Elucigene® CF Iberian Panel Instructions for Use
Contents
1. Uh Ceerettnrtes Lleeitied R 2s 2 Fie Yew Pojet Gopiketions Toos Hep E RRS TE iT z oe ree LE Sire Templars Fetree i i Moye Projat tMauuolsceba b Ge mage v ADIA AEII DI 1016 A EM Ar ARNI t0 tre Coceermcn B ON no anim ANCE u Padigies Fle Name Match Fic Name Group Tuol lumna MLPA Ansys Conven lert to Earary Free eel Mar Auasi 2 Show cast Evert 3 ae ae ae Mt 1000 1200 1400 ae Set 2003 2233 249 Ie ee faite Rup 49 460 4 50 500 S200 S400 SEE SESE COST SAO E400 SEE Glee 7000 n 49 Tan 7 w 33 5 49 AMR ASERI 2094 10 16_16 19_0GE _ ALOE ho 3714S x t sos JA L 2 Select Import Panels from the File drop down menu Figure 15 Figure 15 Importing Panels File Tools Help D Create New Panel I v mevr we Ss X Delete Current Panel Marker AFLPPanel fel Save Changes Ctri S E Save Changes with Signal Info Save As New Panel limpet Panes Import Pre defined Panels Import ABI Panels Import MRC Holland Panels Export Panel Exit Eg microsat_panel Ve Samples amp Sampl poe Left Range Right Range Allele Name Control Distance kb Chromosome Position Recombination Frequencs B A01_Run_AB31304_201 4 B B01 Run AB31 304 2014 1 Blue Blue 35 8 04 04 3 0 0 00 1 00 ies g 2 Blue Bluel 3 9 04 04 F 0 0 00 1 00 CO1_Run_AB31304_2014 B nin T 3 Blue Bluel 37 8 04 04 38 0 0 00 1 00 4 Blue Blue 41 6 04 02. Elucigene CF Iberian Panel Products Instructions for Use Importing CF Iberian Panel Raw Data Files into GeneMapper 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 6 Figure 6 Samples have been added to list and are ready to add to project File Edit Analysis View Tools Help Of ear B g gt Table Setting QSTR Table Settings v02 BPE SER Sample File Sample Name Sample ID Comments Sample Type SFN Analysis Method Panel Size Standard Matrix SNP Set Run Name m Add Samples to Project Edit View Files GM Database Samples To Add Fr ERUN AB3130A_2014 03 19_15 10_0020 a E i Run_AB3130A_2014 03 19_15 10_0020 Jo Run_AB3130A_2014 04 10_09 20_0032 AQ1_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa Run_AB3130A_2014 04 14_14 24 0033 AO2_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa J Run_AB3130A_2014 04 14_14 24_0034 BO1_Run_AB3130A_2
3. Elucigene CF Iberian Panel Products Instructions for Use Run Wizard Additional Settings There are no additional settings required Figure 18 1 Click OK to continue Figure 18 Run Wizard Additional Settings Run Wizard Additional Settings Fragment Animal Analysis Set additional options related to the different analysis type Allelic Ladder NONE Allele Evaluations Peak Score Reject lt 10 00 Check 20 00 lt Pass E 30 lt lt Back Cancel Data Processing Box After clicking OK in the Run Wizard Additional Settings box the Data Processing box appears Figure 19 The raw data is processed and sized then the filtering parameters and the selected Panel are applied 1 Click OK in the Data Processing box when analysis is complete Figure 19 Data Processing Box Events Checking options Processing Samples A01_Run_AB3130A_2014 10 16_16 19_0265_2014 10 16 fsa Completed BO1_Run_AB3130A_2014 10 16_16 19_ 0285 2014 10 16 fsa Completed CO1_Run_AB3130A_2014 10 16_16 19_ 02685 2014 10 16 fsa Completed Checking Calibration 3 samples processed Analysis Time 0 40s CFIBNBYEN 001 Jun 2015 Page 38 of 42 Elucigene CF Iberian Panel Products Instructions for Use Main Analysis Window The Main Analysis window Figure 20 of GeneMarker has an easy to use layout This layout includes The sample files list displayed on the left
4. PCFZEU_3100_gmas001 a Recent Items _ anan000_gmas002 _ ananO00_gmps002 1 ananQ00_grmps003 _ ananQ00_gmsz002 ananQ00_gmts002 _ ananO03_gupt0o2 _ ananO13_gupf001 _ anan018_gupf001 _ ananO21_gupf002 _ ananOpl_gupf004 K A _ ananOxy gupf00s a anan500_gusz001 Computer _ anan600_qusz001 _ CF2EU_3130_grmas001 CF2EU_3500_grmas001 CF2EU_grsz001 CF2EU_gmts001 _ CF2EU_Plot_PT off001 _ CF2ZEU_Plot_PT on001 _ CF_EU POPS _ CF_EU POP cfcf004_gmas003 cfcf004_grnps003 cfcf004_gmsz001 cfcf004_gmts002 THFO3_gmas001 Ll 4 TE File name CF Iberian Panel Analysis Method xml Network Files of type XML Files xml 4 Repeat the process selecting the appropriate tab and importing the corresponding file for e Table Settings e Plot Settings e Size Standard Note Table Settings Plot Settings and Size Standard are the same as used in CF EU2v1 Operators currently using CF EU2v1 can apply these settings without importing CFIBNBYEN 001 Jun 2015 Page 23 of 42 Elucigene CF Iberian Panel Products Instructions for Use Importing CF Iberian Product Bin and Panel Files in Panel Manager Before first use of the kit it is necessary to import the CF Iberian product bin and panel files for GeneMapper This process is controlled through the Panel Manager interface CF Iberian GeneMapper bin and panel files are available from the Elucige
5. Service CFIBNBYEN 001 Jun 2015 Page 5 of 42 Elucigene CF Iberian Panel Products 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 Store below temperature shown x Use before date shown REF Catalogue code Lot or batch number In Vitro Diagnostic Medical Device CFIBNBYEN 001 Jun 2015 Page 6 of 42 Elucigene CF Iberian Panel Products Instructions for Use Materials Provided Each kit contains Elucigene CF Iberian Panel TA Part Code 450215 1 x 120ul 25 tests Primer Mix containing primers to amplify the following wild type and mutant alleles A1006E 1812 1G gt A A561E 1069delCA 2869insG K710X R709X 2184insA 712 1G gt T H199Y P205S and V232D This mix also contains primers for identification of 2 hypervariable short tandem repeat STR markers PCR Master Mix TM Part Code 404480 1 x 400ul vial of containing HotStart Taq DNA Polymerase and deoxynucleotide triphosphates in buffer DNA Control DC Part Code 40448 1 x 50ul vial DNA Control at 6ng ul normal for the mutations detected by Elucigene CF Iberian Panel Kit Preparation and Storage Storage of the reagents should be in an area free from contaminating DNA or PCR product Store all components below 20 C in the dark The fluorescent dyes used in this product are photosensitive minim
