GB - Elucigene
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1. D21511 D2151409 D2151442 D21851280 D21 1246 D2152055 02181411 CONCLUSION CHECKED 5 The calculated ratios for each marker will now be shown in the data table to the left The data table can be printed or saved as a new file for each specific sample 6 In order to process subsequent samples it is important that all data is fully cleared from the report template In order to do this click the CLEAR DATA button located underneath the raw data table within the QST R 21 Euplex Report Template New sample data can now be imported following the procedure outlined above ANE21BYEN 002 OCT 2015 Page 24 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Scoring the Report 1 Trisomy is determined by either a Two peaks of uneven height due to one of the peaks representing two alleles which are common to both parents In this case the ratio between the two peaks will be classed as 2 1 or 1 2 Where A1 A2 will give a result in the region of 1 8 to 2 4 when the peak representing the shorter length allele is greater in area than the peak representing the longer length allele or where A1 A2 will give a result in the region of 0 45 to 0 65 when the peak representing the shorter length allele is smaller in area than the peak representing the longer length allele In both cases Ratio will appear in the Warning colu2. ANE21BYEN 002 OCT 2015 Page 34 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Previewing the GeneMarker Report Click Preview to view the GeneMarker report Figure 25 From this window the operator can review and print each sample s peak data from across all markers providing a simple one or two page sample report Figure 25 GeneMarker Report Concuson Comments Panel QSTR 21 Euplex Exp Time 08 11 2015 08 56 34 gt 08 11 2015 09 28 38 pas pp 2250 32133688 087 a a fozisize0_ 2___ asa60__ aasoseze ha __ nsus p nezz ems hoo _ pasan p sese rarse pa mo qms T S pastas p zezs sserses ose E pziszoss p isses fesoa a _ The GeneMarker report includes the following features e Report Header Contains information about the analysis project sample and parameters e Signature Box Date and initial space for report reviewers e Electropherogram Similar to the Trisomy analysis window displays all dye colours of the sample trace e Report Table Displays selected peak and marker values for the current sample Trisomy calls are highlighted grey An additional check column is provided for reviewer initials e Corrected Ratio Plot Contains the entire dataset s plot points for all markers in the dye colour Symbol shapes represent different markers and can be deciphered from the Symbol row in the Report Table Yellow filled symbols rep
3. 02151446 D2181442 D2182055 D2181411 02181409 22181280 C01 Run AB3130D21511 vio b o w z 3 t 4 For more detailed information regarding Trisomy analysis features and their use please refer to the GeneMarker Manual ANE21BYEN 002 OCT 2015 Page 33 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use GeneMarker Report GeneMarker includes a report template that is compatible with Elucigene QST R kits To access the report click the Print icon located on the toolbar at the top left of the Trisomy Analysis Window im Sa This will open the Trisomy Print Settings window Figure 24 Trisomy Print Settings Window The Trisomy Print Settings detail the options for including and visualising sample data in the GeneMarker report select the options as shown in Figure 24 Ensure that Custom Size Range is set to 98bp Start and 510bp End Figure 24 Trisomy Print Settings Window Trisomy Print Settings Samples Selected Samples x Markers Ratio Plot fe All Markers fe Show Population Selected Markers Show Sample Only Report Table W Show Allele Mame jw Show Peak Height Area lw Show Peak Ratio J Show Trisomy Scores W Show Allele Iv Show Observation Electrapheragram Size Range Show Curent Size Range Show All Markers Size Hange f Show Custom Size Range start bp 88 End bn 51 0 J Scale data to highest peaks within size range eb Ok x Cancel
4. Figure 4 6 Click Apply then click OK ANE21BYEN 002 OCT 2015 Page 17 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Figure 3 Importing QST R 21 Euplex GeneMapper Panel File Tete SE sane EUH Ree Sin Salma fb Logged ncs 10 lc File dit Analyss View Toots Help SOM Se MM Bw WED gt seee cst rate moo B Peb zo Satus Samole File Sample Name sample 10 Comments _ Sample Type sFN Analysis Method 1 2 3 4 5 5 7 1 9 10 Lookin Genemapper Files 5 QSTR 21 Euplex Bins ey LEGERE Recent Items Desktop i My Documents A Computer ia File rame QSTR 21 plex Panel txt Network ris of type Ad Fies Figure 4 Importing QST R 21 Euplex GeneMapper Bin File File Edit Analysis View Tools Help n SE T Su f l m3 B qd Tabe Setting QSTR Tabie Settings v02 8 b i2o Project Samples nes 1 2 3 ja c E EN n Tr 10 ad ANE21BYEN 002 OCT 2015 Page 18 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Modifying the Analysis Parameter file It may be necessary to modify the default Analysis Ranges in the QST R analysis settings to account for local variance in run conditions The minimum analysis range will depend on the capillary and polymer being used during data collection To view current analysis settings
5. Open GeneMapper Manager by clicking on the icon 2 Select the Analysis Methods tab The Imported QST R file will be listed as QSTR Analysis v02 Click on QSTR Analysis v02 The row will now appear highlighted Click the Open button and select the Peak Detector tab Figure 5 By default the analysis ranges are set to start at 2 000 and finish at 18 000 Figure 5 Analysis Ranges pP m GeneMapper Manager Local Southern Method Global Southern Method Note For 35XX series data collection normalization only Factory Defaults mnm 28 Cluster Plot Settings Matrices Size Standards SNP Sets Report Settings j Analysis Methods Table Settings Plot Settings Analysis Method Editor Microsatellite BS jm General Allele Peak Detector Peak Quality Quality Flags E Peak Detection Algorithm Advanced L Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Partial Range All Sizes B 50 R 50 Start Pt 2000 Start Size 0 uL cm G 50 P 50 Stop Pt 18000 Stop Size 000 Y 50 O 20 rSmoothing and Baselining I Min Peak Half Width 2 pts Smoothing None Light Polynomial Degree Heavy Peak Window Size 15 pts Baseline Window 51 pts Slope Threshold Peak Start 0 0 zs edges Peak End 0 0 2nd Order Least Squares SEEN a 3rd Order Least Squares Size Standard Normalization F Cubic Spline Inter
6. Pul up Correction V Spike Removal Local Region gt 35 Local Max Size Call WV Stutter Peak Filter Iw Plus A Filter Left 64 Right 32 i lt lt Back Cancel Note For 3500 data increase the Minimum Intensity to 150 ANE21BYEN 002 OCT 2015 Page 29 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Run Wizard Additional Settings There are no additional settings required when performing QST R analysis Figure 19 1 Click OK to continue Figure 19 Run Wizard Additional Settings Run Wizard Additional Settings Fragment Animal Analysis Set additional options related to the different analysis type Allelic Ladder NONE Allele Evaluation Peak Score Reject lt 1 00 Check 7 00 Pass a 30 x Back i Cancel Data Processing Box After clicking OK in the Run Wizard Additional Settings box the Data Processing box appears Figure 20 The raw data is processed and sized then the filtering parameters and the selected QST R Panel are applied 1 Click OK in the Data Processing box when analysis is complete Figure 20 Data Processing Box Events Checking options Processing Samples C11 Run AB3130A 2014 10 24 15 12 0299 2014 10 24 fsa Completed Checking Calibration 1 samples processed Analysis Time 0 14s ANE21BYEN 002 OCT 2015 Page 30 of 40 Elucigene QST R 21 Euplex Assay Inst
