ABA iplex Pro SMN1/TREC/KREC Panel Protocol

Transcription

1 ABA iplex Pro SMN1/TREC/KREC Panel Protocol This document gives the assay protocol for the Assays by Agena (ABA) iplex Pro SMN1/TREC/KREC Panel. The SMN1/TREC/KREC Panel is a DNA test used to detect SMN1 double deletions and T-cell receptor excision circle (TREC) levels, as well as kappa-deleting recombination excision circle (KREC) levels. For complete information on equipment and reagents required and setting up the experiment in the software, see the iplex Reagents User Guide. For detailed instructions on desalting and dispensing analyte and acquiring data, see the applicable instrument user guide. All user guides are available on AgenaCx.com. Contents Required Reagents, Equipment, and Software... 2 Assay Protocol... 3 A. Preparing Sample DNA Plate... 4 B. PCR Amplification... 4 C. SAP Reaction... 6 D. iplex Pro Extension Reaction... 7 E. Water Addition... 8 Analyte Desalting, Dispensing, and Data Acquisition... 9 Generating Reports... 9 A. Generating the SMN1_TREC_KREC Report... 9 B. Reading the SMN1_TREC_KREC Report USG-CUS-106 R01 1

2 Required Reagents, Equipment, and Software The SMN1/TREC/KREC Panel may be run in 96 and 384-well formats. The following items (Table 1) are required. Upon receipt, store the items as described in Table 1. Table 1. Required Agena Reagents for the SMN1/TREC/KREC Panel Materials Provided Number of Reactions Sufficient For Shipping Conditions Storage Temperature Storage Location (see Table 4) iplex Pro SMN1/TREC/KREC Panel SMN1/TREC/KREC PCR Primer SMN1/TREC/KREC Extend Primer 960 Dry Ice -10 to -25 C Lab Area 2 SMN1/TREC/KREC Competitor Mix iplex Pro Master Mix Reagent Kit iplex Pro Master Mix, PCR Mix iplex Pro Master Mix, SAP Mix iplex Pro Master Mix, SBE Mix Large: 3840 Small: 960 Dry Ice -10 to -25 C Lab Area 2 3-Pt Calibrant 3840 Dry Ice -10 to -25 C Lab Area 3 10x384: 3840 SpectroCHIP Arrays 10x96: 960 2x384: 768 Ambient Temperature Room Temperature Lab Area 3 2x96: 192 Clean Resin (28 g or 40g) varies Ambient Temperature Room Temperature Lab Area 3 (paraffincover Clean Resin) In addition, the items in Table 2 are required for the SMN1/TREC/KREC Panel. For complete information on recommended lab set up and recommended vendors for running panels on the MassARRAY System, see MassARRAY System: Recommended Lab Equipment and Set-up, available on AgenaCx.com (Product Support > Applications & Systems > MassARRAY System). USG-CUS-106 R01 2

3 Table 2. Additional Reagents and Equipment Required Item Recommended Suppliers DBS Wash buffer Extracta DBS, Quantabio, cat. no Eppendorf MixMate Mixer Eppendorf ThermoMixer C Orbi-Shaker MP Table 3. Required Software Software Source MassARRAY Typer v or higher Agena Bioscience (download from AgenaCx.com: Product Support > Software > Typer) SMN1/TREC/KREC Report v or higher Agena Bioscience (download from AgenaCx.com: Product Support > Applications and Systems > Hereditary Genetic Testing) R Environment, including RODBC, doby, gap, and RSQLite packages) MassARRAY Analyzer software RT Workstation v3.7 or higher See MassARRAY Typer Release Notes on AgenaCx.com (Product Support > Software > Typer) for instructions for downloading the R software and installing the necessary packages. Agena Bioscience (part of MassARRAY Analyzer and MassARRAY System with Chip Prep Module) Chip Prep Control v1.6 or higher (if using MassARRAY System with Chip Prep Module) Assay Protocol Table 4. Lab Area Activities Lab Area Activities 1 Isolation and dilution of DNA and preparation of the DNA plate. Pre-PCR preparation, including preparation of the PCR reaction plate, preparation of PCR cocktail, and addition 2 of PCR cocktail to the reaction plate. Preparation of the SAP and extension cocktails. Thermocycling the reaction plate after addition of PCR cocktail; addition of the SAP cocktail to the reaction plate 3 and thermocycling; addition of the extension reaction cocktail to the reaction plate and thermocycling; desalting; nanodispensing; and data acquisition. USG-CUS-106 R01 3

