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Biotype ® Template Files for GeneMapper

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1. analyses 400 200 300 400 500 2000 50 0 70 0 90 0 120 0 160 0 190 0 220 0 260 0 300 0 340 0 380 0 425 0 450 0 475 0 500 0 60 0 80 0 100 0 140 0 180 0 240 0 280 0 520 0 60 0 400 0 200 0 Fig 5 Electropherogram of the DNA Size Standard 550 ROX Lengths of Fragments in bp Definition of a New Size Standard To define a new size standard please perform the following steps Tools GeneMapper Manager Select the tab Size Standards New Basic or Advanced Assign a name e g SST ROX_50 500bp and define the size range on the lines of the analysis range of the corresponding test kit See also horizontal scaling in the user s manual Size Standard Dye F r SST ROX Red ausw hlen f r SST BTO Orange SST BTO For Biotype test kits in 5 Color Assay with the fluorescent labels 6 FAM BTG BTY BTR and BTO matrix BT5 the DNA Size Standard 550 BTO in orange Panel is necessary Please use DNA Size Standard 550 BTO template SST BTO 60 500bp to define fragment length for particular test kit GeneMapper ID Template Files March 2010 sd B Panels and BinSets Panels The following features of the test kit s markers STR Loci are depicted Marker Name STR Locus Dye Colour Min Size and Max Size upper and lower allelic range in bp Control A
2. 5ng VWA VEK M91 0 5ng FGA M91 DNA1 0 5ng D18S51 M31 _0 5ng 10851179 M31 DNA1 0 50 D21511 a se 3s T ey AT I T 7 afl sis Fig 20 Output File after Execution of Table for 10 Alleles Table Export Sample Plot with table has to be displayed File Export Table In order to save add txt for tab delimited text or csv for comma delimited text For general instructions for table export please read the chapter Exporting Table Data from GeneMapper ID Software User Guide GeneMapper ID Template Files March 2010 97 5 Check Analysis Data General Procedure for Analysis 1 Check Size Standard 2 Check Allelic Ladder 3 Check Positive Control 4 Check Negative Control 5 Review Sample Data Check Size Standard The first step in any new project with low Size Quality PQV SQ is to check the size standard for the correct fragments named size calling see chapter size standards Size Match Editor File Edit View Tools Sizing Quality 1 0 Override SQ 380400 200 260220 320340360 90 1601 220240 300 5059 89 100120 445 079 LLL 6000 7000 J Apply Fig 21 Checking Size Calling GeneMapper ID Template Files March 2010 28 Check Allelic Ladder Second action
3. BinSets could be generated in the Panel Manager GeneMapper ID Template Files March 2010 1 3 Use GeneMapper ID Manager to import Analysis Methods Table Settings Plot Settings or Size Standards for Biotype products into the GeneMapper ID software Recommendation Further Biotype Template Files like analysis methods Table Settings Plot Settings or Size Standards should be saved as new folders onto the local computer AppliedBiosystems GeneMapper new folder Tools GeneMapper Manager Select the designated tab and click the button Import Analysis Method Import Table Setting Import Plot Setting Import Size Standard Import Imported Analysis Methods Table Settings Plot Settings or Size Standards could be redefined and saved by the user New Analysis Methods Table Settings Plot Settings or Size Standards could be generated in the GeneMapper Manager and should be compiled in Advanced version GeneMapper ID Template Files March 2010 2 Calibration using Allelic Ladders Workings with the GeneMapper ID Software are done with related analysis data i e analyzed DNA samples with allelic ladders of one folder Project To analyse DNA samples with the GeneMapper ID Software calibration with the allelic ladder has to be done The allelic ladder of the used test kit should be analysed ideally at the beginning and the end of the DNA samples For calibration th
4. Cut off Value o 0 o 0 o 0 Minus Ratio o n o 0 o 0 fo 0 lt Distance From o 0 o 0 o 0 o 0 o 0 o 0 fo 0 o 0 Minus Stutter Ratio o 0 o 0 o 0 o 0 Minus Stutter Distance From 25 25 a 25 o 0 75 n 75 s 75 o 0 Plus Stutter Ratio foo joo o 0 o 0 Plus Stutter Distance From o 0 o 0 to foo Amelogenin Cutoff 25 Range Fitter Fig 14 Tab Allele for adjustment of the filter values pre selection No Cut Off filter Changing the Filter Value in the Allele tab To change the lower thresholds please perform the following steps Tools GeneMapper Manager Select Analysis Method and edit via double click The following filter values can be changed in the tab Allele Cut off Value MinusA Minus Stutter Plus Stutter see figure 14 Select the value you would like to change and save changes of the analysis method under a new name GeneMapper ID Template Files March 2010 21 Analysis HID 310 50rfu Analysis Method Editor HID xi General Allele Peak Quality Quality Flags Peak Detection Algorithm advanced T Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Partial Range Sizes x B fo R TEN Start Pt 2000 Start Size fo G 50 0 Stop Pt fi 0000 Stop size fi 000 Y so Smoothing and Baselining Min Peak Half VVidth 2 pts Smoothing C None Light Polynomial D