7. 20ml of amniotic fluid containing fetal cells is drawn off and tested Down syndrome was named after Dr John Langdon Down in 1866 although described earlier It is caused by trisomy for all or part of chromosome 21 2 As well as a cognitive disability of varying degree individuals with Down syndrome typically share a number of common features including hypotonia poor muscle tone a protruding tongue almond shape to the eyes caused by an epicanthic fold upslanting palpebral fissures single palmar crease and shorter than normal limbs They are also at increased risk of congenital heart defects and in later life of developing a form of Alzheimer s ANE21BYEN 002 OCT 2015 Page 2 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Principles of the procedure The method employed by Elucigene QST R kits uses the QF PCR Quantitative Fluorescence Polymerase Chain Reaction technique 3 7 Using PCR amplification fluorescent dye labelled primers target highly polymorphic regions of DNA sequence called short tandem repeats STRs that are located on the chromosomes of interest Each targeted STR marker is specific to the chromosome on which it is located thus the copy number of the STR marker can be diagnostic of the copy number of the chromosome Informative STR markers have been selected that exhibit a high heterogeneity so that copy number can be easily determined A normal diploid sample has the normal complement of two of each of
8. R 21 Euplex GeneMarker Panel Settings It is necessary to import the QST R panel settings for GeneMarker This process is controlled through the Panel Editor interface QST R 21 Euplex GeneMarker panel settings are available from the Elucigene Website http www elucigene com product category rapid aneuploidy analysis 1 Open Panel Editor from the Tools drop down menu Figure 15 Figure 15 Selecting Panel Editor os zz ix T l Au uid Was ANE21BYEN 002 OCT 2015 Page 27 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use 2 Select Import Panels from the File drop down menu Figure 16 Figure 16 Importing Panels 4 Panel Editor Tools Help D Create New Panel X Delete Current Panel Marker al Save Changes Ctrl S E Save Changes with Signal Info Save As New Panel Gar Import Panels S j Import Pre defined Panels Ivar ect 180 200 220 240 260 280 300 320 340 360 380 1 000 800 Import ABI Panels Import MRC Holland Panels ooo Export Panel 400 Exit TA MEO28 PWS AS VslE im 200 a f MSI_Panel P034 DMD Panel OSTR PL m 4 m p c4 amp Samples B amp 01 Run AB31304 2015 No B amp 02 Run AB31304 2015 B B 1 Run AB31304 2015 B B02 Run amp B31304 2015 B CO Run AB31304 2015 B C02 Run AB31304 2015 BB D Run AB31304 2015 B D02 Run AB31304 2015 B EO Run AB31304 2015 B E02 R
9. assigned see above Analysis and Interpretation of Results It is recommended that each laboratory develops its own interpretation and reporting procedures and criteria Best practice guidelines for QF PCR have been documented by the UK s Association for Clinical Genetic Science and are available for reference at www acgs uk com PCR products are observed as a 5 dye labelled system using filter set G5 Filter set G5 detects the 6 FAM blue VIC green NED yellow and PET red labelled fragments plus the Size Standard marker labelled with LIZ orange on an electrophoretogram and in the GeneMapper or GeneMarker program A Guide to Interpretation is available from the Elucigene website http www elucigene com product category rapid aneuploidy analysis Important Note different combinations of instrument polymer and size standard may cause the size calling to vary slightly During validation of the kit users should check that the default bin settings result in accurate peak labelling and adjust if necessary In case of any difficulty please contact Technical Support techsupport elucigene com for advice ANE21BYEN 002 OCT 2015 Page 12 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use General analysis guidelines for QST R 21 Euplex 1 The negative control should show no sharp peaks within the read range of 100 to 510bp The positive control must show the expected results and all peaks must meet the criteria
10. below For analysis of DNA samples at least 1 peak should be observed for each marker tested The acceptable range for marker peaks analysed on the 3130 Genetic Analyzer is between 50 and 6000 relative fluorescent units rfus and for the 3500 Genetic Analyzers is between 175 and 32000 rfus Peak heights falling outside this range must not be analysed Electrophoretograms of poor quality due to excessive bleed through between dye colours also known as pull up or electrophoretic spikes sharp peaks present in more than one dye should not be interpreted The PCR products should be re injected and re analysed Analysis is performed by assessment of peak ratios A1 A2 where A1 is the peak area of the shorter length fragment and A2 is the peak area of the longer length fragment The resulting ratio is indicative of locus copy number For disomic chromosomes heterozygous markers should show two peaks with similar heights A complete analysis of chromosome copy number status is performed by comparison of peak area ratios Heterozygous di allelic i e two alleles markers should fall within a ratio window of 0 8 to 1 4 However for two alleles separated by more than 24bp in size a ratio of up to 1 5 is acceptable Any values falling within this region are referred to as having a ratio of 1 1 If the ratio balance falls out of this window then it may be due to a number of factors such as Whole chromosome trisomy Partial chromosome tr
11. damage to the contents do not use the kit contact Customer Service ANE21BYEN 002 OCT 2015 Page 3 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Symbols used on labels The symbols used on all labels and packaging conform to the harmonised standard ISO 15223 Manufacturer Number of tests See Instructions for Use X C Store below temperature shown Use before date shown REF Catalogue code LOT Lot or batch number In Vitro Diagnostic Medical Device ANE21BYEN 002 OCT 2015 Page 4 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Materials Provided Store all components below 20 C The Elucigene QST R 21 Euplex IVD kit contains 450226 1 x 100ul Reaction Mix TA 404485 1 x 50ul Control DNA DC 450250 10 x 0 2ml PCR vials blue Sufficient for 10 tests Kit Preparation and Storage Upon opening the kit it is recommended that the reaction mix be dispensed into 0 2ml PCR vials provided in 10ul volumes and frozen at 20 C Ensure that vial contents are thoroughly thawed and mixed before dispensing The Control DNA should be frozen at 20 C Materials required but not provided General Laboratory consumables gloves screw capped microfuge tubes 0 2ml PCR vials or microtitre plates recommended by the manufacturer of the thermal cycler used pipette tips Laboratory equipment precision pipettes 2 sets 1 for pre amplification and 1 for post amplification handling pr