6. V232D and 1 was heterozygous for V232D A 2184delA heterozygote was tested and correctly identified by the 2184insA marker All results showed 100 specificity and sensitivity with results previously obtained by an alternative established method Limitations of the Procedure 1 The results obtained from this or any other diagnostic test should be used and interpreted only in the context of the overall clinical picture Elucigene Diagnostics is not responsible for any clinical decisions that are taken 2 The absence of the mutations detected by this kit is no guarantee that other mutations in the CFTR gene are not present Other mutations are possible and are not detected by this kit ci Mutations vary in frequency between different populations Population mutation frequency data is available from The Cystic Fibrosis Genetic Analysis Consortium 6 The user of this kit should emphasise these points when reporting results to the diagnosing Clinician Genetic Counsellor Disclaimer Results from this and other diagnostic assays should be interpreted in conjunction with other laboratory and clinical data available to the Clinician These Elucigene reagents are supplied for In Vitro diagnostic testing CFIBNBYEN 001 Jun 2015 Page 41 of 42 Elucigene CF Iberian Panel Products Instructions for Use References 1 Rosenstein BJ Cutting GR The diagnosis of cystic fibrosis a consensus statement Cystic Fibrosis Foundation Consensus Pane
7. side of the window The Synthetic Gel Image displayed at the top of the window Data Electropherograms below the gel image 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 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 Figure 20 Main Analysis Window iew Project pphcatons oos eg oe gt PEE S B a pE J OB BW Hater iow gt za i bos Nw SGF 4 Srererrrrrrrrw pre SPREEERRRAaa a OR CFIBNBYEN 001 Jun 2015 Page 39 of 42 Elucigene CF Iberian Panel Products Instructions for Use Previewing the GeneMarker Report Click Preview to view the GeneMarker report Figure 21 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 21 GeneMarker Report SoftGenetics Allele Report 6 30 2015 1 50 21 PM GeneMarker V2 6 3 Page 1 Allele Report SoftGenetics Template CF Iberian Panel CF_lIberian Project Comments Sample 1 frag_001_A07 fsa Run date and time 06 22 2015 14 22 52 gt 06 22 2015 15 04 30 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460
8. 014 03 19_15 10_0020_2014 03 19 fsa jJ Run_AB3130A_2014 04 14_14 24_0035 BO2_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa J Run_AB3130A_2014 04 14_14 24 0036 CO1_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa Run_AB3130A_2014 04 14_14 24_0037 CO2_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa Run_AB3130A_2014 04 14_ 14 24 0038 DO1_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa l Run_AB3130A_2014 04 15_09 00_0039 DO2_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa 7 Run_AB3130A_2014 04 15_15 50_0043 E02_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa jJ Run_AB3130A_2014 04 17_08 58_0048 f FO2_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa J Run_AB3130A_2014 04 17_08 58_0049 GO2_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa Run_AB3130A_2014 04 17 08 58 _0050 i HO2_Run_AB3130A_2014 03 19_15 10_0020_2014 03 19 fsa Run_AB3130A_2014 04 17_08 58_0051 Run_AB3130A_2014 04 17_08 58_0052 Run_AB3130A_2014 04 17_16 13_0057 Run_AB3130A_2014 04 17_16 13_0058 Run_AB3130A_2014 04 17_16 13_0059 Run_AB3130A_2014 04 17_16 13_0060 Run_AB3130A_2014 04 23_10 46_0067 J Run_AB3130A_2014 06 10_15 50_0100 Run_AB3130A_2014 06 13_15 16_0102 2 2 2 y t Run_AB3130A_2014 06 16_12 46_0103 J Run_AB3130A_2014 06 17_10 38_0105 J Run_AB3130A_2014 06 17_16 06_0106 Run_AB3130A_2014 06 18_09 12_0107 Run_AB3130A_2014 06 19_17 33_0110 Run_AB3130A_2014 06 20_1
9. 0265 _2014 10 16 fsa Es 4001 Rurn_4637304_2074 10 16_16 13_ 0265 20174 10 16 fsa Remove Remove ll Add Folder Default A Channels Auto Eleyvate OF Cancel 3 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 13 CFIBNBYEN 001 Jun 2015 Page 32 of 42 Elucigene CF Iberian Panel Products Instructions for Use Figure 13 Raw Data Analysis Window UL ceretta Irratia e S i Fie View Project Apebca ons Tools Hep MAIR O AQBS Lay A is bl eet P Gal Image v AN Fur ABit DA ils 21675 1 1 BOLFuc sbi e_22l4 T EE IE B OO Fun sB3 27142 1615 45 OF OF O00 CIN 400 S00 SOE ZOE 2508 2408 TOW Y oa Ru 400 4500 4200 SOO SLO S400 SERO SHO SOLO 829 649 699 6599 99 293 493 ran OH ERO Baw BAN CFIBNBYEN 001 Jun 2015 Page 33 of 42 Elucigene CF Iberian Panel Products Instructions for Use Importing CF Iberian Panel GeneMarker Panel Settings Before first use of the kit it is necessary to import the CF Iberian panel file for GeneMarker This process is controlled through the Panel Editor interface The CF Iberian Panel GeneMarker panel file are available from the Elucigene Website http www elucigene com product category cystic fibrosis 1 Open Panel Editor from the Tools drop down menu Figure 14 Figure 14 Selecting Panel Editor
10. 2 27 0111 J Run_AB3130A_2014 06 20_15 57_0112 J Run_AB3130A_2014 06 20_15 57_0113 J Run_AB3130A_2014 06 20_15 57_0114 Run_AB3130A_2014 06 23_12 09_0115 Run_AB3130A_2014 06 23_14 33_0116 Run_AB3130A_2014 06 23_16 28_0117 Run_AB3130A_2014 06 24_14 53_0118 Run_AB3130A_2014 06 24_14 53_0119 Run_AB3130A_2014 06 25_13 54_0120 J Run_AB3130A_2014 06 25_13 54_0121 Add To List gt gt A Sn CFIBNBYEN 001 Jun 2015 Page 26 of 42 Elucigene CF Iberian Panel Products Instructions for Use Analysing and Genotyping Imported CF Iberian Panel Raw Data Files 1 Inthe main GeneMapper window select the CFEU2 Table Settings as shown Figure 7 Figure 7 Selecting table settings File Edit Analysis View Tools Help r SGOoN u P UEA U gt Tie setina aaben il OS 2 Sse Project Samples Genotypes Run_AB3130B_2014 05 Status Sample File Sampl frag_001_A03 fsa ple Type SFN Analysis Method F CFEU2_ 3130 Analys 1 2 frag_002_A04 fsa CFEU2 3130 Analy C 3 frag_003_B03 fsa 2A ple NA CFEU2_3130 Analys C 4 frag_004_B04 fsa 2B ple NA CFEU2_ 3130 Analy C E fran NNA CNA fsa IRA nle NA CFFH AIBN Anahelf 2 Inthe main GeneMapper window select the first cell under the Analysis Method and select the appropriate analysis method from the drop down menu CF Iberian Panel Analysis Method either 3130 or 3500 3 Repeat th
11. 4 42 0 0 00 1 00 5 Blue Bluel 43 2 0 4 04 43 0 0 00 1 00 6 Blue Bluel 45 2 0 4 1 2 45 0 0 00 1 00 7 Blue Blue1 47 0 04 08 47 0 0 00 1 00 8 Blue Blue1 49 4 0 4 06 49 0 0 00 1 00 3 Blue Bluel 52 1 1 0 1 0 52 0 0 00 1 00 10 Blue Bluel 54 5 04 04 55 0 0 00 1 00 11 Blue Blue1 56 4 0 4 04 56 0 0 00 1 00 12 Blue Blue 57 9 0 4 04 58 0 00 1 00 13 Blue Blue 59 9 1 0 1 0 60 0 0 00 1 00 1 Blue Blue1 63 7 1 0 07 64 0 0 00 1 00 Panel Name AFLPPanel Ploidy 2 3 Navigate to and import the panel CF Iberian Panel GeneMarker Panel File xml 4 Repeat the process as required for other relevant panel files CFIBNBYEN 001 Jun 2015 Page 34 of 42 Elucigene CF Iberian Panel Products Instructions for Use Importing CF Iberian Panel GeneMarker Size Standard Settings Before first use of the kit it is necessary to import the appropriate size standard file for GeneMarker This process is controlled through the Size Template Editor interface The CF Iberian Panel GeneMarker size standard file is available from the Elucigene Website http www elucigene com product category cystic fibrosis 5 For the Size Standard File open Size Template Editor from the Tools drop down menu 6 Select Size Standard from the File drop down menu 7 Navigate to and import the CF Iberian Panel Size Standard file CF Iberian Panel GeneMarker Size Standard xnil Processing Data After the raw data files have been uplo