5. 500bp B Panels Biotype Panels BinSets Biotype Bins C Analysis Method Analysis HID 310 Analysis HID 3130 Analysis HID 310 50rfu Analysis HID 3130 50rfu D PlotSettings Plots 2dyes Plots 3dyes Plots 4dyes PlotsBT5_4dyes Plots 5dyes E Table Settings Table for 2 Alleles Table for 10 Alleles Panels and BinSets always need to be executed whereas the use of additional Template Files is facultative GeneMapper ID Template Files March 2010 A Size Standards SST ROX To check the correct assignment of the labels to the sample click the colour panel of the Size Standard red icon in the upper toolbar of the GeneMapper ID Software Thus the red panels DNA Size Standard of all samples are displayed Compare the sample fragments sizes with the sizes of the DNA Size Standard 550 ROX that should be 50 60 70 80 90 100 120 140 160 180 190 200 220 240 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp If sizes differ further analysis should be performed with GeneMapper ID Software If necessary create a new size standard definition within the GeneMapper ID Software Adjustment of the basic template The basic template SST ROX 50 500 bp defines all fragments from 50 bp to 500 bp see figure 5 If for example only 400 bp are necessary for analysis of a particular test kit define fragment length just up to 400 bp The new template could be saved as e g SST ROX 50 400bp and used for further
6. Biotype Template Files tor cenevapper D Software Introduction Biotype Template Files are software sets for the GeneMapper ID Software in order to simplify the evaluation of analysis data Biotype Template Files may be used with ABI PRISM single and multi capillary instruments of Applied Biosystems GeneMapper ID Software with Biotype Template Files assigns the analyzed DNA fragments relative to their length to the allele designation of the STR loci Optionally the corresponding fragment length of peaks in base pairs or peak height in relative fluorescent units RFU can be indicated These data genotypes can be tabulated and exported Biotype Template Files are available for all STR kits from GeneMapper ID software version 3 1 The following documentation shows the analysis of PCR products of the Mentype Nonaplex 6 FAM HEX NED PCR Amplification Kit using ABI PRISM 310 Genetic Analyzer and Biotype s DNA Size Standard 550 ROX consistently Analyses were performed with GeneMapper ID Software version 3 2 1 and Mentype Nonaplex I Template File For general introductions of the software please read the GeneMapper ID software User Guide More info about Human Identification products HID from Applied Biosystems https products appliedbiosystems com ab en US adirect ab Products Human Identification and Forensic DNA Software Validity for HID The Biotype STR Kits require a calibration with an allelic la
7. dder Therefore the software used must be compatible with human identification HID products for forensic applications This Biotype Template Files are valid with GeneMapper ID software Ordering Information Biotype Template Files CD ROM Cat No 49 10900 0000 for Windows Content 1 Setup on the Analysis Computer WMO GI EE 1 2 Panel 1 3 GeneMapper Manager 2 Calibration using Allelic Ladders enne 3 Evaluation of Analysis Data ssseennnnnnnes 4 Template Files for Biotype Test Kits A Size Standards 0 cece B Panes and BOES oce ioo dee SAN AIYSISIMGMNOGS crn ccna Gooch D Plot Settings E Table Settings 5 Check Analysis Data ort eto ee e tete 6 Print Options and Page Setup sse Troubleshooting Guide GeneMapper ID Template Files March 2010 1 Setup on the Analysis Computer Biotype Template Files for GeneMapper ID Software are available as free downloads from our homepage www biotype de or as CD ROM on request Before using the GeneMapper ID Software for the first time Biotype Template Files either have to be saved to the local analysis computer Windows PC or can be imported directly from intranet or CD ROM Panels and BinSets are imported by the Panel Manager Other template fi
