Biotype ® Template Files for Genotyper
Contents
1. Fig 3 Plot Window after Execution of Macro Label 20 Filter Labelling of all relevant Peaks Peak designations below 20 of the height of the highest peak of the category STR loci are not shown in the macro Label 20 Filter For changing the preselected 20 filter of the macro see Changing Filter Value p 12 Filter options are integrated in order to avoid stutter peaks and template independent nucleotide addition minusA peaks Peaks in the allelic range labelled with allele can not be assigned to an allele size These labels have to be checked manually and may be deleted by clicking on see Troubleshooting on page 18 If necessary scale down the window Genotyper Template Files March 2010 New Peak Designations Labels To change peak designations in the macro please perform the following steps Unlock the Template File File gt Unlock Select the macro which has to be adjusted Label Open step window View Show Step Window cx Genotyper 3 7 Steps Nonaplex _v3c_dmi gta EL File Edit Analysis Category Table views Macro Window Help Macro Label 20 Filter 4 Preselected 20 Fitter i Clear Labels Select blue lanes y Select categories Amelogenin 0391358 THO1 SE33 viark selected categories Select blue lanes Remove labels from peaks whose height is less than 20 of the highest pe2. If different colours of the panels are displayed one below the other the Genotyper Software is not able to indicate categories such as Amelogenin D3S1358 THO1 SE33 any more Genotyper Template Files March 2010 E Horizontal Table for 2 Alleles Ctrl 8 Description To analyze samples of one DNA e g for a DNA database Generates a table after the macro Label has been performed Displays the allele designation of up to two peaks per category STR locus for each sample in one line For genetical homozygote peaks the allele designation will be displayed twice with exception of X and Y chromosomal test kits To discriminate between individual samples the field Sample Info of each sample colour has to be filled in with an appropriate description Views Show Dye Lanes Window Use Table Update Table to take over the current allele designation of the peaks to the table Misnomers resulting from pull up peaks between different panels can be deleted manually by clicking onto E Genotyp ab onaple D 310 9ta IES A File Edit Analysis Category Table Views Macro Window Help la x Sample Info Amelogenin 1 Amelogenin 2 D3S1358 1 D351358 2 THO1 1 THO1 2 SE33 1 SE33 2 vWA 1 DNAL x Y 17 18 6 9 3 17 21 2 DNA318 x 15 18 19 30 2 DNA319 14 17 3 19 30 2 DNA320 E 14 15 19 29 2 DNA321 15 17 23 2 26 2 DNA322 16 18 22 2 27 2 Fig 10 Output File after Execution of the Macro Horizontal Table for 2
3. Possible Cause The allelic ladder file was not loaded The allelic ladder file was not identified Not all four dye colours blue green yellow red have been imported Bubbles in the capillary lead to pull up peaks in all colour panels spikes that result in allele misnomer 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 The DNA size standard 550 ROX or BTO was not defined or was identified incorrectly Data from different analysers like ABI 310 and ABI 3130 have been saved within one gta file The point alleles were not separated in the GeneScan analysis Mentype is a trademark of Biotype Diagnostic GmbH 6 FAM HEX NED ROX ABI PRISM GeneScan Genotyper and Applied Biosystems are trademarks of Life Technologies Macintosh is a trademark of Apple Computers Inc Windows and Excel are trademarks of Microsoft Corporation Genotyper Template Files Comments Import the file with the allelic ladder For every colour of the allelic ladder the term ladder has to be listed in Views Show Dye lanes Window in the text field Sample Info Choose all dye colours via Edit gt Set Preferences Import Colours and import them once again Repeat electrophoresis to confirm results Reduce the
4. at the left top of the main window The red panels of all samples in the plot window will be displayed Now compare the sizes of the sample fragments 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 a further analysis should be performed with GeneScan Software If necessary create a new size standard definition using GeneScan Software T T T T T T T T T T T T T T T T T T T T T T T T T B0 g0 100 120 140 160 180 200 220 0 260 280 300 320 0 360 30 400 40 440 40 480 500 52 540 DNA Size Standard 550 fsa 17 Red poo ano L200 30 00 20 00 140 00 180 00 220 00 250 00 3001 00 3401 00 3801 00 425 00 460 00 475 00 500 00 528 00 i Booo 80 00 16000 t9000 240 00 280 00 320 00 250 00 400 00 550 00 7o00 100 00 200 00 720 00 Fig 1 Electropherogram of the DNA size standard 550 ROX Lengths of Fragments in bp 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 icon Q is necessary Please use DNA Size Sta
