WIXLIB

One Lambda, Inc. Application Note: AllType FASTplex NGS Assay on Illumina Sequencing Systemsc) STOP Program: 68 C for 10 min., 25 C holdd) Library Amplification Program:TEMPERATURETIMECYCLE72 C98 C98 C10 min3 min15 sec1164 C72 C72 C4 C30 sec1 min5 minHOLD15116. Pulse-spin kit components. Liquids can condense or shift locations inside containers during shipment orstorage. Before every use of the Sample Plate, and before dispensing from reagent tubes, pulse-spin ina suitable centrifuge to gather the reagents at the bottom of the well or tube. If the kit components freezeduring shipment or storage, thaw, mix, and pulse-spin before use.7. Equilibrate Paramagnetic beads to room temperature. Paramagnetic beads can be stored for up to 2weeks at room temperature or for longer periods at 2 - 8 C. If stored cold, set the beads at roomtemperature for 30 minutes and vortex them thoroughly to resuspend before use. Pipette slowly and donot pre-wet pipette tips to transfer volumes accurately.8. Check the Stop Solution for precipitate before use. If a precipitate is visible, incubate the tube at 37 C for5 minutes. Mix gently by inversion until the precipitate dissolves (do not vortex).Note: Stop Solution contains SDS and will precipitate if stored below room temperature. Overly vigorousmixing will cause foaming.9. The FASTplex Barcoding Buffer is viscous and should be stored at room temperature when in use. Totransfer Barcoding Buffer accurately, pipette slowly and do not pre-wet pipette tips. While addingBarcoding Buffer to reactions, mix in the Barcoding Buffer completely by pipetting up and down severaltimes with the same pipette tip(s) that was used for addition.10. Prepare 80% ethanol fresh daily.11. Use FASTplex DNA Suspension Buffer supplied in the kit to resuspend purified amplicons, and in allother elution steps. Do not use EDTA-containing solutions (e.g., TE buffer) to dissolve or dilute inputDNA because EDTA can inhibit enzymatic activity.12. Safe-stopping points are indicated in the procedure. For optimal results, proceed directly to the next stepunless an optional safe-stopping point is indicated.E. Barcoding Strategy1. Plan sample barcoding before starting the assay. The AllType FASTplex NGS 11 Loci Flex Kit – 96 usesa combinatorial dual indexing strategy. The kit contains a total of 24 unique Sample Barcodes (SB) (i7indices) and 4 unique FASTplex UB (i5 indices), to allow up to 96 samples per run. Each library (or pool)can contain between 8 to 24 samples and is barcoded with one unique UB. When planning a librarypreparation, select non-overlapping SB if the sample size is between 8-24. For libraries that containmore than 24 samples, refer to section 3b below to prepare libraries with more than one UB with samplesize recommendations.The following figure provides an example layout for a 96-sample library – i.e., four batches of 24samples. A01 through H03 represents the Sample Barcode indices, while F1 through F4 represents theUB indices.TDX-OLI-DMR-PS-3213, Rev 02Page 6 of 27