8. dder are miscalled the ladder can not be used for the analysis One peak of the allelic ladder is below the Peak Detection Value 50 200 RFU of the analysis method used which is not identified One peak of the allelic ladder is not identified because it is outside the expected size range of the software in bp GeneMapper ID Template Files Comments Setting from the PC has to be edit to English United States First delete all Panels in the Panel Manager then import Panels Bin Sets from the Biotype first Compatible versions of Panels and BinSets have to be saved within one folder Open panel txt data in Windows Explorer and change the penta repeat motive of D10S2325 from 5 to 4 Then import the new panel data again Alternatively install at least version 3 2 of GeneMapper ID Software Install GeneMapper ID Software Select the actual BinSet For Biotype Templates Files Panels and BinSets always use the same version e g v3 Data from different analysers always have to be saved within different run folders Use a different injection file of the allelic ladder and check firstly the data of the analysed sizes from the Size Standard in bp of the allelic ladder Always use the DNA Size Standard 550 for Biotype test kits The allelic ladder has to be load with higher concentration onto the analysis instrument Alternatively data of the allelic ladder can be analysed with a ower Pea
9. e measured allele sizes are transferred automatically to the expected sizes within the project In general calibration bases on one appropriate run of the allelic ladder If more runs will be used calibration is implemented with the means of all allelic ladders The correct assignment of the alleles should be reviewed If not DNA samples should be further runs with an appropriate allelic ladder Calibration using Multi Capillary Analysers To ensure a reliable allelic assignment on multi capillary analysers a number of allelic ladders should be run on different capillaries The room temperature might influence the running performance of PCR products that might result in split peaks especially at low temperatures or an altered run velocity of DNA fragments Please pay attention to keep conditions recommended by the instrument manufacturer System Parameters Different analysis instruments DNA size standards or polymers may result in different fragment lengths Thus DNA samples and allelic ladders from one sample set should be analysed using the same system parameters GeneMapper ID Template Files March 2010 3 Evaluation of Analysis Data Open GeneMapper ID Software and login Import data File Add Samples to Project Select Analysis Data Files fsa Add Samples Add to List Add Select Table Setting via pull down menu at the top of the centre use Table for 2 Alleles for samples with one DNA use Table for 10 Alle
10. egree 3 C Heavy Peak Window Size 11 pts Baseline Window 51 pts Slope Threshold Peak Start Size Calling Method Peak End fo 0 C 2nd Order Least Squares 3rd Order Least Squares C Cubic Spline Interpolation Local Southern Method Global Southern Method Factory Defaults Cancel Fig 15 Tab Peak Detector pre selection 50rfu filter Changing the Filter Value in the Peak Detector tab Peak Amplitude Threshold describes the minimal peak height which can be detected with the GeneMapper ID Software Common values are 50 200 RFU and should be determined individually be the laboratory Recommendation The minimal peak height should be three times higher then the background noise of the baseline GeneMapper ID Template Files March 2010 29 D Plot Settings Plots 2dyes Display of two colour panels B R Plots 3dyes Display of three colour panels B R Plots 4dyes Display of four colour panels B G Y R PlotsBT5 4dyes Display of four colour panels B G Y O Plots 5dyes Display of five colour panels B G Y R O Description To compare samples with the appropriate allelic ladder Shows the peak designation e g allele with the chosen analysis method Designations of alleles can be changed allelic ladders and defined control samples are fixed in the upper part of the window In the lower part DNA samples will be displayed Zoom up the appropriate area
