AllType FASTplex NGS 11 Loci Flex Kit - 96

Product definition

Official Product Name AllType™ FASTplex™ NGS 11 Loci Flex Kit – 96 Catalog Information Catalog ID ALL-FAST11LF Intended Use AllType™ FASTplex™ NGS HLA Typing products are for in vitro diagnostic use and professional use of HLA typing for HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, - DQB1, -DPA1, and -DPB1 loci. Intended Purpose AllType FASTplex Flex NGS HLA Typing products are for in vitro diagnostic use, by trained Healthcare professionals, for HLA typing for HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, and -DPB1 loci using next generation sequencing by synthesis, Cyclic Reversible Termination (CRT) for transplant diagnostics. The AllType FASTplex Flex NGS HLA Typing products are qualitative tests for amplification and library preparation and sequencing of DNA extracted from whole blood or buccal swabs. The test is not automated, and for use in screening and determination of the safety and compatibility with potential transplant recipients. Product Overview AllType FASTplex NGS HLA Flex Typing products include reagents to enrich the 11-loci HLA genes for HLA-A, B, C, DRB1, DRB345, DQB1, DPB1, DPA1, and DQA1 by a long-range multiplex polymerase chain reaction (PCR) method, purify the amplification products, and construct libraries containing up to 96 samples for sequencing on the Illumina systems AllType FASTplex NGS HLA Flex Typing products consist of pre-optimized HLA gene amplification primer solution in aqueous liquid format, PCR enzyme, 5X reaction buffer, and deoxyribonucleotides (dNTP) provided at recommended storage temperature at -20°C. The amplification primer design is based on the known DNA sequences reported in public DNA sequence databases such as Genbank (http://www.ncbi.nlm.nih.gov/genbank/) and IMGT (http://www.ebi.ac.uk/ipd/imgt/hla/). AllType FASTplex NGS HLA Flex Typing products provide a 96-well plate containing unique Sample Barcoding reagents in each well and a single Universal Barcoding reagent (see Product Identification). When combined, a user can prepare a library for 8 to 96 samples with unique i7 barcodes and a common i5 sequence for subsequent library amplification. The library amplification primer mix included in the kit identifies the P7 and P5 common sequences at the ends of DNA fragments and amplifies all properly barcoded library fragments. Additional reagents needed throughout library preparation, including barcoding buffer, stop solution, and paramagnetic beads are also provided in the kit. Throughput for AllType FASTplex NGS HLA Flex Typing Products with Illumina Sequencers Illumina Number of Samples Sequencer Illumina Reagent Kit Maximum Throughput MiSeq Reagent Kit v2 (300 cycle) 96 MiSeq MiSeq Reagent Micro Kit v2 (300 cycle) 24 MiSeq Reagent Nano Kit v2 (300 cycle) MiSeq i100 Series 25M Reagent Kit (300 cycle) 96 *MiSeq i100 Series MiSeq i100 Series 5M Reagent Kit (300 cycle) 24 MiniSeq High Output Kit (300 cycles) 96 MiniSeq MiniSeq Mid Output Kit (300 cycles) 72 iSeq iSeq 100 i1 Reagent v2 (300 cycles) 24 *Includes both instruments the MiSeq i100 or MiSeq i100 Plus Principles of the Method Typing of Human Leukocyte Antigen (HLA) genes in the major histocompatibility complex (MHC) in humans is an essential diagnostic test for organ and bone marrow transplantation, various disease associations, and pharmacogenetics to screen for drug hypersensitivity [2]. Due to the high diversity of [1] polymorphisms and homologous sequences in HLA genes, next generation sequencing (NGS) technologies that allow for clonal sequencing of full, or near-full, genes are becoming a necessary method for providing the highest resolution typing results. The AllType FASTplex NGS product generates target-specific amplification products for multiple HLA genes in a single multiplex polymerase chain reaction (PCR). DNA sequencing data for HLA typing are obtained by processing the amplified product with a fragmentation library workflow to prepare a library that can be sequenced using Illumina sequencer platforms. The AllType FASTplex NGS 11 Loci Flex Kit has been designed and optimized to produce nearly equimolar quantity of each target to provide adequate sequence coverage. Resulting data are analyzed using TypeStreamTM Visual NGS Analysis Software to generate high resolution HLA allele assignments. WARNINGS WARNING: Refer to the Safety Data Sheet for detailed information. Individual Safety Data Sheets can be downloaded at www.onelambda.com and/or www.thermofisher.com. MATERIALS PROVIDED Package Contents and Storage The AllType FASTplex NGS 11 Loci Flex Kit is comprised of two separate boxes: The AllType FASTplex NGS 11 Loci Flex Kit – 96 (Box 1 of 2) will be stable until the expiry date when stored unopened at -20°C. Once opened, this product will be stable for up to 12 freeze thaws or until expiry, whichever comes first, if handled properly and stored tightly capped at -20°C. Store product upright when not in use. This product is shipped under dry ice conditions. The AllType FASTplex NGS Kit (Box 2 of 2) will be stable until the expiry date when stored unopened at +2 to 8°C. Once opened, the FAST-BEAD and FAST-SUSP will be stable until expiry if handled properly and stored tightly capped at +2 to 8°C. Once opened, the FAST- STOP and FAST-BBUF will be stable until expiry if handled properly and stored tightly capped at +20 to 25°C. Store product upright when not in use. This product is shipped under refrigerated conditions. CAUTION: Upon receipt, keep each box intact and stored until ready to use. Avoid unnecessary handling. A full list of the provided materials and their appropriate storage conditions can be found in the Components Provided in the AllType FASTplex NGS 11 Loci Flex Kit section. The product label also contains individual component storage conditions. Components Provided in the AllType FASTplex NGS 11 Loci Flex Kit Box 1 of 2 Component Catalog ID Quantity Amount (µL)

Storage

AllType FASTplex 11 Loci Primer Flex Mix ALF-P11F 1 vial 600 AllType FASTplex 11 Loci Exon 1 Primer Flex Mix ALF-P11FE1 1 vial 240 AllType dNTPs ALL-NTP 1 vial 200 AllType Buffer ALL-BUF 1 vial 500 AllType LR Polymerase ALL-LRPOL 1 vial 100 –20°C FASTplex Sample Flex Plate 96 FAST-SFP96 1 vial 6 per well FASTplex Univ Barcode Flex A FAST-UBFA 1 vial 48 FASTplex Library Primer Flex Mix FAST-LPFM 1 vial 100 FASTplex Library Amp Mix FAST-LAM 1 vial 900 Box 2 of 2 Component Catalog ID Quantity Amount (µL) FASTplex Barcoding Buffer FAST-BBUF 1 vial 1500 +2 to 25°C FASTplex Stop Solution FAST-STOP 2 vials 1200 per vial FASTplex Paramagnetic Beads FAST-BEAD 1 vial 7,700 +2 to 8°C FASTplex DNA Suspension Buffer FAST-SUSP 1 vial 8,000 Instability Indications MATERIALS – REQUIRED but NOT PROVIDED Supplies and Consumables Description Supplier Catalog No. 96-Well, 0.2 mL, PCR Plates MLS* 96 Well Plastic/Foil Plate Seals MLS Thermo Scientific Nunc™ 96-Well Polypropylene MicroWell™ Plates Fisher Scientific 12-565-369 PCR Plate & 0.2 mL Tube Cooler MLS Eppendorf DNA LoBind Tubes, 5.0 mL Eppendorf 0030108310 Eppendorf DNA LoBind Tubes, 2.0 mL Eppendorf 022431048 PCR tube 0.2 mL MLS 50mL Conical Tubes Nuclease free MLS 15mL Conical Tubes Nuclease free MLS Serological Pipettes, 10 or 25 mL MLS Pipette Controller MLS Full array of manual single channel pipettes (20 µL, 200 µL, 1 mL) MLS 20 µL and 200 µL manual multi-channel pipettes MLS Full array of filtered, pre-sterilized, pipette tips MLS Sterile Reagent Reservoirs MLS Strip Tubes / Caps MLS Thermal Cycler PCR Pressure Pad MLS PhiX Control Kit Illumina FC-110-3001 MiSeq Reagent Kit v2 (300 cycle) Illumina MS-102-2002 MiSeq Reagent Nano Kit v2 (300 cycle) Illumina MS-103-1001 MiSeq Reagent Micro Kit v2 (300 cycle) Illumina MS-103-1002 iSeq 100 i1 Reagent v2 (300 cycles) Illumina 20031371 MiniSeq High Output kit (300 cycles) Illumina FC-420-1003 MiniSeq Mid Output kit (300 cycles) Illumina FC-420-1004 MiSeq i100 Series 5M Reagent Kit (300 cycles) Illumina 20126565 MiSeq i100 Series 25M Reagent Kit (300 cycles) Illumina 20126568 *MLS: Major Laboratory Supplier EQUIPMENT – REQUIRED but NOT PROVIDED Equipment Description Supplier Catalog No. TypeStream™ Visual NGS Analysis Software (Version 2.0.1 or One Lambda. Inc. TSVPGR higher) iSeq® 100 System Illumina, Inc FS10000717 MiniSeq® System Illumina, Inc MN01037 MiSeq® System Illumina, Inc M01817 MiSeq i100 Illumina 20115694 MiSeq i100 Plus Illumina 20115695 Magnetic-Ring Stand (96 well) Thermo Fisher Scientific AM10027 or AM10050 2.0 mL Tube Magnet Stand Thermo Fisher Scientific 12321D Veriti 96-Well Thermal Cycler* Thermo Fisher Scientific 4375786 Qubit® Fluorometer or equivalent Thermo Fisher Scientific Q33216 or Q33226 Qubit dsDNA HS Assay Kit Thermo Fisher Scientific Q32854 Qubit Assay Tubes Thermo Fisher Scientific Q32856 Plate Centrifuge capable of 1,500 x g MLS** Mini/Micro Centrifuge MLS Vortex MLS *The Veriti 96-Well Thermal Cycler may be substituted with a thermal cycler capable of temperature settings optimized for the PCR reaction – 0.8 °C per second heating rate and 1.6°C per second cooling rate – and reaction volumes of 100 µL. **MLS: Major Laboratory Supplier

Precautionary statements

Wear gloves and appropriate PPE CAUTION: Follow clean bench work techniques to reduce the risk of contamination. Clean the workbench thoroughly with a DNA-removing agent (e.g., DNA Away™, Termi-DNA-Tor, 10% bleach followed by 70% alcohol, or equivalent) to reduce the risk of sample contamination. While working at the bench, wipe gloves frequently with a DNA-removing agent to reduce the risk of sample and reagent cross-contamination. Alternatively, change gloves frequently. IMPORTANT GUIDELINES General Laboratory Practice Use of manual multi-channel pipettes is recommended. Single channel pipettes are only recommended for workflows containing a small quantity of samples. All instruments and pipettes should be calibrated and maintained per the manufacturer’s guidelines. Technical and Equipment Use of dedicated equipment in both the pre- and post-PCR environment is advised. For convenience, set up all the thermal cycler programs before starting. Use the Veriti 96-Well Thermal Cycler or a model capable of 9600 emulation speed or +0.8°C/sec heating and -1.6°C/sec cooling and a heated cover at 105°C or equivalent for selected programs. If a thermal cycler other than the Veriti-96 will be used, validate the thermal cycler. If a quantification method other than a Qubit™ Fluorometer will be used, validate the quantification method. Reagents The items in Box 1 of 2 of the kit should be kept on ice during use and returned to -20°C immediately after use. Store pre-amplification (Box 1 of the AllType FASTplex NGS 11 Loci Flex Kit) and post-amplification reagents separately. Master mix preparation and dispensation should be done on ice and at a reasonably fast pace to avoid unintended results. Use FASTplex DNA Suspension Buffer for all gDNA and amplicon dilution and library elution. Do not use EDTA-containing