One Lambda, Inc. Application Note: AllType FASTplex NGS Assay on Illumina Sequencing Systems24 SamplesBatch 1A01 A02 A03B01 B02 B03C01 C02 C03D01 D02 D03E01 E02 E03F01 F02 F03G01 G02 G03H01 H02 H0324 SamplesBatch 2A01 A02 A03B01 B02 B03C01 C02 C03D01 D02 D03E01 E02 E03F01 F02 F03G01 G02 G03H01 H02 H0324 SamplesBatch 3A01 A02 A03B01 B02 B03C01 C02 C03D01 D02 D03E01 E02 E03F01 F02 F03G01 G02 G03H01 H02 H0324 SamplesBatch 4A01 A02 A03B01 B02 B03C01 C02 C03D01 D02 D03E01 E02 E03F01 F02 F03G01 G02 G03H01 H02 H03Univ Barcode F1Univ Barcode F2Univ Barcode F3Univ Barcode F4Note: Each batch, or pool, of samples does not need to be the same size but should be as equal aspossible. The minimum number of samples per pool is 8 samples. When performing multiple batch runs,the number of samples per pool (using different UB) must be equal to the number recommended by theAllType FASTplex NGS 11 Loci Flex Kit Calculator. Refer to section 3b below to calculate samplenumbers when the total number of samples is above 24.2. To perform a sequencing run with sample sizes above 24, enter the total number of samples to cell F6 ofthe “Pool Configuration” tab in the AllType FASTplex NGS 11 Loci Flex Kit Calculator. The samplenumbers for each pool will be shown in the Recommended Samples for each pool in cell D8, G8, J8 andM8. Use of the recommended sample sizes is to ensure good index read balance for healthy index readbalance. Examples of pool sizes for various sample numbers are listed in table below, RecommendedBarcoding and Pooling for Various Sample Sizes.3. Alternatively, to combine libraries of different sample sizes, follow the instructions below.a) For the libraries with sample sizes between 8-24 and shares the same UB, make sure that the SBdoes not overlap. Skip to step 3-c below.b) For the libraries with sample sizes between 8-24 with non-overlapping UB, proceed to step 3-c.c) As described in Direction for Use, section D-4-c, Quantification of Final Library, step (16), adjust theconcentration of the libraries to an appropriate concentration for the platform used (Table 4-c-15).Mix libraries using volumes that are proportional to the sample size of the libraries. For example, tomix two libraries of a sample size of 8 and 24 for MiSeq, adjust the concentrations to 1.35 ng/µL. Mix8 parts and 24 parts of the libraries such as 8 and 24 µL. Proceed to the next step after section D-4c- (16).Recommended Barcoding and Pooling for Various Sample SizesSample Size fora Single Run8- 24Multiples of 2425294955657385Recommended SampleSizes for PoolUse the whole sampleper poolUse 24 sample size per apool13 1215 1417 16 1619 18 1822 21 2219 18 18 1922 21 21 21Number of Unique UBReagents Required1Use 1 unique UB per24 sample2233344F. Reagent Volumes for Various Sample Sizes1. The AllType FASTplex NGS 11 Loci Flex Kit accommodates up to 96 samples using a dual indexingmethod – i.e. a combination of 24 sample barcodes and 4 UBs. When processing smaller sampleTDX-OLI-DMR-PS-3213, Rev 02Page 7 of 27

One Lambda, Inc. Application Note: AllType FASTplex NGS Assay on Illumina Sequencing Systemsbatches, different volumes for each of the following reagents will be used: (1) FASTplex ParamagneticBeads, (2) FASTplex Barcoding Buffer, and (3) FASTplex UB reagents (F1, F2, F3, F4). Note that thevolumes of reagents, diluted amplicons, SB reagent, and Barcoding Buffer for the sample-barcoding(SB) reactions remains the same for all sample numbers.2. Use the AllType FASTplex NGS 11 Loci Flex Kit Calculator to display the required reagent volumes. Inthe “Pool Configuration” tab, enter the total number of samples into cell F6, and enter sample IDs intothe appropriate column(s). In the “SB – UB -Lib. Amp” tab, the required reagent volumes are displayedin per pool.3. The kit contains enough barcoding reagents, Barcoding Buffer and Stop Solution to process 12 runs of 8samples per run. The required volume of reagents for smaller sample pool sizes are reduced as shownbelow.Important: AllType FASTplex NGS 11 Loci Flex Kit contains enough reagents for the followingcombinations of sample size and run.SampleSizeNumber of BatchAccommodatedVolume of PooledSB ReactionVolume ofUB Reagentper SampleVolume ofBarcodingBuffer for OLI-DMR-PS-3213, Rev 02Volume ofStopSolution forUB43414038Volume ofStopped UBReaction130122119115Page 8 of 27

