RUO Edition 2.1. RUO Edition 2. RUO Edition 1

Transcription

1

2 IMPORTANT NOTES AND UPDATES RUO Edition 2.1 HLA-typing analysis of DRB3/4/5 has been optimized in NGSengine v2.1 (and higher). NGSengine applies a split-analysis of the individual DRB3/4/5 loci for improved HLA typing. A new NGSgo pooling calculation sheet can be downloaded from the website ( The following sections have been updated: 4. Warning and Precautions Product use limitations have been updated for DRB3/4/5. 9. Protocol 2B. Pooling of amplicons Amplicon pooling strategy for DRB3/4/5 recommends using NGSengine v2.1 or higher. 11. Troubleshooting guide Troubleshooting guide recommends using NGSengine v2.1. for analysis of DRB3/4/5. RUO Edition 2 Minor textual changes throughout the entire IFU. The following sections have been updated: 3. Shipping and storage Information on stability of the NGSgo products is added. 4. Warning and Precautions Product use limitations have been updated. 8. Equipment and Reagents NGSgo-AmpX and NGSgo-LibrX are now also available in a 96 reaction format. All NGSgo-AmpX loci, including HLA-G, are also available separately. 9. Protocol 2A. DNA quantification. Correction of dilution factor. 9. Protocol 2B. Pooling of amplicons. Amplicon pooling strategy has been optimized for all loci. 9. Protocol 3B and 3D. Clean-up and size selection with 0.45x/0.6x SPRI beads. Protocol is updated for clarification of incubation times. 9. Protocol 4A. Library pooling. Flow cell capacity guidelines have been updated. 11. Troubleshooting guide. New chapter on troubleshooting has been added. RUO Edition 1 The workflow no longer requires BSA in the fragmentation reaction for improved SPRI bead elution. The workflow has an optimized denaturation protocol for improved cluster density. IFU RUO Edition /06 2 OF 28

3 IFU RUO Edition /06 3 OF 28

4 CONTENTS 1. Key to symbols 5 2. Kit content 5 3. Shipping and storage 6 4. Technical assistance 6 5. Warning and precautions 6 6. Principle 7 7. Procedure 8 8. Equipment and reagents 9 9. Protocols 11 protocol 1. HLA locus-specific amplification 11 protocol 2A. DNA quantification 13 protocol 2B. Pooling of amplicons (optional) 14 protocol 3A. Fragmentation and adapter ligation 15 protocol 3B. DNA clean-up and size selection with 0.45x SPRI beads 17 protocol 3C. Indexing PCR 18 protocol 3D. DNA clean-up and size selection with 0.6x SPRI beads 19 protocol 4A. Library pooling 20 protocol 4B. Library quantification 21 protocol 4C. Sample sheet set-up 23 protocol 4D. Library denaturation Appendix A. Contamination control Troubleshooting guide Limited license agreement 28 Ordering information 29 DISCLAIMER has made every effort to ensure that this IFU is accurate. Information in this IFU is subject to change without notice. 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 2016 IFU RUO Edition /06 4 OF 28

5 1. KEY TO SYMBOLS Material number Components Batch code / Lot number Catalogue number Consult Instructions For Use Contains reagents for N tests Store at -20 C VOL Legal manufacturer Store at -20 C Volume Add liquid 2. KIT CONTENT The NGSgo workflow consists of three parts (see 8 Equipment 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 and 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, datailing, adapter ligation, dual-indexing PCR) from HLA amplicons. The DNA libraries are compatible with Illumina NGS platforms. NGSgo-LibrX products are available for 48 and 96 reactions (RUO). 3. NGSgo-IndX for Illumina consists of an adapter and a panel of indices for the dual-indexing of libraries, for sample identification and sequencing on the Illumina NGS platform. Three NGSgo-IndX products (RUO) are available: NGSgo-IndX Set A (4x 24), enabling multiplexing of 24 libraries. NGSgo-IndX Set A+B (2x 96), enabling multiplexing of 96 libraries. NGSgo-IndX Set A+B+C+D (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. IFU RUO Edition /06 5 OF 28