solutions (e.g., TE buffer or Low TE buffer) post amplification, as it may inhibit the reactions in this assay. See below, genomic DNA may be diluted in Low (0.1mM or below) EDTA DNA Suspension Buffer. Safe Stopping Point For optimal results, perform all the library preparation steps in one day. But if more time is needed, look for the SAFE STOPPING POINT to store an intermediate material at -20°C. Due to an unknown amount of left-over enzyme activities, do not to keep the materials for more than 72 hours at -20°C; longer storage time would require validation. SAMPLE COLLECTION and SAMPLE PREPARATION Sample Type Specimens, including anticoagulated peripheral blood, cultured lymphocytes, and buccal epithelial cells, which have been validated using the AllType FASTplex NGS 11 Loci Flex Kit. It is recommended that samples be processed within 2 to 3 days of collection (4, 5). DNA may be kept at the storage condition and length as validated by the individual laboratory. Sample Storage Whole blood must be collected in ACD or EDTA anticoagulated tubes. Buccal swab must be collected using swabs that are validated for epithelial cell collection and should be stored in a sealed container to avoid excessive drying. DNA Extraction Methods The following extraction methods have been validated with the AllType FASTplex NGS 11 Loci Flex Kit: Maxwell 16 Blood DNA Purification Kit (Promega Corporation, Cat No. AS1010) QIAamp DNA Blood Mini Kit (QIAGEN, Cat No./ID: 51104) Automated MagMAX™ DNA Multi-Sample Ultra Kit (Thermo Fisher Scientific, Cat. No. A25597) using KingFisher Flex Purification System (Thermo Fisher Scientific, Cat. No. 5400630, 5400610). Other DNA extraction methods can be used, but the methods and equipment must be validated by the end-user. Make sure the samples are free from any inhibitors of DNA polymerase. Do not resuspend the samples in solutions containing chelating agents, such as EDTA, above 0.1 mM in concentration. Resuspend the DNA samples to be used for PCR in Low (below 0.1mM) EDTA DNA Suspension buffer, sterile water or in 10 mM Tris-HCl, pH 8.0 at an optimal concentration of 25 ng/µl with a fluorometric method such as Qubit Fluorometer and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Other specifications can be used but must be validated by the laboratory. IMPORTANT: Only in the dilution of the genomic DNA can the above reagents be used. In all subsequent steps, when DNA Suspension Buffer is listed, only the FASTplex DNA Suspension Buffer can be used. DNA Storage Use DNA samples immediately after isolation or store DNA at -20°C or below. Ship DNA samples at 4°C or below to preserve their integrity during transport. Extracted DNA has been validated that has been stored up to 14 days using the AllType FASTplex NGS 11 Loci Flex Kit. Additional DNA storage may be validated by the individual laboratory. WORKFLOW for the ASSAYS Total time 200 minutes; hands on time 50 minutes HLA 11-Loci Amplicon AMPLIFICATION Amplicon Multiplex Purification Quantification Dilution Amplification Total time 100 minutes; hands on time 20 minutes Sample SAMPLE Sample Barcoding Reaction Stop Purification BARCODING Reaction Pooling Universal Purification Reaction Stop Barcoding Reaction Total time 65 minutes; hands on time 5 minutes Library LIBRARY Final Library Amplification AMPLIFICATION Reaction Purification Quantification Total time 20 minutes; hands on time 15 minutes PREPARATION for ILLUMINA PLATFORM Sample Sheet Pool Library Preparation SEQUENCING: MiSeq, MiSeq i100 Series MiniSeq, or iSeq100 GENERAL PREPARATION for the ASSAYS 1. Separate pre- and post-PCR areas. 2. Gather the following items and reagents so that they are accessible throughout the workflow: A full range of filtered pipette tips Single and multi-channel pipettes 96-Well 0.2 mL PCR Plates Eppendorf LoBind® tubes 0.2 mL PCR tubes Magnetic racks for a 96-well plate and a 2.0 mL tube Nuclease-free water Ice PCR plate cooler Tube-labeling supplies FASTplex DNA Suspension Buffer 200-proof Ethanol Sterile reagent reservoirs 3. Resuspend DNA samples in FASTplex DNA Suspension Buffer, Low (below 0.1mM) EDTA DNA Suspension buffer, sterile water, or in 10 mM Tris-HCl, pH 8.0 at an optimal concentration of 25 ng/µl quantified with a fluorometric method such as Qubit Fluorometer and Qubit dsDNA HS Assay Kit. Optical density (OD) methods should not be used to measure DNA concentration; the readings could be overestimated. An acceptable range of genomic DNA concentrations is 3.00 ng/µL to 50 ng/µL. The expected A260/A280 ratio is in the range of 1.65 - 1.80, if optical density (OD) method is used to measure DNA quality. 4. Record each sample location, the volume of the FASTplex Sample Flex Plate 96 (Refer to Appendix 2 for a diagram of the plate.), and the FASTplex Univ Barcode Flex A (UB) reagent to be used for library prep. 5. Before starting the procedure, label the PCR 8-tube strip(s) or 96-well plate clearly. 6. Keep track of which wells from the FASTplex Sample Flex Plate 96 and how much UB reagent has been consumed in the library preparation. Users may refer to the plate grid at the end of this document. 7. Do not discard reagents in the kit until empty since they are intended for multiple uses. 8. Avoid contamination of all kit reagents/components. 9. Return the unused portions to storage at the temperature indicated on the label. 10. Take precautions to avoid well-to-well cross-contaminations. The FASTplex Sample Flex Plate supplied in the kit contains enough volume to prepare a 96-sample library once or up to twelve 8-sample libraries. The FASTplex kit is also compatible with any library size between 8 and 96 samples. The plate is designed to allow for columns of barcodes (wells) to be separated; the remaining contents of the FASTplex Sample Plate should be stored at -20°C until the next use. Care must be taken to avoid well-to-well cross-contaminations. 11. Program the thermal cycler(s). 12. Set up all the thermal cycler programs before starting. 13. Use the following programs: HLA 11-Loci Amplification Illumina Program: Temperature Time Cycle 94°C 2 min 98°C 10 sec 30 69°C 3 min 4°C HOLD 9600 emulation speed and heated cover on TAG Program: 55°C for 15 min., 25°C hold, heated cover on STOP Program: 68°C for 10 min., 25°C hold, heated cover on FASTplex Illumina Library Amplification Program: Temperature Time Cycle 72°C 10 min 98°C 3 min 98°C 15 sec 64°C 30 sec 12 72°C 1 min 72°C 5 min 4°C HOLD 9600 emulation speed and heated cover on IMPORTANT: At the completion of each PCR step, select the next thermal cycler program to be used, so the thermal lid is prewarmed. The thermal cycler lid must be at the appropriate target temperature before placing the reaction. Avoid placing reactions onto a cold thermal cycler. 14. Pulse-spin the kit components in a suitable centrifuge to gather the reagents at the bottom of the well or tube before every use of the FASTplex Sample Plate and before dispensing from the reagent tubes, since liquids can condense or shift locations inside containers during shipment or storage. 15. If the Room Temperature kit components freeze during shipment or storage, perform the following steps before use: a) Thaw the kit components. b) Mix the kit components. c) Pulse-spin the kit components. 16. Equilibrate FASTplex Paramagnetic Beads to room temperature. FASTplex Paramagnetic Beads can be stored for up to 2 weeks at room temperature or for longer periods at 2 - 8°C. If the FASTplex Paramagnetic Beads are stored cold, perform the following steps before use: a) Set the beads at room temperature for 30 minutes. Vortex the beads thoroughly to resuspend. IMPORTANT: To transfer volumes accurately, do not pre-wet pipette tips and pipette slowly. 17. Check the Stop Solution for precipitate before use. If a precipitate is visible, perform the following steps: a) Incubate the tube at 37°C for 5 minutes. b) Mix gently by inversion until the precipitate dissolves. IMPORTANT: Do not vortex. Stop Solution contains SDS and will precipitate if stored below room temperature. Overly vigorous mixing will cause foaming. 18. Store the FASTplex Barcoding Buffer at room temperature when in use because it is viscous. IMPORTANT: To transfer Barcoding Buffer accurately, do not pre-wet pipette tips and pipette slowly. While adding Barcoding Buffer to reactions, mix in the Barcoding Buffer completely by pipetting up and down several times with the same pipette tip(s) that was used for addition. 19. Prepare 80% Ethanol daily. 20. Plan sample barcoding before starting the assay. 21. When planning for sample barcoding, keep the following in mind: The kit contains a total of 96 unique Barcodes as Sample Barcodes (SB) and a unique FASTplex Universal Barcode (UB) to prepare up to a 96-sample library. The AllType FASTplex NGS 11 Loci Flex Kit (Cat. No. ALL-FAST11LF) has been tested using Illumina MiSeq v2 Standard (up to 96 samples), MiSeq v2 Micro (up to 24 samples), MiSeq v2 Nano (up to 8 samples), MiSeq i100 Series 25M Reagent Kit (up to 96 samples), MiSeq i100 Series 5M Reagent Kit (up to 24 samples), MiniSeq High Output (up to 96 samples), MiniSeq Mid Output (up to 72 samples), and iSeq v2 (up to 24 samples). A library can contain between 8 to 96 samples and is barcoded with one unique UB. Each well contains enough barcoding reagents for one reaction. Figure 1 provides the layout for a 96-sample library. Illumina barcode IDs and their positions are shown. Figure 1. Barcode sample plate layout The TypeStream Visual (TSV) NGS Calculator has a tab for the sample sheet that reflects this barcode order. IMPORTANT: Alternative configurations, kits, and sequencing systems are not supported by this application and must be determined and validated by the user. 22. If working with various sample sizes, keep the following in mind for dealing with reagent volumes: When processing different sample sizes, different volumes of the pooled SB reaction and of the FASTplex Paramagnetic Beads will be used after Sample Barcoding step. The TSV NGS Calculator displays the required reagent volumes. In the “SB UB Lib Amp” tab, the total number of samples will automatically update based on the number of samples added in the “Pool Configuration” tab. In the “SB – UB-Lib. Amp” tab, the required reagent volumes are displayed. To ensure sample IDs are properly populated throughout, please refer to the TypeStream Visual NGS Analysis Software User Manual. The kit contains enough barcoding reagents, barcoding buffer and stop solution to process 12 runs of 8 samples AMPLIFICATION Materials and Equipment Diluted genomic DNA (25 ng/µL) Eppendorf DNA LoBind Tubes, 2.0 mL 96-Well, 0.2 mL, PCR Plates AllType FASTplex 11 Loci Primer Flex Mix (Cat. ID ALF-P11F) AllType FASTplex 11 Loci Exon 1 Primer Flex Mix (Cat. ID ALF-P11FE1) AllType Buffer (Cat. ID ALL-BUF) AllType dNTPs (Cat. ID ALL-NTP) AllType LR Polymerase (Cat. ID ALL-LRPOL) Nuclease Free Water FASTplex Paramagnetic Beads (Cat. ID FAST-BEAD) FASTplex DNA Suspension Buffer (Cat. ID FAST-SUSP) 200 Proof Ethanol, Molecular Biology Grade Nunc 96-Well Polypropylene MicroWell Plates Magnetic Plate Stand 50 mL Polystyrene Reagent Reservoirs Qubit dsDNA High Sensitivity Assay Kit Qubit Fluorometer Full array of manual single channel pipettes (20 µL, 200 µL, 1 mL) Full array of filtered, pre-sterilized, pipette tips Serological Pipettes, 10 or 25 mL PCR Plate Centrifuge Mini Centrifuge Vortex Ice or Cold Block Thermal cycler PCR Pressure Pad Veriti 96-Well Thermal Cycler or equivalent TSV NGS Calculator Part 1: Sample Amplification 1. Turn on a thermal cycler. 