SeCore. GSSP Kits. Instructions for Use. 1 Research Use Only

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1 SeCore GSSP Kits Instructions for Use 1

2 SeCore GSSP Kits Instructions for Use Invitrogen SeCore GSSP (Group Specific Sequencing Products) are designed to resolve ambiguous HLA heterozygous combinations, and are used in conjunction with SeCore Sequencing Kits. The reagents are for post-amplification sequencing only. For instructions on how to set up the locus specific genomic DNA amplification, users should refer to Sections 1-6 of SeCore Sequencing Kits Instructions for Use. Each SeCore GSSP test utilizes a group specific sequencing primer mix, Big Dye Mix, and Sequencing PPT Buffer. The ExoSAP-IT treated amplicon, prepared from the SeCore Sequencing Kit, is the template for GSSP reactions. The data generated from the GSSP is analyzed with the original data from the corresponding SeCore Sequencing Kit to determine the sequence result. This product is for. The performance characteristics of this product have not been established. In this manual: Section Description Page 1. Kit Components 3 2. Materials, Reagents, and Equipment not Supplied 3 3. Sample Preparation 4 4. Sequencing Reaction 4 5. Ethanol Precipitation of Sequencing Reaction Products 6. Electrophoresis of Sequencing Reaction Products 6 7. Data Analysis 6 8. Limitations and Cautions 7 9. Troubleshooting Licenses and Trademarks 9 5 2

3 1 Kit Components: Invitrogen SeCore GSSP kits are supplied as individual loci and/or groups. Class I products are designated by a Z number and Class II kits are designated as DR loci/groups. See specific kits for loci/group identification. Description Quantity Storage Sequencing Primer Mix 1 vial of 100 µl -20 C in a Non-Frost Free Freezer Big Dye Mix 1 vial of 100 µl -20 C in a Non-Frost Free Freezer Sequencing PPT Buffer 1 vial of 50 µl -20 C in a Non-Frost Free Freezer Note: Big Dye Mix containing Dye terminators is light sensitive and should be protected from light when stored and during usage. 2 Materials, Reagents, and Equipment not supplied: well thermal cycler with heated lid: SeCore Sequencing Kits have been tested on the following thermal cyclers: MJ Research PTC 225 DNA Engine Tetrad, Applied Biosystems Gene Amp 9600, Gene AMP 2700 and Gene AMP Automated DNA sequencer and accessories: SeCore Sequencing Kits have been tested on the Applied Biosystems ABI Prism 3100 sequencer. Use of other DNA sequencers requires validation by user ABI 3100 Users: 36-cm 3100 Capillary Array for ABI 3100, Applied Biosystems product code MicroAmp 96-well Tray/Retainer Set, Applied Biosystems product code MicroAmp 0.2-ml Reaction Tubes, Reaction Tubes (8 tubes/strip) and Caps (8 caps/strip), Applied Biosystems product code N , N and N MicroAmp Optical 96-Well Reaction Plate and 9600 Full Plate Cover, Applied Biosystems product code N and N Table top centrifuge with plate adapters for 96-well plates. The centrifuge must reach a force of 2500 x g 2.7 Pipettors and tips: 1-10 µl, µl, µl 2.8 Dispensing electronic pipettors: capable of dispensing µl aliquots. 3

4 2.9 Multichannel (8 or 12 channels) pipettors: µl adjustable volume 2.10 CoolSafe System to fit 0.2 ml tubes, Diversified Biotech product code CSAF Reagents for Capillary Sequencer ABI 3100: POP-6 polymer, Applied Biosystems product code Use of POP-4 and POP-7 polymers must be validated by user X Genetic Analyzer Buffer, Applied Biosystems product code Hi-Di Formamide, Applied Biosystems product code Absolute Ethanol, Sigma Aldrich product code L 2.13 Sequencer software: 2.14 Analysis Software For use with ABI 3100 ABI Prism Data Collection Software v1.1 or higher (Win NT) Sequencing Analysis Software v3.7 or higher (Win NT) Invitrogen utype SBT software, product code or (CE). 3 Sample Preparation: 3.1 Locus specific genomic DNA amplifications purified with ExoSAP-IT from SeCore Sequencing Kits can be used with this kit. Note: ExoSap-IT treated amplicons can be stored at 20ºC for up to one week. Note: Change pipet tips in between the pipetting of each sample and each different mix or reagent to prevent cross contamination. The same pipet tip may be used to dispense the same mix or reagent into multiple tubes provided the tip does not come into contact with genomic DNA or PCR product. If there is any question that this may have occurred, change the pipet tip to prevent contamination. 4 Sequencing Reaction: 4.1 Preparation of sequencing reactions: Note: Sequencing reaction mixtures should be kept as cold as possible, by either using the recommended CoolSafe system or ice. 4

