Devyser QF-PCR. Guide to Sample Runs, Data Analysis & Results Interpretation

Transcription

1 Devyser QF-PCR Guide to Sample Runs, Data Analysis & Results Interpretation Version

2 Contents 1. Setting up a sample run on an ABI Genetic Analyzer Introduction Workflow Create a Run Module Create an Instrument Protocol Set up a Plate for run GeneMapper Analysis Introduction Analysis Workflow Importing Panels and Bin Set in the Panel Manager Deleting old Panel and Bin Settings in the Panel Manager Importing new Panel and Bin Settings in the Panel Manager Importing GeneMapper Analysis Settings in GeneMapper Manager Deleting old Analysis Settings in the Panel Manager Importing new Analysis Settings in the Panel Manager Performing Data Analysis ABI 3500 Normalization Reviewing QF-PCR data Failed analysis due to low sizing quality Using the GeneMapper report format (only in GeneMapper versions ) Modifying the Analysis Method Setting Missing allele calls - Analysis Method and Bin Set connection lost Results Interpretation Normal Male (46, XY) Normal Female (46, XX) Chromosome Trisomy Patau Syndrome, Trisomy Edwards Syndrome, Trisomy Down Syndrome, Trisomy Turner Syndrome (45, X) Klinefelter Syndrome (47, XXY) , XYY Syndrome Triple X Syndrome (47, XXX) Triploidy Version Page 2 of 43

3 1. Setting up a sample run on an ABI Genetic Analyzer 1.1 Introduction Before starting the electrophoresis for fragment analysis on the ABI Genetic Analyzer the following settings need to be set up in the instrument s Data Collection Software; Run Module, Instrument Protocol and Plate. The instructions below are from an ABI 3130 Genetic Analyzer with Devyser Compact v3 as an example (Dye set: Any5Dye, DEV-5). The procedure is however similar to the other instruments. For further details, refer to the User Guide for the instrument used. 1.2 Workflow 1.2 Create a Run Module 1.3 Create an Instrument Protocol 1.4 Set up a Plate for Run Version Page 3 of 43

4 1.3 Create a Run Module 1. In the left navigation window select Module Manager and New. Version Page 4 of 43

5 1. Fill out the Run Module Editor according to the kit instructions for use (IFU) a. Name: Enter a name of the Run Module b. Type: Regular c. Template: FragmentAnalysis36_POP7 (default template for the capillary array and polymer used) d. Specific parameters (e.g. Injection Voltage, Injection Time and Run Time) for each Devyser product can be found in the product s IFU) e. Click OK Note! In case peak signals are too high or too low the injection time/voltage can be decreased or increased by creating an additional Run Module and an Instrument protocol. The increase/decrease in injection time/voltage is typically proportional to the measured signal intensity (peak area and peak height). Increasing the injection time >30 seconds might however decrease the peak resolution. A too high injection voltage can also cause sloping and broadening of longer length fragments. Version Page 5 of 43

6 1.4 Create an Instrument Protocol 1. From the left navigation window select Protocol Manager and New. Version Page 6 of 43

7 2. Fill out the Protocol Editor a. Name: Enter a name of the Run Module b. Type: Regular c. Run Module: Select the Run Module created in step 1.3: POP7_36cm_1.5kV_20s d. Dye Set: Any5Dye e. Click OK Note! Consult the Instructions for Use (IFU) to select the correct Dye Set for each Devyser product. Version Page 7 of 43

8 1.5 Set up a Plate for run 1. From the left navigation window select Plate Manager and New Version Page 8 of 43

9 2. Fill out the New Plate Dialog a. Name: Enter a name of the plate b. Application: GeneMapper-Generic (used if data is analyzed on a separate computer) c. Plate type: 96-Well d. Owner Name: enter the name of the owner e. Operator Name: enter the name of the operator f. Click OK Version Page 9 of 43

10 3. Fill out the GeneMapper Plate Editor a. Sample name: Enter the sample names b. Comment: optional c. Result Group 1: location where the raw data files (.fsa files) will be saved d. Instrument Protocol 1: Select the instrument protocol that you created in step 1.4 e. Click OK Version Page 10 of 43

11 4. From the left navigation window, select Run Scheduler, select Find All. Version Page 11 of 43

12 5. Select the plate created in Step 3 (status pending). Link the plate by clicking on the yellow plate position indicator, which will turn green when linked. Start the run on the green arrow. 6. The Process Plates dialog box appears. Click OK to start processing the plate. Version Page 12 of 43