2. Program the thermal cycler to run the following HLA 11-loci Amplification Illumina Program: HLA 11-Loci Amplification Illumina Program Temperature Time Cycle 94°C 2 min 98°C 10 sec 30 69°C 3 min 4°C HOLD 3. Use 9600 emulation speed or +0.8°C/sec heating and -1.6°C/sec cooling with the heated cover set to 105°C IMPORTANT: Check the thermal cycler lid temperature before adding the reaction. Make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. 4. Use the “gDNA Dilution” tab in the TSV NGS Calculator to calculate the original sample volumes and DNA Suspension Buffer volumes to prepare 25 ng/µL dilutions. Note: To ensure sample IDs are properly populated throughout, please refer to the TypeStream Visual NGS Analysis Software User Manual. 5. Thaw AllType FASTplex 11 Loci Primer Flex Mix, AllType FASTplex 11 Loci Exon 1 Primer Flex Mix, AllType dNTP, and AllType Buffer at room temperature. 6. Keep the AllType LR Polymerase tube on ice. Briefly vortex and spin down all reagents EXCEPT polymerase. 8. Keep them on ice until ready for use. In a 96-Well 0.2 mL PCR Plate, aliquot 4.0 µL of genomic DNA with the concentration adjusted to 25 ng/µL. 10. Go to the “Amp-Clean Up” tab of the TSV NGS Calculator, which will automatically calculate the volumes of each amplification reagent based on the number of samples input into the “gDNA Dilution” tab. 11. Check the “Exon 1” box if using the Exon 1 primer mix. 12. In a 2.0 mL LoBind Tube, prepare an Amplification Master Mix based on the reagent volumes shown in the “Amp Clean Up” tab of the TSV NGS Calculator. 13. Prepare reagents in the order shown in the “Amplification” table of the “Amp-Clean Up” tab in the TSV NGS Calculator. IMPORTANT: At this stage, only prepare the Amplification Mix inclusive of the first five components. Do not add the Polymerase at this stage. 14. Vortex and pulse-spin the mix for 10 seconds. 15. Keep the mix on ice. 16. Quick spin the Polymerase. 17. Add the Polymerase to the Master Mix according to the “Amplification” table in the TSV NGS Calculator. 18. Mix well by pipetting 15-20 times with the pipet volume set to half the total volume of the Master Mix. 19. Aliquot 16 µL of the Master Mix to each well with DNA. 20. Seal the plate with a tray seal. 21. Pulse-spin the plate. 22. Load the plate into a pre-warmed thermal cycler. 23. Cover the plate with a pressure pad. 24. Run the HLA 11-loci Amplification program. IMPORTANT: Check the thermal cycler lid temperature before adding the reaction. Make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. SAFE-STOPPING POINT: Proceed immediately to the next step or store the amplicon at -20°C. Part 2: Amplicon Purification Equilibrate the FASTplex Paramagnetic Beads to room temperature for 30 minutes. 2. Prepare fresh 80% Ethanol solution by measuring Ethanol and nuclease-free water separately and mixing. Refer to the TSV NGS Calculator and see the “Amp Clean Up” tab in the Amplification Purification section. 3. Vortex the FASTplex Paramagnetic Beads at medium speed for 30 seconds to fully resuspend the beads. In a Nunc LoBind 96-well plate, aliquot 12 µL of the beads to each well. Use a multi-channel pipette and reagent reservoir for convenience. 5. Transfer all amplified product (~20 µL) to the corresponding wells. 6. Mix by pipetting up and down 10 times. IMPORTANT: Change tips for each sample and avoid producing bubbles. 7. Incubate at room temperature for 5 minutes. 8. Place the plate on the magnetic stand. Adjust the plate as needed so the beads form a pellet on one side of each well. Let stand for ~3 minutes or until clear. 9. Using a pipette set to 28 µL, carefully remove and discard the supernatant from each well without disturbing the bead pellet. 10. With the plate on the magnet, add 100 µL of 80% Ethanol to each well. 11. Incubate at room temperature for 30 seconds or longer until the solution clears. 12. With a pipette set to 110 µL, remove and discard the supernatant. IMPORTANT: Be careful not to disturb the bead pellet. 13. Repeat steps 10 through 12 above for a second Ethanol wash. 14. With the plate on the magnet, remove visible Ethanol residue with a P-20 pipette. 15. Air-dry the beads at room temperature for up to 3 minutes on the magnet. IMPORTANT: Avoid over-drying. 16. With the plate on magnet, add 27 µL of FASTplex DNA Suspension Buffer to each well. 17. Remove the plate from the magnet. 18. Mix each sample by pipetting up and down 10 times avoiding making any bubbles. 19. Incubate at room temperature for 5 minutes. 20. Place the plate back on magnet for ~3 minutes or until clear. 21. With a pipette volume set to 25µL, transfer the supernatant to a new 0.2 mL 96-well PCR plate without disturbing the beads. SAFE-STOPPING POINT: Proceed immediately to the next step or store the purified products at -20°C. Part 3: Amplicon Quantification 1. Label Qubit Assay Tubes for each sample and two additional tubes for Qubit Standard #1 and #2. 2. Prepare a Working Qubit Solution using the volumes per sample shown below plus 15% overage by mixing the equivalent of: a) 199 µL of Qubit dsDNA HS Buffer (Component B) per sample. b) 1 µL of Qubit dsDNA HS Reagent (Component A) per sample. Vortex prior to use. 3. Vortex the Working Qubit Solution and protect from light. Use within 2 hours. 4. Add 198 µL of Working Solution to each labeled Qubit Assay Tube. 5. Add 190 µL of Working Solution to the two tubes for the Standards. 6. Add 2 µL of the corresponding purified amplicon to each labeled tube. 7. Add 10 µL of the corresponding Standard to the appropriate, labeled tube. 8. Briefly vortex and spin all tubes. 9. Protect all tubes from light. 10. Incubate at room temperature for 2 minutes. 11. Turn on the Qubit Fluorometer. 12. Select DNA from the home screen. 13. Select dsDNA High Sensitivity. 14. Press the appropriate button to begin reading the standards. 15. Measure Standards 1 and 2 to complete the calibration. 16. Begin reading the amplicon tubes. 17. When prompted, set the volume of sample used to 2 µL and the units to ng/µL. 18. Record the concentration of sample. 19. Repeat steps 16 through 18 for all samples. 20. If any sample exceeds the upper detection, perform the following steps: a) Dilute the samples. b) Remeasure the diluted samples. c) Multiply the reading by the dilution factor. See the Qubit protocol for additional clarification. Part 4: Amplicon Dilution Lower concentration samples may not contain an adequate quantity of the amplicons. The following steps in this section will help calculate a volume of FASTplex DNA Suspension Buffer to dilute amplicons. 1. Use the “Amplicon Dilution” and “Plate Layout” tabs of the TSV NGS Calculator for amplicon dilution calculation. Functions of the “Amplicon Dilution” and “Plate Layout” Tabs Calculation Tool Tab Name Functions Calculation of 96 (max) amplicon dilution factors and diluent volumes to Amplicon Dilution prepare 3-30 ng input DNA for Sample Barcoding reaction View the result of input DNA and FASTplex DNA Suspension Buffer Plate Layout volumes in an 8 x 12 plate format 2. Open the TSV NGS Calculator. Sample IDs will be automatically populated from the sample IDs input into the “gDNA Dilutions” tab. 3. In the “Amplicon Dilution” tab, enter the amplicon concentrations in the column labeled “Amplicon Conc. (ng/µL).” • The table will display a volume of FASTplex DNA Suspension Buffer to add to 2 µL of each amplicon [“Sample (µL)” column]. • Samples whose amplicon concentrations, after dilution, fall outside of the desired 3-30ng input into Library Preparation will be highlighted in orange (“High”) or red (“Low”). • The “Range” column will show either “High” or “Low” for the respective sample, and volume of sample and buffer to use. • The stock sample volume cell in the calculator is adjustable if more volume is needed. 4. In a clean 96-well plate, add the indicated volume of FASTplex DNA Suspension Buffer and 2 µL of the stock amplicon. • For any outlier samples, add volumes of the FASTplex DNA Suspension Buffer and stock amplicon that are indicated in the Sample (µL) and FASTplex DNA Suspension Buffer (µL) columns for each sample. The optimal input amount is 10 ng in a 4 µL volume at 2.5 ng/µL. • Use the “Plate Layout” tab to locate positions of outlier samples in the plate IMPORTANT: For any samples that fall outside the normal range of concentrations, it is important to add the indicated volume. 5. Carefully mix the diluted amplicon well by pipetting. Avoid cross-contamination. 6. Seal the plate 7. Pulse-spin the plate. SAMPLE BARCODING Materials and Equipment • Diluted amplicons • Eppendorf DNA LoBind Tubes, 2.0 mL • PCR tube 0.2 mL • 96-Well, 0.2 mL, PCR Plates • 2.0 mL Tube Magnet Stand • Serological Pipettes, 10 or 25 mL • FASTplex Sample Flex Plate 96 (Cat. ID FAST-SFP96) • FASTplex Univ Barcode Flex A (Cat. ID FAST-UBFA) • FASTplex Barcoding Buffer (Cat. ID FAST-BB) • FASTplex Stop Solution (Cat. ID FAST-STOP) • FASTplex Paramagnetic Beads (Equilibrated to room temperature) (Cat. ID FAST-BEAD) • FASTplex DNA Suspension Buffer (Cat. ID FAST-SUSP) • 200 Proof Ethanol, Molecular Biology Grade • Full array of manual single channel pipettes (20 µL, 200 µL, 1 mL) • Full array of filtered, pre-sterilized, pipette tips • 8 tube strips • Thermal cycler PCR Pressure Pad • Veriti 96-Well Thermal Cycler or equivalent • TSV NGS Calculator Part 1: Sample barcoding (SB) Reaction, Reaction Stop, Sample Pooling, and Purification IMPORTANT: Before starting this step, make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. IMPORTANT: Before starting this step, ensure the “SB UB Lib Amp” tab of the TSV NGS Calculator has been populated. Useful Tip: In the following Sample Barcoding section below through step 20, the individual sample must be treated separately. An 8-channel pipette and use of an 8-tube strip can make this step faster and less error-prone. Common reagents, Barcoding Buffer and Stop Solution, can be aliquoted into an 8-tube strip or equivalent for use with the 8-channel pipette. The volume of reagents to aliquot using an 8-tube strip are summarized below. Barcoding and Stop Buffer Volume (µL) per Well in 8-Tube Strip (w/Overage) Reagent 8 Samples 16 Samples 24 Samples 48 Samples 96 Samples Barcoding Buffer 7.0 14.0 21.0 42.0 84.0 Stop Solution 10.0 20.0 30.0 60.0 120.0 1. Pulse-spin the FASTplex Sample Flex Plate 96 in a centrifuge. 2. After centrifugation, carefully remove the plate seal from the FASTplex Sample Flex Plate. 