5. Alleles Table Export To export the table to Microsoft Excel please perform the following steps Table Export to File save with extension xls Open Excel software and load table open Excel software load table and follow the conversion procedure up to step 3 Step 3 Click Button gt Advanced Decimal separator gt dot Thousands separator gt space Genotyper Template Files March 2010 F Vertical Table for 10 Alleles Ctrl 9 Description To analyze stains or mixed samples of different DNAs Generates a table after the macro Label has been performed Shows the allele designation of up to ten alleles in each category STR locus and generates a vertical table The categories are listed in one line e g Nonaplex I all categories x 10 alleles In Sample Info the File Name name of the sample is displayed automatically For genetical homozygote peaks of a category the allele designation will be displayed twice with exception of X and Y chromosomal test kits Use Table Update Table to take over the current allele designation of the peaks to the table Misnomers resulting from pull up peaks between different panels can be deleted manually by clicking onto E Genotyper 3 7 Table MT Nonaplex I NED 310 4 J l8 x EID File Edt Analysis Category Table Views Macro Window Hel
6. by the user itself Alternatively the macro Label 10 Filter can be used Genotyper Template Files March 2010 c Label 10 Filter Ctrl 4 Description To analyze stains or mixed samples of different DNAs e g for forensic case work Peaks will be labelled with the appropriate allele A plot window will open automatically with the following entries DNA samples allelic ladder DNA size standard ROX if imported Genotyper 3 7 Plots Nonaplex _v3b_Genotyper_Abb gta GiB Ele Edt Analysis Category Table Views Macro Window Help 8x T T T T T T rr T T 7 T rT T 80 too 0 o mo io 200 O20 O20 280 280 00 320 3o 30o 38o 040722_Gednap28_Spur fsa 30 Blue 2o00 E Hooo fe E OENES Kl 13 fe A 17 20 30 2 i 1 040722_Gednap28_Spur2 fsa 30 Green 040722_Gednap28_Spur2 fsa 30 Yellow 11 13 e 14 EJ 040722_Gednap28_Spur2 fsa 30 Red 1600 1000 600 60 00 100 00 120 00 140 00 160 00 180 00 200 00 22000 240 00 Booo 220 00 40 00 60 00 260 00 20 00 190 00 260 00 280 00 Fi
7. injection time up to 1 second reduce the amount of the PCR amplification product for analysis or reduce the quantity of DNA for PCR Click on icon R for ROX or 0 for BTO in the left upper corner of the Genotyper software and display the red or orange fragments of all samples in the Plot Window Data from different analysers always have to be saved within different gta files Check the settings of the GeneScan analysis parameters Change the value of the Peak Window Size to 11 pts and save Then analyse the DNA samples with the new parameters again March 2010
8. Biotype Template Files tor cenotyper software Introduction Biotype Template Files are software sets macros for the Genotyper Software in order to simplify the analysis of GeneScan data Biotype Template Files may be used with ABI PRISM single and multi capillary instruments of Applied Biosystems Genotyper Software with Biotype Template Files assign the analyzed DNA fragments relative to their length to the allele designation of the STR loci Optionally the corresponding fragments 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 Genotyper Version 2 5 Macintosh From Genotyper Version 3 7 Windows Note Both Genotyper and GeneScan Software applications from Applied Biosystems are scheduled for discontinuation and will be unavailable for purchase as of June 1 2004 Applied Biosystems have provided support ending June 1 2009 The following documentation shows the analysis of PCR products of the Mentype Nonaplex 6 FAM HEX NED PCR Amplification Kit using the ABI PRISM 310 Genetic Analyzer and Biotype s DNA size standard 550 ROX consistently Analyses were performed with Genotyper Software version 3 7 for Windows and the Mentype Nonaplex I Template File For general introductions of the software please read the Genotyper S
9. ak in a category s range Remove labels from peaks followed by a 666 higher labeled peak within 0 00 to 5 00 bp Find all ladder in sample info in blue dyeAanes Clear common labels within tolerance 0 50 bp Label category peaks with the category s name Remove labels from peaks whose height is less than 20 of the highest peak in a category s range Select all categories Unmark selected categories 4 Fig 4 Step Window of Macro Label 20 Filter Select the step you want to adjust by clicking on the appropriate line in the step window e g Label category peaks Figure 4 Edit this step Macro Edit Step Genotyper Template Files March 2010 Click the desired peak designation e g size in bp peak height peak area in the appearing window and fix the adjustment with Replace Label peaks within marked categories with IV the size in bp T rounded to integer J divided by scale factor the peak area I divided by scale factor the scan number Dtetet IV the category s name I label peak source the peak modulation score Coreei Fig 5 Window in the Step Window to edit Peak Designations Repeat the same procedure with all categories you want to adjust After performing the changes lock the Template File File gt Lock and save it File Save as Note The Macro Label 20 filter should not be used for the analysis of DNA mixtures New filter options has to be defined