4 A. Preparing Sample DNA Plate IMPORTANT Perform this procedure in Lab Area 1. The SMN1/TREC/KREC Panel requires at least 6 ng of DNA input per well. It has been developed for use with dried blood spots (DBS) as template, specifically one appropriately-washed 2 mm punch per well. A DNA dilution may also be used, for example for controls for SMN1. Control DNA may be purchased from Coriell (e.g., Coriell catalog no. NA00232 [0 SMN1, 1+ SMN2], or NA03814 [1 SMN1, 1+ SMN2]). Prepare a dilution of DNA of at least 3 ng/µl; 2 µl of this will be used for each well. 1. For sample wells: Prepare a 2 mm DBS punch in each sample well. a. Punch a 2 mm DBS disc directly into a sample well in a new MTP plate. b. Add 100 µl of wash buffer to each well. c. Cover the plate with adhesive film and centrifuge to submerge disc. d. Shake plate at 1500 RPM for 20 minutes at room temperature. e. Briefly centrifuge the plate to bring contents to bottom of wells. f. Remove the adhesive film. g. With a multichannel pipette and filtered tips, remove and discard wash buffer, changing tips with each sample. Take care to leave the washed DBS disc (now almost white) in the plate. h. Add 2 µl HPLC-grade water to each sample well. 2. For any positive control wells, dispense 2 µl of DNA dilution (at least 3 ng/µl). 3. Visually inspect the individual wells from the bottom of the plate to confirm uniform and adequate DNA sample is present in every well before continuing. 4. Seal the plate and centrifuge at 2,000 x g for one minute. 5. Proceed immediately to PCR amplification. B. PCR Amplification IMPORTANT Prepare the PCR cocktail and add to the reaction plate in Lab Area 2. Thermocycle the PCR reaction plate in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure reagents are homogenized before taking aliquots. USG-CUS-106 R01 4

5 1. Prepare the PCR cocktail in a 1.5 ml tube placed on ice by adding reagents in the order in which they are listed in Table 5. Prepare more cocktail than the number of PCR reactions to be performed. Either prepare for one extra reaction or use a percentage extra to ensure sufficient overage is present to overcome typical pipetting variation. Table 5: PCR Reaction Reagent Per Well (µl) Final Concentration HPLC-grade water 6.48 n/a PCR Master Mix X SMN1/TREC/KREC PCR Primer nm SMN1/TREC/KREC Competitor Mix 1.0 1X PCR Cocktail Final Volume 13.0 DNA 2.0 PCR Reaction Final Volume 15.0 Note: The SMN1/TREC/KREC Competitor Mix is used for quantification of Albumin QC, TREC, and KREC levels and should only be used at the indicated volume to ensure proper quantification. 2. Vortex the tube for 3 seconds and briefly centrifuge. 3. Dispense 13 µl PCR cocktail into each sample well of the sample plate, for a final PCR reaction volume of 15 µl. 4. Seal the PCR reaction plate, pulse vortex 5 times, then centrifuge at 3200 x g for 5 seconds. 5. Visually inspect the individual wells from the bottom of the PCR reaction plate to confirm uniform and adequate cocktail solution is present in every well before continuing. 6. Thermocycle the PCR reaction plate using the following conditions: 95 C 20 minutes* 1 cycle Initial Denaturation 95 C 30 seconds 60 C* 30 seconds 45 cycles Amplification 72 C 1 minute 72 C 5 minutes 1 cycle Final Extension 10 C Hold *Note differences from typical iplex Pro themocycling conditions. The initial denaturation stage has been optimized for use with DBS and allows for release of DNA from the filter paper and successful PCR amplification. USG-CUS-106 R01 5