3. Discard the plate seal. IMPORTANT: Do not reuse the plate seal. 4. Place the FASTplex Sample Flex Plate on ice. 5. Equilibrate the FASTplex Paramagnetic Beads at room temperature. 6. Set up the Sample Barcoding reaction at ambient temperature. 7. Transfer 6 µL of the reagent from the FASTplex Sample Flex Plate 96 to a pre-labeled PCR plate or an 8-tube PCR strip(s) using an accurate pipette. IMPORTANT: Use clean tips for each transfer. 8. Visually confirm that the volume of reagents in the plate appears equal. 9. Return the plate to the freezer (-20°C storage) if not all 96 samples are used. 10. Transfer 4 µL of diluted input DNA (2.5 ng/µL) to each well (one sample per well) or tube using an accurate pipette. 11. Mix the DNA thoroughly with the sample barcode reagent in each tube by pipetting up and down ten times at 5μL. IMPORTANT: Be careful not to introduce excessive foams. 12. Use clean tips for each sample. 13. Carefully pipette 5 µL of Barcoding Buffer to each well or tube using new pipette tips for each transfer. 14. Mix thoroughly but slowly by pipetting up and down ten times at 6 μL. IMPORTANT: Be careful not to introduce excessive foams. Barcoding Buffer is very viscous and is subject to foaming. 15. Carefully and completely seal or cap the SB reactions to prevent evaporation. 16. Pulse-spin the plate. 17. Transfer to a thermal cycler. 18. Place a PCR pressure pad over the plate. 19. Run the following program (TAG) with heated cover on: TAG program 55°C for 15 min., 25°C hold, Reaction volume 15 µL IMPORTANT: Check the thermal cycler lid temperature before adding the reaction. Make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. 20. Immediately take the plate out of the thermal cycler, pulse-spin. 21. Add 7.5 µL of FASTplex Stop Solution to each SB reaction. 22. Slowly pipette up and down 5 times to mix, being careful not to introduce excessive bubbles. IMPORTANT: Use clean tips for each transfer. 23. After mixing with the Stop Solution, securely re-seal the PCR plate. 24. Pulse-spin the PCR plate. 25. Transfer the PCR plate to a thermal cycler 26. Run the following STOP program with the heated cover on: STOP program 68°C for 10 min., 25°C hold, Reaction volume 22.5 µL IMPORTANT: Check the thermal cycler lid temperature before adding the reaction. Make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. 27. Vortex (or vigorously pipette) room temperature FASTplex Paramagnetic Beads to ensure that the beads are fully resuspended. 28. After thermal cycling, pulse-spin the PCR plate containing the stopped SB reactions. 29. Using a 10-20 µL pipette, transfer 15 µL of each SB reaction into a single 2 mL LoBind tube, label this tube as “Tube A” and gently mix by pipetting up and down slowly. The final volume of the combined SB reaction pool is determined by the batch size. 30. Once pooling is complete, refer to the “Sample and Universal Barcoding” section of the “SB UB Lib Amp” tab of the TSV NGS Calculator for the volume of FASTplex Paramagnetic Beads to be added to the pooled SB reaction. 31. In a new 2.0 mL LoBind tube, transfer the volume specified in the calculator cell “Volume of stopped, pooled SB reaction” from “Tube A” over to “Tube B”. Not all of Tube A volume will be used. 32. Label this tube as “Tube B.” 33. Proceed to the next step with “Tube B” and its contents Volumes of the pool and paramagnetic bead for common sample sizes are listed in the following SB Reaction Pool and FASTplex Paramagnetic Bead Volumes table. The volumes for different sample sizes can be displayed using the TSV NGS Calculator. SB Reaction Pool and Paramagnetic Bead Volumes (µL) Batch Size (Samples per Pool) 8 Samples 16 Samples 24 Samples 48 Samples 96 Samples Volume of stopped SB 80.0 160.0 240.0 480.0 960.0 reaction pool Add FASTplex 72.0 144.0 216.0 432.0 864.0 Paramagnetic Beads 34. Go to the “SB UB Lib Amp” tab to find the volumes of the pool, paramagnetic bead, and the reagents for the rest of workflow. 35. Add the appropriate volume of FASTplex Paramagnetic Beads to the stopped SB reaction pool in ”Tube B”. 36. Mix thoroughly by pipetting up and down 10x. 37. Incubate in a tube rack (non-magnetic) on the bench for 5 minutes to allow the DNA to bind. 38. Transfer the tube to a magnetic stand for ~ 5 minutes or until clear. A bead pellet will form. 39. Slowly remove the supernatant with a pipette and discard. IMPORTANT: Be careful not to disturb the bead pellet. 40. With the tube in the magnetic stand, add 2.0 mL of 80% Ethanol to completely immerse the bead pellet without disturbing the beads. IMPORTANT: Be careful not to disturb the beads. 41. After 30 seconds, slowly remove and discard the supernatant. IMPORTANT: Be careful not to disturb the bead pellet. 42. Repeat steps 40 and 41 for a total of two washes with 80% Ethanol. Use a small pipette (e.g., P20) to remove the residual Ethanol after the second wash. 43. Air-dry beads by leaving the tube uncapped on the magnetic stand for 2 minutes. 44. Check to ensure that there are no visible Ethanol droplets in the tubes at end of 2 minutes. • If Ethanol droplets are still visible, air-dry the beads longer. CAUTION: Do not dry the pellet longer than 3 minutes in total or the DNA recovery will be compromised. 45. Remove the tube from the magnetic stand. 46. Pipette the volume of FASTplex DNA Suspension Buffer provided in the “SB UB Lib Amp” tab of the TSV NGS Calculator. This volume is directly dependent on the number of samples and will vary accordingly. Volumes of buffer for common sample sizes are listed in the following SB Reaction Pool and Paramagnetic Bead Volumes table. SB Reaction Pool and Paramagnetic Bead Volumes (µL) Batch Size (Samples per Pool) 8 Samples 16 Samples 24 Samples 48 Samples 96 Samples Volume of FASTplex DNA 50.0 96.0 144.0 288.0 576.0 Suspension Buffer to elute 47. Pipette the buffer-bead solution along the inner wall of the tube multiple times to thoroughly mix and resuspend the bead pellet. 48. Incubate the tube in a tube rack (non-magnetic) on the bench for at least 5 minutes to elute the purified SB reaction pool from the beads. 49. Return the tube to a magnetic stand and allow a bead pellet to reform on the inner wall of the tube ~ 2 minutes or until clear. 50. When the supernatant has cleared completely, carefully transfer the full library eluate to a new 2.0 mL tube. The transferred eluate contains the purified SB reaction pool. SAFE-STOPPING POINT: Proceed immediately to the next step or store the purified products at -20°C. Part 2: Universal barcoding (UB) reaction, reaction stop, and purification IMPORTANT: Before starting this step, make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. 1. Set up a UB reaction in a 0.2 mL PCR tube using 48 µL of the purified SB reaction pool from the last step. For all library pools > 8 samples, there will be excess purified SB reaction pool. 2. Add the UB Reagent and Barcoding Buffer by using the TSV NGS Calculator or according to the amounts in the following table: UB Reagent and Barcoding Buffer Volumes (µL) UB Reagent and Barcoding Buffer Volume Purified SB reaction 48.0 FASTplex Univ. Barcode Flex A 4.0 FASTplex Barcoding Buffer 26.0 Total Reaction Volume 78.0 3. Using a pipette set at 50 μL, mix the UB reaction thoroughly by pipetting up and down 10 times. 4. Cap the PCR tube containing the UB reaction. 5. Pulse-spin the PCR tube. 6. Transfer the tube to a thermal cycler. 7. Use a PCR pressure pad. 8. Run the following program (TAG) with appropriate reaction volume and lid-heating on: TAG program 55°C for 15 min., 25°C hold, Reaction Volume 78 µL for all library sizes IMPORTANT: Check the thermal cycler lid temperature before adding the reaction. Make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. 9. After the TAG program, remove the tube from thermal cycler. 10. Add 39 μL Stop Solution. 11. With the pipette set at 50 μL, mix thoroughly by pipetting up and down 10 times. IMPORTANT: Be careful not to produce excessive foaming. 12. Re-cap the PCR tube containing the UB reaction. 13. Pulse-spin the PCR tube. 14. Transfer the tube to a thermal cycler. 15. Run the following program (STOP) with appropriate reaction volume and lid-heating on: STOP program: 68°C for 10 min., 25°C Hold. Use 100 µL volume in the PCR program. IMPORTANT: Check the thermal cycler lid temperature before adding the reaction. Make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. 16. Transfer the entire volume of the stopped UB reaction to a single 2.0 mL LoBind tube. 17. Vortex (or vigorously pipette) room temperature Paramagnetic Beads to ensure they are completely resuspended. The UB reaction volume should be 117 μL, but evaporation during the reaction may reduce its volume. 18. Measure the volume of UB reaction using a pipette. 19. Add an equal volume as measured in step 18 of paramagnetic beads in the 2.0 mL LoBind tube 20. Mix thoroughly by pipetting. 21. Incubate at room temperature in a tube rack (non-magnetic) on the bench for 5 minutes to allow the DNA to bind. 22. Transfer the 2.0 mL LoBind tube to a magnetic stand. 23. Let the beads separate completely for 3 minutes. A bead pellet will form along one side of the tube and the supernatant should appear completely clear after ~3 minutes. 24. Slowly remove the supernatant with a pipette and discard. IMPORTANT: Be careful not to disturb the bead pellet. 25. With the tube in the magnetic stand, add 500 µL of 80% Ethanol, ensuring the bead pellet is submerged. IMPORTANT: Be careful not to disturb the pellet. 26. Slowly remove and discard the supernatant after 30 seconds or until clear. IMPORTANT: Be careful not to disturb the bead pellet. 27. Repeat steps 25 and 26 for a total of 2 washes with 80% Ethanol. 28. Use a large pipette to remove most of the Ethanol waste. 29. Use a smaller pipette (e.g., P20) to remove the residual Ethanol that collects at the bottom of the tube. 30. Air-dry beads by leaving the tube uncapped on the magnetic stand for 2 minutes. 31. Check to ensure that there are no visible Ethanol droplets in the tube at end of 2 minutes. • If Ethanol droplets are still visible, air-dry the beads longer. CAUTION: Do not dry the pellet longer than 3 minutes in total or the DNA recovery will be compromised. 32. Remove the tube from the magnetic stand. 33. Add 20 µL of FASTplex DNA Suspension Buffer. • Pipette the liquid along the inside of the tube several times to thoroughly resuspend the bead pellet. 34. Incubate in a tube rack (non-magnetic) on the bench for at least 5 minutes to elute the purified DNA from the beads. 35. Return the tube to the magnetic stand. 36. Allow the bead pellet to form on the inner wall of the tube for ~2 minutes or until clear. 