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4 IMPORTANT NOTES AND UPDATES This workflow does not use BSA in the fragmentation reaction for improved SPRI bead elution. This workflow has a new denaturation protocol for improved cluster density. RUO Edition 1, 2015/06 4 OF 36

5 CONTENT 1 KEY TO SYMBOLS 6 2 KIT CONTENT 7 3 SHIPPING AND STORAGE 7 4 TECHNICAL ASSISTANCE 8 5 WARNING AND PRECAUTIONS 8 6 PRINCIPLE 9 7 PROCEDURE 10 8 EQUIPMENT AND REAGENTS 10 9 Protocols 13 PROTOCOL 1. HLA LOCUS-SPECIFIC AMPLIFICATION 13 PROTOCOL 2A. DNA QUANTIFICATION 16 PROTOCOL 2B. POOLING OF AMPLICONS (OPTIONAL) 18 PROTOCOL 3A. FRAGMENTATION AND ADAPTER LIGATION 19 PROTOCOL 3B. DNA CLEAN-UP AND SIZE SELECTION WITH 0.45x SPRI BEADS 21 PROTOCOL 3C. INDEXING PCR 23 PROTOCOL 3D. DNA CLEAN-UP AND SIZE SELECTION WITH 0.6x SPRI BEADS25 PROTOCOL 4A. LIBRARY POOLING 27 PROTOCOL 4B. LIBRARY QUANTIFICATION 28 PROTOCOL 4C: SAMPLE SHEET SET-UP 31 PROTOCOL 4D. LIBRARY DENATURATION APPENDIX A: CONTAMINATION CONTROL TROUBLESHOOTING GUIDE LIMITED LICENSE AGREEMENT 35 ORDERING INFORMATION 36 DISCLAIMER GenDx has made every effort to ensure that this IFU is accurate. GenDx disclaims liability for any inaccuracies or omissions that may have occurred. Information in this IFU is subject to change without notice. GenDx assumes no responsibility for any inaccuracies that may be contained in this IFU. GenDx reserves the right to make improvements to this IFU and/or to the products described in this IFU, at any time without notice. If you find information in this manual that is incorrect, misleading, or incomplete, we would appreciate your comments and suggestions. Please send them to info@gendx.com. COPYRIGHT This publication, including all photographs, illustrations, is protected under international copyright laws, with all rights reserved. Neither this manual, nor any of the material contained herein, may be reproduced without written consent of the author. Copyright 2015 RUO Edition 1, 2015/06 5 OF 36

6 1 KEY TO SYMBOLS Material number Components Batch code / Lot number Catalogue number Consult Instructions For Use Contains reagents for N tests Legal manufacturer Store at -20 C VOL Store at -20 C Volume Add liquid RUO Edition 1, 2015/06 6 OF 36

7 2 KIT CONTENT The NGSgo workflow consists of three parts (see 8 Equipments and reagents for product overview and ordering information): 1. NGSgo-AmpX consists of dedicated primer sets for the amplification of individual HLA genes. NGSgo-AmpX products are available for 24 or 96 reactions (CE and RUO), enabling the amplification of the following HLA genes: Class I: HLA-A, -B, -C Class II: HLA-DRB1, -DRB3/4/5, -DPA1, -DPB1, -DQA1, -DQB1, Nonclassical: HLA-G 2. NGSgo-LibrX for Illumina consists of reagents for library preparation (fragmentation, DNA end-repair, da-tailing, adapter ligation, dual-indexing PCR) from HLA amplicons. The DNA libraries are compatible with Illumina NGS platforms. 3. NGSgo-IndX for Illumina consists of an adapter and a panel of indices for the dualindexing of libraries, for sample identification and sequencing on the Illumina MiSeq. Three NGSgo-IndX products are available: NGSgo-IndX (4x 24), enabling multiplexing of 24 libraries. NGSgo-IndX (2x 96), enabling multiplexing of 96 libraries. NGSgo-IndX (1x 384), enabling multiplexing of 384 libraries. IMPORTANT NOTE: Use of all three NGSgo components in the same workflow constitutes a workflow that is currently for research use only. Please see section 5 for product use limitations. 3 SHIPPING AND STORAGE NGSgo is: Shipped on ice packs and should be stored at -20 C upon arrival. Stable until the kit expiration date (kit label) when stored at -20 C. Amplification primers are stable for 5 months after dissolving primers in nuclease free H 2O when stored at -20 C. RUO Edition 1, 2015/06 7 OF 36

8 4 TECHNICAL ASSISTANCE For technical assistance and more information: Website: Phone: +31 (0) or contact your local GenDx distributor ( 5 WARNING AND PRECAUTIONS Product Use Limitations The NGSgo workflow is for Research Use Only and not to be used in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. To ensure the best performance, please use the NGSgo products and the NGSgo workflow for HLA typing by NGS with the materials, reagents, and equipment recommended in section 8 Equipment and Reagents. Use of materials other than specified, must be validated by user! Reconstitution or dilution of reagents in volumes other than described in this IFU can lead to performance errors and is strongly discouraged! Please take special note of Appendix A: Contamination control. Before implementing the NGSgo workflow for HLA typing by NGS in your laboratory, please perform a validation of sequencing-based typing methods using known molecular typed samples. Such samples may be obtained from the International Workshop Reference Cell Panel or the UCLA DNA Reference Panel. Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from Product Application The NGSgo workflow for HLA typing is designed for high-resolution identification of Human Leukocyte Antigen (HLA) alleles by means of Next-Generation Sequencing (NGS). RUO Edition 1, 2015/06 8 OF 36