5 4.1.1 For Class II reactions only, if the ExoSAP-IT treated amplicons are used, they have been diluted for the initial sequencing, therefore, no further dilution is needed. If they have not been diluted, dilute to 2x the amplicon volume For Class I and Class II reactions, add 2 µl of ExoSAP- IT treated PCR amplicons to the appropriate tube or well Mix the contents of the vials containing Big Dye mix and the Sequencing Primer Mix. Note: After mixing, the primer/big Dye vial should be stored at -20 C up to the expiration date on the kit label. 4.2 Sequencing profile for Class I & II: Add 8 µl of each Sequencing Primer/Big Dye mix to the appropriate tube or well Cover the tubes or plate, vortex briefly to mix and centrifuge the tubes or plate to get the liquid to the bottom of the wells Place the tubes or plate in the thermal cycler and run the profile described in Section 4.2. Step Number of Cycles Temperature Time 95 C 20 sec Cycle C 15 sec 60 C 60 sec Soak 1 4 C infinite The total reaction time is ~1.5 hrs. 5 Ethanol Precipitation of Sequencing Reaction Products: After cycle sequencing, an ethanol precipitation is performed to remove excess terminators. The instructions are identical for Class I and Class II sequencing reactions. 5.1 Add 2 µl of Sequencing PPT Buffer to each sequencing reaction mixture. 5.2 Centrifuge briefly to ensure all contents are mixed and at the bottom of the well. 5.3 Add 40 µl of 100% ethanol to each mixture. 5.4 Cover the plate and vortex well for about 60 seconds. Note: Make sure the contents of all tubes or wells are mixed thoroughly. 5.5 Centrifuge the plate in a centrifuge fitted with a plate-adaptor for 30 minutes at 2,000 x g or greater. 5

6 5.6 Remove cover, cover with a paper towel, and invert. Centrifuge with the paper towel briefly (< 1 min. at 500 x g) in the inverted position to remove as much liquid as possible. 5.7 Add 100 µl of 70% ethanol to the DNA pellets. Do NOT vortex the plate. 5.8 Centrifuge the plate for 5 min at 2,000 x g or greater. 5.9 Remove the supernatant by an inverted spin as in step 8.6. Note: If single tubes are used, centrifuge the tubes for 15 minutes for initial precipitation and 5 minute for washing at ~12,000 rpm. Remove the supernatant by a brief inverted-spin or aspiration. Note: DNA pellets can be stored at 20 C for a maximum of 7 days. 6 Electrophoresis of Sequencing Reaction Products 6.1 Preparation of Loading Samples Add 15 µl of Hi-Di Formamide (Applied Biosystems product code ) to each DNA pellet. Note: If single tubes are used up to this step, transfer the samples to an Optical 96-Well Plate (ABI product code N ). Use the sample layout suggested in sections Cover the plate and briefly centrifuge Denature the samples at 95 C for 2 minutes in a thermal cycler Centrifuge briefly to remove any air bubbles in the samples. Note: If air bubbles get into the capillary, they will cause damage to the capillary Place the plate in the sequencer autosampler. 6.2 Electrophoresis Conditions on an ABI Prism 3100 Capillary Sequencer Parameters Dye Set Mobility File Basecaller Run Module Injection Time Rum Time Settings E KB_3100_POP6_BDTv1.mob Kb.bcp RapidSeq36_POP6 10 seconds 1800 seconds 7 Data Analysis: 6

7 7.1 Refer to utype SBT software Instruction for Use to process the sequence files and create an HLA report. Note: It is strongly recommended that the most recent European Bioinformatics IMGT/HLA database ( allele database is implemented for sequence data analysis using utype. Data generated from the GSSP kit is analyzed together with the original data from the SeCore Sequencing Kit. 8 Limitations and Cautions 8.1 To ensure the best performance of SeCore GSSP Kits, use the products with the materials, reagents, and equipments recommended in Section 2 of this document. 8.2 The SeCore GSSP Kits are for research use only. The performance characteristics of this product have not been established. 8.3 These products are for professional use only. 8.4 GSSP primers may detect polymorphic sites from untargeted alleles. Any peaks detected from untargeted alleles generally exhibit peak heights less than 33% of the peak height produced by the targeted allele. 8.5 Best alignment of sequence data to the library is achieved with less than 300 bases of sequence. 9 Troubleshooting: Problems Possible Causes Solutions Excessive background (baseline noise) Poor or incorrect matrix Poor injection (ABI 3100) ABI 3100: Injection time set too high. ABI 3100: Repeat the spectral calibration and reinject samples. Reinject samples. ABI 3100: Reduce injection time and reinject. Signal strengths of are optimal. (Samples of poor quality may have lower signal strengths but may still be analyzed and typed. Some samples may have signals that are over 3000 and will not have excess background.) 7