One Lambda, Inc. Application Note: AllType FASTplex NGS Assay on Illumina Sequencing SystemsD. Direction for Use1. Materials: Genomic DNA Amplification, Purification, Quantification, and DilutionRequired materials and equipment Diluted genomic DNA (25ng/uL)Diluted 11-Loci amplicons (3-30ng)Eppendorf DNA LoBind Tubes, 2.0 mL96-Well, 0.2mL, PCR PlatesAllType FASTplex BufferAllType FASTplex dNTPsAllType FASTplex Primer MixAllType FASTplex PolymeraseNuclease Free WaterParamagnetic BeadsAllType FASTplex DNA Suspension Buffer200 Proof Ethanol, Molecular Biology GradeNunc 96-Well Polypropylene MicroWell PlatesMagnetic Plate Stand50mL Polystyrene Reagent ReservoirsQubit dsDNA High Sensitivity Assay KitQubit FluorometerFull array of manual single channel pipettes (20 µL, 200 µL, 1 mL)Full array of filtered, pre-sterilized, pipette tipsSerological Pipettes, 10 or 25mLPCR Plate CentrifugeMini CentrifugeVortexIce or Cold BlockThermal cycler PCR Pressure PadVeriti 96-Well Thermal Cycler2. Procedure: Genomic DNA Amplification, Purification, Quantification, and Dilutiona) Sample Amplification1) Turn on and program a thermal cycler to run the HLA 11-loci Amplification Program below. Besure to use 9600 emulation speed or 0.8 C/sec heating and -1.6 C/sec cooling.HLA 11-LOCI AMPLIFICATION ILLUMINA PROGRAM (9600 EMULATION)TEMP OCTIMECYCLESO2 min1O98 C10 sec69OC3 min4OCforever94 C3012) Thaw genomic DNA diluted at 25 ng/µL (as described in Procedure – Section C, Before Startingthe Procedure), AllType FASTplex 11 Loci Primer Flex Mix, AllType dNTP and AllType Buffer atroom temperature. Keep the AllType Polymerase tube on ice. Briefly vortex all reagentsEXCEPT polymerase. Spin down all reagents and keep on ice until ready for use.3) In a 96-Well 0.2mL PCR Plate, aliquot 4.0 µL of genomic DNA. Record the sample IDs and wellpositions in the PCR plate.4) In a 2.0 ml LoBind Tube (or higher volume tube for 96 samples), prepare an Amplification MasterMix based on the recipe shown in the “Amp-Clean Up-Quant” tab of the AllType FASTplex NGS11 Loci Flex Kit Calculator (Calculator). Use the Calculator to display the volumes of theTDX-OLI-DMR-PS-3213, Rev 02Page 9 of 27