9 6 PRINCIPLE The GenDx NGSgo workflow for HLA typing enables the high-resolution identification of Human Leukocyte Antigen (HLA) alleles by means of Next-Generation Sequencing (NGS). NGSgo is compatible with Illumina sequencing platforms. HLA locus-specific amplification with NGSgo-AmpX NGSgo-AmpX consists of dedicated primer sets, enabling the amplification of HLA genes. HLA locus-specific amplification is performed in a thermal cycler, using the amplification primer mix, template genomic DNA and the QIAGEN LongRange PCR kit. Each HLA locus is amplified separately. The HLA amplicons are verified on an agarose gel and the amplicon concentration is determined with a suitable DNA quantification method, such as Qubit or Nanodrop. The DNA quantification protocol described in this IFU refers to the Qubit DNA BR Assay kit which measures dsdna using the fluorescent signal of a DNA-intercalating dye. Alternatively the Nanodrop can be used, but this method is less accurate as it does not distinguish dsdna from other components. After quantification, the amplicons of different HLA loci per sample can be pooled in equimolar ratios prior to library preparation to increases the sample throughput. Fragmentation and adapter ligation with NGSgo-LibrX and NGSgo-IndX NGSgo-LibrX consists of library preparation reagents for the HLA amplicons. In the first reaction, different enzymes are used for the random fragmentation, end-repair and datailing of the HLA amplicons. A-tailing is required to create binding sites for the Illuminacompatible adapter, which is ligated to the DNA fragments, using ligation reagents from the NGSgo-LibrX kit and the adapter from the NGSgo-IndX kit. The adapter creates binding sites for the Illumina-compatible indices and creates binding sites for the sequencing primers. After adapter ligation, the DNA is size-selected and cleaned-up using magnetic beads. DNA fragments of ~400 bp and larger are selected and any shorter fragments or remaining enzymes and salts are removed. Indexing PCR with NGSgo-LibrX and NGSgo-IndX The adapter-ligated DNA fragments are dual-indexed during the indexing PCR using the HiFi PCR Mix from the NGSgo-LibrX kit and indices from the NGSgo-IndX kit. The indices are composed of index 1 (i7) and index 2 (i5) primers, containing sequences required for cluster formation and sample identification. After indexing, a second size-selection and cleanup step is performed using magnetic beads. The indexed libraries are pooled to create one library pool, representing multiple HLA loci of multiple samples. The library concentration is determined to achieve an optimal cluster density on the Illumina flowcell. The library quantification protocol described in this IFU refers to the KAPA Library Quantification kit, which is qprc-based using adapter-specific primers to accurately quantify the HLA library. Next-generation sequencing and data analysis The libraries are sequenced on an Illumina NGS platform. The FastQ data can be analysed with the software package NGSengine to determine the HLA genotype. RUO Edition 1, 2015/06 9 OF 36

10 7 PROCEDURE Indicated times are for preparing 96 individual libraries simultaneously. Total hands-on time: 3-4 hours Total time: hours NGSgo- AmpX Protocol 1 HLA locus-specific amplification Hands-on time: 60 minutes Total time: 5-7 hours Protocol 2A Protocol 2B DNA quantification Hands-on time: 30 minutes Total time: 30 minutes Pooling of amplicons (optional) Hands-on time: 15 minutes Total time: 15 minutes NGSgo- LibrX & NGSgo- IndX Protocol 3A Protocol 3B Protocol 3C Protocol 3D Fragmentation and adapter ligation Hands-on time: 20 minutes Total time: 70 minutes No safe stopping point DNA clean-up and size selection with 0.45x SPRI beads Hands-on time: 15 minutes Total time: 1 hour Indexing PCR Hands-on time: 15 minutes Total time: 1 hour DNA clean-up and size selection with 0.6x SPRI beads Hands-on time: 15 minutes Total time: 45 minutes Protocol 4A Protocol 4B Protocol 4C Library pooling Hands-on time: 10 minutes Total time: 10 minutes Library quantification Hands-on time: 15 minutes Total time: 1.5 hour Sample sheet set-up Hands-on time: 15 minutes Total time: 15 minutes Protocol 4D Library denaturation Hands-on time: 5 minutes Total time: 15 minutes Illumina NGS run RUO Edition 1, 2015/06 10 OF 36

11 8 EQUIPMENT AND REAGENTS Table 1. NGSgo product overview Product description Number of Catalogue number reactions CE RUO Supplier HLA locus-specific amplification (NGS platform-independent) NGSgo-AmpX HLA-A GenDx NGSgo-AmpX HLA-B GenDx NGSgo-AmpX HLA-C GenDx NGSgo-AmpX HLA-DPB GenDx NGSgo-AmpX HLA-DQB GenDx NGSgo-AmpX HLA-DRB GenDx NGSgo-AmpX HLA-DRB3/4/ GenDx NGSgo-AmpX HLA-DQA GenDx NGSgo-AmpX HLA-DPA GenDx NGSgo-AmpX HLA-G GenDx NGSgo-AmpX HLA-A, B, C, DRB1, DQB GenDx NGSgo-AmpX HLA-DPB1, DPA1, DQA1, DRB3/4/ GenDx NGSgo-AmpX HLAGeneSuite* GenDx Library preparation, compatible with Illumina NGS platform NGSgo -LibrX Library 48 n.a Preparation for Illumina 96 n.a GenDx 4x24 n.a NGSgo -IndX Adapter & 2x96 n.a Indices for Illumina 1x384 n.a GenDx NGSgo-IndX caps 24 (purple) n.a GenDx 24 (white) n.a GenDx *Contains amplification primers for HLA-A, B, C, DRB1, DQB1, DPB1, DPA1, DQA1, DRB3, DRB4 and DRB5 For a complete overview of reagents and equipment required for the NGSgo workflow, please see RUO Edition 1, 2015/06 11 OF 36