8 Weak Signal Excessive Dye Blobs Poor sequencing reaction due to error in pipetting Incorrect mobility file was chosen- peaks will be shifted or will be on top of each other. Excess 100% EtOH was added. Sequencing reactions were not vortexed thoroughly after the addition of the Sequencing PPT Buffer and EtOH. Injection time needs to be increased. The sequencing PPT buffer was not added to the sequencing reactions prior to the addition of EtOH. Failure to wash the sequencing reactions with 70% EtOH. Poor sequencing reaction due to error in pipetting or weak amplification product. Failure to remove all of the remaining EtOH during the precipitation. EtOH used in wash step was too dilute Be sure that both the ExoSAP- IT treated PCR product and the correct sequencing mix are added and combined. Choose correct mobility file. Re-sequence. Check to be sure pipet is set at correct volume. Repeat sequencing reactions. Vortex thoroughly (50-60 seconds) after the addition of EtOH. Be sure all reactions are mixing. Repeat sequencing reaction(s) and increase injection time. Repeat sequencing reaction. Add the sequencing ppt buffer prior to the addition of EtOH. Repeat sequencing reactions without omitting the 70% EtOH wash step. Be sure that both the ExoSAP- IT treated PCR product and the correct sequencing mix are added and combined. In the case of a weak amplification, confirm the intensity of the amplification product by running an agarose gel. Repeat sequencing reactions. The centrifuge step of 500 x g is very important in removing all remaining ethanol in the reaction tubes. Repeat sequencing reactions and confirm that 70% EtOH is being used for the wash step. 8

9 Excessive Signal Strength ABI 3100: Injection time set too high. ABI 3100: Reduce injection time and reinject. Signal strengths of are optimal. (Samples of poor quality may have lower signal strengths but may still be analyzed and typed.) Random sequence failures Poor sequencing reaction due to error in pipetting Be sure that both the ExoSAP- IT treated PCR product and the correct sequencing mix are added and combined. 9

10 10 Licenses and Trademarks NOTICE TO PURCHASER: LIMITED LICENSE A license under the process claims of U.S. Patents; 151,507;5,171,534; 5,332,666; 5,242,796; 5,306,618; 5,366,860; 5, 821, 058 and or their foreign counterpart claims, has an up-front fee component and a running royalty component. The purchase price of this kit includes limited non-transferable rights under the running-royalty component to use only this amount of the product to practice the DNA sequencing process described in said patents when this product is used in a conjunction with an Authorized DNA Sequence Analysis instrument whose use is covered under the up-front fee component of these patents. No other rights are granted expressly, by implication, or by estoppel, or under any other patent rights owned or licensable by Life Technologies Corporation (Invitrogen Corporation). Further information on purchasing licenses to perform DNA sequencing may be obtained by contacting the Director of Licensing at Life Technologies Corporation, 5791 Van Allen Way Carlsbad CA NOTICE TO PURCHASER: LIMITED LICENSE The purchase price of this kit includes a limited, non-transferable right, non-exclusive license (without the right to resell, repackage, or sublicense) under the process claims of one or more U.S. Patents 5,800,996, 5,863,727, and 5,945, 526, and corresponding claims in foreign counterpart patents and patent applications to use this product solely with a Life Technologies (Applied Biosystems LLC) commercial automated DNA Sequencing machine that have been authorized under these patents by Life Technologies. No license is herby granted for the use of this kit or the reagents therein in any other automated sequencing machine. For information concerning the availability of additional licenses to practice the patented methodologies, Contact: Director of Licensing, Life Technologies Corporation, 5791 Van Allen Way Carlsbad CA NOTICE TO PURCHASER: ABOUT LIMITED LICENSE This kit is also sold pursuant to a limited sublicense from Amersham International plc under one or more U.S. Patent Nos. 5,498,523 and 5, 614,365;, and corresponding foreign patents and patent applications. The purchase of this kit includes a limited non-exclusive sublicense (without the right to resell, repackage, or sublicense) under such patent rights to use this reagent for DNA sequencing solely with a Life Technologies (Applied Biosystems LLC) commercial automated DNA Sequencing machine. No license is herby granted for the use of this kit or the reagents therein in any other automated sequencing machine. No other license is granted expressly, impliedly, or by estoppel. For information concerning the availability of additional licenses to practice the patented methodologies, Contact: Director of Licensing at Life Technologies Corporation Trademarks SeCore is a registered trademark of Life Technologies Corporation Invitrogen is a trademark of Life Technologies Corporation ABI PRISM, Applied Biosystems, BigDye are registered trademarks of Applied Biosystems LLC or its subsidiaries in the U.S and certain other countries. FastStart is a trademark of Roche Molecular Biochemicals ExoSap-IT is a trademark of USB Corporation utype is a registered trademark of Life Technologies Corporation All other trademarks are the sole property of their respective owners. PR038 Revision 06 Print 7/09 10

HARPS Instructions for Use Version No: 1.0 Issue Date: October 2017

HARPS Instructions for Use Version No: 1.0 Issue Date: October 2017. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or