12. 480 500 520 540 560 580 600 620 1_A1006E_MUT 1_A100E_WT 5_2869 nsG_WT 8_2184insA WT 10_H199Y_WT 12_V232D_WT 2_1812_1G_A WT 6 _K710X_WT 9 712_1G T WT 7_R709X_WT Sample 2 frag_002_A08 fsa Run date and time 06 22 2015 14 22 52 gt 06 22 2015 15 04 30 5 000 Soa lw a a a G 4 000 3 000 2 000 1 000 0 L j a a i 1 L La i 9712 1d T MUT 1_A100E_WT 2869insG_WT 8_2184insA_WT 10_H199Y_WT 12_V232D_WT 2_1812_1G_A_WT 6_K710X_WT 9 712_1G T WT 3_AS61E_WT 7_R709X_WT 11_P205S_WT 4 1609delCA_WT The GeneMarker report includes the following features e Report Header Contains information about the analysis project sample and parameters e Electropherogram Similar to the Trisomy analysis window displays all dye colours of the sample trace CFIBNBYEN 001 Jun 2015 Page 40 of 42 Elucigene CF Iberian Panel Products Instructions for Use Performance Characteristics Thirty five DNA samples extracted from whole blood were tested blind using Elucigene CF Iberian Panel All samples tested gave interpretable results Of those results 16 were normal 1 was heterozygous for A1006E 2 were heterozygous for 1812 1G gt A 1 was heterozygous for A561E 2 were heterozygous for 1609delCA 3 were heterozygous for 2869insG 2 were heterozygous for K710X 1 was heterozygous for R709X 1 was heterozygous for 2184insA 2 were heterozygous for 712 1G gt T 1 was heterozygous for H199Y 1 was a compound heterozygote for P205S and
13. 5 kvolts Injection_Time 600 sec Voltage _Number_of_Steps 100 nk Voltage_Step_Interval 60 sec Data_Delay_Time 3600 sec Run_ Voltage 15 kvolts Run_Time 300 14000 sec COON O1 8 WO DY Note Required run time will vary dependent on the ambient temperature of the location in which the Genetic Analyzer has been installed For more information on creating run modules please refer to the Applied Biosystems 3130 Genetic Analyzer User Manual Create the CF Iberian Panel protocol in the Protocol Manager ensure the following are selected e Type Regular e Run Module CF_Iberian see run module above Dye Set G5 To run the samples create a sample sheet using the Plate Manager ensure the correct protocol has been selected for the instrument protocol see above Note For more information on instrument setup operation and troubleshooting please refer to the Applied Biosystems 3130 Genetic Analyzer User Manual CFIBNBYEN 001 Jun 2015 Page 13 of 42 Elucigene CF Iberian Panel Products Instructions for Use ABI3500 GENETIC ANALYZER An Instrument Protocol needs to be created which can then be used for both the CF Iberian Panel and CF EU2v1 Create the Instrument Protocol through the 3500 Instrument Protocols library Ensure the following are selected e Run Module FragmentAnalysisS5O POP7 e Enter the settings detailed in the image below Edit Instrum
14. CF Iberian Panel Products 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 100bp GS600v2 LIZ peak Orange as a guide decide on a data point approximately 100 data points smaller Figure 11 Ensure the maximum analysis range encompasses the largest peak of the size standard 600bp for GS6OO0LIZv2 Input the new values into the Analysis file accessing it as described above Figure 11 Finding the minimum range using sample raw data oo z sba Project info Raw Data EPT Data Run_AB3130B_2014 05 frag_001_A03 fsa frag_002_A04 fsa frag_003 B03 fsa 0 1000 2000 3000 4000 5000 frag_004_804 fsa frag_005_C03 fsa frag_006_C04 fsa frag_007_D03 fsa frag_008_D04 fsa frag_009_ 03 fsa frag_010_ 04 fsa frag_011_F03 fsa Q frag_012_F04 fsa frag_013_G03 fsa Tag_Vio_GU5 TSa frag_014_G04 fsa 100bp frag_015_H03 fsa 7000 120bp 180bp 6 09_016_H04 fsa 140bp 160bp 200bp 80bp 6000 5000 A j J natar A Fanaa NAAA n onan hE Y ET Oe tiie gt akadai Aiii ad a dered Pach ieee NT NR OP ROT x T 4000 2000 2000 1000 IC UD 0 UA ANAY Ng fl on A aad AP r i CFIBNBYEN 001 Jun 2015 Page 30 of 42 Elucigene C
15. Elucigene CF Iberian Panel Products Instructions for Use Elucigene Diagnostics Elucigene CF Iberian Panel Instructions for Use s Product si Size Catalogue Code Elucigene CF Iberian Panel 25 tests CFIBNB1 For In Vitro Diagnostic Use Oth serv tactured 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 CFIBNBYEN 001 Jun 2015 Page 1 of 42 Elucigene CF Iberian Panel Products Instructions for Use Elucigene CF Iberian Panel Intended Use For the simultaneous detection of the following human Cystic Fibrosis Transmembrane conductance Regulator CFTR gene mutations in DNA extracted from whole blood EDTA preserved HGVS Nomenclature Intron Exon Name eBNAname _Proteinname Legacy Inron Exon Inron Exor 712 1G gt T Intron Exon 62 Exon 6a v232D ceast gt A oNVal232Aso Exon6a Exon 10 18124G gt a c1ssoigea non Exon 12 Exon 1 Exon 1 Exon 13 Exon 1 A1006E 0 3017C gt A 3 Ala1006Glu Exon 17a 80 1G gt T 95C gt 1 695T gt A 80 1G gt A insA gt I with reference to Mutation Nomenclature in Practice Findings and Recomme
16. F Iberian Panel Products Instructions for Use GeneMarker Analysis Guide Introduction The following section describes the procedures for sample analysis of Elucigene CF Iberian Panel results using SoftGenetics GeneMarker software package version 1 95 or greater Note screenshots in this document are taken from GeneMarker v 2 6 3 Analysis Process The process for analysis of samples is summarised in the flow diagram shown below Run Amplified Samples on Genetic Analyzer Open GeneMarker Software Add Sample FSA Files to Project Select Relevant CF Iberian Panel amp Confirm Analysis Settings Run Sample Analysis Select the Application ARMS Comparative Analysis Select Preview CFIBNBYEN 001 Jun 2015 Page 31 of 42 Elucigene CF Iberian Panel Products Instructions for Use Adding Sample Files to GeneMarker Open the GeneMarker 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 12 Figure 12 Samples added to the Data File list Open Data Files Data File List E 4 4401_Fun_4631304_ 2074 10 16 16 15 0285 2014 10 16 fsa Ew B0 Rurn_4637304_20174 10 16_16 13_