13 2. GeneMapper Analysis 2.1 Introduction Before importing the raw data generated by capillary electrophoresis it is necessary to import all the settings needed for analysis into GeneMapper. GeneMapper settings for the Devyser products are available for download from the Devyser website at To ensure that the latest versions of the GeneMapper settings are used, always download the files from the website. 2.2 Analysis Workflow Amplified samples are detected on an ABI Genetic Analyzer The latest version of GeneMapper settings for kits and size marker are downloaded from the Devyser website and imported into GeneMapper Sample raw data files (.fsa) are imported into GeneMapper Data analysis of the.fsa files is performed using the imported GeneMapper settings Data is reviewed and interpreted in the electrophoretic trace and in the GeneMapper Report Version Page 13 of 43

14 2.3 Importing Panels and Bin Set in the Panel Manager GeneMapper settings for individual kits and size marker may be downloaded from If previous versions of Panels and Bin Set have already been imported in GeneMapper, delete these before importing the latest version by following section 2.3.1, steps 1-3. If previous versions do not exist in GeneMapper, go directly to section 2.3.2, steps Deleting old Panel and Bin Settings in the Panel Manager 1. Delete previous versions of Panels and Bins in the Panel Manager by selecting Tools and Panel Manager in the GeneMapper main menu or by clicking on the icon. or 2. Highlight the kit to be deleted on the left navigation window. The selected kit will now appear highlighted in blue. 3. Select Edit and Clear Kit. Both the Panels and the Bin Set will now be deleted from the Panel Manager. Version Page 14 of 43

15 2.3.2 Importing new Panel and Bin Settings in the Panel Manager 1. Open the Panel Manager by selecting Tools and Panel Manager in the GeneMapper main menu or by clicking on the icon. or 2. Import panels by clicking on the Panel Manager on the left navigation window. Panel Manager will now appear highlighted in blue. 3. Select File and Import Panels. Version Page 15 of 43

16 4. Navigate to and select the appropriate (kit specific) Panel file (.txt file) on your computer. Click Import. 5. The imported panel will now be displayed in left navigation window. 6. Add a Bin Set to the imported panel by highlighting the panel in the in left navigation window. 7. Select File and Import Bin Set. Version Page 16 of 43

17 8. Navigate to and select the appropriate (kit specific) Bin Set file (.txt file) on your computer. Click Import. 9. The imported Bin Set will appear in the Bin Set drop-down list. Click Apply and OK to confirm the imported panel and bin settings. Version Page 17 of 43

18 2.4 Importing GeneMapper Analysis Settings in the GeneMapper Manager The following settings are important tools for data analysis in GeneMapper; Analysis Methods, Plot Settings, Table Settings, Report Settings and Size Standards. These settings are imported through the GeneMapper Manager tool. If previous versions of these settings have already been imported in GeneMapper, delete these before importing the latest version by following section 2.4.1, steps 1-4. If previous versions do not exist in GeneMapper, go directly to section 2.4.2, step Deleting old Analysis Settings in the GeneMapper Manager 1. Delete previous versions of any of the above mentioned GeneMapper settings by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking on the icon. or 2. Select the corresponding tab for the setting to be deleted (in this example Analysis Method). 3. Highlight the setting to be deleted and select Delete and Done. The deleted setting will disappear from the Analysis Methods tab. 4. Repeat this procedure for any other settings to be deleted. Version Page 18 of 43

19 2.4.2 Importing new Analysis Settings in the GeneMapper Manager 1. Open the GeneMapper Manager by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking on the icon. or 2. In the GeneMapper Manager select the Analysis Methods tab and then click Import. 3. Navigate to and select the appropriate (kit specific) Analysis Methods file (.xml file) on your computer. Click Import. Version Page 19 of 43

20 4. The imported Analysis Method will now be listed in the Analysis Methods tab. 5. Repeat the process, selecting the appropriate tab and importing the corresponding file for; Plot Settings Table Settings Report Settings Size Standards (560 SIZER ORANGE). Downloaded separately from the website Note: The Table Settings and Report Settings are imported in order to generate a GeneMapper Report in the data analysis. This is applicable in GeneMapper versions only (see section 2.9 on page 28-29). 6. Select Done to confirm the imported analysis settings. Note: A matrix file (.mtx file) needs to be generated or imported in the Matrices tab when analyzing raw data generated from capillary electrophoresis using the ABI 310 Genetic Analyzer. Instructions on how to generate a matrix file can be found in the Devyser instruction DEV-5 Matrix file generation for ABI 310 that is available for download from the website Version Page 20 of 43