37. When the supernatant has cleared completely, carefully transfer 17 µL of DNA eluate to a clean 0.2 mL PCR tube. The transferred eluate contains the DNA purified from the UB reaction and is now ready for library amplification. SAFE-STOPPING POINT: Proceed immediately to the next step or store the purified products at -20°C. LIBRARY AMPLIFICATION Materials and Equipment • UB Barcoded library • 0.2 mL PCR tube • FASTplex Library Amp Mix (Cat. ID FAST-LAM) • FASTplex Library Primer Flex Mix (Cat. ID FAST-LPFM) • FASTplex Paramagnetic Beads (Cat. ID FAST-BEAD) • FASTplex DNA Suspension Buffer (Cat. ID FAST-SUSP) • Manual single channel pipettes (20 µL, 200 µL, 1 mL) • Filtered, pre-sterilized, pipette tips • 200 Proof Ethanol, Molecular Biology Grade • Veriti 96-Well Thermal Cycler or equivalent • 2.0 mL Tube Magnet Stand • Nuclease Free Water • Eppendorf DNA LoBind Tubes, 2.0 mL • Qubit® Fluorometer or equivalent • Qubit dsDNA HS Assay Kit • Qubit Assay Tubes • Calculation Tool Part 1: Library Amplification Reaction IMPORTANT: Before starting this step, make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. Reagent Volumes for the Steps Below (µL) Reagent Volume FASTplex Library Primer Flex Mix 8.0 Barcoded Library from step 37 in Part 2 of the 17.0 Sample Barcoding section FASTplex Library Amp Mix 75.0 1. As described in the table above, add 8 µL of Library Primer Mix to the 17 µL Barcoded Library eluate in the 0.2 mL PCR tube from step 37 in Part 2 of the Sample Barcoding process. 2. Add 75 µL of Library Amp Mix. 3. Mix well by pipetting. 4. Cap the PCR tube and pulse spin. 5. Use an appropriate PCR pressure pad. 6. Run the following FASTplex Illumina Library Amplification PCR program with a heated cover on and 9600 emulation mode or +0.8°C/sec heating and -1.6°C/sec cooling with the heated cover set to 105°C. The total reaction volume is 100 µL. • FASTplex Illumina Library Amplification Program: TEMPERATURE TIME CYCLE 72°C 10 min 1 98°C 3 min 1 98°C 15 sec 12 64°C 30 sec 72°C 1 min 72°C 5 min 1 4°C HOLD 1 9600 emulation speed and heated cover on IMPORTANT: Check the thermal cycler lid temperature before adding the reaction. Make sure the thermal cycler lid is at the appropriate target temperature. Do not place the reaction onto a cold thermal cycler. 7. After PCR, pulse-spin the library amplification reaction. 8. Transfer 95 µL of the library amplification reaction to a 2.0 mL LoBind tube. • If it’s less than 95 µL, note the volume for PART 2: Purification of the Amplified Library, step 1. IMPORTANT: The volume will normally decrease due to evaporation during thermal cycling, so it is important to measure the volume prior to the purification steps. SAFE-STOPPING POINT: Proceed immediately to the next step or store the purified products at -20°C. Part 2: Purification of the Amplified Library 1. In a new 2.0 mL LoBind tube, dilute the library amplification reaction to a final volume of 200 µL by adding 105 µL (or more in case of evaporation) of FASTplex DNA Suspension Buffer to the amplified product. 2. Vortex room temperature FASTplex Paramagnetic Beads to ensure they are completely resuspended. 3. Add 160 µL of FASTplex Paramagnetic Beads (0.8x volume equivalents) to the diluted multiplexed library. 4. Mix thoroughly by pipetting up and down. 5. Incubate in a tube rack (non-magnetic) on the bench for 5 minutes to allow the DNA to bind. 6. Transfer the 2.0 mL LoBind tube to a magnetic stand. 7. Let the beads settle completely for approximately 3 minutes. A bead pellet will form along one side of the tube and the supernatant should appear completely clear blue after ~3 minutes. 8. Slowly remove the supernatant with a pipette and discard. IMPORTANT: Be careful not to disturb the bead pellet. 9. With the tube in the magnetic rack, add 500 µL of 80% Ethanol. IMPORTANT: Be careful not to disturb the beads. 10. Incubate for 30 seconds, until the solution clears. 11. Keep the tube on the magnetic rack. 12. Carefully remove and discard the supernatant. 13. Repeat steps 9 through 12 for a total of two washes with 80% Ethanol. 14. Use a small pipette (e.g., P20) to remove the residual Ethanol after the second wash. 15. Air-dry the beads by leaving the tube uncapped on the magnetic stand for 2 minutes. 16. Check to ensure that there are no visible Ethanol droplets in the tubes after 1 minute. • If Ethanol droplets are still visible, air-dry the beads longer. CAUTION: Do not dry the pellet longer than 3 minutes in total or the DNA recovery will be compromised. 17. Remove the tube from the magnetic stand. 18. Add 35 µL of FASTplex DNA Suspension Buffer. 19. Pipette the liquid along the inside of the tube several times to thoroughly disperse the beads. 20. Incubate for 5 minutes in a non-magnetic tube rack on the bench to elute the multiplexed library from the magnetic beads. 21. Return the tubes to magnetic stand. 22. Allow the bead pellet to form on the inner wall of the tube for ~2 minutes or until clear. 23. When the supernatant has cleared completely, carefully transfer 33 µL of DNA eluate to a new 2.0 mL LoBind tube. The transferred eluate contains the final library. SAFE-STOPPING POINT: Proceed immediately to the next step or store the purified products at -20°C. Part 3: Quantification of the Final Library 1. Label five Qubit Assay Tubes – three for triplicate quantification of the library and two additional tubes for Qubit Standard #1 and #2. 2. Prepare a Working Qubit Solution in a 5 mL tube for 10 samples (to account for the measurement in step 16 and step 23 for pre-dilution and post-dilution samples) by mixing the equivalent of: a) 199 µL of Qubit dsDNA HS Buffer (Component B) per sample. b) 1 µL of Qubit dsDNA HS Reagent (Component A) per sample. Vortex before use. 3. Vortex the Working Qubit Solution. 4. Cover with foil. IMPORTANT: Use within 2 hours. 5. Add 198 µL of Working Solution to the three Qubit Assay Tubes used for triplicate quantification of the library. 6. Add 190 µL of Working Solution to the two tubes used for the Qubit Standards. 7. Add 2 µL of the purified library to the three Qubit Assay Tubes used for triplicate quantification of the library. 8. Add 10µL of the corresponding Qubit Standard to the two tubes used for the Qubit Standards. 9. Briefly vortex and spin all tubes. 10. Cover the tubes with foil. 11. Incubate the tubes at room temperature for 2 minutes. 12. Turn on the Qubit Fluorometer, 13. Select DNA from the home screen. 14. Select dsDNA High Sensitivity. 15. Press the appropriate button to begin reading the standards. 16. Measure Standards 1 and 2 to complete the calibration. This calibration can be used for quantification of the final library in step 23 of this section, if the final quantification is performed within 2 hours. 17. Begin reading the three library tubes. 18. When prompted, change the volume of sample used to 2 µL and the units to ng/µL. 19. Record the concentration of the library in triplicate. 20. In the TSV NGS Calculator, open the tab named “Final Quant” 21. Choose the appropriate Instrument from the drop-down menu in the upper left-hand corner of the tab. Different sequencing systems require different final library concentrations. The following table lists the concentrations for the MiSeq, MiSeq i100 Series, MiniSeq, and iSeq sequencing systems. Final Library Concentrations Sequencing System Library Concentration MiSeq 2.1 ng/µL MiSeq i100 Series 0.53 ng/µL MiniSeq 0.53 ng/µL iSeq 0.056 ng/µL 22. Input the average concentration of the three triplicate readings that was determined by Qubit into the TSV NGS Calculator “Pool Quant Values (ng/µL).” The calculator will calculate the amount of FASTplex DNA Suspension Buffer and sample pool volume that should be combined in a new 2.0 mL LoBind tube to achieve the proper library concentration. The “Diluted Pool Volume (µL)” can be adjusted as needed, but the final desired target concentration cannot be edited in the calculator. It may be helpful to make an intermediate dilution, especially for libraries that are to be sequenced on the iSeq as the final library concentration is low. 23. Quantify the diluted library in triplicate using the Qubit Working Solution in order to verify that it is at the appropriate concentration. Qubit Working Solution Volumes per Sequencing System Sequencing System Qubit Working Solution Volume Library Volume MiSeq 198 µL 2 µL MiSeq i100 Series 198 µL 2 µL MiniSeq 198 µL 2 µL iSeq 195 µL 5 µL PREPARING for ILLUMINA PLATFORM SEQUENCING Materials and Equipment • Final library with the concentration adjusted for appropriate Illumina sequencing kit • Illumina Sequencing Kit(s) of choice (see Required Materials and Equipment Not Provided above for compatible kits) • PhiX Control Kit • NaOH Solution ≥1.0M, Molecular Biology Grade (for MiSeq and MiniSeq only) Part 1: Preparing PhiX Control 1. In a new 2.0 mL LoBind tube, combine 2μL of 10nM PhiX stock to 3 μL of FASTplex DNA Suspension Buffer to dilute the PhiX to 4nM. 2. Vortex and pulse-spin. 3. To the same tube containing 4 nM PhiX, add 5μL of freshly prepared 0.2M NaOH solution 4. Vortex and pulse-spin. 5. Incubate at room temperature for 5 mins. 6. When the 5-minute incubation is complete, add 990μL of HT1/Hybridization buffer to the denatured PhiX to bring the concentration down to 20 pM. 7. Vortex and pulse-spin the 20 pM PhiX. 20 pM PhiX can be used immediately in the following library preparation or stored at -20°C for up to 2 weeks. Part 2: MiSeq Sequencing – Preparing the Sample Sheet 1. Open the “Sample Sheet” tab in the TSV NGS Calculator. 2. If using MiSeq with Windows 7 Operating System, choose “MiSeq” from the drop-down menu in the top left corner of the calculator. The Sample Sheet “Sample_Name” and “Sample_ID” columns will automatically populate. 3. If using MiSeq with Windows 10 Operating System, choose “MiSeqV2” from the drop-down menu in the top left corner of the calculator. The Sample Sheet “Sample_Name” and “Sample_ID” columns will automatically populate. 4. Fill in Experiment Name in the highlighted cell (yellow). The Date will automatically populate, but it can be edited. 5. Export the completed sample sheet using the “Export” button 6. Name the sample sheet. 7. Copy the file on a USB memory drive. Part 3: MiSeq Sequencing – Preparing the Pool Library IMPORTANT: Prior to beginning the next series of steps, fully thaw the MiSeq Reagent Cartridge by placing it in room temperature water up to the water line for at least an hour. Once fully thawed, the cartridge can be stored at 4°C until ready for sequencing. 1. In addition to thawing the Reagent Cartridge, thaw the HT1/Hybridization Buffer that is included with the MiSeq Reagent Kit. Once thawed, keep the HT1/Hybridization Buffer on ice. 2. In a new 1.5 mL LoBind tube, combine 5 μL of the 2.1 ng/μL (or other value used) library pool with 5 μL of freshly prepared 0.2 M NaOH solution. 3. Vortex and pulse-spin. 4. Incubate at room temperature for 5 mins. 5. When the 5-minute incubation is complete, add 990 μL of HT1/Hybridization buffer to the denatured library to bring the concentration down to 20 pM. 6. Vortex and pulse-spin. 7. Add 25 μL of 20 pM PhiX to the 1 mL denatured and diluted library. Vortex, and pulse-spin. 8. Obtain the thawed MiSeq Reagent Cartridge. 9. Invert the MiSeq Reagent Cartridge 10 times to mix. 10. Gently tap the cartridge in the upright position on a countertop. 