11. eight f 00 0 rHeterozygote balance Min peak height ratio oz Peak morphology Max peak width basepairs 5 Pull up peak Pull up ratio 0 05 number Max expected alleles b Factory Defaults Fig 12 Tab Peak Quality for adjustment of the peak quality parameters All settings here are defaults from Applied Biosystems for HID analysis GeneMapper ID Template Files March 2010 Analysis_HID_310 Analysis Method Editor HID x General Allele Peak Detector Peak Quality Quality weights are between 0 and 1 Quality Flag Settings Spectral Pull up os Control Concordance 0 Broad Peak fos Low Peak Height Out of Bin Allele jos Off scale os Overlap os Peak Height Ratio POY Thresholds PassRange 0 Low Qualty Range Sizing Quality From oss 1010 From 0 0 to 025 Genotype Quality From 75 to1 0 FromO 0to 0 25 Factory Defaults Fig 13 Tab Quality Flags for weighting of the peak quality parameters All settings here are defaults from Applied Biosystems for HID analysis GeneMapper ID Template Files March 2010 20 Sensitive HID analysis method for stains and DNA mixtures Analysis HID 310 50rfu Analysis Method Editor HID E X General Allele Peak Detector Peak Quality Quality Flags Bin Set Biotype Bins v3 v Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Penta Hexa
12. el 3 None Invert mutant labels Label 4 Allele Call Label Color Dye Color Border Area Height Data Point Size Comment Fig 17 Plot Setting Tab Labels for Designation of the Peaks Designation of peaks which have to be displayed in the table is defined in the tab Sizing Table see Figure 18 Select the value which has to be changed or added and save the changing with OK Plot Settings Editor General Sample Header Genotype Header Sizing Table Labels Display Settings Sizing Table Settings E _____ cm Sample File Size 2 fla Fig 18 Plot Setting Tab Sizing Table for Table Display GeneMapper ID Template Files March 2010 24 E Table Settings Table for 2 Alleles For samples with one DNA Table for 10 Alleles For samples with DNA Mixtures Table for 2 Alleles To analyze samples of one DNA e g for a DNA database Displays the allele designation of two peaks of each marker Generates a table where the analysis of each marker STR Locus is displayed in one line e g Nonaplex I all markers x 2 alleles The following columns are displayed in the tab Genotypes Sample Name Marker Allele 1 Allele 2 as well as the quality values AE OS BIN PHR SPU AN GQ PQV explanation in table 2 7 ID v3 2 1 QC_M91 gmid Is Logged In E ioj xi File Edit Analys
13. eted Fig 3 Start Analysis Annotation Use the function Fill Down in order to analyse all samples with the same parameters Select the parameter 0 Nonaplex Panels v2 mark the top column via the pull down menu and press Strg D or do Edit Fill Down GeneMapper ID Template Files March 2010 Project Analysis Select data Edit Select All Analysis Display Plot Select Plot Setting via pull down menu 0 Plot_4dyes for the blue green yellow red panel Scaling of the Analysis Range In order to scale up the analysis range of the test kit g Nonaplex I ca 75 405 bp click the magnifying glass icon atop the horizontal scale in front of the first possible allele and pull it behind the last possible allele To return to basic setting double click onto the scale CI ox File Edit View Tools Alleles Help Plot Setting Plots_4ayes Panes f M E fig iia fer as PE el n Sample Name Pma 22 25 28 oJzs 2e 2 21 24 27 so 23 2 Fig 4 Scaling of the Analysis Range in Sample Plot GeneMapper ID Template Files March 2010 4 Template Files for Biotype Test Kits A SizeStandards SST ROX 50 500bp SST BTO 60
14. in order to simplify allocation see Figure 4 on page 9 Plot Settings are initialised without tables ipi xi File Edt View Tools Aleles Pt Setting Pits_aayes z E Pones E ctn ETT ut d 1118 fier o 2 es o 12 1 16 18 20 17 19 21 23 25 27 29 1 0 12 1 16 18 20 22 24 10 2 13 2 118 2 6 12 14 20 186 41 Y 4038 Peak Data Point 4326 Size 186 78 Height 3754 Allele 20 Bin 20 Marker FGA Fig 16 Plot Window with allelic ladder and DNA Sample AEN GeneMapper ID Template Files March 2010 23 New Peak Designations Labels To change peak designation please perform the following steps Tools GeneMapper Manager Select Plot Setting and edit via double click The following designations can be adjusted in the tab Labels Allele Call Height Size see Figure 17 Select the value which has to be changed or added and save the changing with OK Plot Settings Editor General Sample Header Genotype Header Sizing Table Labels Display Settings Show Labels When opening the Plot Window Label AleleCal v Show data type prefixes Label 2 Show type of edit Lab