17. aded 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 16 To access the Run Wizard simply click the Run Project icon in the main toolbar Run Wizard creating a Run Template lt is necessary to create a run template the first time this software is used to analyse CF Iberian Panel data This is done through the Run Wizard 1 To access the Run Wizard simply click on the Run Project icon in the main toolbar 2 Assigna Template Name e g CF Iberian 3 Select the Panel Size Standard Standard Colour and Analysis Type as shown in Figure 16 below 4 Click Save to store the template for future analyses 5 Click Next to continue CFIBNBYEN 001 Jun 2015 Page 35 of 42 Elucigene CF Iberian Panel Products Instructions for Use Figure 16 Run Wizard Template Selection Window Run Wizard Template Selection Set the template of the project f Select an existing template or create one H AFLP Template Name CF Iberian 1 BHS_MPX1 Panel CF_Iberian UL a 4 CF Iberian A Fragilex Size Standard CFEU2 Size Standard Y u 4 LOH Standard Color Orange 4 Microsatellite _Dinucleoti H MLPA Analysis Type Fragment Animal E mn Firari 4 Ly Save Dele
18. 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 8ul of size standard 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 Sample injection settings can be modified to suit the amount of amplicon produced during the PCR which can vary due to amount of inout 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 time of injection Previously amplified samples can be re injected multiple times for re analy
19. culty please contact Technical Support techsupport elucigene com for advice CFIBNBYEN 001 Jun 2015 Page 15 of 42 Elucigene CF Iberian Panel Products Instructions for Use General analysis guide for CF Iberian Panel In the CF Iberian Panel both mutant specific and wild type specific primers are combined in the same mix The presence of a CF mutation is identified by the presence of a blue peak and the presence of a wild type normal allele is identified by the presence of a green peak Individuals who carry both a mutant copy and a wild type copy heterozygote of a specific allele will show both a blue and a green peak for that allele Individuals who carry two mutant copies of a specific allele homozygous mutant will show a single blue peak for that allele Individuals who carry two wild type copies of a specific allele homozygous wild type will show a single green peak for that allele Hypervariable STR markers red are also included in this mix These STR markers are the same as those included in the CF EU2v1 A and B mixes which enables the comparison between patient samples amplified with both the CF Iberian Panel and CF EU2v1 A and B mixes This helps to reduce the potential for sample mix up as a different STR profile in any of the three mixes indicates a sample mix up Absence of these STR markers indicates a failed sample Presence of the STR markers at very low rfus indicates a weak sample which should be analysed with ca
20. e that the correct volumes are handled Itis recommended that the PCR Master Mix be added to the previously dispensed primer mixes to ensure all liquid is dispensed from the pipette into the Primer Mix Note Do not use different mixes or components from different lots of CF Iberian Panel kit CFIBNBYEN 001 Jun 2015 Page 10 of 42 Elucigene CF Iberian Panel Products Instructions for Use Table 1 Reaction Mix Formulation Number of Samples to be Tested Toa 12 120 300 600 4 Dispense 10ul of each reaction mix into the bottom of the appropriately labelled 0 2ml PCR vials or plates 5 Using separate pipette tips add 2 5ul of test DNA sample to each of the vials and cap Do not add DNA to the vial for the negative control substitute with 2 5ul of sterile deionised water 6 Briefly centrifuge the vials until all liquid is at the bottom of each vial 7 Place all vials firmly in the thermal cycler block Initiate the 94 C activation program followed by the amplification program see step 1 8 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 CFIBNBYEN 001 Jun 2015 Page 11 of 42 Elucigene CF Iberian Panel Products Instructions for Use Capillary Electrophoresis lt is recommended that each user ensure that the chosen equipment is used according to the manufacturer s instructions
21. ene website http www elucigene com product category cystic fibrosis 1 Open GeneMapper Manager by clicking on the icon Figure 1 Figure 1 Opening GeneMapper Manager File Edit Analysis View Tools Help 2 Select the Analysis Methods tab and then click the import button Figure 2 Figure 2 GeneMapper Manager Interface CFIBNBYEN 001 a m GeneMapper Manager Projects Name Last Saved Instrument CF IBN 2014 07 08 12 44 03 0 CFEU2 FAM bg 2012 01 23 14 49 58 0 QSTR Analysis Settings 3501 2011 11 03 12 15 02 0 QSTR Analysis Settings 3131 2011 11 03 12 10 17 0 Primer QC Analysis 2009 11 13 10 55 19 0 CF4 2 Analysis Method 2009 10 27 09 08 51 0 QSTR Analysis v02 2005 12 09 00 23 06 0 CFEU2 3500 2010 05 07 09 06 10 0 QSTR 3500 normalisation 2010 04 21 09 10 58 0 CFEU2_3130 low size 2011 09 08 12 48 45 0 CFEU2_3130 Analysis Methe 2008 07 26 12 52 32 0 QSTR Analysis 3500 2011 11 03 12 26 44 0 Jun 2015 Page 22 of 42 Elucigene CF Iberian Panel Products Instructions for Use 3 Navigate to and import the CF Iberian Panel Analysis Method 3130 or 3500 Figure 3 Figure 3 Importing Analysis Methods im Import Analysis Method Look in Supporting Files from Web di Official QSTR report templates L di ld report templates
22. ent Protocol CFEU2 Setup an Instrument Protocol Application Type Fragment a Capillary Length 50 x cm Polymer POP7 Dye Set G5 v Instrument Protocol Properties Run Module FragmentAnalysis50_POP7 v Protocol Name CFEU2 Description Oven Temperature C 60 Run Voltage kVolts 19 5 PreRun Voltage kVolts 15 Injection Voltage kVolts 3 Run Time sec 1200 PreRun Time sec 180 Injection Time sec 12 Data Delay sec 1 gt Advanced Options Close To run the samples create a sample plate by clicking on Create Plate from Template in the Dashboard ensure the correct Instrument Protocol has been assigned see above CFIBNBYEN 001 Jun 2015 Page 14 of 42 Elucigene CF Iberian Panel Products Instructions for Use Analysis and Interpretation of Results General Interpretation of Results 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 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 diffi