11. Wipe off any liquid or condensation from the cartridge using a lab wipe. 12. Pierce well 17 of the MiSeq Reagent Cartridge using the 1mL pipette tip. 13. Discard the pipette tip once the MiSeq Reagent Cartridge is pierced. 14. Transfer 600μL of the combined library and PhiX solution to well 17 using a pipette. 15. Make sure that the solution is dispensed to the bottom of the well. 16. Press the Sequence button on the instrument interface to walk through the remainder of the sequencing run setup. 17. When prompted to insert the flow cell, remove the flow cell from its container. 18. Clean the flow cell by thoroughly rinsing it with lab grade water. 19. Once rinsed, thoroughly dry the flow cell surface by blotting gently with a lab wipe. 20. Ensure that all visible smudges, dust, and lab wipe lint are removed from the flow cell surface. 21. Insert the flow cell into the MiSeq flow cell compartment and close the latch. 22. Proceed with initiation of the sequencing run on MiSeq sequencer screen. 23. After loading the sequencing cartridge and Incorporation Buffer, load the sample sheet made in the previous section, Part 2: MiSeq Sequencing – Preparing the Sample Sheet, as a sample sheet from the USB memory drive by using the browse button. 24. Press the Restart/Check button to proceed with the sequencing run setup. 25. When the run is complete, clean the instrument in a timely fashion per the manufacturer’s recommendations. Part 4: MiSeq i100 Series Sequencing – Preparing the PhiX Control 1. In a new 1.5 mL LoBind tube, combine 2.4 µL of 10 nM PhiX stock with 37.6 µL of FASTplex DNA Suspension Buffer to dilute the PhiX control library to 0.6 nM. 2. Vortex and pulse-spin. 3. Store the diluted PhiX on ice until ready to use. IMPORTANT: The 0.6 nM PhiX control can be used immediately or stored at -20°C for up to 2 weeks Part 5: MiSeq i100 Series Sequencing – Preparing the Sample Sheet 1. Open the “Sample Sheet” tab in the TSV NGS Calculator. 2. Users of the MiSeq i100 Series, choose “MiSeq i100” from the drop-down menu in the top left corner of the calculator. The Sample Sheet “Sample_Name” and “Sample_ID” columns will automatically populate. 3. Fill in the Experiment Name in the highlighted cell (yellow). The Date will automatically populate, but the date can be edited. 4. Export the completed sample sheet clicking the “Export” button. 5. Name the sample sheet. 6. Copy the file on a USB memory drive. 7. From the instrument Home Screen, select the menu icon in the upper-left corner. 8. Select Runs, followed by the Planned tab. 9. Select “Import Sample Sheet” and then browse and the select the completed FASTplex MiSeq i100 Series Sample Sheet (.csv file). 10. After the Sample Sheet validation is complete, select “Next” to review the imported run details. 11. Confirm the run parameters and select “Save as Planned” to finalize the run setup. Part 6: MiSeq i100 Series Sequencing– Preparing the Pool Library IMPORTANT: MiSeq i100 Series consumables are shipped and stored at ambient temperature. Thawing is not required. Before loading libraries into the dry cartridge, dilute FASTplex libraries to the appropriate concentration and optionally spike-in 0.6 nM PhiX control. Libraries are denatured onboard the instrument. Keep the wet cartridge in the foil packaging until it is ready to be loaded on the instrument. The wet and dry cartridges must be used within 4 hours of opening the foil packaging. 1. Remove the Library Denaturation Buffer (KLD, blue cap) tubes from the wet cartridge foil packaging. Set the tubes aside at room temperature. 2. In a new 1.5 mL LoBind tube, combine 30 µL of the 0.53 ng/µL (or other value used) library pool and 1 µL of the 0.6nM PhiX control library to obtain a final volume of 31 µL, resulting in ~2% PhiX spike-in. IMPORTANT: PhiX control spike-in is optional. If not adding PhiX, transfer 30 µL of diluted library pool to a new 1.5 mL tube. 3. Add 270 µL of Library Denaturation Buffer to the 30 µL of library mixture to achieve a final concentration of 100 pM (0.053 ng/uL). 4. Vortex and pulse-spin. 5. Keep the 100 pM (0.053 ng/uL) on ice until ready to load. IMPORTANT: The diluted library solution is stable for up to 6 hours when stored on ice or at 4ᵒC. 6. Remove the dry cartridge from its foil packaging. Handle the dry cartridge by the sides only and avoid touching the flow cell. 7. Using a clean pipette tip, pierce the foil seal covering the reagent well labeled “Library”. 8. Load 250 µL of the 100 pM (0.053 ng/uL) diluted library pool into the “Library” well. 9. Load the dry and wet cartridges onto the MiSeq i100 Series instrument according to the onscreen prompts. Note: Sequencing files may be saved locally by creating and selecting an output folder on an external hard drive. Alternatively, data may be stored in BaseSpace when the instrument is connected to a network. 10. On the review run screen, verify the run and consumables, and then select “Start Run”. Note: Instrument pre-run checks take ~ 15 minutes to complete. The sequencing run will begin automatically after checks are complete. Part 7: MiniSeq Sequencing – Preparing the Sample Sheet 7. Open the “Sample Sheet” tab in the TSV NGS Calculator. 8. Users of the MiniSeq, choose “MiniSeq” from the drop-down menu in the top left corner of the calculator. The Sample Sheet “Sample_Name” and “Sample_ID” columns will automatically populate. 9. Fill in the Experiment Name in the highlighted cell (yellow). The Date will automatically populate, but the date can be edited. 10. Export the completed sample sheet clicking the “Export” button. 11. Name the sample sheet. 12. Copy the file on a USB memory drive. 13. Log into Local Run Manager. 14. Click the blue “Create Run” button on the top right of the page. 15. Choose “GenerateFASTQ”. 16. Click the “Import Sample Sheet” button on the top left side of the page. 17. Navigate to the location of the sample sheet. 18. Open the sample sheet. The Run Name should automatically update with what was entered in cell B3 of the Sample Sheet. The Library Prep kit should automatically update to “Custom.” 19. Scroll to the bottom of the page. 20. Click the “Save Run” button. Part 8: MiniSeq sequencing – Preparing the Pool Library IMPORTANT: Prior to beginning the next series of steps, fully thaw the MiniSeq Reagent Cartridge by putting it in room temperature water up to the bottom of the handle for 3+ hours. Once fully thawed, the cartridge can be stored at 4°C until ready for sequencing. Equilibrate the flow cell, which is stored separately at 4°C, to Room Temperature for 30 minutes before sequencing. 1. In addition to thawing the reagent cartridge, thaw the HT1/Hybridization Buffer that is packaged separately from the cartridge. 2. Once thawed, keep the HT1/Hybridization Buffer on ice. 3. In a new 1.5 mL LoBind tube, combine 5 μL of the 0.53 ng/μL library pool with 5μL of freshly prepared 0.1M NaOH solution. IMPORTANT: MiniSeq denaturation uses a lower concentration of NaOH than MiSeq. 4. Vortex and pulse-spin. 5. Incubate at room temperature for 5 mins. 6. When the 5-minute incubation is complete, add 5 μL of FASTplex DNA Suspension Buffer to the denatured library to bring the concentration down to 333 pM. 7. Vortex and pulse-spin. 8. Add 985 μL of HT1/Hybridization Buffer to the denatured library to bring it down to 5 pM. 9. Vortex and pulse-spin. 10. Add 2.5 μL of prepared 20 pM PhiX to the denatured and diluted library. 11. Add 180 μL of the denatured and diluted library to 320 μL of HT1/Hybridization Buffer to bring it down to the final loading concentration of 1.8 pM. 12. Vortex and pulse-spin. 13. Obtain the thawed MiniSeq Reagent Cartridge. 14. Invert 10 times to mix. 15. Gently tap the cartridge in the upright position on a countertop. 16. Wipe off any liquid or condensation from the cartridge using a lab wipe. 17. Pierce well 16 of the MiniSeq Reagent Cartridge using a 1mL pipette tip. Discard the tip once pierced. 18. Transfer 500μL of the combined library and PhiX solution to well 16 using a pipette. 19. Make sure the solution is dispensed to the bottom of the well. 20. Press the Sequence button on the instrument interface to walk through the remainder of the sequencing run setup. The interface will prompt the user to insert the flow cell. 21. Inspect for the flow cell carefully for any dust or marks. 22. Ensure that all visible smudges, dust, and lab wipe lint is removed from the flow cell surface. 23. Insert the flow cell into the receptacle, secure it, and close the lid. 24. Proceed with initiation of the sequencing run on MiniSeq sequencer screen. 25. Load the MiniSeq Reagent Cartridge containing the prepared sequencing library. Note: When the run is complete, the MiniSeq is capable of auto-cleaning if the option is chosen. Part 9: iSeq 100 sequencing – Preparing the Sample Sheet 1. Open the “Sample Sheet” tab in the TSV NGS Calculator. 2. If using iSeq, choose “iSeq” from the drop-down menu in the top left corner of the calculator. The Sample Sheet “Sample_Name” and “Sample_ID” columns will automatically populate. 3. Fill in the Experimental Name in the highlighted cell (yellow). The date will automatically populate, but the date can be edited. 4. Export the completed sample sheet using the “Export” button and name the sample sheet. Copy the file on a USB memory drive. 5. Log into Local Run Manager and click the blue “Create Run” button on the top right of the page and choose “GenerateFASTQ”. 6. Click the “Import Sample Sheet” button on the top left side of the page, navigate to the location of your sample sheet, and open it. The Run Name should automatically update with what was entered in cell B3 of the Sample Sheet. The Library Prep kit should automatically update to “Custom”. 7. Scroll to the bottom of the page. 8. Click the “Save Run” button. Part 10: iSeq 100 Sequencing – Preparing the Pool Library IMPORTANT: Prior to beginning the next series of steps, fully thaw the iSeq Reagent v2 Cartridge thawed by submerging it in room temperature water for at least 6 hours. Alternatively, it can be thawed overnight at 4°C and brought to room temperature for ~ 1 hour before use. 1. In addition to thawing the reagent cartridge, take the flow cell cartridge from the 4°C storage and allow it to equilibrate at room temperature for at least 15 minutes. 2. Add 20 μL of 20 pM PhiX to 100 μL of the 0.056 ng/μL library pool. 3. Vortex and spin-down. 4. Invert the bagged cartridge 5 times to mix reagents. 5. Tap the cartridge with the label facing upwards several times to bring reagents down from the wall of the wells. 6. Remove the cartridge from the bag. 7. Open the flow cell package. 8. Remove the flow cell package from the plastic tray. 9. Inspect the flow cell for any damage. 10. Insert the flow cell into the cartridge so that the label is visible from outside of the cartridge and the glass portion of the flow cell is visible through the opening in the cartridge. There will be an audible click when the flow cell is properly inserted. 