12 Table 2. Equipment and reagents per protocol Equipment and reagents Catalogue number Supplier General equipment and reagents Pipettes and tips (hydrophobic filters) N.A. Multiple Multi-channel pipettes (10 µl and 300 µl) N.A. Multiple Ice or cooling block (4 C) N.A. Multiple Reaction tubes and/or 96-wells reactions plates N.A. Multiple PCR tubes, strips or plates N.A. Multiple Adhesive seals or 8-strip caps N.A. Multiple Micro centrifuge N.A. Multiple Centrifuge N.A. Multiple Thermal cycler N.A. Multiple Vortex N.A. Multiple MilliQ water N.A. Multiple Protocol 1: HLA locus-specific amplification NGSgo-AmpX See Table 1 GenDx QIAGEN LongRange PCR Kit GenDx Agarose gel electrophoresis system N.A. Multiple Gel Loading Dye, Orange (6x) B7022S New England Biolabs Quick-Load 1 kb DNA ladder N0468S; N0468L New England Biolabs Protocol 2: DNA quantification Qubit DNA BR Assay Kit (500 assays) Q32853 Life Technologies Qubit Fluorometer Q33216 Life Technologies Qubit Assay Tubes Q32856 Life Technologies Protocol 3: HLA library preparation NGSgo-LibrX and IndX for Illumina See Table 1 GenDx NGSgo-IndX caps See Table 1 GenDx Magnetic SPRI beads (50 ml); A63880 (5 ml), A63881 (60 ml), A63882 (460 ml) Magnetic stand-96 AM10027 Ambion Shaker (1500 rpm) N.A. Multiple 100% ethanol N.A. Multiple Macherey-Nagel or Beckman Coulter Elution buffer (10 mm Tris-HCl, 0.1% Tween20 (ph N.A. Multiple 8.0) or 0.1x TE) 25 ml reagent reservoir AVR Protocol 4: Library pooling, quantification and denaturation KAPA library quantification kit for Illumina KK4824 KAPA Biosystems Real-time PCR instrument N.A. Multiple Optical tubes/plates and seal (real-time instrumentdependent) N.A. Multiple 1 M NaOH N.A. Multiple Next-generation sequencing and NGS data analysis Illumina NGS platform See Illumina website Illumina Illumina MiSeq reagents MS (standard), Illumina MS (micro), MS (nano) NGSengine HLA typing software See GenDx website GenDx RUO Edition 1, 2015/06 12 OF 36

13 8 PROTOCOLS PROTOCOL 1. HLA LOCUS-SPECIFIC AMPLIFICATION Important notes before starting: Blood samples should be collected in tubes with ACD or EDTA as an anticoagulant. Do NOT use heparinized samples. Purified DNA should have an A260/A280 ratio between 1.7 and 1.9. To streamline the process, validate your DNA purification procedure so that you can use a set volume corresponding to ng DNA. If necessary, DNA should be diluted in nuclease free H2O before use. The optimal amount of template DNA to use in the NGSgo-AmpX amplification reaction is 100 ng, as determined with a Nanodrop. However, template DNA in the range of ng (in 1 4 μl) can be used without affecting results. Centrifuge all NGSgo-AmpX tubes containing the amplification primers (red caps) for at least one minute before opening for the first time to ensure that the pellet is at the bottom of the tube. Resuspend each primer in 27 µl nuclease free H2O (24 reaction products) or 108 µl nuclease free H2O (96 reaction products). Invert the tubes a couple of times to ensure that the primer is completely dissolved, thoroughly vortex each tube and centrifuge the tubes for one minute. Prepare a volume of reaction mix at least 10% greater than required for the total number of assays to be performed. Protocol: 1. Set up all reactions on ice or on a cooling block (4 C). 2. Prepare a separate reaction mix for each amplification primer. 3. Thaw 10x LongRange PCR Buffer, dntp mix, nuclease free H2O, and primer solutions. Mix and centrifuge briefly. 4. HLA-DQB1 requires the addition of Q-Solution and double the amount of LongRange enzyme per reaction, Table 3. It is extremely important to include at least one negative control in every PCR setup that lacks template nucleic acid to detect possible contamination. 5. Mix the reaction mix thoroughly, and centrifuge briefly. RUO Edition 1, 2015/06 13 OF 36

14 6. Dispense the reaction mix into each PCR tube. The appropriate volume is 25 μl minus the amount of DNA to be added in the next step. Table 3. Composition of the NGSgo-AmpX reaction mix for HLA locus-specific amplification Component All loci (except DQB1) DQB1 Nuclease free H2O μl μl Q-Solution (5x) - 5 μl LongRange Enzyme Mix (5 U/μl) 0.4 μl 0.8 μl LongRange Buffer (10x) 2.5 μl dntp mix (10 mm each) 1.25 μl AmpX primer (red cap) 1 μl Template DNA 1 4 μl Total Volume 25 μl 7. Add 1 4 μl template DNA ( ng) to each tube containing reaction mix. 8. Program the thermal cycler according to the manufacturer s instructions, using the conditions outlined in Table 4. Important: For an artificial hot start, place the tubes immediately into a thermal cycler that is heated to 95 C and start the cycling program as outlined in Table 4. Use the artificial hot start to ensure PCR specificity. Table 4. Cycling protocol for NGSgo-AmpX amplification Step Temp Time Initial denaturation 95 C 3 min 3-step cycling Denaturation 95 C 15 sec Annealing 65 C 30 sec Elongation 68 C 6 min 35 cycles Final elongation 68 C 10 min Cooling 15 C Safe stopping point: After amplification, the amplicons can be stored at 2 8 C. RUO Edition 1, 2015/06 14 OF 36