23. ftware The following steps can be taken to increase signal e Increase injection time see Capillary Electrophoresis section e Re extract sample and elute in reduced volume 50ul of water CFIBNBYEN 001 Jun 2015 Page 9 of 42 Elucigene CF Iberian Panel Products 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 94 C for 20 minutes linked to an amplification cycling program of 1 minute at 94 C denaturation 2 minutes at 58 C annealing and 1 minute at 72 C extension for 30 cycles This should be linked to a 20 minutes time delay file at 72 C extension on the final cycle Enzyme Activation Final Extension Ambient Temperature 30 Cycles A negative water control must be included in each PCR run It may also be considered appropriate to include other controls e g positive control DNA control supplied Thaw the Primer Mix and the PCR Master Mix and centrifuge briefly to collect the contents at the bottom of the vials Mix gently by vortexing and briefly centrifuge the vials again Prepare sufficient Reaction Mix for the number of samples and controls to be tested Table 1 Note The PCR Master Mix is viscous and care should be taken to ensur
24. genetics Corporation GENEMAPPER NED VIC PET POP 7 LIZ and HI DI are trademarks of Life Technologies Corporation NOTICE TO PURCHASER LIMITED LICENCE Polynucleotides labeled with VIC NED and PET dyes and or their use may be covered by one or more patents owned by Life Technologies Corp The purchase price of this product includes limited nontransferable rights under certain claims of certain patents owned by Life Technologies Corp to use only this amount of the product solely for activities of the purchaser in detection of Target s within the field of human diagnostics No other rights are conveyed Further information on purchasing licenses relating to the dyes mentioned above may be obtained by contacting the Director of Licensing outlicensing lifetech com Copyright 2015 Delta Diagnostics UK Ltd CFIBNBYEN 001 Jun 2015 Page 42 of 42
25. h is available from the Elucigene website http Awww elucigene com product category cystic fibrosis 234 delG is an example of a deletion detected by the Elucigene CF EU2v1 assay which impacts the exon 14 wild type green peaks of the CF Iberian assay profile as demonstrated below 2347delG Two peaks 1bp apart are observed for CF Iberian wild type green peak 6 K710X peak 7 R709X and peak 8 2184insA in an individual heterozygous for the 2347delG mutation see profile below The presence of the 2347delG mutation will be confirmed if using the CF EU2v1 assay shown as a blue peak at position 16 in the A mix of CF EU2v1 Other Observations H199Y and P205S Interaction The P205S mutation has been identified to partially interfere with amplification of H199Y wild type primer sequence Reduced height of the wild type peak 10 H199Y may be observed in an individual heterozygous for the P205S mutation CFIBNBYEN 001 Jun 2015 Page 18 of 42 Elucigene CF Iberian Panel Products Instructions for Use Cross Reactivity Every effort has been taken during test development to avoid interference of test function by the presence of other reported polymorphisms and mutations in the CFTR gene Evaluation of Known mutations and polymorphisms in the CFTR gene has highlighted the following effects on the Elucigene CF Iberian Panel results 2184delA The 2184insA mutant primer will detect the 2184delA mutation B
26. ian Panel_Panel File CF2EU_Plot_P1 CF Ivacaftor Panel 1 CF Iberian_CF IBN_bins E CF4v2_CF4v2 B _ CF Iberian_Panels E CF4v2_Panels 4 mi r CF Ivacaftor Panel_CF Ivacaftor_bins _ CF_EU2_POP6 acaftor Panel_Panels EU2_ CF Ivacaftor Panel_Panel _ CF_EU2_POP7 L CF UK_CF UK_bins _ CF UK_Panels _ CF2EU_3100_gmas001 _ CF2EU_3130_gmas001 _ CF2EU_3500_gmas001 4 mW _ cfcf004_gmasf cfcf004_gmbft _ cfcf004_gmpfd _ cfcf004_gmps _ cfcf004_gmsz0 T Computer TY Network File name CF Iberian Panel_Panel File txt Files of type all Files b Jun 2015 Page 24 of 42 Elucigene CF Iberian Panel Products Instructions for Use Figure 5 Importing CF Iberian bin file m Panel Manager File Edit Bins View J iama SeSEeaas none S acA _ anan500_gusz001 __ CF2EU_gmbf0 _ anan600_gusz001 CF2EU_gmpf0 _ CF Iberian Panel Analysis Method _ CF2EU_gmsz0 C _ CF2EU_gmts00 _ CF Iberian Panel_Panel File _ CF Iberian_CF IBN_bins _ CF4v2_CF4v2 E __ CF Iberian_Panels CF4v2_Panels CF Ivacaftor Panel_CF Ivacaftor_bins _ CF_EU2_POP6 _ CF Ivacaftor Panel_Panels __ CF_EU2_POP7 _ CF UK_CF UK_bins as _ CF UK_Panels _ CF2EU_3100_gmas001 _ CF2EU_3130_gmas001 _ CF2EU_3500_gmas001 4 CF Iberian Panel_Bin File txt All Files k CFIBNBYEN 001 Jun 2015 Page 25 of 42
27. is process for the Panel by selecting the CF Iberian Panel and similarly for the Size Standard by selecting CFEU2 size standard from the drop down menus 4 Click to initiate sample analysis Assign a project name when prompted CFIBNBYEN 001 Jun 2015 Page 27 of 42 Elucigene CF Iberian Panel Products Instructions for Use Reviewing CF Iberian Panel Trace and Genotype Data 1 Select the sample for analysis highlight sample row AAN 2 Click to Display Plots 3 Use the drop down menu to select the appropriate CF EU2v1 Plot settings Figure 8 Note if analysing CF Iberian Panel data alongside CF EU2v1 data the CF Iberian Panel is compatible with both CFEU2v1 Plot PT on and CFEU2v1 Plot PT off settings Figure 8 Use the drop down menu to select CFEU2 Plot Settings a Samples Plot File Edit View Tools Alleles Help It F I on Plot Setting CFEUZ P L CFEU2 Plot PT off Sample File m pam Panes 2 nA ple Hame Fane 4 The plot window will display sample trace data Figure 9 5 Itis 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 Figure 9 CF Iberian Panel Sample Plot window displaying labelled trace data CFIBNBYEN 001 Jun 2015 Page 28 of 42 Elucigene CF Iberian Panel Products Instructions for Use Modifying the Analysis Paramete
28. ise exposure to light CFIBNBYEN 001 Jun 2015 Page 7 of 42 Elucigene CF Iberian Panel Products Instructions for Use 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 preferably positive displacement pipettes protective clothing vortex mixer microfuge 96 well microtitre plate centrifuge 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 CF Iberian Panel has been validated and shown to perform to specification on the following recommended Thermal Cycler platforms Applied Biosystems GeneAmp 9700 Applied Biosystems Veriti Dx Running in Standard mode Applied Biosystems Veriti Dx Running in 9700 simulation mode Applied Biosystems Proflex Running in Standard mode Note Thermal cycler equipment should be regularly maintained in accordance with the manufacturer s instructions and calibrated to ensure accurate PCR cycling and optimal performance The Elucigene CF Iberian assay was developed on Applied Biosystems Veriti thermal cycler in 9700 mode Other makes and models should be fully tested and eva