6 3. SHIPPING AND STORAGE NGSgo is shipped with ice packs and should be stored at -20 C upon arrival. NGSgo-AmpX can also be shipped at ambient temperatures, when shipped separately. NGSgo is stable until the kit expiration date (kit label) when stored at -20 C. The in use stability of the NGSgo-AmpX kit is one year when stored at -20 C, and can be exposed to at least 25 freeze-thaw cycles. The in use stability of the NGSgo-IndX kit (including the adapter, i5 and i7 indices) is 24 months when stored at -20 C, and can be exposed to at least 17 freeze/thaw cycles. The in use stability of the NGSgo-LibrX kit is 18 months when stored at -20 C, and can be exposed to at least 16 freeze/thaw cycles. NGSgo reagents should be returned to recommended storage temperatures immediately after use. Limit time at ambient temperature. Changes in the physical appearance of the kit reagents may indicate product deterioration and may interfere with performance. 4. TECHNICAL ASSISTANCE For technical assistance and more information: support@gendx.com Website: Phone: +31 (0) or contact your local distributor ( 5. WARNING AND PRECAUTIONS Product Use Limitations For Research Use Only. Not for use 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 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. cannot provide support for any problems resulting from non-adherence to this Instructions for Use document. Always verify the coverage, read depth and allele ratios in a sample. In case of a homozygous typing, please verify that the sample is a truly homozygous sample or whether it possibly contains a second allele underrepresented in the data and therefore below the detection limit of the software. In some cases, allele imbalances can be observed for: NGSgo HLA-DRB1: allele imbalances for DRB1*01, DRB1*04 and DRB1*14 alleles can occur in case of imbalanced amplification. IFU RUO Edition /06 6 OF 28

7 NGSgo HLA-DRB4: allele imbalances for DRB4 exon 2 and exon 3 can occur in case of imbalanced DRB4 amplification. In case of an HLA-DRB4 exon 3 amplicon dropout, limit the analysis to exon 2 only. NGSgo HLA-DRB3/4/5: allele imbalances for heterozygous DRB3/4/5 samples can occur in case of imbalanced amplicon pooling. Analysis of DRB3/4/5 has been optimized in NGSengine v2.1 (and higher), which applies a split-analysis of the individual DRB3/4/5 loci for improved HLA typing. 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). 6. PRINCIPLE The NGSgo workflow for HLA typing enables high-resolution identification of HLA alleles by means of NGS. NGSgo is compatible with Illumina sequencing platforms and has been validated on the Illumina MiSeq. 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 -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. 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 da-tailing of the HLA amplicons. da-tailing is required to create binding sites for the Illumina-compatible 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 qpcr-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. IFU RUO Edition /06 7 OF 28

8 7. PROCEDURE Indicated times are for preparing 24 individual libraries, 5 loci, simultaneously. Total hands-on time: 3-4 hours Total time: hours NGSgo- AmpX Protocol 1 Protocol 2A Protocol 2B HLA locus-specific amplification Hands-on time: 45 minutes Total time: 5-7 hours DNA quantification Hands-on time: 30 minutes Pooling of amplicons (optional) Hands-on time: 10 minutes Total time: 30 minutes Total time: 10 minutes NGSgo- LibrX & NGSgo- IndX Protocol 3A Protocol 3B Protocol 3C 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: 40 minutes Total time: 40 minutes Indexing PCR Hands-on time: 15 minutes Total time: 1 hour Protocol 3D DNA clean-up and size selection with 0.6x SPRI beads Hands-on time: 30 minutes Total time: 30 minutes Protocol 4A Protocol 4B Protocol 4C Protocol 4D Library pooling Hands-on time: 10 minutes Library quantification Hands-on time: 15 minutes Sample sheet set-up Hands-on time: 10 minutes Library denaturation Hands-on time: 5 minutes Total time: 10 minutes Total time: 1.5 hour Total time: 10 minutes Total time: 15 minutes Illumina NGS run IFU RUO Edition /06 8 OF 28