One Lambda, Inc. Application Note: AllType FASTplex NGS Assay on Illumina Sequencing Systemsamplification reagent volumes (with overage) required for the selected sample size. Go to the“Amp-Clean Up- Quant” tab, add the sample size in the cell F3 (Total Number of Samples toAmplify) to show the necessary reagent volumes.5) Prepare reagents in the order shown in the “Amplification” table of the “Amp – Clean Up –Quant” tab in the calculator. At this stage, only prepare the Amplification Mix inclusive of the firstfour (4) components. Do NOT add the Polymerase at this stage. Vortex the mix for 10 seconds,pulse-spin and keep on ice.6) Add Polymerase to the Master Mix according to the “Amplification” table in the calculator. Mixwell by pipetting 15-20 times with the pipet volume set to half the total volume of the Master Mix.7) Aliquot 16 µL of the Master Mix to each well with DNA. Seal the plate with a tray seal, and pulsespin the plate.8) Load the plate into a thermal cycler and cover the plate with a pressure pad. Run the HLA 11loci Amplification program.SAFE-STOPPING POINTProceed immediately to the next step,ORstore the amplified products at -20ºC.b) Amplicon Purification1) Equilibrate the Paramagnetic Beads to room temperature for 30 minutes.2) Prepare fresh 80% ethanol solution enough for 200µL x Number of samples plus 15% overageby measuring ethanol and nuclease-free water separately and mixing.3) Vortex Paramagnetic Beads at medium speed for 30 seconds to fully resuspend the beads. In aNunc LoBind 96-well plate, aliquot 12µL of the beads to each well. Use multi-channel pipetteand reagent reservoir for convenience.4) Transfer all amplified product ( 20µL) to corresponding wells, mix by pipetting up and down 10times. Change tips for each sample and avoid producing foam.5) Incubate at room temperature for 5 minutes. Place the plate on the magnetic ring stand for 3minutes or until the solution clears.6) With a pipette set to 28µL, slowly remove and discard the supernatant from each well withoutdisturbing the bead pellet.7) With the plate on the magnet, add 100µL of 80% ethanol to each well. Incubate at roomtemperature for 30 seconds until the solution clears.8) With a pipette set to 110µL, remove and discard the supernatant without disturbing the beadpellet.9) Repeat steps 7) to 8) above for a second ethanol wash.10) With the plate on the magnet, remove visible ethanol residue with pipette. Air-dry the beads atroom temperature for 5 minutes or less on the magnet.11) With the plate on magnet, add 27 µL of FASTplex DNA Suspension Buffer to each well. Removethe plate from the magnet. Mix each sample by pipetting up and down 10 times and incubate atroom temperature for 3 minutes.TDX-OLI-DMR-PS-3213, Rev 02Page 10 of 27

One Lambda, Inc. Application Note: AllType FASTplex NGS Assay on Illumina Sequencing Systems12) Place the plate back on magnet for 3 minutes to clear the solutions. Set a pipette volume to25µL and transfer the supernatant to a new 0.2 mL 96-well PCR plate without disturbing thebeads.SAFE-STOPPING POINTProceed immediately to the next step,ORstore the purified products at -20ºC.c) Amplicon Quantification1) Label Qubit Assay Tubes for each sample and two additional tubes for Qubit Standard #1 and#2.2) Prepare a Working Qubit Solution in a 10 ml tube for a batch of 24 samples plus 15% overage(28 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 prior to use.3) Vortex the Working Qubit Solution and cover with foil. Use within 2 hours.4) Add 198 µL of Working Solution to each labeled Qubit Assay Tube. Add 190µL of WorkingSolution to the two tubes for the Standards.5) Add 2 µL of the corresponding amplicon to each labeled tube. Add 10 µL of the correspondingStandard to labeled tube.6) Briefly vortex all tubes, spin, cover with foil, and incubate at room temperature for 2 minutes.7) Turn on the Qubit Fluorometer, select DNA from the home screen, and select dsDNA HighSensitivity.8) Press the appropriate button to begin reading the standards. Measure Standard #1 and #2 tocomplete calibration.9) Begin reading of amplicon tubes. When prompted, change the volume of sample used to 2 µLand the units to ng/µL. Record the concentration of sample.10) Repeat for all samples.11) Repeat measurement of all samples that exceed the upper detection limit of 100 ng/µL bymeasuring two-fold diluted samples. Multiply the reading by a factor of 2.d) Amplicon DilutionThe range of valid amplicon input amount into Library Preparation is 3-30 ng. The optimal inputamount is 10 ng in an 8 µL volume at 1.25 ng/µL. Lower concentration sample may not containadequate quantity of the amplicons. The steps below will help calculate a volume of FASTplex DNASuspension Buffer to dilute amplicons.1) The AllType FASTplex NGS 11 Loci Flex Kit Calculator (Excel file) facilitates the amplicondilution, final library pool dilution, and sample sheet preparation. The worksheet contains twotabs that assist calculating dilution factors and the volumes of diluents, “Amplicon Dilution,”“Plate Layout” tabs.TDX-OLI-DMR-PS-3213, Rev 02Page 11 of 27