15 9. Confirm the PCR products using an appropriate detection system such as agarose gel electrophoresis. Prepare a 1% w/v agarose gel, analyse 3 μl of each PCR assay. See for approximate size of PCR products. Table 5. Approximate size of HLA amplicons HLA locus Amplicon size (kb) A 3.1 B 3.4 C 3.4 DRB1 3.7 to 4.8 DQB1 3.7 to 4.1 DPB1 5.0 (exon 1) 5.7 (exon 2-5) DPA1 4.7 DQA1 5.4 to 5.8 DRB3 3.8 DRB4 0.4 (exon 2) 1.3 (exon 3) DRB5 4.0 RUO Edition 1, 2015/06 15 OF 36

16 PROTOCOL 2A. DNA QUANTIFICATION Important notes before starting: The DNA concentration of the HLA amplicons is determined with the Qubit DNA BR Assay Kit. The Qubit DNA Standards are stored at 4 C, and the dye and buffer at room temperature covered from light. Ensure that all Qubit reagents are at room temperature before starting the DNA quantification If you have a large sample panel, it is advised to measure the concentration of each HLA locus for ~3 representative samples, based on the agarose gel results. The average amplicon concentration of each HLA locus is representive for the entire sample panel. Protocol: 10. Set up all reactions at room temperature and shielded from light. Wear gloves when handling the assay tubes. Use only thin-wall, clear 0.5 ml PCR tubes that are appropriate for use in the Qubit Fluorometer. 11. Set up two Qubit Assay Tubes for the two DNA Standards and one Qubit Assay Tube for each DNA sample. 12. Prepare the Qubit Working Solution by making a 1:200 dilution of the Qubit reagent in Qubit buffer according to Table 6. Prepare 200 μl of Working Solution for each DNA Standard and/or DNA sample. Table 6. Composition of the Qubit Working Solution Component Qubit Reagent Qubit Buffer Total Volume Volume 1 l 199 l 200 l 13. Prepare the Qubit Assay Tubes according to Table 7. Table 7. Composition of the Qubit Assay Tubes Component DNA Standard DNA sample Qubit Working Solution 190 l 198 l DNA Standard (from kit) 10 l - DNA sample - 2 l Total Volume 200 l 200 l 14. Vortex all tubes for 2-3 seconds and incubate the tubes for 2 minutes at room temperature, shielded from light. 15. Insert the tubes in the Qubit Fluorometer and take readings. RUO Edition 1, 2015/06 16 OF 36

17 16. Determine the concentration of the amplicons. The concentration can be calculated automatically using the Dilution Calculator feature of the Qubit Fluorometer. To manually calculate the concentration, multiply the measured concentration with the dilution factor (200x) of the diluted DNA sample. RUO Edition 1, 2015/06 17 OF 36

18 PROTOCOL 2B. POOLING OF AMPLICONS (OPTIONAL) Important notes before starting: Do not pool amplicons from different samples, only pool amplicons of the same sample. The optimal way of pooling is to pool equimolar concentrations of HLA amplicons to achieve an equal read depth for all loci. Equimolar pooling takes into account HLA amplicon size and concentration differences. Each pool of amplicons from one sample will later receive unique barcoded sequences, which are implemented during the adapter ligation. It is not needed to label the individual amplicons of one sample, since the the software package NGSengine will automatically identify the sequence data of the different HLA loci per sample. Pooling of HLA-DRB1, -DRB3, -DRB4 and DRB5 may lead to suboptimal NGS results due to the high homology between the DRB loci. It is recommended to make two amplicon pools per sample, in which DRB1 is separate from DRB3/4/5 Protocol: 17. Combine equimolar concentrations of each amplicon of one sample in a nuclease-free tube or 96-well plate, using the Molecular Weights indicated in Table Calculate the (average) concentration of the amplicon pool and proceed to Protocol 3A for fragmentation and adapter ligation. See GenDx website: for an NGSgo pooling calculation sheet. For example, take 4 µl of HLA-A amplicon and use this volume as a reference to calculate the relative volumes of the other HLA loci for equimolar pooling, taking into account MW and concentration differences. When the amplicon yield for all samples is similar, the same pooling strategy can be applied to all samples. Table 8. Approximate molecular weight of HLA amplicons HLA locus A 2.0 B 2.2 C 2.2 DRB1 3.1 DQB1 2.7 DPB1 7.0 DPA1 3.1 DQA1 3.8 DRB3 2.5 DRB4 1.1 DRB5 2.6 Molecular weight (µg/pmol) RUO Edition 1, 2015/06 18 OF 36