9 8. EQUIPMENT AND REAGENTS Table 1. NGSgo product overview Product description Number of Catalogue number reactions RUO Supplier HLA locus-specific amplification primers NGSgo-AmpX HLA-A NGSgo-AmpX HLA-B NGSgo-AmpX HLA-C NGSgo-AmpX HLA-DPB NGSgo-AmpX HLA-DQB NGSgo-AmpX HLA-DRB NGSgo-AmpX HLA-DRB3/4/ NGSgo-AmpX HLA-DQA NGSgo-AmpX HLA-DPA NGSgo-AmpX HLA-G NGSgo-AmpX HLA-A, B, C, DRB1, DQB NGSgo-AmpX HLA-DPB1, DPA1, DQA1, DRB3/4/ NGSgo-AmpX HLAGeneSuite* Library preparation, compatible with Illumina NGS platform NGSgo-LibrX Library Preparation compatible with Illumina x NGSgo-IndX Adapter & Indices 2x compatible with Illumina 1x NGSgo-IndX Caps 24 (purple) (white) *Contains amplification primers for HLA-A, B, C, DRB1, DQB1, DPB1, DPA1, DQA1, DRB3, DRB4 and DRB5 For a complete overview of NGSgo reagents, please see IFU RUO Edition /06 9 OF 28

10 Table 2. Equipment and reagents per protocol Equipment and reagents Catalogue number Supplier General equipment and reagents Pipettes and tips wit hydrophobic filters N.A. Multiple Multi-channel pipettes (20 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 Vortexer N.A. Multiple MilliQ water N.A. Multiple Protocol 1: HLA locus-specific amplification NGSgo See Table 1 -LongRange PCR Kit (250; 1000 rxn) ; 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 See Table 1 Magnetic SPRI beads (50 ml); A63880 (5 ml), A63881 (60 ml), A63882 (460 ml) Macherey-Nagel or Beckman Coulter Magnetic stand-96 AM10027 Ambion Shaker (1500 rpm) N.A. Multiple 100% ethanol N.A. Multiple Elution buffer (10 mm Tris-HCl, 0.1% Tween20 (ph 8.0) or 0.1x TE) N.A. Multiple 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 instrument-dependent) 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 (300 cycle kit, V2) MS (standard); MS (micro); Illumina MS (nano) NGSengine HLA typing software See website IFU RUO Edition /06 10 OF 28

11 9. 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. Heparin has an inhibitory effect on a PCR. Purified DNA should have an A 260/A 280 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 of template DNA. If necessary, template DNA should be diluted in nuclease-free H 2O 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. Prepare a volume of reaction mix at least 10% greater than required for the total number of assays to be performed. NGSgo-AmpX primer preparation Centrifuge all tubes 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 nuclease-free H 2O (provided) in the volume listed below: o 24 reaction tubes: 27 µl o 96 reaction tubes: 108 µl Invert the tubes a couple of times, thoroughly vortex each tube and centrifuge the tubes for one minute. Repeat this step at least two times. 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 H 2O, and primer solutions. Mix and centrifuge briefly. 4. Prepare a reaction mix as shown in Table 3. HLA-DQB1 requires the addition of X-Solution* and double the amount of LongRange Enzyme per reaction. It is extremely important to include at least one negative control in every PCR setup that lacks template nucleic acid to detect possible contamination. *X-Solution: refers to Q-Solution when using LongeRange PCR kit from Qiagen. 5. Mix the reaction mix thoroughly, and centrifuge briefly. 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. IFU RUO Edition /06 11 OF 28

12 Table 3.Composition of the NGSgo-AmpX reaction mix for HLA locus-specific amplification All loci Component DQB1 (except DQB1) Nuclease-free H2O μl μl X-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 NGSgo-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 35 cycles Initial denaturation 95 C 3 min Denaturation 95 C 15 sec Annealing 65 C 30 sec Elongation 68 C 6 min Final elongation 68 C 10 min Cooling 15 C Safe stopping point After amplification, the amplicons can be stored for 3 months at 4 C to 8 C. 9. Confirm the PCR products using an appropriate detection system such as agarose gel electrophoresis. Prepare a 1% w/v agarose gel, and analyse 3 μl of each PCR assay. See 10. Table 5 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 (exon 1) DPB1 5.7 (exon 2-5) DPA1 4.7 DQA1 5.4 to 5.8 DRB (exon 2) DRB4 1.3 (exon 3) DRB5 4.0 G 2.7 IFU RUO Edition /06 12 OF 28