12. present The ratio of the peaks will be classed as 1 1 1 and their values fall within the normal range of 0 8 1 4 although for alleles separated by more than 24bp an allele ratio of up to 1 5 is acceptable If this does not occur then it may be due to one of the factors mentioned in step 6 To interpret a result as normal at least two informative markers consistent with a di allelic genotype are required with all other markers being uninformative A normal result indicates the normal complement of two for the chromosome tested Peak area ratios that fall between the normal and abnormal ranges are classed as inconclusive Inconclusive results may be resolved by using the single chromosome kits If both normal and abnormal allele patterns are obtained for a single chromosome then it is recommended that follow up studies are carried out to identify the reason for the discrepant results prior to any conclusions being reached In rare cases allele size ranges for markers may overlap If this is suspected analysis with the single chromosome kits may resolve this ANE21BYEN 002 OCT 2015 Page 14 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Performance Characteristics INTERNAL VALIDATION Thirty 30 DNA samples extracted from amniotic fluid or chorionic villus and of known chromosome 21 ploidy status were tested using Elucigene QST R 21 Euplex Resultant data was analysed following the recommended methods All thirty samp
13. 0A 2014 06 23 14 33 0116 130A 2014 06 23 16 28 0117 3j Run_AB3130A_2014 06 24_14 53_0118 130A 2014 06 24 14 53 0119 J Run AB3130A 2014 06 25 13 54 0120 Jy Run AB3130A 2014 06 25 13 54 0121 z Ej Run AB3130A 2014 03 19 15 10 0020 A01 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa i A02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa L 4 amp BOi Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa B02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa 4 CO1 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa 4 C02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa D01 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa D02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa t 4 E02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa gt 4 F02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa G02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa i H02 Run AB3130A 2014 03 19 15 10 0020 2014 03 19 fsa Add To List gt gt Add Add amp Anayze Cancel ANE21BYEN 002 OCT 2015 Page 16 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Importing QST R GeneMapper Analysis Settings in GeneMapper Manager It is necessary to import the QST R settings for GeneMapper This process is controlled through the GeneMapper Manager interface QST R GeneMapper settings are available from the Elucigene website http www elucigene com pro
14. 130 RUN MODULE FOR POP7 POLYMER 36cm Capillary Module QSTR Parameter Name Value Range 1 Oven Temperature 60 jnt18 65 Deg C 6 njecion Voltage B O f 15kvols 8 Voltage Number of Steps 20 100nk 9 Voltage Step Interval i5 fl 60 sec 10 Data Delay Time pO t 3600sec ANE21BYEN 002 OCT 2015 Page 11 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use ABI3500 GENETIC ANALYZER A QST R Instrument Protocol needs to be created which can then be used for each QST R run Create the QST R Instrument Protocol through the 3500 Instrument Protocols library Ensure the following are selected e Run Module FragmentAnalysis50_POP7 Enter the settings detailed in the image below Edit Instrument Protocol QSTR Setup an Instrument Protocol K Q Application Type Fragment v Capillary Length 50 cm Polymer POP7 Dye Set G5 Instrument Protocol Properties Run Module FragmentAnalysis50_POP Y Protocol Name OSTR Description Oven Temperature C 60 Run Voltage kVolts 19 5 PreRun Voltage kVolts 15 Injection Voltage kVolts 3 0 Run Time sec 1300 PreRun Time sec 180 Injection Time sec 15 Data Delay sec 1 gt Advanced Options Close Torun the samples create a sample plate by clicking on Create Plate from Template in the Dashboard ensure the correct Instrument Protocol for QST R has been
15. 8 Deutsch S Choudhury U Merla G Howald C Sylvan A Antonarakis SE Detection of aneuploidies by paralogous sequence quantification Journal of Medical Genetics 2004 41 908 915 ELUCIGENE and QST R are trademarks of Delta Diagnostics UK Ltd GENEMARKER is a trademark of Softgenetics Corporation GENEMAPPER GENESCAN NED VIC PET POP 7 LIZ and HI DI are trademarks of Life Technologies Corporation Note to purchaser Limited License This product is sold pursuant to an agreement with Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use only the purchased amount of the product and its components for human in vitro diagnostics solely for the indication described in the accompanying instructions for use For information on obtaining additional rights to use this product or its components please contact outlicensing lifetech com Copyright 2015 Delta Diagnostics UK Ltd ANE21BYEN 002 OCT 2015 Page 40 of 40
16. Elucigene QST R 21 Euplex Assay Instructions for Use Elucigene 9 Diagnostics Elucigene QST R 21 Euplex Instructions for Use Product Size Catalogue Code Elucigene QST R 21 Euplex 10 tests ANE21BX For In Vitro Diagnostic Use ud by Elucigene Diagnostics Citylabs Nelson Street Manchester M13 9NQ For Sales Customer Service and Technical Support T 44 0 161 669 8122 F 44 0 161 669 8129 E enquiries elucigene com E techsupport elucigene com Elucigene Diagnostics is the trading name of Delta Diagnostics UK Limited a company registered in England and Wales registration number 8696299 ANE21BYEN 002 OCT 2015 Page 1 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Elucigene QST R 21 Euplex Intended Use QST R 21 Euplex is a supplemental kit designed to be used in conjunction with QST R or QST Rplusv2 for the routine quantitative in vitro diagnosis of trisomies associated with Chromosome 21 Down syndrome The assay provides additional chromosome testing where necessary or for confirmation of positive results The method employed by these kits is the QF PCR Quantitative Fluorescence Polymerase Chain Reaction technique The devices are intended to be used on DNA extracted from either amniotic fluid or chorionic villus samples CVS taken during amniocentesis The intended target population is pregnant women that have been assessed as being at high risk of carrying an aff
17. Euplex 288 8883 8 i i E01 Run AB3130A 2016 09 11 09 55 1178 201 05 ANE21BYEN 002 OCT 2015 Page 38 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Limitations to the Procedure This test is designed to detect specific chromosomal trisomies and sex chromosome aneuploidies as detailed in the Instructions for Use It may not detect structural rearrangements involving the chromosomes tested and will not detect abnormalities in any other chromosomes Mosaicism for the chromosomes tested may not be detected A QST R 21 Euplex result can only be directly applied to the tissue tested and may not represent the fetal karyotype Maternal cell contamination MCC and confined placental mosaicism CPM may result in discrepancies between the QST R 21 Euplex and karyotype results Note Heterozygosities of the markers used were derived from a random set of samples submitted for routine analysis from a predominantly Northern European Caucasian population Any calculations using these heterozygosities strictly only apply to the population from which the samples were taken A small study using locally derived samples may be carried out as part of a validation study to establish heterozygosities in the population to be tested It is not expected that population variation will significantly alter the overall informativeness of the assay Disclaimer Results from this diagnostic assay should be interprete