29. l J Pediatr 1998 132 589 95 2 De Braekeleer M Allard C Leblanc JP Simard F Aubin G Genotype phenotype correlation in cystic fibrosis patients compound heterozygous for the A455E mutation Hum Genet 1997 101 208 11 3 Drumm ML Konstan MW Schluchter MD Handler A Pace R Zou F Zariwala M Fargo D Xu A Dunn JM Darrah RJ Dorfman R Sandford AJ Corey M Zielenski J Durie P Goddard K Yankaskas JR Wright FA Knowles MR Genetic modifiers of lung disease in cystic fibrosis N Engl J Med 2005 353 1 443 53 4 Goss CH Newsom SA Schildcrout JS Sheppard L Kaufman JD Effect of ambient air pollution on pulmonary exacerbations and lung function in cystic fibrosis Am J Respir Crit Care Med 2004 169 816 21 5 Kerem B Rommens JM Buchanan JA Markiewicz D Cox TK Chakravarti A Buchwald M and Tsui LC Identification of the Cystic Fibrosis Gene Genetic Analysis Science 1989 245 4922 1073 8 6 Cystic Fibrosis Mutation Database www genet sickkids on ca ctftr app 7 Newton CR etal Analysis of any point mutation in DNA The Amplification Refractory Mutation System ARMS Nucleic Acid Res 17 2503 2516 1989 8 Satsangi J et al Effect of heparin on polymerase chain reaction Lancet 343 1509 1510 1994 ELUCIGENE is a trademark of Delta Diagnostics UK Ltd ARMS is a trademark of AstraZeneca UK QIAAMP is a trademark of Qiagen Gmbh PICOGREEN is a trademark of Molecular Probes Inc GENEMARKER is a trademark of Soft
30. luated for optimal performance by the user before reporting results with CF Iberian Panel Capillary Electrophoresis Capillary Electrophoresis POP 7 Polymer ABI Cat No 4352759 10x Genetic Analyzer Buffer ABI Cat No 402824 and Hi Di Formamide ABI Cat No 4811320 GeneScan 600v2 LIZ size standard ABI Cat No 4408399 and DS 33 dye set G5 matrix standard ABI Cat No 4345833 Applied Biosystems ABI 3130 and 3500 Genetic Analyzers with GeneMapper 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 software packages is required GeneMapper 3 7 Applied Biosystems Inc or above or GeneMarker 1 65 SoftGenetics LLC or above CFIBNBYEN 001 Jun 2015 Page 8 of 42 Elucigene CF Iberian Panel Products Instructions for Use Sample Collection and Storage DNA extracted from whole blood EDTA samples have been evaluated to be compatible with this test Sample collection devices have on occasion been reported to be detrimental to the integrity of certain analytes and could interfere with some method technologies 8 It is recommended that each user ensure that the chosen device is used according to the manufacturer s instructions and sample collection devices are compatible with this test Blood samples should be stored at 20 C prior to preparation of DNA Avoid repeated freezing and thawing Pre
31. ndations from the Cystic Fibrosis External Quality Assessment Scheme Berwouts S Morris M Girodon G Schwarz M Stuhrmann M and Dequeker E Human Mutation Vol 00 No 0 1 7 2011 The Elucigene CF Iberian Panel can distinguish between individuals who are heterozygous and homozygous for the all the above mutations and variants CFIBNBYEN 001 Jun 2015 Page 2 of 42 Elucigene CF Iberian Panel Products Instructions for Use Summary and Explanation Cystic Fibrosis CF is the most common life limiting autosomal recessive disorder in the Caucasian population The disease incidence is 1 3200 live births among Caucasians 1 In the Caucasian population the heterozygote frequency is approximately 1 25 Cystic fibrosis affects the epithelia in several organs resulting in a complex multisystem disease that includes the exocrine pancreas intestine respiratory tract male genital tract hepatobiliary system and exocrine sweat glands Disease expression varies by severity of CFTR mutations 2 genetic modifiers 3 and environmental factors 4 The range extends from early childhood death as a result of progressive obstructive lung disease with bronchiectasis to pancreatic insufficiency with gradually progressive obstructive lung disease during adolescence and increasing frequency of hospitalisation for pulmonary disease in early adulthood to recurrent sinusitis and bronchitis or male infertility in young adulthood Most commonly the diagnosi
32. ne website http www elucigene com product category cystic fibrosis Open Panel Manager Program by clicking on the Icon Click Panel Manager on the left navigation window The Panel Manager will now appear highlighted in blue Select File Import Panels Navigate to and import the GeneMapper panel CF Iberian GeneMapper Panel File txt Figure 4 The panel file will now be displayed on the left navigation window Click on the panel file ensuring that it is highlighted blue Select File Import Bin Set Navigate to and import the GeneMapper bin file CF Iberian GeneMapper Bin File txt Figure 5 Click Apply then click OK Figure 4 Importing CF Iberian panel file CFIBNBYEN 001 m Panel Manager File Edit Bins View JE aT EEE Panel Manager Kit Name Kit Type Comment G CF Iberian Panel 1 EF Iberian Panel SNaPshot none S aa Panel Bet 2 IMFI reflex Microsate none 1 MFI reflex CF UK 3 CD QSTR MI 4 _ EQ CF4v2 5 Look in d Supporting Files from Web CQ CFEU2 6 STR 7 _ anan500_gusz001 _ CF2EU_gmbf0 EQ QSTRPL a an ay _ anan600_gusz001 __ CF2EU_gmpf0 Grrr red CF Iberian Panel Analysis Method CF2EU z0 Devyser CFTR Core Ov A 9 F S ean ane ma etho _ gms ECFEU2V1 _ CF Iberian Panel_Bin File _ CF2EU_gmts00 E QSTR MFI L CF Iberian Panel_CF IBN_bins _ CF2EU_Plot_P CF Iberian N CF Iber
33. oth 2184insA and 2184delA mutations are distinguishable based on an amplicon size difference of 2bp The distinction between these mutations is shown in diagrams A and B below A B 2184delA Mutant Peak 8 2184insAd WT 8 2184ina WT A 2184insA mutant amplicon is the same size as the wild type amplicon and is presented as a labelled blue peak significantly overlapping the wild type peak as shown above B 2184delA mutant amplicon is 2bp smaller than the wild type amplicon and is presented as an unlabeled blue peak which falls outside the bin range of the 2184insA panel CFIBNBYEN 001 Jun 2015 Page 19 of 42 Elucigene CF Iberian Panel Products Instructions for Use 2183AA gt G The 2184insA wild type primer will not amplify alleles containing the 2183AA gt G mutation resulting in drop out of the wild type 2184insA peak The example below shows the 2184insA wild type peak at half intensity in the presence of a 2183AA gt G heterozygote 270 310 350 3390 430 6_K710X WT 8_2184insA WT 9_712 1G _ T WT 11 _ P2053 WT _R 09X WT 10_H199 WT Note In the presence of a 2183AA gt G homozygote it is expected that the 2184insA wild type peak would drop out completely due to no amplification of either allele Should this occur follow up studies to test for the presence of 2183AA gt G is recommended The following mutations which have not been checked for possible cross reactivity due to unavailability of relevant samples may interfere with