19 PROTOCOL 3A. FRAGMENTATION AND ADAPTER LIGATION Important notes before starting: The optimal amount of input DNA to achieve the optimal insert size and library concentration is 250 ng. However, (pooled) amplicons with DNA concentrations in the range of 10 ng ng can be used. For optimal fragment sizes and library yield, please perform the fragmentation and adapter ligation (Protocol 3A) and clean-up (Protocol 3B) consecutively. Do not store the samples at 4-8 C at any time point in Protocol 3A and 3B. Samples can be stored after the clean-up (Protocol 3B). Prepare a volume of reaction mix at least 10% greater than required for the total number of assays to be performed. Protocol: 19. Set up all reactions on a cooling block (4 o C) or on ice. Thaw the Fragmentase Enzyme, Fragmentase Buffer, End Prep Buffer, End Prep Enzyme and nuclease-free water. Mix the solutions thoroughly and centrifuge briefly before use. Keep all reagents at 4 o C. 20. Prepare an NGSgo master mix, as described in Table 9. Table 9. Composition of the NGSgo reaction mix for fragmentation, end-repair and da-tailing Component Cap colour Volume HLA amplicon (~250 ng) Variable NGSgo-LibrX Fragmentase Buffer White 2 l NGSgo-LibrX End Prep Buffer Green 3.25 l NGSgo-LibrX Fragmentase Enzyme White 1.5 l NGSgo-LibrX End Prep Enzyme Green 1.5 l Nuclease-free H2O White Variable Total Volume 32.5 l 21. Mix the master mix thoroughly, without HLA amplicons, and centrifuge briefly. 22. Dispense the appropriate master mix volume per sample into each tube or plate well. 23. Dispense the appropriate volume of (pooled) HLA amplicon(s) (~250 ng) into each tube or plate well. 24. Mix the reaction mix thoroughly, and centrifuge briefly. 25. Place the reaction mix in a thermocycler, with the heated lid on, and run the program as described in Table 10. RUO Edition 1, 2015/06 19 OF 36

20 Table 10. Cycling protocol for the NGSgo fragmention, end-repair and da-tailing reaction Step Time Temperature Fragmentation and end-repair 20 min 25 C da-tailing 10 min 70 C Cooling * 15 C * This is not a safe stopping point. Do not store the reactions at 15 C, but immediately proceed to the next step. 26. Thaw the Ligase Mix, Ligation Enhancer, and the Adapter (AD-IL). Mix the solutions thoroughly and centrifuge briefly before use. Keep all reagents at 4 o C. 27. Prepare the NGSgo master mix for adapter ligation in a tube, as described in Table 11. Table 11. Composition of the NGSgo master mix for adapter ligation Component Cap colour Volume NGSgo-LibrX Ligase Mix Red 7.5 μl NGSgo-LibrX Ligation Enhancer Red 0.5 μl NGSgo-IndX Adapter for Illumina (AD-IL) Pink 0.25 μl Nuclease free H2O White 1 μl Total Volume 9.25 μl 28. Mix the reaction mix thoroughly, and centrifuge briefly. 29. Add 9.25 μl of the NGSgo master mix for adapter ligation to the 32.5 μl da-tailed DNA fragments, as described in Tabel 12. Table 12. Composition of the NGSgo reaction mix for adapter ligation Component NGSgo master mix for adapter ligation (Table 11) da-tailed DNA fragments (Step 25) Total Volume 30. Mix the reaction mix thoroughly, and centrifuge briefly. Volume 9.25 μl 32.5 μl μl 31. Incubate at 20 C for 15 minutes in a thermal cycler, according Tabel 13. Table 13. Cycling protocol for the NGSgo adapter ligation reaction Step Time Temperature Adapter ligation 15 min 20 C Cooling * 15 C * This is not a safe stopping point. Do not store the reactions at 15 C, but immediately proceed to the next step. 32. Briefly spin the tubes or plate and immediately proceed to Protocol 3B for the clean-up and size selection of the DNA samples using magnetic SPRI beads. RUO Edition 1, 2015/06 20 OF 36

21 PROTOCOL 3B. DNA CLEAN-UP AND SIZE SELECTION WITH 0.45x SPRI BEADS Important notes before starting: Prepare fresh 80% ethanol from absolute ethanol prior to use. Prepare elution buffer (10 mm Tris-HCl, 0.1% Tween20, ph 8.0) or 0.1x TE prior to use. The magnetic SPRI beads supplied by AMPure XP and Macherey-Nagel have been validated for this protocol. Ensure that the SPRI beads are at room temperature prior to use. Protocol: 33. Set up all reactions at room temperature. 34. Vortex the magnetic SPRI beads thoroughly to resuspend the beads. 35. Add 18.8 µl resuspended beads to the µl NGSgo adapter ligation reaction mixture from Protocol 3A, resulting in a 0.45x beads:dna ratio. 36. Mix well by vortexing or pipetting up and down at least 10 times, and incubate for 5 minutes at room temperature. 37. Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from supernatant. 38. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant by pipetting. 39. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. 40. Repeat step 39 twice, for a total of three washes. 41. Remove all residual ethanol with a 10 μl pipette. 42. Air-dry the beads for 3-5 minutes while the tube is on the magnetic stand with the lid open. Make sure that all ethanol has evaporated in and be sure not to transfer any beads. Trace amounts of bead carry over may affect the optimal performance of the indexing PCR step. RUO Edition 1, 2015/06 21 OF 36

22 43. Remove the tubes from the magnetic stand and elute the DNA target from the beads by adding 12.5 μl of elution buffer (10 mm Tris-HCl, 0.1% Tween20, ph 8.0 or 0.1x TE). 44. Mix well by vortexing thoroughly for seconds. Ensure the beads are in solution. 45. Quickly spin the tube and incubate for two minutes at room temperature. 46. Place the plate on the magnetic stand. 47. After the solution is clear (about 5 minutes), transfer 10 μl of the eluate to a new tube or plate. Make sure not to transfer any beads. 48. Proceed to Protocol 3C for the indexing PCR. Safe stopping point: After cleanup, samples can be stored at 2-8 C. RUO Edition 1, 2015/06 22 OF 36