15. ing of fig 9 13 GeneMapper ID Template Files March 2010 15 Analysis_HID_310 Analysis Method Editor HID Advanced Peak Detection ABI310 with Windows PC 20 Cutoff Filter max 200 rfu Fig 9 Tab General describes the analysis method GeneMapper ID Template Files March 2010 S46 Analysis_HID_310 Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Bin Set 120 000 Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Penta Hexa Cut off Value b2 h2 bh2 foo Minus amp Ratio foo joo joo joo Minus Distance From to foo o foo Minus Stutter Ratio foo foo lo foo Minus Stutter Distance From pas Bas 25 foo to B favs zs foo Plus Stutter Ratio bo foo foo Plus Stutter Distance From joo Amelogenin Cutoff 25 Range Filter Fig 10 Tab Allele Cut off Value Cut off Value 0 2 corresponds to 20 filter in comparison to the highest peak of a marker With this setting all signals with peak heights less than 20 will not shown in plots and tables Method limitation The methods Analysis_HID_310 and Analysis_HID_3130 contain a 20 filter and are unsuitable for the analysis of DNA mixtures GeneMapper ID Template Files March 2010 Analysis_HID_310 Analysis Method Editor HID Ge
16. is View Tools Help fk cR E ih LLL QR 8 Taie seting rabie tor 2 m Samples Genotypes Sample Marker Alele1 Allele2 SPU AN BD o 0 mmm VEK MS DNAI DSng c 0351358 m mm VEK M81 DNA1 D 5ng c THO1 il M81 D5ng c 5 33 mm VEK M81 DNA1 D 5ng c VA m Im VEK M81 DNA1 DS5ng c Fea VEK_M91_DNA1_0 5ng_c 018551 m C M81 DNA1 0 Sng c 0851179 VEK M84 DNA1 Ong c 021511 Y Analysis Completed Fig 19 Output File after Execution of Table for 2 Alleles In GeneMapper ID all quality criteria are pre adjusted by Applied Biosystems for HID products When a colour signal Check Low Quality appears a PQV was not hold Check the reason of the out range PQV in the table use PQV explanation in table 2 Peak quality assessment via different signals Signal Table Pass green Icon square Check yellow icon triangle Low Quality red Icon stop GeneMapper ID Template Files March 2010 25 Table 2 Process Quality Values PQV PQV Definition AE Allele Edit 0S Off scales signals are outside of the scaling pull up peaks in other colors BIN Out of bin allele allele is outside Bin definition PHR Peak Height Ratio indicates if the peak height ratio between the lowest and highest peak is less than defined in the analysis method SPU Spect
17. is to check Allelic ladder for correct allele calling Samples Plot all xd File Edit view Tools Alleles Help Pt Seng Pes_sayeo XII rones 1 NNN ES paf pops ps 16 1 20 22 24 ejr pep fo 1 2 25 ATE 17 19 21 23 2 0 12 1 16 18 2 22 24 232 10 2 13 2 82 X 422 91 Y 583 Fig 22 Checking Allelic Ladder Controls In order to check allele designations please compare the alleles of the allelic ladder and Control DNA of the test kit with data of the latest user s manual We recommend the quality value AN Allele Number for checking the allelic ladder CC Control Concordance for the Positive Control GeneMapper ID Template Files March 2010 29 Check Positive Control VEK_MD1_DNAL_0 5ng_ Nonaplex I Pands v2 E aj 27 0381388 oT ES 100 200 300 400 abd 0 Uh pran 4 La x 17 17 E12 MDl DNAI 05ng I Ponds v A EWA FGA 100 200 300 400 i t 2000 ih ILU 15 18 20 balia DNAI_0 5ng_ Nonaplex I Pands v 51179 1811 100 200 300 400 4000 d m AL n 27 10 J 4 EN Fig 23 Checking Control DNA Positi
18. k Detection Value from GeneMapper ID Software First compare the length of the ragments in bp of the first allele in one colour of the allelic ladder with the corresponding value in the categories hen of the other alleles arch 2010 Observation Single marker is not identified Many peaks are termed OL as off ladder alleles in the samples Point alleles i e alleles with at least 1 bp difference to the next integer allele g THO1 9 3 10 are not found Homocygote alleles are not displayed as duplicates in the table Trademarks 33 Possible Cause Various causes DNA Size Standard 550 ROX or BTO was not defined or identified correctly Too high intensities at the analysis instrument If the peak heights of the samples are outside the linear detection range gt 4000 RFU ABI310 gt 5000 RFU ABI3130 stutters split peaks and artefacts might be increased Bubbles in the capillary lead to pull up peaks in all colour panels spikes that result in allele misnomer Differences in the capillaries run performances of a multi capillary analyser might result in allelic assignment shift The point alleles were not separated in the GeneMapper ID Software Preferences of the GeneMapper ID Software Comments Open the project folder mark the corresponding sample and check Error Message in the tab Info or PQV Click onto the red icon Size Match Editor in the uppe
