window Size Parameters csi dined pada e ed a a dada ate S 8 Peak Detection Slope Threshold for Peak Start and Slope Threshold for Peak End Parameters 000000 16 Baselining Baseline Window Size Parameter 20 SIZ Callers cc denn ege ea pad nde Ree woke eek enka dogs Khe ded 30 This user bulletin supplements the ABI PRISM GeneScan Analysis Software version 3 7 User Guide P N 4308923 to further explain the analysis parameters and size caller available in the Windows NT version of the software The GeneScan Analysis Software v3 7 1 Updater CD P N 4336026 includes new analysis parameter default values For additional information and installation instructions refer to the GeneScan v3 7 1 About file This document is intended for users familiar with the GeneScan an
12. alysis software for the Macintosh operating system who are now using the software on the Windows NT operating system gt Applied KS Biosystems ABI PrisM GeneScan Analysis Software for the Windows NT Operating System GeneScan Analysis Software Process Overview The ABI PrISM GeneScan Analysis Software is available in versions for both the Windows NT operating system and the Macintosh operating system The Windows NT version of the software uses different algorithms and has additional analysis parameters that give users more control with data analysis Flowchart The following flowchart shows how GeneScan analysis software analyzes data Note For multicapillary instruments multicomponenting is performed by the data collection software TF Raw data Limit analysis range t Multicomponent Baseline L Detect peaks l Smooth analyzed electropherogram No Sizecalling needed Yes Match size standard l Quality check L Make sizing curve t Size peaks Analyzed data Figure 1 Simplified GeneScan analysis software flowchart User Bulletin Analysis Parameters Table of Parameters Overview of the Analysis Parameters and Size Caller The following table lists the analysis parameters Parameter Status Parameter Discussed in Unchanged from Macintosh versions e Analysis Range
13. es have been labeled with green dye and the data displayed has been multicomponented but not baselined The electropherogram spans approximately 2800 data points The red blue and black traces depict various reference baselines zero in the analyzed electropherogram that result from different baseline window size settings These reference baselines are subtracted from the sample data during baselining In Figure 15 The red trace depicts the reference baseline that results from an extreme baseline window size value of 2801 At this setting the reference baseline does not touch all peaks resulting in elevated peak heights The blue trace depicts the reference baseline that results from the default value of 51 data points User Bulletin Overview of the Analysis Parameters and Size Caller The black trace depicts the reference baseline that results from an extreme baseline window size value of 5 data points At this setting the peaks are tracked too closely by the reference baseline resulting in significantly reduced peak height ll TMQ 500 1000 1500 2000 2500 3000 Figure 15 Depiction of the baselining of an electropherogram Overview of the Analysis Parameters and Size Caller 21 ABI PRiSM GeneScan Analysis Software for the Windows NT Operating System Baselining Example 1 Initial Electropherogram 22 Figure 16 shows a portion of the electropherogram shown in Figure 15 which depicts various
14. extreme Baseline Window Size value of 5 that appears in Figure 15 as the black trace Five is much smaller than the width in data points for any of the peaks prior to baselining This decrease resulted in a significant decrease in the peak heights ER 2575 3800 3625 3850 3675 3700 3725 3760 3775 3800 3825 3860 3875 3900 3925 3950 3975 4000 4025 4050 4075 4100 4125 4160 4175 4200 4225 4250 4275 400 4925 4350 4975 216 94 B71 18085 l al com 3640 216 97 983 18470 237 585 3686 216 101 f 10 07 492 73 Tia a8 3777 216 107 10 31 200 56 143 307 3666 216 110 10 43 204 84 213 Ba 3910 FAKETI en a E E E 216 120 11 02 225 98 101 284 4132 216 127 a1 37 238 54 OO 389 a6 2146 130 11 48 242 58 33 81 4305 Figure 18 Electropherogram showing significantly reduced peak heights caused by a reduction in the baseline window size value User Bulletin Overview of the Analysis Parameters and Size Caller Baselining Example 2 Initial Figure 19 shows the electropherogram from an analysis of a cluster of Electropherogram peaks using the default Baseline Window Size value of 51 data points 1860 1900 1980 2000 2060 1200 1700 1600 1600 1400 1300 1200 1100 Figure 19 Electropherogram showing a typical result using the default baseline window size value Overview of the Analysis Parameters and Size Caller 25 ABI PRisM GeneScan Analys
15. gher User Bulletin Overview of the Analysis Parameters and Size Caller Guidelines for To detect well isolated base line resolved peaks use polynomial Using These degree values of 2 or 3 For finer control use a degree value of 4 or Parameters greater As a guideline set the peak window size in data points to be about 1 to 2 times the full width at half maximum height of the peaks that you want to detect Examining Peak To examine how GeneScan Analysis software has defined a peak Definitions select View gt Show Peak Positions The peak positions including the beginning apex and end of each peak are tick marked in the electropherogram Effects of Varying Figure 6 depicts peaks detected with a window size of 15 data points the Polynomial and a polynomial curve of degree 2 green 3 red and 4 black Degree The diamonds represent a detected peak using the respective polynomial curves Note that the smaller trailing peak is not detected using a degree of 2 green As the peak detection window is applied to each data point across the displayed region a polynomial curve of degree 2 could not be fitted to the underlying data to detect its structure 2000 Polynomial curve of degree 4 1600 black Polynomial curve of degree 3 red 1000 Polynomial curve of degree 2 green 500 Figure 6 Electropherogram showing peaks detected with the same window size and three different