23 PROTOCOL 3C. INDEXING PCR Important notes before starting: Avoid contamination of indices Do not interchange the caps of the NGSgo-IndX tubes. Caps do not need to be replaced with a new cap after opening the NGSgo-IndX tube, as long as caps are not interchanged. Use a unique combination of i5 and i7 primers for each sample. Protocol: 49. Set up all reactions on a cooling block (4 C) or on ice. 50. Thaw the HiFi PCR Mix, i5 and i7 indices. Mix the solutions thoroughly and centrifuge briefly before use. Keep all reagents at 4 C. 51. Dispense 12.5 μl of HiFi PCR mix in a a new tube or plate well for each sample. 52. Put the i5 and i7 index tubes in the correct order. For example, the index tubes can be placed in a small rack, placing the index tubes at multichannel distance of each other. When removing the caps of the indices, ensure that the caps are put in the same order on the table as the tubes. 53. Dispense 1.25 μl of the i5 indices to each sample. 54. Dispense 1.25 μl of the i7 indices to each sample. 55. Transfer 10 μl of the size-selected DNA fragments to the reaction mix, according to Table 14. Table 14. Composition of the NGSgo reaction mix for indexing PCR Component Cap colour Volume Size-selected, adapter-ligated DNA fragments 10 μl NGSgo-LibrX HiFi PCR Mix Blue 12.5 μl NGSgo-IndX IN-5##-IL White 1.25 μl NGSgo-IndX IN-7##-IL Purple 1.25 μl Total Volume 25 l 56. Mix the reaction mix thoroughly, and centrifuge briefly. 57. Place the reaction mix in a thermocycler, with the heated lid on, and run the program as described in Table 15. RUO Edition 1, 2015/06 23 OF 36

24 Table 15. Cycling protocol for the NGSgo-IndX indexing PCR reaction Step Temp Time Initial denaturation 98 C 30 sec 3-step cycling Denaturation 98 C 10 sec Annealing 65 C 30 sec Elongation 72 C 30 sec 10 cycles Final elongation 72 C 5 min Cooling 15 C 58. Briefly spin the tubes or plate and proceed to Protocol 3D for the clean-up and size selection of the DNA samples using 0.6x SPRI beads. Safe stopping point: After indexing PCR, samples can be stored at 4-15 C. RUO Edition 1, 2015/06 24 OF 36

25 PROTOCOL 3D. DNA CLEAN-UP AND SIZE SELECTION WITH 0.6x SPRI BEADS Important notes before starting: Prepare fresh 80% ethanol from absolute ethanol prior to use. Prepare elution buffer (10 mm Tris-HCl, 0.1% Tween20, ph 8.0) or 0.1x TE prior to use. The magnetic SPRI beads supplied by AMPure XP and Macherey-Nagel have been validated for this protocol. Ensure that the SPRI beads are at room temperature prior to use. Protocol: 59. Set up all reactions at room temperature. 60. Vortex the magnetic SPRI beads thoroughly to resuspend the beads. 61. Add 15 μl resuspended beads to the 25 μl of indexed DNA libraries, resulting in a 0.6x beads:dna ratio. 62. Mix well by vortexing or pipetting up and down at least 10 times, and incubate for 5 minutes at room temperature. 63. Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from supernatant. 64. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. 65. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. 66. Repeat step 65 once, for a total of two washes. 67. Remove all residual ethanol with a 10 μl pipette. 68. Air-dry beads for 3-5 minutes while the tube is on the magnetic stand with the lid open. 69. Remove the tubes from the magnetic stand and elute the DNA target from the beads by adding 16.5 μl of elution buffer (10 mm Tris-HCl, 0.1% Tween20, ph 8.0 or 0.1x TE). RUO Edition 1, 2015/06 25 OF 36

26 70. Mix well by vortexing thoroughly for seconds. Ensure the beads are in solution. 71. Quickly spin the tube and incubate for two minutes at room temperature. 72. Place the plate on the magnetic stand. 73. After the solution is clear (about 5 minutes), transfer 14 μl to a new PCR tube. 74. Proceed to Protocol 4A for the pooling of the DNA libraries. Safe stopping point RUO Edition 1, 2015/06 26 OF 36

27 PROTOCOL 4A. LIBRARY POOLING Important notes before starting: Before pooling the libraries, determine which Illumina NGS flow cell is most suitable. The expected data output can be calculated for any loci/sample. See Table below for an indication of the maximum flow cell capacity, based on a conservative cluster density of 800 K/mm 2, aiming for a median read depth of ~500 reads per locus Protocol: 75. Vortex and spin down the individual DNA libraries. 76. Pool the DNA libraries to be sequenced by combining equal volumes of each library into one tube. The total volume of pooled library should be at least 50 μl. Ideally, libraries are pooled at equimolar levels. Pooling libraries at equal volumes is also possible, as the concentration deviations of different libraries within one experiment are minimal. 77. Vortex the pooled DNA libraries, followed by a quick spin to collect all liquid from the sides of the tube. 78. Proceed to Protocol 4B to quantify the pooled DNA library concentration. Optional: It is recommended to verify the size of the DNA fragments in your library using an appropriate detection system such as agarose gel electrophoresis. A smear of different sized fragments should be observed from 400 bp to about 1000 bp. Prepare a 1% w/v agarose gel according to your laboratory protocol, and analyse 10 μl of the library. Safe stopping point Equimolar HLA library MiSeq reagent kit 300 cycles, V2 MiSeq Flow Cell Type NANO MICRO STANDARD Cluster density (K/mm 2 ) Output (Gb) Maximum number of libraries per flowcell HLA-A, B, C, DRB1, DQB HLA-A, B, C, DRB1, DQB1, DPB HLA-A, B, C, DRB HLA-A, B, C, DRB1, DQB1, DPB1, DPA1, DQA HLA-A, B, C, DRB1, DQB1, DPB1, DPA1, DQA1, DRB3/4/ HLA-DPB1, DPA1, DQA1, DRB3/4/ RUO Edition 1, 2015/06 27 OF 36