19. les like Analysis Methods Table Settings Plot Settings etc are importable to the local PC by the GeneMapper Manager 1 1 Login Open GeneMapper Software and login First Login User Name old Password none Change password Tools Options Users New User Repeat procedure in order to register new users and passwords GeneMapper ID Template Files March 2010 1 2 Panel Manager Use Panel Manager to import Panels and BinSets for Biotype products into the GeneMapper ID software Recommendation For Biotype s Panels and BinSets install a Biotype folder AppliedBiosystems GeneMapper Panels Biotype Content Biotype Panels v3 or current version Biotype Bins v3 or current version Tools Panel Manager File Import Panels Select the folder Panel Manager mark the designated panel version and import File Import BinSet Select the corresponding Panel from the Panel Manager mark the equivalent BinSet version and import see Figure 1 7 Panel Manager File Edit Bins View M CIEE m m cm mm mu E Spare Manager TRE Doeme daoo 2 Biotype_Panels_v3 txt Zuletzt verw amp Desktop ra Eigene Dateien PE Arbeitsplatz Netzwerkum Dateiname Biotype Bins Import Dateityp Alle Dateien z Abbrechen Fig 1 Import of Panels and BinSets Apply OK New Panels and
20. les for DNA mixtures Data Files appear as new project in the Samples tab The table sheet displays the following columns Status Sample Name Sample Type Analysis Method Panel Size Standard Matrix as well as the quality values Q0 SFNF 0 SQ UD1 PQV explanation in table 1 Table 1 Process quality values PQV PQV Definition 500 Sizing Quality Overridden size calling of the sample overridden SFNF Sample File Not Found 0S Off scales signals are outside of the scaling pull up peaks in other colors sa Sizing Quality size calling of the sample or allelic ladder UD1 User Defined Column 1 For more information please check the chapter Process Quality Values of the GeneMapper ID Software User Guide Select Sample Typ via pull down menu 0 Sample Allelic Ladder Positive Control Negative Control etc see Figure 3 One project has to contain at least one allelic ladder Optionally data of the control DNA might serve as positive control data without DNA as negative control GeneMapper ID Template Files March 2010 Select Analysis Method via pull down menu 0 Biotype template Analysis_HID_310 The analysis method is already prepared for evaluation of data from Windows PC Advanced Peak Detection for ABI PRISM 310 instruments There are other methods for ABI PRISM multi capillary instruments like 3130 etc Important Annotation New analysis methods refe
21. llary instruments ABI PRISM single capillary instrument ABI 310 multi capillary instruments ABI 3130 etc Analysis_HID_310 HID analysis method with 20 Filter for samples of one DNA Analysis_HID_3130 HID analysis method with 20 Filter for samples of one DNA Analysis HID 310 50rfu Sensitive HID analysis method for stains and DNA mixtures Analysis HID 3130 50rfu Sensitive HID analysis method for stains and DNA mixtures Note This guide focuses on the HID 310 and HID 310 50rfu examples Analysis HID 310 To analyze samples of one DNA unsuitable for DNA mixtures Label peaks according to filter values used Peaks will be designated with the appropriate allele Filter value pre selection Labels lower then 2096 of the highest peak height of a marker STR locus will not be displayed n plot window Sample Plot the labelled colour panels are displayed Samples Plot laj xj Edt View Tools Help Piot Setting Pots 4dyes 110 rnesk ME e ES faa WA a Fc E SE Fig 8 Sample Plot after Execution of analysis method HID_310 Problems with importing analysis methods To solve import problems please create own analysis methods in your GeneMapper ID software according to Applied Biosystems instructions and fill in the biotype sett
22. lleles alleles of the Control DNA Marker Repeat units of the repeats in bp Marker Specific Stutter Ratio Stutter filter 0 13 corresponds to 13 of the marker s peak height Comments Ladder Alleles alleles of the allelic ladder not shown x Datei Bearbeiten Format Ansicht GenemMapper ID v3 2 1 Last edited 25012010 i GM v3 0 MICROSATELLITE Biotype_Panels_v3 Nonaplex_I_Panels_v2 none blue 86 93 X Y blue 95 148 17 18 blue 149 192 6 9 3 blue 193 389 17 21 green 93 158 15 18 green 159 235 20 26 green 236 379 yellow 115 189 yellow 190 295 HHRHHLHHLEO Fig 6 Structure of a Panel GeneMapper ID Template Files March 2010 BinSets The following features of the allelic designation are depicted Name number of the allele Length of fragments in bp Upper and lower range of tolerance in bp Biotype_Bins_v3 txt Editor jnl x Datei Bearbeiten Format Ansicht 3 2 1 Last edited 25012010 4 GM v3 0 Biotype Panels v3 Biotype Bins v3 Nonaplex I Panels v2 AM 0 5 0 5 D351358 HOL OOOOOOOOO oo 4dRondendununun VON ann 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 THO 0 5 0 5 0 5 0 5 0 4 0 5 0 4 0 5 0 4 00 00 I C on Fig 7 Structure of a BinSet GeneMapper ID Template Files March 2010 C Analysis Methods There different analysis methods for ABI PRISM single and multi capi