6 STOPPING POINT If not proceeding directly to the next step, the reaction plate should be sealed, and stored at 4 C (if storing for less than 24 hours), or at -20 C (if storing for more than 24 hours). Do not store for more than 2 weeks. C. SAP Reaction IMPORTANT Prepare the SAP cocktail in Lab Area 2. Add the SAP cocktail to the reaction plate and thermocycle the plate in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure all reagents are homogenized before taking aliquots. If plates were stored frozen prior to this step, make sure they are thawed completely at room temperature and spun down. 1. Centrifuge the PCR reaction plate at 3200 x g for 5 seconds. 2. Transfer 5 µl from the PCR reaction plate to a new plate. You may keep the remaining PCR analyte in the PCR reaction plate for later use, stored as described in the Stopping Point at the end of this section. 3. Prepare the SAP cocktail in a 1.5 ml tube on ice as shown in Table 6. Prepare more cocktail than the number of SAP reactions to be performed. Either prepare for one extra reaction or use a percentage extra to ensure sufficient overage is present to overcome typical pipetting variation. Table 6: SAP Cocktail Reagent Per Well (µl) Final Concentration HPLC-grade water 1.7 n/a SAP Master Mix X Final Volume SAP Cocktail Vortex the tube for 3 seconds and briefly centrifuge. 5. Dispense 2 µl of SAP cocktail into each sample well of the new reaction plate. 6. Seal the reaction plate, pulse vortex 5 times, then centrifuge at 3200 x g for 5 seconds. 7. Visually inspect the individual wells from the bottom of the reaction plate to confirm uniform and adequate solution is present in every well before continuing. 8. Thermocycle the reaction plates using the following conditions: 37 C 40 minutes 85 C 5 minutes 10 C Hold STOPPING POINT USG-CUS-106 R01 6

7 If not proceeding directly to the next step, the reaction plate should be sealed, and stored at 4 C (if storing for less than 24 hours), or at -20 C (if storing for more than 24 hours). Do not store for more than 2 weeks. D. iplex Pro Extension Reaction IMPORTANT Prepare the extension reaction cocktail in Lab Area 2. Add the extension reaction cocktail to the reaction plate and thermocycle the plate in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure all reagents are homogenized before taking aliquots. If plates were stored frozen prior to this step, make sure they are thawed completely at room temperature and spun down. 1. Prepare the extension cocktail in a 1.5 ml tube on ice, as shown in Table 7. Prepare more extension cocktail than the number of extension reactions to be performed. Either prepare for an extra reaction or use a percentage extra to ensure sufficient overage is present to overcome typical pipetting variation. Table 7. Extension Cocktail Reagent Per Well (µl) Final concentration HPLC-grade water 1.0 n/a Extend Master Mix X SMN1/TREC/KREC Extend Primer 0.8 n/a Extension Cocktails Final Volume Vortex the tube for 3 seconds and briefly centrifuge. 3. Centrifuge the reaction plate at 3200 x g for 5 seconds. 4. Dispense 2 µl of extension reaction cocktail into each well of the reaction plate. 5. Seal the reaction plate, pulse vortex 5 time, then centrifuge at 3200 x g for 5 seconds. 6. Visually inspect the individual wells from the bottom of the reaction plates to confirm uniform and adequate solution is present in every well before continuing. USG-CUS-106 R01 7

8 7. Thermocycle the reaction plate using the following conditions. 95 C 30 seconds 95 C 5 seconds 52 C 5 seconds 80 C 5 seconds 72 C 3 minutes 10 C Hold 5 cycles 40 cycles STOPPING POINT If not proceeding directly to the next step, the reaction plate should be sealed, and stored at 4 C (if storing for less than 24 hours), or at -20 C (if storing for more than 24 hours). Do not store for more than 2 weeks. E. Water Addition 1. Add HPLC-grade water to each well of the reaction plate using a 12-channel multipipettor. a. For 96-well plates, add 41 µl. b. For 384-well plates, add 16 µl. 2. Seal the plate and centrifuge at 3200 x g for 1 minute. STOPPING POINT If not proceeding directly to processing the plate on the MassARRAY System, the reaction plate should be sealed, and stored at 4 C (if storing for less than 24 hours), or at -20 C (if storing more than 24 hours). Do not store for more than 2 weeks. USG-CUS-106 R01 8