21 2.5 Performing Data Analysis 1. Import the raw data files (.fsa files) from the capillary electrophoresis by selecting File and Add Samples to Project or by clicking on the icon. or 2. Navigate to the desired run folder using the folder tree on the left navigation window. Double click or use the plus symbol to display all files contained in the folder. Highlight the complete folder or selected samples and click on Add to List. 3. The added run folder will now appear in the Samples To Add window. Add the samples by clicking on Add. Version Page 21 of 43

22 4. Set the appropriate analysis settings (Analysis Method, Panel and Size Standard) for each sample, alternatively fill down the columns by selecting the settings for one sample, highlighting the desired number of samples and typing Ctrl D keys to copy the settings. Note: Matrix has to be set when analyzing.fsa files from an ABI 310 Genetic Analyzer. 5. Initiate the data analysis by selecting Analysis and Analyze or by clicking on the icon. Assign a project name when prompted. or Version Page 22 of 43

23 2.6 ABI 3500 Normalization 1. When analyzing raw data files from an ABI 3500 Genetic Analyzer, a GeneMapper Alert will show. Click OK to continue. 2. To stop this message from appearing, open the GeneMapper Manager by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking on the icon. or 3. In the GeneMapper Manager select the Analysis Methods tab. Highlight the desired Analysis Method and then click Open. Version Page 23 of 43

24 4. Select the Peak Detector tab and Advanced in the Peak Detection Algorithm drop-down menu. 5. Deselect the Enable Normalization function and then click OK. This action will prevent the GeneMapper Alert from appearing each time samples are analyzed. Version Page 24 of 43

25 2.7 Reviewing QF-PCR data 1. Highlight the sample row for the sample(s) to be selected for analysis in the GeneMapper main window. 2. Display the sample plot by selecting Analysis and Display Plots or by clicking on the icon. or 3. In the Samples Plot window, select the appropriate Plot Setting from the drop-down menu. 4. To add or remove information shown in the peak labels, open the GeneMapper Manager by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking on the icon. or 5. In the GeneMapper Manager select the Plot Settings tab. Highlight the desired Plot Setting and then click Open. Version Page 25 of 43

26 6. Select the Labels tab. In the Show Labels box, select the information to be shown in the labels by using the drop down lists in the Label 1 to Label 4 options. To confirm the modified label format, click OK and Done. Changes in the Plot Settings will be directly applied in the Samples Plot without re-analyzing the samples. 2.8 Failed analysis due to low sizing quality Samples cannot be displayed in the plot if the sizing quality is low. This is indicated by the symbol. Low sizing quality might be caused by insufficient peak height of the size standard fragments or incorrect identification of the fragments by the software due to sample overloading, spectral overlap or pre-terminated runs.. The size standard can be corrected manually in the Size Match Editor. 1. Highlight the sample with the low sizing quality. Select Analysis and Size Match Editor or click on the icon. or 2. The expected size standard pattern for Devyser 560 SIZER ORANGE is shown below. Version Page 26 of 43

27 3. When sizing quality is low, the 560 SIZER ORANGE pattern might not appear as expected, see example below in which multiple size standard fragment have not been correctly identified. 4. Click on the icon to erase all peak sizes or highlight each individual marker to be deleted or changed. 5. By right clicking on a peak, three options will be displayed in a box menu. By selecting Add the peak is assigned a peak size. Add peak sizes as expected for the size marker (e.g 560 SIZER ORANGE, see section 2.8, page 26). Select Delete in order to delete peaks that are not included in the size marker (e.g primer peaks or crosstalk peaks) since these will shift the size pattern. By selecting Change, a peak size can be changed to another predefined size. 6. When the sizes have been corrected according to the expected pattern for the size marker used, select Apply and OK (see step 3). Version Page 27 of 43