One Lambda, Inc. Application Note: AllType FASTplex NGS Assay on Illumina Sequencing SystemsCalculator Tab IDAmplicon DilutionPlate LayoutFunctionsCalculation of 24 amplicon dilution factors anddiluent volumes to prepare 3-30ng input DNA forSample Barcoding reactionView the result of input DNA and FASTplex DNASuspension Buffer volumes in an 8 x 12 plateformat2) Open the AllType FASTplex NGS 11 Loci Flex Kit Calculator. Open the tab named “PoolConfiguration” and enter the total sample numbers to the cell F6. Enter sample IDs in thecorresponding column under Sample ID for each pool. The sample ID entered in this tab willbe automatically copied onto the rest of the worksheet. In the “Amplicon Dilution” tab, enter theamplicon concentrations in the column labeled “Input (ng/µL).”3) The table will display a volume of FASTplex DNA Suspension Buffer to add to 2 µL of eachamplicon [“Sample (µL)” column]. Samples whose amplicon concentrations, after dilution, falloutside 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 respectivesample.4) In a clean 96-well plate, add the indicated volume of FASTplex DNA Suspension Buffer and 2µL of the stock amplicon. Use the Amplicon Dilution - Plate View to locate its positions in theplate.5) For the outlier samples, add volumes of the stock amplicon and FASTplex DNA SuspensionBuffer indicated in the Sample (µL) and FASTplex DNA Suspension Buffer (µL) columns foreach sample. Use the Amplicon Dilution - Plate View to locate its positions in the plate.6) Carefully mix the diluted amplicon well by pipetting. Avoid cross-contamination. Seal the plate,pulse-spin, and store on ice or store at -20 C.SAFE-STOPPING POINTProceed immediately to the next step,ORstore the purified products at -20ºC.3. Sample BarcodingRequired materials and equipment Diluted ampliconsEppendorf DNA LoBind Tubes, 2.0 mLPCR tube 0.2 mL96-Well, 0.2mL, PCR Plates2.0 mL Tube Magnet StandSerological Pipettes, 10 or 25mLFASTplex Sample Flex Plate 24FASTplex Univ Barcode F1, F2, F3, F4FASTplex Barcoding BufferFASTplex Stop SolutionFASTplex Paramagnetic Beads (Equilibrated to room temperature)FASTplex DNA Suspension Buffer200 Proof Ethanol, Molecular Biology GradeFull array of manual single channel pipettes (20 µL, 200 µL, 1 mL)Full array of filtered, pre-sterilized, pipette tipsThermal cycler PCR Pressure PadVeriti 96-Well Thermal CyclerTDX-OLI-DMR-PS-3213, Rev 02Page 12 of 27

One Lambda, Inc. Application Note: AllType FASTplex NGS Assay on Illumina Sequencing Systemsa) Sample Barcoding (SB) Reaction, Reaction Stop, Sample Pooling, and Purification1) Pulse-spin the FASTplex Sample Flex Plate 24 in a centrifuge. After centrifugation, carefullyremove the plate seal from the Sample Flex Plate and discard the plate seal (do not reuse theplate seal) and place it on ice. Equilibrate FASTplex Paramagnetic Beads at room temperature.Useful Tip: The Sample Barcoding step is the only step where an individual sample must betreated separately. An 8-channel pipette and use of an 8-tube strip can make this step faster andless error-prone. Common reagents, Barcoding Buffer and Stop Solution, can be pre-aliquotedinto an 8-tube strip or equivalent for use with the 8-channel pipette. The volume of reagents toaliquot using an 8-

Qubit Fluorometer or equivalent Thermo Fisher Scientific Q33216 or Q33226 Qubit dsDNA HS Assay Kit ThermoFisher Scientific Q32854 Qubit Assay Tubes Thermo Fisher Scientific Q32856 Plate Centrifuge capable of 1,500 x g MLS [1]---Mini/Micro Centrifuge MLS [1]---Vortex MLS [1]---[1]Major Laboratory Supplier C. Before Starting the Procedure 1.