9 Analyte Desalting, Dispensing, and Data Acquisition Follow the instructions in the appropriate instrument user guide to desalt and dispense analyte onto SpectroCHIP Arrays and to acquire data with the MassARRAY Analyzer. Use settings for iplex Pro genotyping. Generating Reports A. Generating the SMN1_TREC_KREC Report 1) Open MassARRAY TyperAnalyzer and in the Project Explorer pane double click on the SpectroCHIP Arrays of interest. The SpectroCHIP Arrays will be added to the Chip List. 2) Load the SpectroCHIP Arrays by checking the box next to the SpectroCHIP Array names in the Chip List. 3) Once the SpectroCHIP Arrays are loaded, select File > Reports > SMN1_TREC_KREC Report-v1 in the MassARRAY TyperAnalyzer menu bar. 4) The SMN1_TREC_KREC Report Settings window will appear (Figure 1). You must select the panel version, specify the mutation in your positive controls, specify common names for control samples, and designate a minimum per sample call rate. See Table 8 for more information. Figure 1. SMN1_TREC_KREC Report Settings Window USG-CUS-106 R01 9

10 Table 8. SMN1_TREC_KREC Report Analysis Settings Setting Description SMN1_TREC_KREC Panel Name of the specific panel being run, either SMN1_TREC or version SMN1_TREC_KREC. Positive Control Samples 1) Enter the common name chosen for your positive control samples. 2) Enter the mutation(s) in the positive control samples. If more than one, separate each entry by a semicolon. The selected mutations should match the mutations associated with the genotyping assays in the panel definition file (SMN1_TREC_KREC_Lookup_Table.csv, located at Typer/bin/Reports/ SMN1_TREC_KREC Report/Panels/PanelName). For example: Negative Control Samples NTC Samples Per Sample Call Rate Enter the common name chosen for your negative control samples. Negative control samples should be negative for SMN2, above threshold for TREC and KREC, and positive for albumin. Enter the common name chosen for your NTC samples. This specifies the threshold assay call rate in order for a sample to pass QC. The recommended default is 0.8 (i.e., a sample with an assay call rate of below 80% will fail QC). 5) Click Run Analysis. The following message will appear. 6) When the report is complete, the results will be made available in a date-and time-stamped folder in the Typer/bin/TyperReports/SMN1_TREC_KREC Report folder. The PDF containing the Run Summary and Sample Results reports will open automatically. USG-CUS-106 R01 10

11 B. Reading the SMN1_TREC_KREC Report The SMN1_TREC_KREC Report folder contains: The SMN1_TREC_KREC Report, in both PDF and CSV formats. These reports, for each sample, the SMN1 double deletion status, TREC level status, and, if the KREC panel was run, the KREC level status, as well as QC results. The Detailed SMN1_TREC_KREC Report in CSV format. This more detailed report provides, in addition to the data above, the results of each assay for each sample. A Details folder with all the raw data from the Typer software. SMN1_TREC_KREC Report This report is provided in both PDF (SMN1_TREC_KREC _report_pdf.pdf) and CSV (SMN1_TREC_KREC _report_.csv) formats. The PDF version will automatically open after the report is run. It provides: 1) A summary of the total number of samples that passed and failed QC, followed by the numbers of NTCs, negative controls, and positive controls that passed and failed QC. USG-CUS-106 R01 11