34. paration of DNA from Whole Blood EDTA Samples Results are consistently obtained with DNA extracted using the QlAamp 96 DNA Blood Kit or the QlAamp DNA Mini Kit using Proteinase K following the protocol as described in the QlAamp Handbook starting with 200ul liquid whole blood and eluting in 200ul of molecular biology grade water DNA Concentration Using the recommended PCR conditions and sample injection settings see note in Capillary Electrophoresis section stated in the capillary column run modules acceptable results are consistently obtained with inout DNA amount of 15ng However interpretable results are obtained with an input DNA range of 5ng to 30ng The quantification of DNA is very important the concentration of every DNA sample to be tested should be measured to ensure optimal results Techniques such as PicoGreen fluorescence or UV absorbance are acceptable Due to variation in DNA quantification techniques the user should consider the following guidance Very high input DNA amounts will increase the likelihood that background peaks are labelled by the analysis software The following steps can be taken to reduce the likelihood that background peaks are labelled e Dilute DNA sample and re amplify e Reduce injection time see Capillary Electrophoresis section e Increase minimum peak amplitude threshold Low input DNA amounts will increase the likelihood that diagnostic peaks are weak and not labelled by the analysis so
35. r feasible nor cost effective and is therefore confined to testing for the most common mutations The Elucigene CF Iberian panel is designed to extend the coverage of Elucigene CF EU2v1 specifically to address the most common mutations found in Spanish and Portuguese populations The Elucigene CF Iberian panel identifies 12 additional mutations in total CFIBNBYEN 001 Jun 2015 Page 3 of 42 Elucigene CF Iberian Panel Products Instructions for Use Principles of the procedure The method employed by the Elucigene CF Iberian Panel uses fluorescent ARMS Amplification Refractory Mutation System allele specific amplification technology which detects point mutations Insertions or deletions in DNA 7 The principle of ARMS is that oligonucleotides with a 3 mismatched residue will not function as Polymerase Chain Reaction PCR primers under specified conditions Selection of appropriate oligonucleotides allows specific mutant or normal DNA sequences to be amplified and detected Amplified sequences amplicons are separated by capillary electrophoresis using an Applied Biosystems Genetic Analyzer instrument Analysis software enables amplicons to be identified and labelled according to their size and dye colour Elucigene CF Iberian Panel is a multiplexed assay comprising a single PCR In a single reaction mix both mutant and wild type alleles are detected for twelve mutations within the CFTR gene In the CF lberian Panel assay mutant alleles are
36. r file It may be necessary to modify the default Analysis Ranges in the CF Iberian Panel Analysis Method settings in order to account for your in run conditions The minimum analysis range will depend on the capillary and polymer being used during data collection To view current analysis settings 1 Open GeneMapper Manager by clicking on the icon 2 Select the Analysis Methods tab The Imported CF Iberian Panel file will be listed as CF Iberian Panel Analysis Method 3 Click on CF Iberian Panel Analysis Method The row will now appear highlighted 4 Click the Open button and select the Peak Detector tab Figure 10 Figure 10 Analysis Ranges Analysis Method Editor SNaPShot Peak Detection Algorithm Advanced Ranges Partial Range Al Sizes w Start Size 0 stop Size 000 Start Ft 1500 Stop Pt 9000 Heavy Baseline Window 5i pts E 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method 5 Global Southern Method CFIBNBYEN 001 Jun 2015 Nt Peak Detection Peak Amplitude Thresholds B 100 R 30 G 0 P 0 Y 100 Om 5 Min Peak Half Width Polynomial Degree Peak Window Size Slope Threshold Peak Start Peak End Size Standard Normalization Enable Normalization Note For 35 XX series data collection normalization only Factory Defaults Page 29 of 42 Elucigene
37. s of cystic fibrosis is established in individuals with one or more characteristic phenotypic features of CF plus evidence of an abnormality in CFTR function based on one of the following presence of two disease causing mutations in the CFTR gene or two abnormal quantitative pilocarpine iontophoresis sweat chloride values gt 60 mEq L or transepithelial nasal potential difference NPD measurements characteristic of CF The CFTR mutation detection rate varies by test method and ethnic background In some symptomatic individuals only one or neither disease causing mutation is detectable in some carriers the disease causing mutation is not detectable CFTR related disorders are inherited in an autosomal recessive manner Siblings of a proband with cystic fibrosis have a 25 chance of being affected a 50 chance of being asymptomatic carriers and a 25 chance of being unaffected and not carriers Molecular genetic testing for disease causing mutation s in the CFTR gene is used for carrier detection in population screening programs Prenatal testing is available for pregnancies at increased risk for CFTR related disorders if the disease causing mutations in the family are Known Since the discovery of the CFTR gene in 1989 5 more than 1700 mutations and variants in the gene have been described 6 Many of these mutations are private having been described only in one patient and or family Routine testing for all possible mutations is neithe
38. sis Post PCR Data Analysis ABI3130 GENETIC ANALYZER The CF Iberian Panel has been designed to be compatible with the Elucigene CFEU2v1 product As such CF Iberian Panel can be run using existing CFEU2v1 Run Modules and settings Alternatively a separate CF Iberian Panel Run Module can be created by the user as follows 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 e Run owner select the default owner for lab e Run Protocol CF_Iberian contains 3130 run module 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 CFIBNBYEN 001 Jun 2015 Page 12 of 42 Elucigene CF Iberian Panel Products Instructions for Use 3130 RUN MODULE FOR POP7 POLYMER 36cm Capillary Module MFI Create the CF Iberian run module in the Module Manager of the 3130 data collection software Ensure the following are selected e Type Regular e Template FragmentAnalysis36_POP7 e Enter the settings detailed in the table below Parameter Name Range Oven Temperature int 18 65 Deg C Poly_fill_ Vol 6500 38000 steps Current Stability int O 2000 uAmps PreRun_ Voltage 0 15 kvolts Pre Run_Time 1000 sec Injection_ Voltage l 1