28 2.9 Using the GeneMapper report format (only in GeneMapper versions ) The Devyser Aneuploidy Report Setting can be used to facilitate the calculation of peak area ratios for the markers included in the analysis. The Devyser Aneuploidy Report Setting is included in all the product settings folders that are available for download from the Devyser website at Use the Devyser Aneuploidy Table Setting with the corresponding Devyser Aneuploidy Report Setting. Report and Table Settings are imported in GeneMapper through the GeneMapper Manager tool according to section 2.4 on page Select the appropriate Table Setting from the Table Setting drop-down menu in the GeneMapper main window. 2. Generate a report by highlighting the Sample file(s) to be analyzed and then selecting Analysis and Report Manager or by clicking on the Report Manager icon. or 3. In the Report Manager window select the appropriate Report Setting from the Report Setting drop-down menu. Version Page 28 of 43

29 4. To review the report data in a samples plot: Highlight a marker to be reviewed and then click on the icon. 5. To edit data in the Samples Plot window: right click on the marker and select the appropriate function in the menu box that appears. Any changes made will appear immediately in the Report. Kits used with the Devyser Aneuploidy report format Devyser Compact v3, Devyser Compact Devyser Complete v2, Devyser Complete Devyser Resolution Devyser Extend Kits used with the Devyser AZF report format Devyser AZF Version Page 29 of 43

30 2.10 Modifying the Analysis Method Setting Depending on the ABI Genetic Analyzer used, it might be necessary to modify the Minimum Peak Height in the Analysis Method. In the Analysis Method provided for each kit, the default Minimum Peak Height is applicable for instruments having a lower detection scale, i.e ABI 310, ABI 3100 and ABI3130 (maximum detection 8100 rfu). When using ABI 3500 or ABI 3730 Genetic Analyzers (maximum detection at rfu), the Minimum Peak Height needs to be adjusted. 1. Open the GeneMapper Manager by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking on the icon. or 2. In the GeneMapper Manager, select the Analysis Methods tab. Highlight the desired Analysis Method and then click Open. Version Page 30 of 43

31 3. Select the Peak Detector tab. The user specified (rfu) values shown below are applicable for the ABI 310, 3100 and 3130 Genetic Analyzers. Click OK and Done after entering the user specified (rfu) values. 4. If using an ABI 3500 or 3730 Genetic Analyzer, the values indicated below are applicable. Click OK and Done after entering the user specified (rfu) values. Version Page 31 of 43

32 2.11 Missing allele calls Analysis Method and Bin Set connection lost Occasionally the Analysis Method looses its connection with the Bin Set. This is likely the explanation when the allele calls and bins cannot be shown. Instead question marks are displayed in the peak labels. Follow the below instructions to make sure that the Bin Set is connected to Analysis Method. 1. To connect the Bin Set to the Analysis Method, open the GeneMapper Manager by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking on the icon. or 2. In the GeneMapper Manager select the Analysis Methods tab. Highlight the desired Analysis Method and then click Open. 3. In the Analysis Method Editor, select the Allele tab. If the Bin Set has been lost, the Bin Set is set to None. Make sure that the correct Bin Set is added (in this example Devyser Complete v2). Click OK and Done. If the Analysis Method has been modified, the samples need to be reanalyzed. Version Page 32 of 43

33 3. Results Interpretation For results interpretation, see Best Practice Guidelines for QF-PCR at To interpret a result as normal for a particular chromosome, at least two informative markers consistent with a normal genotype are required with all other markers being uninformative. To interpret a result as abnormal for a particular chromosome, at least two informative markers consistent with an abnormal genotype are required with all other markers being uninformative. Normal allelic pattern is determined by: Marker showing two peaks of similar height/area and the peak ratio is classified as 1:1. Abnormal allelic pattern is determined by: a) Marker showing two peaks of differing height/area and the peak ratio is classified as 2:1 or 1:2. b) Marker showing three peaks of similar height/area and the peak ratio is classified as 1:1:1. Monosomy X pattern is determined by: a) All X and XY markers showing homozygous allelic pattern. b) The AMELY and SRY peaks are not detected. c) Marker T1 showing two peaks of differing height/area and the peak ratio is classified as 2:1. d) Marker T3 showing two peaks of differing height/area and the peak ratio is classified as 2:1. If peak ratios are classified as inconclusive, results may be resolved by using the Devyser Resolution kits. Marker peaks with sizes outside given marker size ranges may appear and should not be analysed in the context of Devyser QF-PCR products. We recommend the use of Devyser Resolution for the resolution of specific chromosomes. Version Page 33 of 43