18. a g g B Go 9 a B i a B a B Ho a S E go H BH a E E 8 a s gs S 8 Jg m E 5 EN 8 g BO Run AB3130A 2015 08 11 06 55 1178 2015 08 11 1 a 2151409 1260 D2151246 140 160 180 200 220 240 260 290 300 320 34 360 380 00 420 440 460 480 ANE21BYEN 002 OCT 2015 Page 31 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Figure 22 Trisomy Analysis Settings Box Trisomy Analysis Settings Analysis by Classic w BPG f Aneuplomdy Peak Height gt 50 Height Ratio gt 230 00 Max Quantification by f Peak Height Peak Area Trisomy Ratio Me Shorter Length Longer Length J Apply Linear Correction Trisomy 0 80 or gt 1 40 Iw Inconclusive Range gt 0 65 to s O80 or gt 1 40 to lt 1 80 Save Parameters when Save Report Cancel Trisomy Analysis Settings Within the Trisomy Analysis settings box two tabs are available 1 Analysis tab 2 Statistics Plot tab Analysis Tab The Analysis tab provides threshold setting options for Trisomy analysis Ensure that BPG is selected in the analysis box and the following settings displayed e Peak Height 50 Minimum height of 50 for peaks to be called 150 if using 3500 data e Height Ratio 3096 Maximum percentage of the main peak the second peak must reach in order for two alleles to be identified e Quantification by Peak Area e Shorter Length Longer Length selected e Trisomy Rati
19. andard with 250ul Hi Di Formamide and mix thoroughly sufficient mix for 16 wells Dispense 15ul of the mix into the required number of wells of a 96 well optical plate 2 Add 3ul of test sample PCR product to the size standard mix from step 1 already dispensed into the plate and mix using the pipette Seal the plate 3 Denature the PCR product dispensed into the optical plate on a thermal cycler using the following parameters 94 C for 3 minutes linked to 4 C for 30 seconds 4 Centrifuge the plate at 1 000g for 10 seconds to remove any bubbles in the wells and load onto the Genetic Analyzer Note t is essential that unused wells i e wells in which No DNA sample is loaded are still loaded with Hi Di Formamide to ensure that the capillaries do not dry out ANE21BYEN 002 OCT 2015 Page 10 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Post PCR Data Analysis ABI3130 GENETIC ANALYZER Create a sample sheet using the 3130 data collection software with the following settings e Sample Name this must be the same sample specific name or number Run owner select the default owner for lab e Run Protocol QSTR contains QST R 3130 run module see below Note It is necessary to create a run module detailing the instrument settings and subsequently assign this to a Run protocol in which Dye set G5 has been selected For more information on creating run modules please refer to your instrument user manual 3
20. d Template Selection Set the template of the project fe Select an existing template ar create one 1 mFERK 4 MSI 4 MS MLPA 4 QST R PL PELARIS 4 QSTR PL 4 SNaPshot Hd Trisomy 4 wT Use last template Run Wizard Data Process Ul w Fragment Animal La Save XX Delete Template Mame Panel QSTA 21 Euplex Size Standard QSTA Standard Color Analysis Type Cancel The Data Process window of Run Wizard allows the user to select the peak filtering parameters 1 Select the appropriate analysis settings in the Data Process window as shown in Figure 18 2 Click Next to continue Note The analysis range setting within the raw data analysis bow will vary depending on the Polymer used during data collection The operator should select a start data point value that includes the 75bp size standard peak Figure 18 Run Wizard Data Process Window Local Southern Cubic Spline Large Size Load Default Save Default Run Wizard xs Data Process Fragment Animal Analysis Set data process options Raw Data Analysis Allele Call v Auto Range frame tal Auto Range bps 0 10000 sj Start s j End 520 4 iw Smooth Enhanced Smooth Peak Detection Threshold Peak Saturation w Baseline Subtraction Min Intensity 50 Max Intensity E0000 Enh d Baseline Subtracti 5 4 Enhanced Baseline Subtraction Bacon T 21 Global Max j
21. d in conjunction with other laboratory and clinical data available to the clinician These Elucigene reagents are supplied for In Vitro diagnostic testing Further details of Elucigene QST R products are available at http www elucigene com product category rapid aneuploidy analysis ANE21BYEN 002 OCT 2015 Page 39 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use References l CG Antenatal Care Full Guidelines Corrected June 2008 UK National Institute for Health and Clinical Excellence O Connor C 2008 Trisomy 21 Causes Down Syndrome Nature Education 1 1 Mansfield E S Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and s mall tandem repeat polymorphisms Human Molecular Genetics 1993 2 1 43 50 Mann K Fox SP Abbs SJ Yau SC Scriven PN Docherty Z Mackie Ogilvie C Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis The Lancet 2001 358 9287 1057 1061 Mann K Donaghue C Fox SP Docherty Z Mackie Ogilvie C Strategies for the rapid prenatal diagnosis of chromosome aneuploidy European Journal of Human Genetics 2004 12 907 915 Mackie Ogilvie C Donaghue C Fox SP Docherty Z Mann K Rapid prenatal diagnosis of an aneuploidy using Quantitative Fluorescence PCR QF PCR Journal of Histochemistry and Cytochemistry 2005 53 3 285 28
22. dit View Samples To Add Files GM Database BRun_AB3s H i Run_AB3 J Run AB3 Run AB3 J Run AB3 z ui Run AB3 jj Run AB3 1 Run AB3 Jy Run AB3 Run AB3 J Run AB3 k Run AB3 J Run AB3 do Run AB3 L Run AB3 Run AB3 Run AB3 Run AB3 Run AB3 Run AB3 p Run AB3 Run AB3130A 2014 06 10 130A_2014 03 19_ 15 10 0020 Run_AB3130A_2014 04 10_09 20_0032 Run_AB3130A_2014 04 14_14 24_0033 130A 2014 04 14 14 24 0034 1j Run AB3130A 2014 04 14 14 24 0035 130A 2014 04 14 14 24 0036 130A 2014 04 14 14 24 0037 130A 2014 04 14 14 24 0038 130A 2014 04 15 09 00 0039 130A 2014 04 15 15 50 0043 Run AB3130A 2014 04 17 08 58 0048 _ Run AB3130A 2014 04 17 08 58 0049 130A 2014 04 17 08 58 0050 Run_AB3130A_2014 04 17_08 58_0051 130A_2014 04 17_08 58_0052 Run_AB3130A_2014 04 17_16 13_0057 130A 2014 04 17 16 13 0058 130A 2014 04 17 16 13 0059 J Run AB3130A 2014 04 17 16 13 0060 130A 2014 04 23 10 46 0067 15 50 0100 130A 2014 06 13 15 16 0102 j Run AB3130A 2014 06 16 12 46 0103 130A 2014 06 17 10 38 0105 130A 2014 06 17 16 06 0106 j Run AB3130A 2014 06 18 09 12 0107 130A 2014 06 19 17 33 0110 F 3j Run_AB3130A_2014 06 20_12 27_0111 130A 2014 06 20 15 57 0112 4 j Run AB3130A 2014 06 20 15 57 0113 130A 2014 06 20 15 57 0114 130A 2014 06 23 12 09 0115 Run AB313