28 PROTOCOL 4B. LIBRARY QUANTIFICATION Important notes before starting: Ensure that all components of the Illumina library quantification kit, including the standards, are completely thawed and thoroughly mixed prior to use. The KAPA kit is supplied with 6 standards. It is sufficient to generate a reliable standard curve by only measuring the four standards with the highest concentration. The KAPA assay is the preferred method to quantify the library concentration. Alternatively, the DNA concentration of the library can be quantified using the Qubit DNA quantification procedure described in Protocol 2A. Qubit DNA quantification for library quantification is less accurate, as it does not distinguish the functional DNA library from other dsdna in the sample. Validate your Qubit quantification procedure to achieve an optimal cluster density in the MiSeq run. When using a qpcr instrument that requires a reference dye please add 0.4ul of 50x ROX to each reaction. See instrument compatibility table from KAPA Biosystems for further details. Protocol: 79. For first time use of a new kit: Prepare the qpcr/primer mix by adding 1 ml of Illumina Primer Premix (10X) to 5 ml bottle of KAPA SYBR FAST qpcr Master Mix (2X) and mix well. 80. Prepare a 100x dilution of the pooled DNA library, as described in Table 16, and mix well. Table 16. Composition of the 100x diluted DNA library Component Pooled indexed DNA libraries (Protocol 4A) Nuclease free H2O Total Volume Volume 10 l 990 l 1000 l 81. Prepare a 1000x dilution of the pooled DNA library, Table 17,and mix well. Table 17. Composition of the 1000x diluted DNA library Component 100x diluted DNA library Nuclease free H2O Total Volume Volume 10 l 90 l 100 l 82. Prepare a 2000x, 4000x, 8000x, 16000x, and 32000x dilutions of the DNA library, as described in Table 18. RUO Edition 1, 2015/06 28 OF 36

29 This is done by adding 10 µl of the pooled library (1000x diluted) to 10 µl of nuclease-free water to generate a 2000x dilution. Vortex the reaction mix thoroughly, followed by a quick spin to collect all liquid from the sides of the tube. Next, add 10 µl of the pooled library (2000x diluted) to 10 µl of water to generate a 4000x dilution. Vortex the reaction mix thoroughly, followed by a quick spin to collect all liquid from the sides of the tube. Proceed with this 2x dilution series to generate the 8000x, 16000x and 32000x, as described in Table 18, and make sure to vortex and spin-down in between each dilution step. Table 18. Preparation of 2x dilution series of the DNA library Final dilution factors Components 2000x 4000x 8000x 16000x 32000x Library dilution to be diluted 1000x 2000x 4000x 8000x 16000x Volume for the indicated library dilution 10 l 10 l 10 l 10 l 10 l 10 mm Tris-HCl, ph % Tween l 10 l 10 l 10 l 10 l Total Volume 20 l 20 l 20 l 20 l 20 l 83. Prepare the qpcr tubes or plate, using the 2000x, 4000x, 8000x, 16000x and 32000x dilutions of the DNA libraries, according to Table 19. Table 19. Composition of the KAPA qpcr reaction mix Components KAPA SYBR FAST qpcr Master Mix containing Primer Premix Nuclease free water Diluted library DNA or DNA Standard (1-4) Total Volume Volume 12 l 4 l 4 l 20 l 84. Ensure that the qpcr tubes or plate are sealed. Collect all components in the bottom of the wells by brief centrifugation. 85. Run the KAPA cycling protocol according to Table 20 on the laboratory s qpcr instrument and analyze the data. The qpcr instrument must acquire at the SYBR Green channel. Table 20. Cycling protocol for the KAPA assay Step Temp Time Initial denaturation 95 C 5 min 2-step cycling Denaturation 95 C 30 sec Annealing, extension, data 60 C 45 sec acquisition 35 cycles 86. Calculate the library concentration by generating a standard curve using the DNA Standards described in Table 21. RUO Edition 1, 2015/06 29 OF 36

30 Multiply the concentration of the diluted libraries with the dilution factor to calculate the library concentration. Only include library dilutions in the calculation that fall within the DNA Standards region. Convert the intermediate library concentration to the final library concentration, compensating for the median fragment size of 600 bp. This is done by multiplying the intermediate library concentration with factor Table 21. DNA Standards in KAPA kit Standards dsdna concentration (pm) RUO Edition 1, 2015/06 30 OF 36

31 PROTOCOL 4C: SAMPLE SHEET SET-UP A sample sheet has to be created for the Illumina MiSeq instrument to operate the machine and to identify the samples by their index sequences. 87. Download the sample sheet template and instruction manual from the GenDx website, on the following location: Fill in the sample sheet according the instructions in the online manual. 89. Proceed to Protocol 4D to prepare the DNA library for the Illumina MiSeq run. RUO Edition 1, 2015/06 31 OF 36

32 PROTOCOL 4D. LIBRARY DENATURATION In this procedure, the pooled DNA libraries are denatured and diluted in hybridization buffer, in preparation for the cluster generation prior to loading the Illumina MiSeq reagent cartridge. Important notes before starting: The HT-1 buffer from the Illumina MiSeq reagent kit should thaw completely at room temperature prior to use. After the buffer has thawed, put the HT-1 buffer at 4 C to prechill before starting this protocol. The MiSeq reagent cartridge should be thawed completely prior to use. The 0.2 M NaOH solution should always be freshly prepared. The cluster density on the Illumina MiSeq instrument depends on the library concentration. The DNA concentrations mentioned in this protocol are based on the KAPA quantification. When using a different DNA quantification method, e.g. Qubit or the Bioanalyzer, the required input DNA concentration to reach an optimal cluster density may be different. Optimally, this protocol should result in a cluster density of K/mm². Protocol: 90. Dilute the pooled DNA library from Protocol 4A in nuclease-free water to a final concentration of 4 nm. 91. Prepare a fresh solution of 0.2 M NaOH from a 1 M NaOH stock, according to Table 22, and mix well. Table 22. Composition of the 0.2 M NaOH solution Component Volume Laboratory-grade water 800 μl 1 M NaOH 200 μl Total Volume 1 ml 92. Add the pooled DNA library (4 nm) to the 0.2 M NaOH solution in a tube, according to Table 23. Table 23. Composition of the denatured DNA library solution Component Volume 4 nm pooled DNA library 5 μl 0.2 M NaOH 5 μl Total Volume 10 μl 93. Vortex briefly and spin-down the DNA library solution. RUO Edition 1, 2015/06 32 OF 36