12 2) For each sample, a report with the sample results, including QC messages. Specifically each Sample Report lists: Sample location (well and plate). SMN1 Double Deletion Status: Either Detected, Not Detected, Indeterminate, or No Call. All three SMN assays must give the same result in order for a call of Detected or Not Detected to be made. If all three SMN assays do not agree, a result of Indeterminate will be given. If the sample fails QC, a result of No Call will be given. TREC Level Status and KREC Level Status (if KREC panel was run): Either Above Threshold (> 40 copies detected), Indeterminate (20-40 copies detected), Below Threshold (< 20 copies detected), or No Call (if the sample failed QC). The exact number of copies detected is reported, and the allele frequency is also included in the CSV version of the report. Control Assays Status: For samples, the result will be Passed (if albumin assay detects 1900 or more DNA copies) or No Call (if < 1900 copies were detected). NTC samples should have a result of Failed (0 copies). USG-CUS-106 R01 12

13 QCLabel: QC status (PASS or FAIL). o o o o Samples will fail if the Control Assays are labeled No Call. Samples will fail if the sample call rate falls below the minimum specified in the SMN1_TREC_KREC Report Settings window (default 0.80). Positive control samples will fail if the expected mutation, as entered in the SMN1_TREC_KREC Report Settings window, was not detected, or if unexpected mutations were found. Negative control samples will fail if a mutation was found. o NTC samples will fail if there is assay extension above a default threshold (10%). QC message(s): The following QC messages may be reported: Type Message Control assay Albumin_QC had an extension rate below the required threshold. Control assay Albumin_QC had a product peak intensity below the required threshold. Control assay Albumin_QC did not produce any significant peaks. Control assay Albumin_QC did not detect a sufficient number of DNA copies to proceed (XX < 1900). Call rate of 0.XX is below the minimum of 0.8. On positive control, extension of [assays] are not expected but relevant peaks were observed. On positive control, extension of [assays] are expected but relevant peaks were not observed. On negative control, extension of [assays] are not expected but were observed. On NTC, extension of [assays] were not expected but were observed. Sample Warning Sample could not be called for SMN1 Double Deletion due to the failure of all relevant assays. Sample Warning Sample could not be called for SMN1 Double Deletion due to contradictory assay outcomes. Sample Warning TREC [or KREC] Level Assay produces an indeterminate number of DNA copies. If not done so already, sample needs to be repeated. Sample Warning TREC [or KREC] Level Assay provided a "No call". Detailed SMN1_TREC_KREC Report This report (SMN1_TREC_KREC _report_detailed.csv) gives, for every sample on the selected plates, results for each assay for that sample. USG-CUS-106 R01 13

14 For the sample: Location (well and plate). SMN1 Double Deletion Status TREC Level Status KREC Level Status Control Assay Status QC Label and Messages For each assay for each sample: Mutation name Assay name Outcome o For SMN1 assays: MUTATION, WT, or NO_CALL. o Location For TREC, KREC, and Albumin_QC assays: The allele frequencies and number of copies of the wild-type allele are reported, followed by the frequency of the mutant allele. No Call will be reported if there were no qualified peaks (peaks that meet or exceed the minimum intensity and probability thresholds). USG-CUS-106 R01 14

15 Patents and Trademarks MassARRAY, iplex, SpectroCHIP, and Agena Bioscience are registered trademarks of Agena Bioscience, Inc. All other trademarks or service marks set forth herein are the property of their respective owners. Agena Bioscience s patented nucleic acid analysis by mass spectrometry methods and products are protected under United States patent rights including but not limited to; 6,440,705; 6,558,623; 6,730,517; 6,979,425; 6,994,969; 7,019,288; 7,025,933; 7,332,275; 7,390,672; 7,501,251; 7,888,127; 7,917,301; 8,003,317; 8,315,805; 8,349,566; 9,249,456; 9,310,378; 9,394,565; 9,669,376; and 9,896,724, and patents pending including but not limited to US , and foreign counterparts including but not limited to EP B1, EP B1, EP B1, EP B1, EP B1, EP B1, and EP B1. [0818] The MassARRAY System and the iplex Pro SMN1/TREC/KREC Panel are For Research Use Only. Not for use in diagnostic procedure. 10/18/18 USG-CUS-106 R01 15