39. t blue peaks should be located to the left of the wild type green peaks th 1_A1006E WT E AS61E WT 4 1609deICA WT 5_2869insG WT 6_K710X WT 8_2184insA WT 9_712 1G T WT 11_ P2058 WT 12_ 232D WT 2_1812 1G_A Mut _R709X WT 10_H199 WT 2_1812 1G_4 WT Compound Heterozygote The example below shows a compound heterozygote result for mutations P205S and V232D Both mutant P205S and V232D alleles are indicated by the labelled blue peaks to the left of their respective wild type allele peaks labelled green peak 1600 1200 800 i as a l L meee d i ALY N 1_A41006E WT 3_A561E WT 4 1609deICA WT 5_2869insG WT 6_K710X WT 8 2184insd WT 9 712 16 _ T WT 11_ P2058 Mut 12_ 232D Mut 2_1812 1G_A WT 7_R709X WT 10_H199Y WT 12_ 232D WT 11 _P205S WT P2058 WT CFIBNBYEN 001 Jun 2015 Page 17 of 42 Elucigene CF Iberian Panel Products Instructions for Use Insertions and Deletions Due to the nature of the design of the Elucigene CF Iberian Panel the presence of insertions or deletions between two opposing primers will result in size changes to all of the amplicons produced between these two primers Therefore in addition to the 12 mutations detected by the CF Iberian Panel any insertions and deletions within the amplified target sequences can be detected by the change in expected amplicon size of the wild type green peaks These have been described in the Table of Insertions and Deletions whic
40. te f Use last template a es CFIBNBYEN 001 Jun 2015 Page 36 of 42 Elucigene CF Iberian Panel Products Instructions for Use Run Wizard Data Process 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 the figure below Figure 17 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 60bp size standard peak Figure 17 Run Wizard Data Process Window Run Wizard Data Process Fragment Animal Analysis Set data process options Ram Data Analysis Allele Call W Auto Range frame tal M Auto Range bps 0 e0000 oo Ww Smooth Enhanced Smooth Peak Detection Threshold W Peak Saturation v Baseline Subtraction Mir Intensity 100 Max Intensity 30000 Enh d Baseline Subtract LT ea eae eS eee Percentage gt Global Max We Pullup Correction w Spike Removal Local Region gt Local Max W Stutter Peak Filter 72 We Plus 4 Filter Left 30 Right 40 Size Call f Local Southern C Cubic Spline Large Size Load Default Save Default lt 4 Back Cancel Note For 3500 data increase the Minimum Intensity to 150 CFIBNBYEN 001 Jun 2015 Page 37 of 42
41. test function G194R G194V H199R H199Q P205R L233F Q493X Q493P Q493R 1612delTT R560S 1813insC R709Q 2868G gt A S912L 912X 11005R This list is neither exclusive nor exhaustive CFIBNBYEN 001 Jun 2015 Page 20 of 42 Elucigene CF Iberian Panel Products Instructions for Use GeneMapper Software Analysis Guide Introduction The following section describes the procedures for sample analysis of Elucigene CF lberian Panel results using Applied Biosystems GeneMapper software packages version 3 7 or greater Note screenshots in this document are taken from GeneMapper v5 Analysis Process The process for analysis of samples is summarised in the flow diagram shown below Run Amplified Samples on Genetic Analyzer Open GeneMapper Software Add Sample FSA Files to Project Select Appropriate Analysis Method Panel amp Size Standard Run Sample Analysis Save GeneMapper Project Select Sample amp Open Plot Window Review Trace amp Genotype Data Score Sample Genotypes Print Elucigene CF Iberian Panel Sample Traces CFIBNBYEN 001 Jun 2015 Page 21 of 42 Elucigene CF Iberian Panel Products Instructions for Use Importing CF Iberian Panel GeneMapper Analysis Settings Before first use of the kit it is necessary to import the CF Iberian Panel settings for GeneMapper This process is controlled through the GeneMapper Manager interface CF Iberian Panel GeneMapper settings are available from the Elucig
42. ution Markers Detected The table below summarises the markers detected by CF Iberian Panel mix Markers are listed according to the size range of PCR product observed Markers Detected Marker Marker Product bp Size Range Peak No 3130 POP7 data A1006E 145 154 1812 1G gt A 175 182 5 A561E 183 191 1609deICA 198 213 2869insG 228 233 K710X 261 268 R709X 270 277 2184insA 332 338 712 1G gt T 383 395 H199Y 400 408 P205S 415 423 V232D 496 505 Note Marker sizes may vary due to the instrument and polymer used CFIBNBYEN 001 Jun 2015 Page 16 of 42 8 Elucigene CF Iberian Panel Products Instructions for Use Examples of Interpretation Normal Result In a normal result all alleles detected are homozygous wild type which is indicated by the presence of labelled single green peaks for all 12 markers The STR markers should be unlabelled and the marker sizes pattern should be concordant with the marker sizes pattern of the CF EU2v1 A and B mixes for a given sample An example of a normal result is shown below tot MALLA aA 1_Al1006E WT 3_A561E WT 4 1609deICA WT 5_2869insG WT 6_K710X WT 8_2184isA WT 9_712 1G_T WT 11_P2058 WT 12_ 232D WT 2_1812 1G_ A WT 2 _R709X WT 10_H199Y WT Heterozygous Mutation The example below shows an 1812 1G gt A heterozygote The mutant 1812 1G gt A allele is indicated by the labelled blue peak to the left of the wild type A1006E allele labelled green peak In all cases the mutan
43. visualised as blue amplicon peaks and wild type alleles are visualised as green amplicon peaks The internal amplification control markers non cystic fibrosis found in CF EU2v1 are also included in the CF Iberian Panel mix These are visualised as red amplicon peaks and are used to monitor the efficiency of sample amplification and aid patient identification when running the CF Iberian Panel alongside the Elucigene CF EU2v1 assay CFIBNBYEN 001 Jun 2015 Page 4 of 42 Elucigene CF Iberian Panel Products Instructions for Use 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 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 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 In line 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 If kit box is damaged there may be damage to the contents do not use the kit contact Customer
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