16. he sole property of their respective owners Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For sales office locations and technical support please call our local office or refer to our web site at www applied biosystems com www appliedbiosystems com AS Applied sw Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in USA 06 2002 Part Number 4335617 Rev A Stock Number 106UB35 01 an Applera business
17. igned to allow the exclusion of one of the listed values to obtain a better match To use an excluded fragment try the steps outlined in Figure 27 User Bulletin Base pair Data point Overview of the Analysis Parameters and Size Caller 400 Basepair 50 100 200 400 X 2x 4x Scan 100 200 400 800 50 100 200 400 Base 59 100 200 pair xX 2x 4x Data 100 200 400 800 point 100 200 400 500 ao 400 a 5 30 rod Qa se 8 200 Pal a a 100 a A 0 tt oo 0 100 200 300 400 500 600 700 800 900 Data point Defining the Size Standard from a list of sizes 50 100 200 and 400 used to calculate the expected ratios in red Data 100 200 400 and 800 shown with anomalous peak dotted Assigning Peaks that match the 800 expected ratio The anomalous peak is ignored Generating the Size Standard Curve when a good ratio match is found Figure 26 Peak identification with GeneScan analysis software for the Windows NT operating system Overview of the Analysis Parameters and Size Caller 33 ABI PRisM GeneScan Analysis Software for the Windows NT Operating System GeneScan Macintosh version GeneScan Windows NT version All peaks present in size standard are detected 4 Information in the size standard definition is used to select the peaks of the size standard 10 data points from the position defined in the Size Standard file lee
18. is Software for the Windows NT Operating System Effects of Extreme Decrease of the Baseline Window 26 Size Figure 20 shows the re analysis of the electropherogram shown in Figure 19 with an extreme Baseline Window Size value of 5 All peaks within the cluster have been baselined and have a reduced peak height 1850 1900 1950 2000 2060 900 700 Figure 20 Electropherogram showing dramatically reduced peak heights caused by a reduction in the baseline window size value User Bulletin Overview of the Analysis Parameters and Size Caller Baselining Example 3 Raw Data The data in the electropherogram has been multicomponented but not baselined There are two pull down peaks in the blue trace below the two major green peaks see arrows 1000 _ Figure 21 Electropherogram showing raw data that has been multicomponented but not baselined Overview of the Analysis Parameters and Size Caller 27 ABI PRisM GeneScan Analysis Software for the Windows NT Operating System Raised Baseline 28 After analyzing with a baseline window size of 251 data points the low points represented in the blue trace within this 251 data point window are set to zero This results in a raised baseline between these points Figure 22 Electropherogram showing a raised baseline User Bulletin O
19. malous peaks outside of a 10 data point bin are ignored but those within the bins can be incorrectly called resulting in an incorrect size curve In that case you must redefine a new size standard for that particular sample User Bulletin yestr petteri CCECI P OCCEE yetedi weet Data Point 100 Base Pair 50 Overview of the Analysis Parameters and Size Caller Defining the Size Standard The boxes show a 10 data point range used to identify PILILLA bereerea VALLEEEEER ENE VERRAAI UILEN size standard peaks in YILLLLLR YILLLLIE YILLLLLIL FACEEEEA YOCEEEE A YOCEEEE CES su b seq uent runs FAASCESA RAACEECA PACAAAEECE ELLLLLLA ULLAL teeeddidds is Data 100 200 400 and 800 shown with anomalous peak 200 400 800 dotted 100 200 Assigning Peaks that fall into the correct range The anomalous peak is ignored Data Point 100 200 400 800 500 5 a 400 we Generating the Size Standard ail ae Curve for sample file using a g specified sizing method e g 200 wee Local Southern ca a 100 en P 0 O 100 200 300 400 500 600 700 800 900 Data point Figure 24 Peak identification with GeneScan analysis software for the Macintosh operating system Overview of the Analysis Parameters and Size Caller 31 ABI PrisM GeneScan Analysis Software for the Windows NT Operating System 32 Windows NT Version GeneScan analysis software for the Windows NT operating
20. meters How These Parameters Work How to Use These Parameters Use the Polynomial Degree and the Peak Window Size settings to adjust the sensitivity of the peak detection You can adjust these parameters to detect a single base pair difference while minimizing the detection of shoulder effects or noise Sensitivity increases with larger polynomial degree values and smaller window size values Conversely sensitivity decreases with smaller polynomial degree values and larger window size values The peak window size functions with the polynomial degree to set the sensitivity of peak detection The peak detector computes the first derivative of a polynomial curve fitted to the data within a window that is centered on each data point in the analysis range Using curves with larger polynomial degree values allows the curve to more closely approximate the signal and therefore the peak detector captures more peak structure in the electropherogram The peak window size sets the width in data points of the window to which the polynomial curve is fitted to data Higher peak window size values smooth out the polynomial curve which limits the structure being detected Smaller window size values allow a curve to better fit the underlying data Use the table below to adjust the sensitivity of detection To Polynomial Window Size ac Degree Value Value Increase sensitivity use Higher Lower Decrease sensitivity use Lower Hi