34 3.1 Normal Male (46, XY) o o o o All informative autosomal STR markers demonstrate a normal 1:1 marker ratio. The presence of AMELY and SRY is consistent with male gender. The X chromosome counting markers (T1 and T3) demonstrate a 2:1 marker ratio and all the X chromosome STR markers are uninformative, in line with the expected dosage of X chromosomes in a normal male. The 1:1 marker ratio of the informative pseudoautosomal XY chromosome STR markers (XY2 and XY3) and the non-polymorphic XY markers (AMELXY and ZFYX) confirms a normal male sex chromosomal dosage. Version Page 34 of 43

35 3.2 Normal Female (46, XX) o o o o o All informative autosomal STR markers demonstrate a normal 1:1 marker ratio. The presence of AMELX and the absence of AMELY and SRY is consistent with female gender. The X chromosome counting markers (T1 and T3) demonstrate a 1:1 marker ratio, in line with the dosage of two X chromosomes. The presence of informative X chromosomal STR markers with normal marker ratios confirms the dosage of two X chromosomes. The presence of informative pseudoautosomal STR markers (XY2 and XY3) with normal marker ratios confirms the dosage of two sex chromosomes. Version Page 35 of 43

36 3.3 Chromosome trisomy Patau Syndrome, Trisomy 13 o In this example: 47, XY, +13. o All informative autosomal STR markers on chromosome 13 demonstrate abnormal 1:2, 2:1 or 1:1:1 marker ratios consistent with trisomy 13. Version Page 36 of 43

37 3.3.2 Edwards Syndrome, Trisomy 18 o In this example: 47, XY, +18. o All informative autosomal STR markers on chromosome 18 demonstrate abnormal 1:2, 2:1 or 1:1:1 marker ratios consistent with trisomy 18. Version Page 37 of 43

38 3.3.3 Down Syndrome, Trisomy 21 o In this example: 47, XY, +21. o All informative autosomal STR markers on chromosome 21 demonstrate abnormal 1:2, 2:1 or 1:1:1 marker ratios consistent with trisomy 21. Version Page 38 of 43

39 3.4 Turner Syndrome (45, X) o o o o All informative autosomal STR markers demonstrate a normal 1:1 marker ratio. The presence of AMELX and the absence of AMELY and SRY confirm female gender. The X chromosome counting markers (T1 and T3) demonstrate an abnormal female 2:1 marker ratio consistent with the dosage of a single X chromosome. All X and XY STR markers are uninformative (X1, X3, X9, XY2, XY3), consistent with the dosage of a single X chromosome. Version Page 39 of 43

40 3.5 Klinefelter Syndrome (47, XXY) o All informative autosomal STR markers demonstrate a normal 1:1 marker ratio. o The AMELXY marker demonstrates an abnormal AMELX to AMELY (2:1) marker ratio and pseudoautosomal markers are trisomic (XY2 and XY3). o The X chromosome counting markers (T1 and T3) demonstrate an abnormal male 1:1 marker ratio consistent with the dosage of two X chromosomes. o The presence of an informative X chromosomal STR marker confirms the dosage of two X chromosomes. o AMELY and SRY confirm the presence of a Y chromosome. o The abnormal ZFYX marker ratio (1:2) confirms the dosage of three sex chromosomes. Version Page 40 of 43

41 3.6 47, XYY Syndrome o o Informative STR markers on all autosomal chromosomes demonstrate a normal 1:1 marker ratio. Markers AMELXY, XY2 and ZFYX demonstrate abnormal (1:2 and 2:1) marker ratios consistent with the dosage of two Y chromosomes. Version Page 41 of 43

42 3.7 Triple X Syndrome (47, XXX) o Informative STR markers on all autosomal chromosomes demonstrate a normal 1:1 marker ratio. o Informative pseudoautosomal STR markers (XY2 and XY3) demonstrate an abnormal (2:1) marker ratio. o Informative X chromosomal STR markers (X1, X2, X3, X9) demonstrate abnormal (1:2, 2:1 or 1:1:1) marker ratios, consistent with the dosage of three X chromosomes. o The abnormal marker ratio (1:2) of the X chromosome counting markers (T1 and T3) is consistent with an abnormal female X chromosome dosage. Chromatogram kindly provided by Erasmus, Rotterdam, Netherlands Version Page 42 of 43

43 3.8 Triploidy o All informative STR markers on chromosomes 13, 18, 21, X and Y demonstrate abnormal 1:2, 2:1 or 1:1:1 marker ratios consistent with triploidy. Version Page 43 of 43