23. neral Allele Peak Quality Quality Flags Peak Detection Algorithm advanced ied Peak Detection Ranges Analysis Sizing Partial Range bd All Sizes v Start Pt 2000 Start Size Stop Pt fi 0000 Stop Size 000 Smoothing and Baselining C None Light C Heavy Baseline Window 51 pts Smoothing Size Calling Method C 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method C Global Southern Method Peak Amplitude Thresholds B R G 0 Y 200 Min Peak Half Victth 2 pts Polynomial Degree Peak Window Size 11 pts Slope Threshold Peak Start 0 Peak End foo Factory Defaults OK Fig 11 Tab Peak Detector For an improved peak detection especially for detection of point alleles i e alleles with at least 1 bp difference to the next integer allele the value for the Peak Window Size can be minimized to 11 pts For improved peak detection minimize the Peak Window Size further on Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis GeneMapper ID Template Files March 2010 18 Analysis_HID_310 Analysis Method Editor HID x General Allele Peak Detector Quality Flags r Signal level Homozygous min peak height 200 0 Heterozygous min peak h
24. ngth via Custom GeneMapper ID Template Files March 2010 31 Change Panels and BinSets Panels BinSets data are saved locally on Windows PC within the folder AppliedBiosystems GeneMapper Panels Biotype In order to modify open them with the Windows Explorer via double click see chapter 4B Change date and version of the Panels and BinSets Important Version number of Panels and BinSets have to be identical in one version e g v3 Choose the allele and change its range of tolerance or fragment length File Save as Load the new version of Panels and BinSets in the Panel Manager and delete the old version Annotation New categories can be created in the same way e g for newly found point alleles SE33 20 1 etc The tolerance range of 0 5 bp for all alleles and 0 4 for point alleles respectively should be entered Biotype assumes no liability for changing of Panels and BinSets The actual versions for all test kits can be downloaded from the homepage www biotype de 6 Print Options and Page Setup In Samples Plot the following print options can be chosen via File Page Setup Table to edit i e size or typeface Plot to choose one of four different settings Honor plots per pane to print data and or plots onto one page Small Medium Large to scale the plot size per page GeneMapper ID Template Files March 2010 32 7 Troubleshooting Guide General Help in software Click the me
25. nu Help GeneMapper ID 3 2 1 Help in GeneMapper ID software and search the User Guide for your specific theme Observation Error message during saving data Error message during import Panels and BinSets unable to save panel data java SQLException ORA 00001 unique constraint IFA CKP UK violated Error message during import Panels Invalid marker repeat value in line for marker D10S2325 Valid marker repeat values are 2 3 4 amp 9 Error message during import Analysis Method Plot Setting Table Setting Size Standard Essor message during analysis There are samples that do not meet analysis requirements Please see Error Message in the info view of each sample Injection file of the allelic ladder not appropriate Possible Cause Country setting Path on your computer AppliedBiosystems GeneMapper and check text file Panel Import Log for error message Panels and BinSets are not identified correctly GeneMapper ID v3 1 does not identify penta repeat motives 5 bases like the marker D10S2325 of the Humantype Chimera test kit GeneMapper Software is not GeneMapper ID Software An invalid BinSet is preselected in Analysis Method Data from different analysers like ABI 310 and ABI 3130 have been saved within one run folder An additional signal can be identified as peak of the allelic ladder because of dysfunctions during the electrophoresis If peaks of the allelic la