23. duct category rapid aneuploidy analysis Open GeneMapper Manager by clicking on the icon Select the Analysis Methods tab and then click the import button Navigate to and import the required QST R Analysis Settings file s 3130 or 3500 Repeat the process selecting the appropriate tab and importing the corresponding file for p I p e Table Settings e Plot Settings e Size Standards Note Cluster Plot Settings Matrices SNP Sets and Report Settings do not require file imports Importing QST R 21 Euplex Panel settings in Panel Manager It is necessary to import the QST R 21 Euplex panel and bin settings for GeneMapper This process is controlled through the Panel Manager interface QST R 21 Euplex specific GeneMapper panel and bin settings files are available from the Elucigene website http www elucigene com product category rapid aneuploidy analysis 1 Open Panel Manager Program by clicking on the icon 2 Click Panel Manager on the left navigation window Panel Manager will now appear highlighted in blue 3 Select File Import Panels Navigate to and import the GeneMapper panel file QSTR 21 Euplex GeneMapper Panel File txt Figure 3 4 The panel file will now be displayed on the left navigation window Click on the panel file ensuring that it is highlighted blue 5 Select File Import Bin Set Navigate to and import the GeneMapper bin file QSTR 21 Euplex GeneMapper Bin File txt
24. e irre Samples Plot File Edit View Toots Alleles Help E 2 NN NR Bo ee sae ar 5223 2 8 3 81 8 Manual Editing of Profiles WARNING GeneMapper will only assign labels for up to 2 peaks per marker Manual editing of profiles may therefore be required i e when assigning labels to 3rd peaks if present or removing labels from stutter peaks To add a peak label left click on the unlabelled peak You will get the option to add allele comment Click OK The peak will now be labelled with its size in base pairs and its peak area The table will automatically incorporate the newly labelled peak To remove a peak label left click on the peak label You will get the option to delete allele comment Click OK The peak label will now be removed The table will automatically remove the deleted peak data Copying Table Data 1 Highlight all rows with the table at the bottom of the plot window 2 Copy the selected rows by typing Ctrl C keys ANE21BYEN 002 OCT 2015 Page 22 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use QST R 21 Euplex Report Template The associated QST R 21 Euplex report template can be used to determine the peak ratios for a marker and assist in analysis of data The QST R 21 Euplex specific Report Template is available from the website http Awww elucigene com product category rapid aneuploidy analysis 1 Open
25. e Marker Datasets4Fl P sEran MA ANA fsa E Channels OK Cancel Remove Click OK button in the Open Data Files box and the samples will be uploaded to GeneMarker The software will then automatically open the Raw Data Analysis window Figure 14 ANE21BYEN 002 OCT 2015 Page 26 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Figure 14 Raw Data Analysis Window Sample File Tree Report Table Synthetic Gel Image Electropherogram 4 GeneMarker Untitled lU File View Proj ct Applications Tools Help gt NMRB n amp HB 9 9 lE DEW I o Maker None v 3 ta Untitled ER SE Report 2 Help p e Raw Data gt Gel Image C Allele Call xp m MERE ID 1 4 A03 17 QSTR QC 22102009 1 jaos 17 osTR oc 2 ES B 803 18 QSTR QC 22102009 2 Bos 1e osTR OC 44g 142 B C03 19 QSTR QC 22102009 B cos 1 osT oc 44 E D03 20 QSTR QC 22102009 4 pos zo osr ac 2 E03 21 QSTR QC 22102003 M BB F03 22 QSTR QC 22102008 B gos 21 osTR oc 44 G03 23 QSTR QC 22102003 e Fo 22 osTR oc 2f B H03 24 QSTR QC 22102009 TUI B E02 13 QSTR QC 22102009 e Hos z4 osrR oc 4 Hoge pesar 2 gozas oer oc df H02 16 QSTR QC 22102009 Eo roz 14 ostr OC MN 150 200 250 300 350 400 450 500 a A03 17 QSTR QC 22102009 A fsa x AAAS 1 187 1 3921 S1 40GHRM M1 3259 D21 1 44299 858168562865533 200 300 400 500 omn ons Wn PageUp Page Down Importing QST
26. ected foetus by either biochemical or ultrasound diagnostic procedures or assessed to be at risk due to either previous family history or maternal age The device is intended to be used in conjunction with other diagnostic procedures to support or discount the proposed clinical diagnosis The device is for Professional Use only within a molecular or cytogenetics laboratory environment Summary and Explanation The risk of giving birth to a Down syndrome child increases significantly with the age of the mother from approximately 1 1600 at age 20 24 to 1 200 at age 35 and 1 19 at age 45 and older Pregnant women are routinely offered screening for Down syndrome in the first trimester of pregnancy A standard test is the so called OSCAR test One Stop Clinical Assessment of Risk that is carried out at between 10 and 13 5 weeks of pregnancy This combines two biochemical markers free beta hCG human Chorionic Gonadotrophin and PAPP A Pregnancy Associated Plasma Protein A with measurement of the thickness of the fetal nuchal fold nuchal translucency 1 Combining the results of these three tests gives an overall risk figure Women identified as being at increased risk of giving birth to a Down syndrome child are then offered an amniocentesis to test directly for the abnormality Amniocentesis is an invasive test and involves introducing a needle guided by ultrasound into the amniotic sac surrounding the fetus A small sample typically 10
27. eferably positive displacement pipettes protective clothing vortex mixer microfuge 96 well microtitre plate centrifuge DNA Extraction DNA Preparation InstaGene Matrix Bio Rad Laboratories Cat No 732 6030 sterile de ionised water PCR Amplification Thermal cycler to accommodate 96 well microtitre plates or 0 2ml vials with a temperature accuracy of 1 C between 33 C and 100 C and static temperature uniformity of 1 C QST R 21 Euplex has been validated on and shown to perform to specification on the following Thermal Cycler platforms Life Technologies GeneAmp 9700 Running in Standard mode Life Technologies Veriti Dx Running in Standard mode Life Technologies Veriti Dx Running in 9700 simulation mode Life Technologies Proflex Running in Standard mode ANE21BYEN 002 OCT 2015 Page 5 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Capillary Electrophoresis Capillary Electrophoresis GeneScan 500 LIZ size standard ABI Cat No 4322682 POP 7 Polymer ABI Cat No 4352759 DS 33 dye set G5 matrix standard ABI Cat No 4345833 10x Genetic Analyzer Buffer ABI Cat No 402824 and Hi Di Formamide ABI Cat No 4311320 Applied Biosystems ABI 3130 and 3500 Genetic Analyzers with current Data collection software 36cm capillary array 50cm capillary array for 3500 Genetic Analyzer 96 well optical plates 96 well septa 96 well cassettes Data Analysis One of the following data analysis soft
28. f pre aliquoted QST R 21 Euplex reaction mix for the number of samples and controls to be run see note under Materials Provided and centrifuge the vials at 12 000g for 10 seconds Using separate pipette tips add 2 5ul of test DNA to a sample vial containing 10ul QST R 21 Euplex reaction mix and mix by pipetting up and down Do this for all samples to be tested Do not add DNA to the PCR vial for the negative control instead add 2 5ul of sterile distilled water Note care must be taken not to contaminate the PCR reaction with any InstaGene resin Briefly centrifuge the vials until all liquid is at the bottom of each vial Place all vials firmly in the thermal cycler block Initiate the 95 C activation program followed by the amplification program see step 1 On completion of the amplification program the samples may be stored at room temperature overnight or at 2 8 C for up to 7 days before analysis by capillary electrophoresis ANE21BYEN 002 OCT 2015 Page 9 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Capillary Electrophoresis It is recommended that each user ensure that the chosen equipment is used according to the manufacturer s instructions and is compatible with this test In this context the key parameters are the polymer and the capillary array Optimal results can be obtained using the following capillary electrophoresis conditions on an ABI3130 or ABI3500 Genetic Analyzer 1 Combine 6 85yl of size st