11. Pierce the foil in the orange box marked “Library.” 12. Add 20 μL of your library plus PhiX to the bottom of the Library reservoir. 13. Proceed with initiation of the sequencing run on the iSeq sequencer screen. 14. Follow the instructions on the screen to proceed. 15. When the run options screen is presented, choose “1” under the “Read Index” option for single index sequencing. 16. Proceed with sequencing. RESULTS Data Acquisition Upon completion of sequencing, the sequencer system software demultiplexes all sequence reads using the barcodes and generates a read 1 and 2 file per sample. Refer to user manual of the sequencer system software to download sequence data in its default or specified format such as a FASTQ file for each sample. HLA TypeStream Visual NGS Analysis Software imports a FASTQ file from the sequencer for analysis. Sample ID can be embedded in the data file from the sequencer software or can be added at the time of data acquisition through TypeStream Visual NGS Analysis Software. Data Calculation Please see TypeStream Visual NGS Analysis Software User Manual for details. The TypeStream Visual NGS Analysis Software provides all the necessary quality metrics depth of coverage, quality score for the base reads, read alignment and variant call, and the laboratory should determine the acceptable values for each quality metric in order to assure an accurate result. Data Analysis TypeStream Visual NGS Analysis Software sorts imported sequence reads using the locus-specific reference sequences from the IPD-IMGT/HLA Database, which provides a specific database for sequences of the human major histocompatibility complex, identifies reference alleles that best match the sample reads, quality-trims, maps and assembles contiguous sequences from the mapped reads, and assigns alleles per locus based on the assembled consensus contiguous sequences. The use of other analysis software may lead to incorrect typing and is not supported. LIMITATIONS of the PROCEDURE A. Clinical laboratories are governed by ASHI, CLIA, and EFI guidelines mandating laboratories to make clinical decisions based on multiple sources. Solid organ transplants require confirmatory testing derived from multiple sources to determine donor suitability. A transplant decision will not be made solely on the results of the test in question being either positive or negative. B. Sub-optimal sample or library quality and/or quantity may cause test failures. The causes of such failures may include low sample quantity and quality, contamination, presence of inhibitors, random enzymatic reaction failures, uncalibrated and malfunctioning instruments, use of expired reagents or third-party reagents, incorrect reagent maintenance, protocol modification, and incorrect quantification or calculation. C. Sample DNA should be quantified with a fluorometer and be free from any known PCR inhibitor. PCR inhibitors may be introduced from the original sample source or from various DNA extraction methods. Reference samples should be validated for amplification using the AllType FASTplex NGS 11 Loci Flex Kit reagents. D. The PCR and library preparation workflow described in this protocol requires highly controlled conditions. Follow the standard PCR guidelines listed in the section above, GENERAL PREPARATION for the ASSAYS to minimize contaminations. E. The AllType FASTplex NGS 11 Loci Flex Kit - 96 has been tested for use with the Applied Biosystems Veriti 96-Well Thermal Cycler (Cat. No. 4375786), a model capable of 9600 emulation speed or +0.8°C/sec heating and -1.6°C/sec cooling and a heated cover at 105°C or equivalent for all programs. Other thermal cyclers considered for use require end-user evaluation and validation. F. The AllType FASTplex NGS 11 Loci Flex Kit - 96 (Cat. No. ALL-FAST11LF) has been tested using Illumina the MiSeq Sequencing System (300 cycles) using the MiSeq Reagent Kit v2 (≤96 samples), Micro Kit v2 (≤24 samples) kit, Nano Kit v2 (≤8 samples), MiSeq i100 Series Sequencing System using the 25M Reagent Kit (≤96 samples) and the 5M Reagent Kit (≤24 samples), MiniSeq Sequencing System using the High (≤96 samples) and Mid Output Kit (≤72 samples), and iSeq Sequencing System using the 100 i1 Reagent v2 Kit (≤24 samples). Alternative configurations, kits, and sequencing systems are not supported by this application and must be determined and validated by the user. G. The minimum sample size per run is eight. H. The AllType FASTplex NGS 11 Loci Flex Kit - 96 (Cat. No. ALL-FAST11LF) has not been tested using any protocol that deviates from the one described above and may lead in erroneous results. I. HLA typing at high resolution using the NGS technology is a complex process that requires qualified personnel to review data and final HLA allele assignments. J. This test must not be used as the sole basis for making a clinical decision. K. Please refer to the AllType FASTplex NGS 11 Loci Flex Kit - 96 Resolution Limitations – Ambiguities List for a known list of lot-specific allele assignment ambiguities for polymorphisms located outside of the amplified region. Any allele listed may produce incorrect results and needs to be evaluated before assigning a result. o AllType FASTplex NGS 11 Loci Flex Kit - 96 was designed to detect DRB4*03:01N and DQB1*03:276N, using TypeStream Visual 2.1 or later. L. Genotype ambiguities are expected from the limitation in the primer design and heterozygous genotype phasing due to limitations in sequencing read length and associated sequence alignment. M. The AllType FASTplex 11 Loci Primer Flex Mix for HLA-DRB1, -DQB1 and -DPB1 primers do not amplify exon 1. The AllType FASTplex Exon 1 Primer Flex Mix must be used to resolve Exon 1 ambiguities for these genes. When using the AllType FASTplex 11 Loci Exon 1 Primer Flex Mix with low quality or highly fragmented DNA, users may experience low uniformity across sequence reads. N. In rare cases, unknown sequence variants at the amplification primer binding sites in the untranslated regions (UTRs) may affect amplification efficiency of molecular typing reagents listed above. Homozygous typing needs be confirmed by a secondary method before assigning a result. O. The AllType FASTplex primer mixes were tested using alleles identified in the Nomenclature List in brackets in Index 4 for AllType (e.g., A*01:01[1234]). The reactivity of alleles that were not available has been predicted from its available sequence and may produce false reactions and needs to be evaluated before assigning a result. P. The AllType FASTplex primer mixes were tested based on Field-3 (6-digits HLA types) only. There are no performance claims outside this field. Q. Software analysis for this kit is only supported with the TypeStream Visual NGS Analysis Software 2.0.1 or above using AllType FASTplex NGS 11 Loci Flex Kit catalog file. The use of other software is not supported. R. For lot specific limitations, please review the TSV catalog File. S. Failure to completely read and explicitly follow all the instructions contained herein may result in invalid test results, damage to the product(s), injury to persons, including to users or others, and damage to other property. One Lambda, Inc. does not assume any liability arising out of the improper use of the product(s) described herein (including parts thereof or software). EXPECTED VALUES Sample Amplification The AllType FASTplex NGS 11 Loci Flex Kit is expected to amplify and produce HLA locus-specific amplification products consisting of ~5000 base pairs in averaged length (subject to change per lot). Physical separation and monitoring of contamination in a pre-amplification laboratory and equipment is highly recommended. Exon 1 amplicons will range from 1.6-2.2 kb. Exon imbalance and higher Exon 1 reads may occur when testing with Exon 1 primer mix. This does not impact typing results or dropouts. Library Preparation The instruction provided in these instructions for use has been validated to produce a final sequence- compatible barcoded library using amplified DNA from the AllType FASTplex NGS 11 Loci Flex Kit for the final concentration of 2.1 ng/µL, 0.53 ng/µL, and 0.056 ng/µL for Illumina MiSeq, MiSeq i100 Series, MiniSeq, and iSeq sequencing systems, respectively. The expected final library concentration is 10 ng/µL or above, as measured in LIBRARY AMPLIFICATION, Part 3, step 19. A lower concentration library may give incorrect or ambiguous typing due to lower reads. The input sample specifications and the procedures described in this instructions for use have been validated to produce a library that meets the target fragment size of 250bp to 2500bp with the mode of 650 to 950bp. DNA Sequencing The AllType FASTplex NGS 11 Loci Flex Kit has been validated using up to 96 samples per sequencing run to ensure a minimum average read depth of 50 reads or greater per allele when using Illumina MiSeq Reagent Kit V2 with 2x150bp (300 cycle) output, 24 samples with V2 Micro Reagent Kit, and 8 samples with V2 Nano Reagent Kit. Ninety-six (96) samples have been validated for the MiSeq i100 25M Reagent Kit and 5M Reagent Kit with 2x150bp (300 cycle). Ninety-six (96) samples with the MiniSeq High Output kit and 72 samples for the MiniSeq Mid Output Kit. Twenty-four samples have been validated for the iSeq 100 i1 Reagent V2 kit. Alternative sample-per-run throughputs are not supported by this application and must be determined and validated by the user. The following sequencing metrics in the Illumina BaseSpace Sequencing Hub report have been validated to produce reliable HLA typing results for the 96-sample run. %Q30 Reads is the number of reads that meets the sequencer QC filters and is a good metric to assess if the run produced enough reads. Mean read length indicates if the sequenced reads meet the minimum length needed to phase the sequence for high resolution typing. %CV of Reads Identified (PF) Per Index indicates if the library contains an even number of reads for each sample. A high %CV with samples with significantly lower reads indicates poor sample input or normalization that may lead to sample-specific typing issues. A deviation of a single metric does not necessarily indicate a sequencing error. A combination of low total reads and high barcode read balance %CV due to multiple low read samples, or low read length may lead to ambiguous or incorrect typing due to insufficient reads. QC metrics for Sequencing: • %Q30 Reads: ≥70% for MiSeq, MiSeq i100 Series, MiniSeq, >60% for iSeq • Mean Read Length: ≥130 bases • %CV of Reads Identified (PF) Per Index: ≤30% without excessively low reads sample(s) SPECIFIC PERFORMANCE CHARACTERISTICS Performance characteristics of AllType FASTplex NGS 11 Loci Flex Kit was studied in internal verification testing as described below. Internal verification tested performance of the AllType FASTplex NGS 11 Loci Flex Kit using six categories of test including accuracy, precision (reproducibility and repeatability), measuring range, sample preparation and interference substance. The results of these tests demonstrated that AllType FASTplex NGS 11 Loci Flex Kit product when used according to these instructions for use produces accurate and high Field-3 resolution (6 digits) resolution HLA typing results that are reproducible and repeatable between operators and three separately manufactured lots. Accuracy / Trueness of measurement was established against well characterized DNA panel. 