13 PROTOCOL 2A. DNA QUANTIFICATION Important notes before starting It is recommended to quantify the DNA concentration of the HLA amplicons before proceeding to the library preparation step. For the NGSgo workflow, the Qubit DNA quantification method is described and validated. 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. For DRB3/4/5, ensure that the selected amplicons show a positive band. Protocol 11. 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. 12. Set up two Qubit Assay Tubes for the two DNA Standards and one Qubit Assay Tube for each DNA sample. 13. 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. It is recommended to make a master mix with a sufficient volume for all samples and Standards. Table 6. Composition of the Qubit Working Solution Component Volume Qubit Reagent 1 l Qubit Buffer 199 l Total Volume 200 l 14. 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 15. Vortex all tubes for 2-3 seconds and incubate the tubes for 2 minutes at room temperature, shielded from light. 16. Insert the tubes in the Qubit Fluorometer and take readings. 17. 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 (100x) of the diluted DNA sample. IFU RUO Edition /06 13 OF 28

14 PROTOCOL 2B. POOLING OF AMPLICONS (OPTIONAL) Important notes before starting Do not pool amplicons from different samples, only pool amplicons of the same sample. For consistent and balanced read outputs for all loci, amplicon concentration variation between samples should be limited. In case of amplification dropouts, repeat the amplification. The optimal way of pooling is to combine equimolar concentrations of HLA amplicons to achieve an equal read depth for all loci and all samples. Equimolar concentrations can be determined using the NGSgo Pooling Calculation Sheet. ( that takes the HLA amplicon size and concentration differences into account. Once the amplification procedure has been optimized, resulting in reproducible amplicon yields, it is also possible to pool amplicons using volume ratios established in previous experiments. For amplicon pools containing all 11 HLA loci, it is recommended to always quantify the amplicon concentration beforehand to achieve equimolar pooling. Ensure that the NGS software used for HLA typing is optimized for analysis of pooled amplicons. Analysis of amplicon pools containing DRB3/4/5 has been optimized in NGSengine v2.1 and higher. Protocol 18. Determine the (average) amplicon concentration for each locus in ng/µl. This can be done by filling in the measured DNA concentrations in the NGSgo Pooling Calculation Sheet. 19. Pool the amplicons for each sample according to the volumes calculated by the NGSgo Pooling Calculation Sheet. When the amplicon yield for all samples is similar, you can apply the same pooling strategy to all samples. For weak amplicons (<50 ng/µl), compensate by doubling the amount of volume of the weak amplicon in the pool, or quantify the concentration and calculate the exact volume required for equimolar pooling using the calculation sheet. Table 8. Example of NGSgo-AmpX amplicon pooling strategy for 6 loci. HLA amplicon: A B C DRB1 DQB1 DPB1 Average concentration (ng/µl) Equimolar pooling factor Volume in amplicon pool (µl) Proceed to Protocol 3A, using ~250 ng of the amplicon pool as DNA input for library preparation. IFU RUO Edition /06 14 OF 28

15 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 50 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 21. 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 H 2O. Mix the solutions thoroughly and centrifuge briefly before use. Keep all reagents at 4 o C. 22. 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 23. Mix the master mix thoroughly, without HLA amplicons, and centrifuge briefly. 24. Dispense the appropriate volume of master mix per sample into each tube or plate well l 25. Dispense the appropriate volume of (pooled) HLA amplicon(s) (~250 ng) into each tube or plate well. It is recommended to use a minimum DNA volume of 1 l. 26. Mix the reaction mix thoroughly, and centrifuge briefly. 27. Place the reaction mix in a thermal cycler, with the heated lid on, and run the program as described in Table 10. Table 10. Cycling protocol for the NGSgo fragmention, end-repair and da-tailing reaction Step Temperature Time Fragmentation and end-repair 25 C 20 min da-tailing 70 C 10 min 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. IFU RUO Edition /06 15 OF 28