26. r to the last BinSets used of the GeneMapper ID Software For Biotype products please select the updated Biotype BinSets 0 Biotype_Bins_v3 from the tab Allele see Figure 2 Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Bin Set 0710 0007 Fig 2 Analysis Method tab Allele check here Adjustment of the BinSet Select Panel via pull down menu g Nonaplex Panels v2 Select Size Standard via pull down menu 0 SST ROX_50 400bp for the DNA Size Standard 550 ROX Matrix for PRISM 310 samples is shown not for ABI PRISM multi capillary instruments e g 3130 samples Click onto the green arrow Icon in order to start Analysis name the project and save it If the analysis worked well the icon disappears from the column Status see Figure 3 F GeneMapper ID v3 2 1 QC_M91 gmid Is Logged In ioj x File Edt Analysis Tools Help oS ih LU E Taie setting rabie tor 2 Aretes E IB amp m Serr Samples Genotypes Status Sample Name Sample Type Analysis Method Panel Size Standard Matrix VEK_ALM_M91 Ladder Analysis HD 310 Nonapiex Panels _v2 SST ROX 50 4006p A1 _Pictypet M91 DNA1 0 5ng b Positive Control Analysis HD 310 Nonaplex_ _Panels_v2 SST ROX_50 400bp 1 _Biotypet _DNA1_0 Sng_c Sample Analysis HID 310 Analysis Compl
27. r toolbar or the GeneMapper ID Software Check he red or orange fragments of all samples Always use the internal DNA Size Standard 550 for Biotype test kits Reduce the injection time up to 1 second reduce the amount of the PCR amplification product for analysis or reduce the quantity of DNA for PCR Repeat electrophoresis to confirm results For reliable allelic assignment on multi capillary analysers a number of allelic ladder should be run for further information see chapter Electrophoresis of the test kits manuals Check the settings of the analysis method Lower the Peak Window Size value to 11 pts Adjustment can be changed via Tools Options choose the tab Analysis and the setting Duplicate homozygous alleles so that e g the homocygote allele 18 is displayed as 18 18 6 FAM HEX NED ROX ABI PRISM GeneScan Genotyper GeneMapper and Applied Biosystems are trademarks of Life Technologies Windows is a trademark of Microsoft Corporation GeneMapper ID Template Files March 2010
28. ral Pull Up spectral pull peaks in another colour channel AN Allele Number BD Broad Peaks the allele peak is wider as expected default 1 5 bp CC Control Concordance comparison with an internal control e g Control DNA GQ Genotype Quality quality of the DNA profile or allelic ladder For more information please check the chapter Process Quality Values of the GeneMapper ID Software User Guide GeneMapper ID Template Files March 2010 26 Table for 10 Alleles To analyze stains or mixed samples of different DNAs Displays the allele designation of up to ten peaks of each marker Generates a table where the analysis of each marker STR Locus is displayed in one line g Nonaplex all markers x 10 alleles The following columns are displayed in the tab Genotypes Sample Name Marker Allele 1 Allele 10 as well as the quality values AE 0 BIN PHR SPU GQ PQV explanation in table 2 7 ID v3 2 1 QC_M91 gmid Is Logged In File Edit Analysis View Tools Help e i 8 Marker Dye Allele 1 Allele 2 Allele 3 Allele 4 AM ALM M91 0351358 ALM M81 THO ALM M81 SE33 VEK ALM M81 ALM M91 FGA VEK ALM M81 018551 ALM M81 0851179 ALM M81 021911 VEK M91 DNA1 0 5ng AM M91 DNA1 0 5ng 10351358 M91 DNAT _0 5ng THD1 VEK M31 DNA1 0 50 SE33 VEK M81 DNA1 D
29. ve Control We recommend the following quality value Control Concordance for the Positive Control CC Negative Control The negative control contains no DNA and is a helpful information for background signals of the current analysis conditions GeneMapper ID Template Files March 2010 30 Review Sample Data aloe File Edt View Tools Alleles Help masa XIBN I ENS Ind SER ER E 16 18 20 22 2 4 26 2 elish 17 19 21 23 26 27 29 1 Gonza 23 2 10 2 13 2 AA FGA 18551 Rename Allele gt History Izle Fig 24 Changing the Allele Designation Off Ladder Allele Peaks labelled with OL Off Ladder could not be assigned to an allele size These labels have to be checked manually and may be deleted or redefined by Clicking on Delete Allele In order to change the allele designation of unrealized peaks click onto the OL icon below the peak icon gets bold Open pull down menu by clicking onto the right mouse button and chose Delete Allele Thereafter window Add Allele Comment opens in order to take notes regarding to the changes Rename Allele Inside the pull down menu known alleles of the marker can be chosen from the Bins Predefine a new allele based on its le

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