320 DNA samples were sequenced on 3 different sequencing platforms. AllType FASTplex NGS 11 Loci Flex Kit – 96 met the acceptance criteria of 95% or higher concordance on the Illumina MiSeq Standard, Micro, and Nano v2 flowcells; MiSeq i100 Series (25M and 5M) reagent kits; MiniSeq Mid and High Output kits; and the iSeq 100 sequencing platforms, with the average concordance of any test being 99.2% using the Field-3 resolution (6 digits) based on LB Clopper-Pearson method. Discordant calls were attributed to limited resolution of reference typing and technician error. Repeatability and reproducibility were established by determining lot to lot variability using multiple lots within one run, between runs, operator, days, and sequencers. The studies demonstrated assay can withstand variabilities when assay protocol is followed as described in this Instruction for use. Robustness of the product was also demonstrated in the sample preparation, measuring range interference substance tests that showed that the product performance was unaffected. Sample Preparation studies were performed using two biological sources (Blood and Buccal Swabs) with three different DNA extraction methods, studies conclude that the expected concordance was met. Measuring Range of the assay was tested at four DNA concentrations showing that the acceptable DNA input is 3ng/µl and 50 ng/µl with the optimal concentration of 25 ng/µl. The Interference Substances test demonstrated that the presence of commonly found blood serum factors or a potential PCR inhibitor (at tested concentration) did not affect the performance of the AllType FASTplex NGS 11-Loci Flex Kit. Test Details – Accuracy for MiSeq, MiniSeq, and iSeq % Number of Concordanc Total Number Number of Discordant Total Number % e of Samples Number of Number No Allele Allele of Allele Concordanc LB Clopper- Tested Assay of Users Calls e Pearson 730 22 4 2 13,108 99.9% 99.8% Test Details – Precision for MiSeq, MiniSeq, and iSeq Number of Total % Test Samples Number Number of No Allele Discordant Number of Concordanc Category per Assay of Assay Users Calls Allele Calls e Reproducibility 48 15 3 9 12,900 99.8% Repeatability 24 9 1 0 3,888 100% Test Details – Clinical Accuracy and Reproducibility Clinical Accuracy and Reproducibility testing was conducted for Alltype FASTplex NGS Flex Assay Kits. Testing demonstrated the typing concordance percentage for each site, user, and day greater than 95.0% and the lower bound of the 95% confidence interval for each site and kit greater than 95% Clinical Accuracy for MiSeq, MiniSeq, and iSeq % Total Number Number of Concordanc of Samples/ Number of Discordant Total Number % e Sequencing Samples No Allele Allele of Allele Concordanc LB Clopper- run Analyzed Calls e Pearson 288 14 32 5,158 99.36-99.42% 98.94-99.02% Clinical Reproducibility for MiSeq, MiniSeq, and iSeq % Total Concordance Number of # of Total # of Number of % LB Clopper- sites tested samples replicates users days Allele Calls Concordance Pearson 3 of 8 3 24 2 5 12,960 100% 99.86% samples CONTACT INFORMATION Manufacturer One Lambda, Inc. 22801 Roscoe Blvd, West Hills, CA 91304, USA T 747.494.1000 | F 747.494.1001 Technical Assistance For technical questions or customer support, please contact us at: One Lambda Technical Support North America: + 1 747-494-1000 Option #2 (PST) North America Toll Free: +1 800-822-8824 Option #2 (PST) International: +49 3302883-426 (CET) International Toll Free: 00800 6200 0000 (CET) Web: www.onelambda.com Email: 1lambda-TechSupport@thermofisher.com NOTIFICATIONS of SERIOUS INCIDENT Should the user observe any serious incident that has occurred in relation to this in vitro medical device, the user shall report the serious incident to the manufacturer, any local regulatory agency, and the competent authority of the Member State in which the user is established. APPENDICES Appendix 1: PCR Program Reference Guide The following is a list of PCR programs used for the assays: A. HLA 11-Loci Amplification Illumina Program: Temperature Time Cycle 94°C 2 min 1 98°C 10 sec 30 69°C 3 min 4°C HOLD 1 9600 emulation speed and heated cover on B. TAG Program: 55°C for 15 min., 25°C hold, heated cover on C. STOP Program: 68°C for 10 min., 25°C hold, heated cover on D. FASTplex Illumina Library Amplification Program: Temperature Time Cycle 72°C 10 min 1 98°C 3 min 1 98°C 15 sec 64°C 30 sec 12 72°C 1 min 72°C 5 min 1 4°C HOLD 1 9600 emulation speed and heated cover on Appendix 2: FASTplex Sample Flex Plate 96 Worksheet H G F E D Figure 2. FASTplex Sample Flex Plate 96 TDX-OLI-DMR-PS-4201, Rev 05 Issue Date: 09 Sep 2026 1 2 3 4 5 6 7 8 9 10 Appendix 3: Quick Guide (Refer to the Instructions for Use for more details.) Illumina – AllType FASTplex NGS Assay Quick Guide • After amplification of DNA sample, purify with magnetic beads, and measure concentration with standard Qubit protocol. • Enter the Qubit values into the TSV NGS Calculator. Dilute the amplicons with the FASTplex DNA Suspension Buffer from kit as indicated. • Diluted amplicons are ready for library preparation Sample Barcoding Universal Barcoding Library Amplification 60 Minutes 40 Minutes 65 Minutes 1. FASTplex Reaction 1 1. FASTplex Reaction 2 1. Library Amplification 2. Stop 2. Purify 3. Pool 3. Purify 3. Quantify 4. Purify Note: Before beginning, thaw reagents and keep on ice. Place FASTplex Paramagnetic Beads at room temp for at least 30 minutes. Ensure that Buffers contain NO crystals IMPORTANT: Only use FASTplex DNA Suspension Buffer Sample Barcoding Steps: • Centrifuge FASTplex Sample Plate and transfer 6 µL barcoding reagent into a fresh plate • Add 4 µL diluted amplicon and pipette mix 10x • Add 5 µL FASTplex Barcoding Buffer and pipette mix 10x • Seal Plate and pulse centrifuge. Run FASTplex TAG program. • Add 7.5 µL FASTplex Stop Solution and pipette mix 5x. • Seal plate and pulse centrifuge. Run FASTplex STOP program. • Pool 10 µL from each sample barcoding reaction well. • Complete Sample Barcode (SB) purification following TSV NGS Calculator • Elute in volume of DNA suspension Buffer according to the TSV NGS Calculator. • Transfer 48 µL Purified SB pool to 0.2 mL PCR tube Universal Barcoding Steps: • Add 4 µL FASTplex Univ Barcode Flex A reagent to 48µL Purified SB Pool • Add 26 µL FASTplex Barcoding Buffer, pipette mix and pulse centrifuge. Run FASTplex TAG program. • Add 39 µL FASTplex Stop Solution, pipette mix, and pulse centrifuge. Run FASTplex STOP program. • Complete Univ Barcode (UB) Purification (1 volume equivalent) • Elute in 20 µL of FASTplex DNA Suspension Buffer • Transfer 17 µL Purified UB pool to 0.2 mL PCR tube Library Amplification Steps: • Add 8 µL FASTplex Library Primer Flex Mix to 17ul purified UB pool, • Add 75 µL FASTplex Library Amp Mix and pipette to mix • Run FASTplex Illumina Library Amplification Program • Dilute Library – 95 µL Library in 105 µl FASTplex DNA Suspension Buffer • Complete Library Purification with 160 µl of FASTplex Paramagnetic Beads (0.8x volume equivalent) • Elute in 35 µl of FASTplex DNA Suspension Buffer • Transfer 33 µL of final library to a 2mL LoBind Tube • Dilute final library for sequencer loading: Miseq 2.1 ng/µL, MiSeq i100 Series & MiniSeq 0.53 ng/µL and iSeq 0.056 ng/µL BIBLIOGRAPHY 1. Jonathan C. Barone, Katsuyuki Saito, Karl Beutner, Maria Campo, Wei Dong, Chirayu P. Goswami, Erica S. Johnson, Zi-Xuan Wang, Susan Hsu, “HLA-genotyping of clinical specimens using Ion Torrent-based NGS,” Tissue Antigens (2015) 76:903-909 2. Takashi Shiina, Kazuyoshi Hosomichi, Hidetoshi Inoko and Jerzy K Kulski: “The HLA genomic loci map expression, interaction,diversity and disease,” Journal of Human Genetics (2009) 54 15-39 3. SGE Marsh, ED Albert, WF Bodmer, RE Bontrop, B Dupont, HA Erlich, M Fernández-Vina, DE Geraghty, R Holdsworth, CK Hurley, M Lau, KW Lee, B Mach, WR Mayr, M Maiers, CR Müller, P Parham, EW Petersdorf, T Sasazuki, JL Strominger, A Svejgaard, PI Terasaki, JM Tiercy, J Trowsdale “Nomenclature for factors of the HLA system,” 2010. Tissue Antigens (2010) 75 291-455 4. American Society for Histocompatibility and Immunogenetics Laboratory Manual, 4th Edition Volume 1, American Society for Histocompatibility and Immunogenetics (2000) 5. EFI Standards for Histocompatibility & Immunogenetics Testing, Version 7.0The European Federation for Immunogenetics, Strasbourg, France (2017) TRADEMARKS All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. “Eppendorf” “and “LoBind” are trademarks of Eppendorf AG. “MiSeq,”, ”MiSeq i100 Series”, “MiniSeq,” and “iSeq” are trademarks of Illumina, Inc. DISCLAIMERS All One Lambda products are designed to assist personnel experienced in HLA analysis by suggesting typing results or antibody assignments. All test results must be carefully reviewed by qualified personnel to assure correctness. Specifications, terms, and pricing are subject to change. Not all products are available in all countries. Please consult the local sales representative for details. EUROPEAN AUTHORIZED REPRESENTATIVE MDSS GmbH, Schiffgraben 41, 30175, Hannover, Germany EXPLANATION of SYMBOLS Reference EN ISO 15223-1: Medical Devices – symbols to be used with medical device labels, labeling and information to be supplied. Symbol Description Symbol Description Consult instructions for Contains sufficient for <n> use. An eIFU indicator can tests be a manufacturer's ISO 7000 Reg No. 1641 website URL or some other ISO 7000 Reg No. 0518 appropriate indication that the instructions for use are available in an electronic format. Catalog number Manufacturer ISO 7000 Reg No. 2493 ISO 7000 Reg No. 3082 In vitro diagnostic medical Authorized representative device in the European Community Caution Date of Manufacture ISO 7000 Reg No. 0434A ISO 7000 Reg No 2497 Temperature limitation Use by Date ISO 7000 Reg No 2607 ISO 7000 Reg No. 0632 Upper limit of Temperature Batch code ISO 7000 Reg No. 2492 ISO 7000 Reg No. 0633 Other Symbols Irritant (Skin, Eyes) Carcinogen The batch field on the label is for traceability of a manufacturing event. For Summary of Safety and Performance, contact TDX Customer Service. EXPLANATION of HELPFUL SYMBOLS Icon Description Safe Stopping Point Note Useful tip REVISION HISTORY Revision Date of Issue Revision Description 03 03 Nov 2025 Clarification to specimen storage. Update Intended Purpose, and Performance Characteristics to comply 04 09 MAR 2026 with IVDR 05 Current Inclusion of the MiSeq i100 Series © 2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Specifications, terms, and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.