10. ategory THOL 7 F i r 80 100 120 140 180 180 200 220 240 260 280 300 320 340 380 380 lA 0351358 THOI SE 7 NP1_Ch 05 09 1_End n fsa 6 Blue ladder IU AU AN UNNA E 1 Ezlie 25 272 31 2 34 6 18 222 28 2 33 13 3 7 3 10 19 10 va ial eal 2 29 2 36 17 12 19 2 24 2 30 2 36 2 15 2 20 25 2 32 38 20 2 28 2 32 2 21 2 33 33 2 m 03 1368 THOT SEN S 91_Gednap 28_Spur 3 tsa 9 Blue soo 600 ia LI U0 AT ge El 15 3 H 17 273 I Y E Fig 9 Plot Window after Execution of the Macro Blue Panels with allelic ladder and DNA Sample Show Categories STR Loci To display all categories STR loci in the panel of one colour set a tick at the check box Category boundaries Views Plot Options Plot Window Upper Panel and Plot Window Lower Panel In Plot Window Upper Panel the allelic ladder fixes at the top of the window In Plot Window Lower Panel the DNA samples are shown at the bottom of the window
11. bility for changing of Template Files The actual versions for all test kits can be downloaded from the homepage www biotype de Genotyper Template Files March 2010 6 Troubleshooting Guide Observation Error message during program start Could not start the application because there is not enough memory The template file cannot be opened Error message during analysis Could not complete your request because the labeled peak could not be found Possible Cause Both Genotyper and GeneScan Software applications have been scheduled for discontinuance and will be unavailable for purchase as of June 1st 2004 Applied Biosystems stopped support for these software applications in June 2009 The Genotyper Software has not the Macintosh version 2 5 or not the Windows version Kae Genotyper Software was not installed on the local computer Template Files or the CD ROM were damaged An additional signal can be identified as peak of the allelic ladder because of dysfunctions during the electrophoresis If peaks of the allelic ladder are miscalled the ladder can not be used for the analysis One peak of the allelic ladder is smaller than the peak detection value 50 200 RFU of the GeneScan Analysis parameter used and is not identified as a peak One peak of the allelic ladder was not identified because the size is not in the range in bp expected by the software Deviant fragme
12. ed detection of categories STR Loci such as Amelogenin D3S1358 THO1 SE33 select the step Remove Labels by clicking on the appropriate line in the step window see Figure 7 Edit this step Macro Edit Step ES Filter Labels I Remove labels from peaks in the size range 0 00 to 100 00 MV Remove labels from peaks whose height is less than of the highest peak in a category s range I Remove labels from peaks preceeded by higher labeled peak within 0 00 to 1 60 bp Higher by at least 5 I Remove labels from peaks followed by higher labeled peak within 0 00 to 4 50 bp V Higher by at least 666 Cancel Replace Fig 8 Window in the Step Window to edit Filter Values In Filter Labels fill in the new value for lower threshold of the filter see black shaded field in Figure 8 Fix the adjustment with Replace lock the Template File File gt Lock and save it File gt Save as Genotyper Template Files March 2010 D Blue Green Yellow Panels Ctrl 5 6 7 Description To compare samples with the appropriate allelic ladder Shows the labels generated with macro Label see chapter B C To simplify allocation zoom up the appropriate area Views Zoom Zoom To e g 150 to 190 bp Genotyper 3 7 Plots Nonaplex _v3b_Genotyper_Abb gta Eek M File Edit Analysis Category Table Views Macro Window Help ox peak Scan 4106 Size 159 00 Height 1009 rea 6677 C