29. isomy including sub microscopic duplications Mosaicism Contaminating second genotype e g maternal twin external otutters causing skewing Preferential amplification of one allele causing skewing Primer site polymorphisms Somatic microsatellite mutations The Guide to Interpretation gives examples of typical profiles for many of these Homozygous markers are uninformative since a ratio cannot be determined f To interpret a result as abnormal i e trisomy present at least two informative markers consistent with a tri allelic genotype are required with all other markers being uninformative It is not recommended to interpret a result as abnormal based on information from only one marker ANE21BYEN 002 OCT 2015 Page 13 of 40 10 11 Elucigene amp QST R 21 Euplex Assay Instructions for Use Trisomy is determined by either 7 1 Two peaks of uneven height due to one of the peaks representing two alleles which are common to one or both parents In this case the ratio between the two peaks will be classed as 2 1 or 1 2 such that A1 A2 will give a result in the region of 1 8 to 2 4 when the peak representing the shorter length allele is greater in area than the peak representing the longer length allele or where A1 A2 will give a result in the region of 0 45 to 0 65 when the peak representing the shorter length allele is smaller in area than the peak representing the longer length allele 7 2 Three peaks of comparable height
30. ize Standard heading Each time remember to fill down by pressing Ctrl D to ensure each setting is applied to the full list of samples 3 Click to initiate sample analysis Assign a project name when prompted Reviewing QST R 21 Euplex Data 1 Select the sample for analysis highlight sample row iii 2 Click to Display Plots 3 Select the QST R Plot settings Figure 8 Figure 8 QST R Plot settings drop down menu Samples Plot File Edit View Tools Alleles Help Plot Setting QSTR Plot Settings Y E ait ju ia FRE iu dle ig d sui 4 The plot window will display sample profile with the tabulated data Figure 9 A maximum of two peaks for each marker will be labelled automatically by GeneMapper f three alleles are present for a marker the third unlabelled peak should be manually labelled see Manual Editing of Profiles below Note Allele size ranges for each marker are based on previously observed data Rare alleles may fall outside the given marker size range and it may be necessary to adjust the bin set accordingly 5 It is recommended that the plot window has Single click editing activated To do this select Alleles set click editing and ensure that this option is checked ANE21BYEN 002 OCT 2015 Page 21 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Figure 9 Sample Plot window displaying labelled trace data and correlating genotype tabl
31. les amplified satisfactorily on the first attempt of these 25 were determined to be normal and 5 indicated the presence of a Trisomy 21 All results showed 100 concordance with results previously obtained by alternative established methods ANE21BYEN 002 OCT 2015 Page 15 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use GeneMapper Analysis Guide v3 7 and above Note The following GeneMapper screen shots are taken from GeneMapper v5 0 Importing and Analysing QST R Files 1 Open the GeneMapper Program file 2 Click in order to add data files to a new project Navigate to where the raw fsa data files are stored highlight the appropriate data files and click the Add to List gt gt button 3 The run folder will now appear in the Samples to Add window Double clicking on the run folder icon in this window will show each fsa file to be imported Samples are then added by clicking at the bottom of the screen The data files now appear within the GeneMapper main screen figure 2 Figure 2 Samples ready to add to project File Edit Analysis View Tools Help Gog r aa EPE TER Samples B u B Tablesetting QsTR Table Settings vo2 Status Comments Sample Type SFN Analysis Method Panel Size Standard Matrix SNP Set Run Name Sample File Sample Name Sample ID Add Samples to Project E
32. mn b Three peaks of comparable height present The ratio of the peaks will be classed as 1 1 1 and their values fall within the normal range of 0 8 1 4 although for alleles separated by more than 24bp an allele ratio of up to 1 5 is acceptable In this case 3 Alleles will appear in the Warning column 2 To interpret a result as abnormal i e trisomy present at least two informative markers consistent with a tri allelic genotype are required with all other markers being uninformative It is not recommended to interpret a result as abnormal based on information from only one marker 3 To interpret a result as normal at least two informative markers consistent with a diallelic genotype are required with all other markers being uninformative A normal result indicates the normal complement of two for the chromosomes tested 4 Peak area ratios that fall between the normal and abnormal ranges are classed as inconclusive Inconclusive results may be resolved by using the single chromosome kits 5 Inthe absence of any peak data for a marker Absent will be displayed in the warning column This warning will be routinely observed in the absence of Y chromosome markers ANE21BYEN 002 OCT 2015 Page 25 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use GeneMarker Analysis Guide Note The following screen shots have been taken from GeneMarker v2 6 3 Adding Sample Files to GeneMarker Open the GeneMarker
33. nd the pellet Note this additional washing step helps to lyse the red blood cells and remove haem that could inhibit PCR 8 Add 200yl of InstaGene matrix from step 1 to the samples using a pipette tip with a large bore such as 1 000yl Note to optimise the extraction protocol the added volume of InstaGene matrix Chelex 100 resin can be varied using 100ul of InstaGene matrix for small AF cell pellets barely visible or 300ul for large pellets covering the base of the tube CV and tissue samples Record the amount of InstaGene matrix added to each sample 9 Thoroughly mix the samples by vortexing and incubate at 100 C for 8 minutes in a hot block or water bath 10 Thoroughly mix again by vortexing at high speed for 10 seconds 11 Centrifuge the samples at 12 000g for 3 minutes The supernatant contains the extracted DNA 12 Proceed to PCR set up or store the extracted DNA at 20 C until required ANE21BYEN 002 OCT 2015 Page of 40 Elucigene QST R 21 Euplex Assay Instructions for Use DNA Concentration lt is recommended that alternative DNA extraction methods and sample types are thoroughly evaluated with the Elucigene QST R test prior to the results being used for diagnostic use Under optimal PCR conditions and using the recommended sample injection settings stated in the capillary column run module page 11 acceptable results are consistently obtained with input DNA amounts of 1 25ng to 10ng Note sample i