33 94. Incubate the solution for 5 minutes at room temperature to denature the DNA into single strands. 95. Stop the reaction by adding 990 µl of pre-chilled HT-1 buffer, as described in Table 24 the concentration of the denatured library is 20 pm. Table 24. Composition of the denatured DNA library solution (20 pm) in HT-1 buffer Component Denatured DNA library solution (4 nm) Pre-chilled HT-1 buffer Total Volume 96. Invert several times and spin-down the DNA library solution. Volume 10 μl 990 μl 1000 μl 97. Dilute the denatured library further in pre-chilled HT-1 buffer to the desired concentration using the example in Table 25. The concentrations described in this protocol are a guideline, but optimal conditions can deviate from instrument to instrument. Optimization of the DNA library concentration to get the optimal cluster density may be required. When starting optimization, it is recommended to start with a medium DNA library concentration (e.g. ~12 pm). Based on the obtained cluster density, the library concentration in the next NGS run can be adjusted if necessary. Table 25. Library dilutions for MiSeq Final Concentration 6 pm 8 pm 10 pm 12 pm 15 pm 20 pm 20 pm denatured DNA 180 μl 240 μl 300 μl 360 μl 450 μl 600 μl Pre-chilled HT-1 buffer 420 μl 420 μl 300 μl 240 μl 150 μl 0 μl 98. Invert several times and spin-down the DNA library solution. 99. Keep the reaction mix on ice until ready to proceed to the MiSeq Load 600 µl of the final denatured library to the MiSeq reagent cartridge Start the MiSeq, according to the manufacturer s instructions, using the sample sheet from Protocol 4C. RUO Edition 1, 2015/06 33 OF 36

34 9 APPENDIX A: CONTAMINATION CONTROL IMPORTANT: It is extremely important to include at least one negative control in every PCR setup that lacks template nucleic acid to detect possible contamination. General physical precautions Separate the working areas for setting up the PCR amplification mix and DNA handling, including the addition of starting template, PCR product analysis, or plasmid preparation. Ideally, use separate rooms. Use a separate set of pipettes for the PCR amplification mix. Use of pipette tips with hydrophobic filters is strongly recommended. Prepare and freeze small aliquots of primer solutions and dntp mix. Use of fresh nuclease free H2O is strongly recommended. In case of contamination, laboratory benches, apparatus, and pipettes can be decontaminated by cleaning them with a 1% Trigene disinfectant. Afterwards, the benches and pipettes must be rinsed thoroughly with nuclease free H2O. General chemical precautions PCR stock solutions can also be decontaminated using UV light. This method is laborious however, and its efficiency is difficult to control and cannot be guaranteed. We recommend storing solutions in small aliquots and using fresh aliquots for each PCR. Another approach to prevent amplification of contaminating DNA is to treat individual reaction mixtures with DNAseI or restriction enzymes that cut between the binding sites of the amplification primers used, before adding the template DNA sample. 10 TROUBLESHOOTING GUIDE For all you questions and remarks regarding this NGS workflow for HLA typing IFU we currently refer you to our technical support, support@gendx.com. Some additional information like frequently asked question about NGS HLA workflow and product information and updates can be found on our website, RUO Edition 1, 2015/06 34 OF 36

35 11 LIMITED LICENSE AGREEMENT Use of this product signifies the agreement of any purchaser or user of the GenDx NGSgo workflow for HLA typing and NGSgo reagents with the following terms: The workflow and NGSgo kits may be used solely in accordance with the IFU NGSgo workflow for HLA typing, and only with components described in the IFU. GenDx grants no license under any of its intellectual property to use or incorporate the enclosed components of NGSgo with any components not included within this kit except as described in the GenDx NGSgo IFU and additional protocols available at Other than expressly stated licenses, GenDx makes no warranty that NGSgo kit and/or its use(s) do not infringe the rights of third-parties. NGSgo kit and its components are licensed for one-time use and may not be re-used, refurbished, or re-sold. GenDx specifically disclaims any other licenses, expressed or implied other than those expressly stated. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. GenDx may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components. For updated license terms, see Trademarks: Others: NGSgo and NGSengine are registered trade marks of Genome Diagnostics. LongRange PCR kit (QIAGEN), Qubit (Life Technologies), Fragmentase (NEB), MiSeq (IIllumina), KAPA (KAPA Biosystems). All other trademarks are the property of their respective owners. RUO Edition 1, 2015/06 35 OF 36

36 ORDERING INFORMATION GenDx products are supported either directly or by your local GenDx distributor or reseller. Please contact your local GenDx distributor or GenDx Customer Support team at or for any product information or quote request. GenDx Alexander Numan Building Yalelaan CM Utrecht, the Netherlands Phone: +31 (0) Fax: +31 (0) www: GenDx, all rights reserved. RUO Edition 1, 2015/06 36 OF 36