13. er Software will open automatically Import data File gt Import From GeneScan File Select GeneScan Analysis Data Files Add Sample Files gt Add Finish Data Files appear at the top of the window To import choose dye colours of the Biotype test kit and the red colour for the DNA size standard e g B Y G R Edit Set Preferences Import Colours To run macro mark the appropriate macro or double click the macro bottom left of the main window Macro gt Run Macro Note Before analysing data always run macro Calibrate Ladder first see chapter 4A General Procedure for Analysis 1 Check Size Standard 2 Check Allelic Ladder 3 Check Positive Control 4 Check Negative Control 5 Review Sample Data Genotyper Template Files March 2010 4 Macros for Biotype PCR Amplification Kits E F G Calibrate Ladder Label 20 Filter Label 20 Filter incl bp Label 20 Filter incl peak height Label 10 Filter Blue Panels Green Panels Yellow Panels Horizontal Table for 2 Alleles Vertical Table for 10 Alleles Calculate Category Offsets ANHNNNANANNWNNA Macro Calibrate Ladder always needs to be execu macros is facultative Genotyper Template Files rg 0 rg 1 rg 2 rg 3 rg 4 rg 5 rg 6 rg 7 rg 8 rg 9 ted whereas the use of additional March 2010 Size Standard SST ROX To check the correct assignment of the labels to the sample click the icon R
14. g 6 Plot Window after Execution of Macro Label 10 Filter Basic Setting for improved Peak Detection In the macro Label 10 Filter all peak designations higher than 10 of peak height of the highest peak in this category are labelled to achieve an improved peak detection preselected 10 filter Filter options are already integrated in the macros in order to avoid stutter peaks and template independent nucleotide addition minusA peaks Genotyper Template Files March 2010 Nas Changing Filter Value To change the lower threshold for the peak detection preselection filter 20 in the Genotyper macro please perform the following steps Unlock the Template File File gt Unlock Select the macro which has to be adjusted Label Open the step window Views Show Step Window Genotyper 3 7 Steps Nonaplex _v3c_dmi gta BL File Edit Analysis Category Table Yiews Macro Window Help htacro Label 20 Filter Preselected 20 Filter Wi Clear Labels Select blue lanes iy Select categories Amelogenin 03 1358 THO1 E33 viark selected categories PEPA comes Y Threshold Peak Detection Filter Value 20 Label category peaks with the category s name Remove labels from peaks whose height is less than 20 of the highest peak in a cate Remove labels from peaks followed by a 666 higher labeled peak within 0 00 to 5 00 bp Fig 7 Step Window of Macro Label 20 Filter For improv
15. ncl peak height Ctrl 1 2 3 Description To analyze samples of one DNA unsuitable for DNA mixtures Macro Label 20 Filter Peaks are indicated with their allele designation Macro Label 20 Filter incl bp Peaks are indicated with their allele designation and their sizes in base pairs Macro Label 20 Filter incl peak height Peaks are indicated with their allele designation and their peak height in relative fluorescent units RFU A plot window will open up automatically with the following entries DNA samples allelic ladder DNA size standard ROX if imported Genotyper 3 7 Plots Nonaplex _v3x_05 08 1_Abb gta M File Edit Analysis Category Table Views Macro Window Help 0X T T T T T T T T T T T T 80 100 120 140 160 180 200 220 240 260 280 300 920 340 360 380 EK2_MB1_DNAI_0 5ng_a tsa 27 Blue t b EEEN il A A e i Gta ki 13 EK2_M91_DNAI_0 5ng_a fsa 27 Green 13 1a 28 12 14 EK2_M91_DNAI_0 5ng_a fsa 27 Yellow AI 27 ig 28 EK M91_DNAI_0 5ng_a fsa 27 Red Ll ewe elle le ole wl ale oe obrl 0 00 100 00 120 00 140 00 160 00 180 00 200 00 220 00 260 00 280 00 800 00 320 00 340 00 380 00 880 00 20 00 190 00 240 00 X
16. ndard 550 BTO template SST BTO_60 500bp to define fragment length for particular test kit Genotyper Template Files March 2010 A Calibrate Ladder Ctrl 0 Description To calibrate the Template File with a current allelic ladder If Template File will be saved the generated calibration may be used for DNA sample data based on this allelic ladder After calibration the macro Label 20 Filter will run automatically Identification of the Allelic Ladder All colours of the imported allelic ladder have to be defined as ladder in the Dye Lanes window Views Show Dye Lanes Window Therefore add the term ladder to every colour in the text field Sample Info Genotyper 3 7 Dye lanes Nonaplex _v3c_dmi gta BL File Edit Analysis Category Table views Macro Window Help Sample Info Sample Comment ALM _M91_05 09 1 fsa 30 Blue ladder ALM_M91_05 09 1 fsa 30 Green ladder ALM_M91_05 09 1 fsa 30 Yellow ladder ALM_M91_05 09 1 fsa 30 Red ladder Fig 2 Add the Term ladder to every Colour in the Text Field Sample Info Note Peaks in the allelic range which are not identified by the GeneScan Software can not be labelled by the GeneScan Software 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 Genotyper Template Files March 2010 B Label 20 Filter incl bp i