34. njection settings can be modified to suit the amount of amplicon produced during the PCH reaction which can vary due to amount of input genomic DNA added Less amplicon can be applied to the column for analysis by reducing time of injection Conversely more amplicon can be applied to the column for analysis by increasing either time or voltage of injection Previously amplified samples can be re injected multiple times for re analysis ANE21BYEN 002 OCT 2015 Page 8 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Test Protocol Amplification Procedure Note to minimise the risk of contamination steps 3 5 must be carried out in an area free from DNA Steps should also be taken to avoid contamination with PCR product 1 Program the thermal cycler for a single step cycle to activate the DNA polymerase at 95 C for 15 minutes linked to an amplification cycling program of 30 seconds at 95 C denaturation 1 minute and 30 seconds at 59 C annealing and 1 minute and 30 seconds at 72 C extension for 26 cycles This should be linked to a 30 minutes time delay file at 72 C extension on the final cycle Cycling Enzyme Activation Extension Ambient 1 min 30 secs Temperature A negative water control must be included in each PCR run It may also be considered appropriate to include other controls e g positive normal DNA control supplied and positive trisomy control DNA not supplied Thaw sufficient vials o
35. o thresholds are 0 80 1 40 e Apply Linear Correction de selected e Click OK ANE21BYEN 002 OCT 2015 Page 32 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Trisomy Analysis Window The Trisomy Analysis window Figure 23 allows the operator to review QST R sample data and display the ratio of peaks for each marker and access the GeneMarker report There are a number of displays which assist the operator in data analysis they are e Sample List e Electropherogram e Ratio Plot e Report Table Figure 23 Trisomy Analysis Window Sample List Electropherogram Q amp A B S96E 3 Maker Dzis Report Table Sample E 1 A0 Run AB3130A 2015 AD1 Run AB3130A 2015 08 11 08 55 1178 201508 11 fsa 2 A02 Run AB3130A 2015 BOT Run AB3130A 2015 B02 Run AB3130A 2015 C0 Run AB3130A 2015 CO2 Run AB3130A 2015 D0t Run A83130 2015 D02 Run AB31304 2015 E01 Run AB3130A 2015 EAZ Run AB3130A 2015 F 1 Run AB3130A 2015 2 F02 Run AB3130 2015 GM Run A83130 2015 GO2 Run AB83130A 2015 HO1 Flun AB3130A 2015 HOZ Run A83130A 2015 220 225 Ratio Plot o Report Cj i x 1 02181442 D2151246 D2182055 2521511 w 215 232 306 323 w 199 219 2 u o oC o ss 82055 pier E esses enses Run AB313d oe wb 0 Uv D2151446 D2151442 2181246 D2151411 D2151409 D2151280 w vo amp wo amp
36. polation n ee ANE21BYEN 002 OCT 2015 Page 19 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use To find the correct analysis range for your laboratory 1 From the main GeneMapper window double click on the imported Run Folder to view the list of fsa files it contains Select an fsa file Clicking the Raw data tab will display the electropherogram of the raw data Using the first peak of the size standard e g 75bp of GS500LIZ as a guide select on a data point approximately 100 data points larger Figure 6 This determines the lowest point in the analysable range Ensure the maximum analysis range encompasses the largest peak of the size standard e g 500bp of GS500LIZ or 600bp of GS600LIZv2 Input the new values into the QST R Analysis file accessing it as described above Figure 6 Finding the minimum range using sample raw data RY 40mm onm ANE21BYEN 002 OCT 2015 Page 20 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Analysis of Imported QST R 21 Euplex Files 1 In the main GeneMapper window select the QST R Table Settings v02 Figure 7 Figure 7 Setting QST R table settings bs Table Setting QSTR Table Settings v02 zi 2 Under Analysis Method select QSTR Analysis v02 Fill down each column by pressing Ctrl D Repeat this process selecting QSTR 21 Euplex for under the Panel heading and QSTR under the S
37. program file and when prompted select Open Data The Open Data Files box will appear Click the Add button The Open dialog will appear Navigate to directory containing raw data files 1 Select all files by CTRL A or use CTRL and or SHIFT keys to select individual samples 2 Click Open button in the Open dialog The files selected will appear in the Data File List field Figure 13 Figure 13 Samples added to the Data File list Open Data Files Data File List C SoftGenetics Gene Marker Datasets 4FLP frag_001_H01 fsa C SoftGenetics Gene MarkersD atasets FLP frag 002 _G01 fsa C SoftGenetics Gene MarkersDatasetsy amp FLP frag 0 3 F T fsa C SoftGenetics Gene Marker Datasets 4FLP rag_004_E01 fsa C SoftGenetics Gene MarkersDatasetsV FLP frag 005 DOT fsa C SoftGenetics Gene MarkersDatasetsy amp FLP frag O05 CUT fsa C SoftGenetics Gene MarkersDatasetssAFLP frag 07 BU T fsa R All C SoftGenetics Gene MarkersDatasetss FLP frag OO8 amp UT fsa adii C SoftGenetics Gene MarkersDatasetsv amp FLP frag O03 HO3 fsa C SoftGenetics Gene MarkersDatasetss amp FLP frag 10 G 3 fsa C SoftGenetics Gene Marker Datasets 4FLP rag_011_FO3 fsa C SoftGenetics Gene Marker Datasets 4FLP rag_012_E03 fsa C SoftGeneticssGene Marker Datasets AFLPYfrag_013_D03 fsa Add Folder C SoftGenetics Gene MarkersDatasetss FLP frag 14 CO3 fsa C SoftGenetics Gene MarkersDatasetss amp FLP frag 015 BOS fsa 7 S nfl aenehiessf3en
38. resent the current sample s data points Red outlined symbols represent trisomy calls Note 7he Corrected Ratio Plot appears on a second page for each sample only when Ratio Plot is selected in the Trisomy Print Report Settings box ANE21BYEN 002 OCT 2015 Page 35 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use APPENDIX 1 Dye Labels The markers are labeled as follows NED D21S2055 D2151411 See Appendix 2 for further details of the STR markers including size ranges ANE21BYEN 002 OCT 2015 Page 36 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use APPENDIX 2 Table of Maker location Observed Heterozygosity allele size range Note the NED dye used in the kits is identified spectrally as a yellow dye It is conventionally displayed in black type for clarity D2151280 21022 11 320 0 380 0 Red D2151246 21q022 2 410 0 470 0 D2152055 21q022 2 140 0 230 0 Yellow D2151411 21022 3 278 5 348 5 Yellow D2151446 21q22 3 198 5 239 5 Green Observed heterozygosities are based on number of alleles observed with an Elucigene Diagnostics testing panel These figures may therefore differ from published data and may also vary according to the population being tested ANE21BYEN 002 OCT 2015 Page 37 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use APPENDIX 3 GeneMapper Profile GeneMapper normal male profile showing relative positions of the markers detected by QST R 21
39. ructions for Use Main Analysis Window The Main Analysis window Figure 21 of GeneMarker has an easy to use layout This layout includes e The sample files list displayed on the left side of the window e The Synthetic Gel Image displayed at the top of the window e Data Electropherograms below the gel image e A Report Table displayed on the right side of the window In this window it is important to check that all the appropriate peaks in each profile have been called correctly 1 Double click on each sample in turn in the sample file tree on the left hand side of the screen Right click on any peaks in question and choose from the options in the dialogue box e g edit or delete allele confirm or unconfirm as appropriate 2 From the Main Analysis window select the Applications drop down menu option from the top of the screen Select Trisomy Analysis The Trisomy Analysis Settings box will then open Figure 22 Figure 21 Main Analysis Window Sample File Tree Synthetic Gel Image Report Table dl GeneMarker Untitled on xX File View Project ApplfiBtions Tools Help ob WEB B a P 5IiE J E E m lA Maker None aa Uriiled 4 j Report Hep es feni id Gel Image I Jozisizse 2152 I I 2151411 YT gs S ES 8S F za B 402 g i a B EB 21 JY BO 5 3 ES c 8 c Me B dE 4 B i B i BS 2 BB on g g s 23 B oo ss e B to 6 Bh cc a E SN E a d a ao S H B Go g 8g