16 28. 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. 29. 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 30. Mix the reaction mix thoroughly, and centrifuge briefly. 31. Add 9.25 μl of the NGSgo master mix for adapter ligation to the 32.5 μl da-tailed DNA fragments, as described in Table 12. Table 12. Composition of the NGSgo reaction mix for adapter ligation Component Volume NGSgo master mix for adapter ligation (Table 11) 9.25 μl da-tailed DNA fragments (Step 26) 32.5 μl Total Volume μl 32. Mix the reaction mix thoroughly, and centrifuge briefly. 33. Incubate at 20 C for 15 minutes in a thermal cycler, according Table 13. Table 13. Cycling protocol for the NGSgo adapter ligation reaction Step Temperature Time Adapter ligation 20 C 15 min 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. 34. 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. IFU RUO Edition /06 16 OF 28

17 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. Magnetic SPRI beads supplied by Beckman Coulter (AMPure XP) and Macherey-Nagel have been validated. Ensure that the SPRI beads are at room temperature prior to use. Protocol 35. Set up all reactions at room temperature. Vortex the SPRI beads thoroughly to resuspend the beads. 36. 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. 37. Mix well by vortexing or pipetting up and down at least 10 times. Incubate for 5 minutes at room temperature, preferably on a shaker (1500 rpm). 38. Quickly spin (3-4 seconds at 500 rpm) to collect liquid at bottom of the tube. The beads have to remain in solution. 39. Place the plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant by pipetting. 40. 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. Repeat this step twice, for a total of three washes. 41. Remove all residual ethanol with a 10 μl pipette. Visually check that all ethanol is removed. 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. Do not over-dry the beads to prevent SPRI beads from clumping together. 43. Remove the tube or plate from the magnetic stand. 44. Elute the DNA from the beads by adding 12.5 μl of elution buffer. 45. Mix well by vortexing thoroughly for 30 seconds. Ensure that the beads are in solution. 46. Incubate for two minutes at room temperature, preferably on a shaker (1500 rpm). 47. Quickly spin the tube (3-4 seconds at 500 rpm) to collect liquid at bottom of the tube. The beads have to remain in solution. 48. Place the plate on the magnetic stand. Meanwile, prepare the reaction mix for indexing PCR (Protocol 3C), while leaving the plate on the magnet. After the solution is clear (about 5 minutes), transfer 10 μl of the eluate directly in the indexing PCR mix. Optional: for storage of the eluate, transfer the eluates to a new plate or tube. Safe stopping point. After clean-up, samples can be stored at 4-8 C. IFU RUO Edition /06 17 OF 28

18 PROTOCOL 3C. INDEXING PCR Important notes before starting Avoid contamination of indices: Do not interchange the caps of the NGSgo-IndX tubes. Caps can be reused, 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. Thaw the HiFi PCR Mix, i5 and i7 indices. Mix the solutions thoroughly and centrifuge briefly before use. Keep all reagents at 4 C. 50. Dispense 12.5 μl of HiFi PCR mix in a new tube or plate well for each sample. 51. Put the i5 and i7 index tubes in the correct order. This can be done by placing the index tubes in a small rack, at multi-channel 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. 52. Dispense 1.25 μl of the i5 indices to each sample. 53. Dispense 1.25 μl of the i7 indices to each sample. 54. Transfer the 10 μl eluate (size-selected DNA fragments) from Protocol 2B directly to the indexing PCR reaction mixes, according to Table 14. Table 14. Composition of the NGSgo reaction mix for indexing PCR Component Cap colour Volume Size-selected DNA fragments 10.0 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 55. Mix the reaction mix thoroughly, and centrifuge briefly. 56. Place the reaction mix in a thermal cycler, with the heated lid on, run the program as described in Table 15. Table 15. Cycling protocol for the NGSgo-IndX indexing PCR reaction Step Temp Time Initial denaturation 98 C 30 sec Denaturation 98 C 10 sec 10 cycles Annealing 65 C 30 sec Elongation 72 C 30 sec Final elongation 72 C 5 min Cooling 15 C 57. 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 IFU RUO Edition /06 18 OF 28