17. nt lengths because of unsuitable size standard Genotyper Template Files Comments Use appropriate windows computer Maybe a computer with less than 1 GB memory works with Genotyper software Upgrade the Genotyper Software The old Macintosh Version 2 1 can be updated to Version 2 5 by free download rom the homepage www appliedbiosystems com nstall Genotyper Software on he local Computer Download the files from the homepage www biotype de again or order the CD ROM again Use a different injection of the allelic ladder The analysed ragments of the internal size standard in bp of the allelic adder file should be examined irst A larger quantity of the allelic adder has to be loaded again to he instrument Alternatively the allelic ladder ile can be analysed again with a smaller peak detection value in the GeneScan Software First compare the length of ragments in bp of the first allele in one colour of the allelic adder with the corresponding value in the categories then of he other alleles Always use the internal DNA size standard 550 for Biotype est kits March 2010 Observation Error message during analysis Could not complete your request because no dye lanes are selected There are many peaks of the sample termed allele Point alleles i e alleles with at least 1 bp difference to the next integer allele e g THO1 9 3 10 are not found Trademarks
18. oftware User s Manual Ordering Information Biotype Template Files CD ROM Cat No 49 10900 0000 for Macintosh and Windows Contents 1 Setup on the Analysis COMpPUtEL ceccssssccssesssssessesseesesseesssssessreseenss 2 Calibration using Allelic Ladders cccccccsccsescscseececsseecssssessssessseeseeas 3 Evaluation of Analysis Data ccsccsssccescseecsseeceeees 4 Macros for Biotype PCR Amplification Kits Calibrate Ladder Ctrl O cccccccsssccsssscsecsssscsssecsssseessseesees Label 20 Filter incl bp incl peak height Ctrl 1 2 3 ccceeeees 8 Label 10 Filter Ctrl 4 Blue Green Yellow Panels Ctrl 5 6 7 Horizontal Table for 2 Alleles Ctrl 8 Vertical Table for 10 Alleles Ctrl 9 Calculate Category Offsets cccccsssesseteseneeees Adjustment of Categories 6 TrOUBIESHOOTING Guide Ss ssiessccooesneerh ssc sear arte uv eee eh io QnNNI OWL oO Genotyper Template Files March 2010 1 Setup on the Analysis Computer Biotype Template Files for Genotyper Software are available as free downloads from our homepage www biotype de or as CD ROM on request Before using the Genotyper Software for the first time Biotype Template Files have to be saved inside a folder to the local analysis computer Windows or Macintosh e g for Mentype Nonaplex Mentype_Nonaplex _v3c or current version For Macintosh computers please use the CD ROM in order to avoid n
19. on conformities during conversion 2 Calibration using Allelic Ladders Workings with the Genotyper Software are done with related analysis data i e analyzed DNA samples with allelic ladders as gta file To analyse DNA samples with the Genotyper Software calibration with a current run of the allelic ladder has to be done The allelic ladder of the used test kit should be analysed at the beginning and the end of the DNA samples For calibration the measured allele sizes are transferred automatically to the expected sizes within the categories of the Template File In general calibration bases on one current run of the 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 might result in different fragment lengths Thus DNA samples and allelic ladders from one sample set should be analysed using the same system parameters Genotyper Template Files March 2010 3 Evaluation of Analysis Data Open Template File of the Biotype test kit Genotyp
20. p 18 xl File Name Sample Info Category Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 5 N91_Spurl fsa Spurl Anelogenin X Y N91_Spurl fsa Spuri D351358 14 17 Es M91_Spurl fsa Spurl THOL 6 8 9 3 E M91_Spurl fsa Spurl SE33 24 2 22 2 28 2 iS M91_Spurl fsa Spurl vA 15 18 20 epi M91_Spurl fsa Spur1 FGA 19 21 22 N91_Spurl fsa Spuri D18551 14 15 16 17 M91_Spurl fsa Spurl D8S1179 10 13 14 M91_Spurl fsa Spurl D21511 28 29 32 2 Fig 11 Output File after Execution of the Macro Vertical Table for 10 Alleles G Calculate Category Offsets Macros for calibration of individual categories They are used to Calibrate Ladder and should not be implemented separately Genotyper Template Files March 2010 5 Adjustment of Categories Open Template File of the Biotype test kit Genotyper Software will open automatically Unlock the Template File See lock symbol at the lower left sidebar File Unlock Open categories Views Show Categories Select allele to be adjusted from table Category Edit Category Change allele features tolerance range fragment length Replace File Save Annotation For newly found intermediate alleles additional categories can be created in the same way Here a tolerance range of 0 45 bp should be chosen However these added categories are not evaluated by the automatic calibration of the macro Calibrate Ladder Biotype Diagnostic GmbH assumes no lia
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