40. the QST R 21 Euplex Report Template file QSTR 21 Euplex Report Template xlsm 2 Ifthe Report Template displays a warning indicating that macros have been disabled click the Enable Content button to enable macros Figure 10 Figure 10 Enabling macro function in QST R 21 Euplex Report Template T HOME INSERT PAGELAYOUT FORMULAS DATA REVIEW VIEW dh Cut ES Copy Arial l10 Paste i Format Painter Clipboard E p s Alignment D SECURITY WARNING Macros have been disabled Enable Content 3 Ifa security warning is shown as seen in Figure 11 click Yes in order to proceed Figure 11 Allowing security access Security Warning A Do you want to make this file a Trusted Document This file is on a network location Other users who have access to this network location may be able to tamper with this file What s the risk E Do not ask me again for network files 4 Paste the data copied from the GeneMapper data table see above Copying Data Table using Ctrl V into the top left cell in the outlined area See Figure 12 ANE21BYEN 002 OCT 2015 Page 23 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Figure 12 Importing raw GeneMapper data into the QST R 21 Euplex Report Template semen coora QST R 21 Euplex Report Template Software GeneMapper PASTE DATA INTO THE CELL BELOW 1 Check 2 Check 3 Check Comment
41. the somatic chromosomes thus two alleles of a chromosome specific STR are determined by the QF PCR technique as two peaks in a 1 1 ratio The observation of an extra STR allele as either a three peak pattern in a 1 1 1 ratio or two peak pattern in a 2 1 or 1 2 peak ratio is diagnostic of the presence of an additional sequence which in turn may represent an additional chromosome as in the case of a trisomy Amplified products of the QF PCR technique are analysed quantitatively on a capillary electrophoresis Genetic Analyzer to determine the copy number of the analysed STR markers Warnings and Precautions 1 The normal DNA Control provided in the kits has been independently tested and found to be negative for Hepatitis B Virus HBV Hepatitis C Virus HCV and Human Immunodeficiency Virus HIV 1 and 2 2 Care should be taken when handling material of human origin All samples should be considered potentially infectious No test method can offer complete assurance that HBV HCV HIV or other infectious agents are absent 3 Handling of samples and test components their use storage and disposal should be in accordance with the procedures defined by the appropriate national biohazard safety guideline or regulation 4 Inline with current good laboratory practice laboratories should process their own internal QC samples of known type in each assay so that the validity of the procedure can be assessed 5 f kit box is damaged there may be
42. un AB31304 2015 BB FOI Run amp B31304 2015 1 B F02 Run AB31304 20154 B GUI Run AB31304 2015 B GU2 Run AB31304 2015 BB HO1 Run AB31304 2015 B H02 Run AB31304 2015 Panel Name QSTR 21 Euplex Ploidy 3 3 Navigate to and import the panel QSTR 21 Euplex xml 4 Repeat the process as required for other relevant panel files Processing Data After the raw data files have been uploaded to GeneMarker they are ready to be processed The processing step includes application of a sizing standard filtering of noisy peaks and comparison to a known allelic panel if desired GeneMarker combines all these steps in one simple tool called the Run Wizard Figure 17 To access the Run Wizard simply click the Run Project icon in the main toolbar Run Wizard creating a Run Template It is necessary to create a run template the first time this software is used to analyse QST R 21 Euplex data This is done through the Run Wizard To access the Run Wizard simply click on the Run Project icon in the main toolbar Assign a Template Name e g QSTR 21 Euplex 1 2 3 Select the Panel Size Standard Standard Colour and Analysis Type as shown in Figure 17 4 Click Save to store the template for future analyses 5 Click Next to continue ANE21BYEN 002 OCT 2015 Page 28 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use Figure 17 Run Wizard Template Selection Window Run Wizar
43. ware packages is required GeneMapper 3 7 Applied Biosystems Inc or above or GeneMarker 1 65 SoftGenetics LLC or above Additional Elucigene QST R Documentation These Instructions for Use include a basic section on interpretation of the results obtained in addition to a guide to software analysis with both the GeneMapper and GeneMarker packages A supplemental Guide to Interpretation with examples and glossary are available from the Elucigene website http www elucigene com product category rapid aneuploidy analysis Sample Collection and Storage Chorionic Villus CV or Amniotic Fluid AF samples should be used Sample collection devices have on occasion been reported to be detrimental to the integrity of certain analytes and could interfere with some method technologies It is recommended that each user ensure that the chosen device is used according to the manufacturer s instructions and both sample collection devices and DNA preparation methods are compatible with this test ANE21BYEN 002 OCT 2015 Page 6 of 40 Elucigene QST R 21 Euplex Assay Instructions for Use DNA Extraction Elucigene QST R kits are validated on the InstaGene matrix method of DNA extraction and can be performed in a single tube eliminating the necessity for tube to tube transfers Other extraction methods have been shown to provide equally reliable results e g Qiagen QlAamp kits The InstaGene method of DNA extraction is described below Instagene E
44. xtraction Method Amniotic Fluid AF Approximately 1 2ml of amniotic fluid should be used Chorionic Villus CV CV samples should be carefully cleaned to remove any adhering maternal decidua It is important that cells from more than one region of the sample are tested and that cells from the mesenchyme core are represented A small aliquot of the cell suspension as prepared for conventional cell culture set up is recommended for QST R analysis This ensures that the QST H result is obtained from the same population of cells used for karyotype analysis 1 Resuspend the InstaGene matrix on the magnetic stirrer and set at a medium speed for at least 5 minutes 2 Centrifuge the sample AF or CV at 12 000g for 1 minute in order to pellet the cells 3 Remove the samples from the centrifuge and visually check the pellet for blood staining Make a note of the percentage of blood staining if any 4 Carefully remove and discard the supernatant from the pellet ensuring that the pellet is undisturbed Leave approximately 10 20ul of supernatant behind to re suspend the pellet 5 Thoroughly mix the sample by vortexing 6 If greater than 50 bloodstaining is observed proceed to Step 7 If less than 50 blood staining is observed proceed to Step 8 7 Add 200ulI of sterile deionised water to the cell pellet Thoroughly mix by vortexing Centrifuge at 12 000g for 1 minute remove the supernatant leaving 10 20ul of supernatant behind to resuspe
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