21. o zo ats neo zw 21e0 zoo za00 amo gmo 2m0 zao 260 m00 270 2280 290 2000 78102020 Figure 14 Electropherogram showing the effect of changing the slope threshold for peak end Overview of the Analysis Parameters and Size Caller 19 ABI PRISM GeneScan Analysis Software for the Windows NT Operating System Baselining Baseline Window Size Parameter About This Parameter How This Parameter Works 20 Guidelines for Using This Parameter Baselining Example Use the Baseline Window Size parameter to control the baseline for a group of peaks The software determines a reference baseline value for each data point In general the software sets the reference baseline to be the lowest value that it detects in a specified window size in data points centered on each data point A small baseline window relative to the width of a cluster or grouping of peaks spatially close to each other can result in shorter peak heights Larger baseline windows relative to the peaks being detected can create an elevated baseline resulting in peaks that are elevated or not baseline resolved As a guideline choose a value that encompasses the width in data points of the peaks being detected while preserving a qualitatively smooth baseline The trade off for a smoother baseline that touches all peaks is a reduction in peak height Figure 15 depicts an allelic ladder containing clusters of alleles The allel
22. polynomial degrees Overview of the Analysis Parameters and Size Caller 9 ABI PrisM GeneScan Analysis Software for the Windows NT Operating System Effects of Figure 7 shows the same peaks that are shown in Figure 6 However Increasing the in this depiction both polynomial curves have a degree of 3 and the Window Size window size value was increased from 15 red to 31 black data Value points As the cubic polynomial is stretched to fit the data in the larger window size the polynomial curve becomes smoother Note that the structure of the smaller trailing peak is no longer detected as a distinct peak from the adjacent larger peak to the right 2000 1500 Window size value of 31 black ey Window size value of 15 red 500 Figure 7 Electropherogram showing the same peaks as in Figure 6 after increasing the window size value while keeping the polynomial degree the same 10 User Bulletin Overview of the Analysis Parameters and Size Caller Optimizing Peak Detection Sensitivity Example 1 Initial Figure 8 shows two resolved alleles of known fragment lengths that Electropherogram differ by one nucleotide detected as a single peak The analysis was performed using a polynomial degree of 3 and a peak window size of 19 data points R so aso wo 4170 giso gioo a200 4210 4220 4230 4240 4250 4200 4270 4280 4200 4300 4310 4920 4000 4340 4350 400 4070 4360 4300 4400 44104420
23. system uses ratio matching to identify the size standard fragments Ratio matching does not rely on the manual assignment of size standard definitions in base pairs to their associated data points within a run or a previous run Selecting a peak in the electropherogram to enter an associated value in the Size column now serves only as a guide Simply listing the values to be used for sizing as an array of numbers without regard to the highlighted peak is sufficient E 2900 3950 4000 4060 4100 4160 4200 4260 4300 2400 2000 1600 1200 Bi Template File Info File PP Sample_2fsa Run Date Fri Jan 01 1904 Run Time 12 00 00 AM Dye R Parameters lt Analysis Parameters Figure 25 Electropherogram showing a selected peak and the associated value in the Size column The size caller ignores anomalous peaks that do not match the expected ratio The size caller constructs a best fit curve using the data points of each size standard fragment detected A comparison between the sizes calculated from the best fit curve and the matched peaks from the size standard definition using the array of numbers is performed Size calling will fail if significant differences are found or if no match can be made based on the expected ratios In Figure 26 that is x 2x and 4x Additionally you may find that one of the size fragments has not been identified even though it was listed as part of the definition The size caller has been des
24. verview of the Analysis Parameters and Size Caller Eliminating After re analyzing with a baseline window size of 51 data points a Raised Baseline window size range between the pull down peaks the raised baseline is eliminated This results in a more accurate baseline EN 5860 5700 5750 8800 5250 5000 5050 6000 400 360 300 Figure 23 Electropherogram showing a more accurate baseline Overview of the Analysis Parameters and Size Caller 29 ABI PRISM GeneScan Analysis Software for the Windows NT Operating System Size Caller About the Size Caller How the Size Caller Works Macintosh Version 30 The size caller matches size standard peaks with a quality check The way in which the fragment sizes are calculated has not changed from previous versions of the software e g local southern However the way in which the Windows NT version of the software identifies the size standard is different from previous versions Method for Identifying the Size Standard Macintosh Versions Windows NT Version User assigns fragment sizes to Software matches the size particular peaks based on scan standard fragments by ratio number matching based on relative distance between neighboring peaks In GeneScan analysis software for the Macintosh operating system the size standard peaks are identified based on their assignment within a run or a previous run Ano
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