19 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. Magnetic SPRI beads supplied by Beckman Coulter (AMPure XP) and Macherey-Nagel have been validated. Ensure that the SPRI beads are at room temperature prior to use. Protocol 58. Set up all reactions at room temperature. Vortex the SPRI beads thoroughly to resuspend the beads. 59. Add 15 μl resuspended beads to the 25 μl of indexed DNA libraries from protocol 3C, resulting in a 0.6x beads:dna ratio. 60. Mix well by vortexing or pipetting up and down at least 10 times. Incubate for 5 minutes at room temperature, preferably on a shaker (1500 rpm). 61. Quickly spin (3-4 seconds at 500 rpm) to collect liquid at bottom of the tube. The beads have to remain in solution. 62. Place the plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. 63. 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. Repeat this step once, for a total of two washes. 64. Remove all residual ethanol with a 10 μl pipette. Visually check that all ethanol is removed. 65. 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. Do not over-dry the beads to prevent SPRI beads from clumping together. 66. Remove the tubes from the magnetic stand and elute the DNA from the beads by adding 16.5 μl of elution buffer. 67. Mix well by vortexing thoroughly for 30 seconds. Ensure that the beads are in solution. 68. Incubate for two minutes at room temperature, preferably on a shaker (1500 rpm). 69. Quickly spin the tube (3-4 seconds at 500 rpm) to collect liquid at bottom of the tube. The beads have to remain in solution. 70. Place the plate on the magnetic stand. After the solution is clear (about 5 minutes), transfer 14 μl of the eluate to a new tube or plate. Make sure not to transfer any beads. 71. Proceed to Protocol 4A for the pooling of the DNA libraries. Safe stopping point After clean-up, samples can be stored at 4-8 C. IFU RUO Edition /06 19 OF 28

20 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 combination. See Table 16 for guidelines of the flow cell capacity. The recommended number of libraries per flow cell type is based on a conservative cluster density of 800 K/mm2, aiming for an average read depth of ~500 per locus to intercept the amplicon input variation and cluster density variation. Although a read depth of >200 reads is optimal for phasing using NGSengine, succesful sequencing and HLA typing has been achieved with read depths as low as 100 bp. Protocol 72. Vortex and spin down the individual DNA libraries. 73. 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. 74. Vortex the pooled DNA libraries, followed by a quick spin to collect all liquid from the sides of the tube. 75. Proceed to Protocol 4B to quantify the pooled DNA library concentration. Optional: It is highly recommended to verify the size of the DNA fragments in your library using an appropriate detection system such as agarose gel electrophoresis, or a Bioanalyzer. DNA fragment sizes are expected to range between 400 bp to 1000 bp. E.g. prepare a 1% w/v agarose gel according to your laboratory protocol and analyse 10 μl of the library. Safe stopping point. Libraries can be stored for 4 months at 4 C to 8 C. Table 16. Recommended MiSeq flow cell capacity. MiSeq reagent kit 300 cycles, V2 MiSeq Flow Cell Type NANO MICRO STANDARD Output (Gb) Equimolar HLA library (examples) Maximum number of libraries per flow cell A, B, C, DRB A, B, C, DQB A, B, C, DRB1, DQB A, B, C, DRB1, DQB1, DQA A, B, C, DRB1, DQB1, DPB A, B, C, DRB1, DQB1, DPB1, DQA A, B, C, DRB1, DQB1, DPB1, DQA1, DPA A, B, C, DRB1, DQB1, DPB1, DQA1, DPA1, DRB3/4/ A, B, C, DRB1, DQB1, DPB1, DQA1, DPA1, DRB3/4/5, G IFU RUO Edition /06 20 OF 28

21 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 4 standards with the highest concentration. The KAPA assay is the preferred method to quantify the library concentration to achieve reproducible cluster densities. 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. When using a qpcr instrument that requires a reference dye please add 0.4 l of 50x ROX to each reaction. See instrument compatibility information from KAPA Biosystems for further details. Protocol 76. 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. 77. Prepare a 100x dilution of the pooled DNA library, as described in Table 17, and mix well. It is advised to do a triplicate measurement of the pooled library, by preparing three individual 100x dilutions of your libraries, to minimize the chance of any dilution errors. Table 17. Composition of the 100x diluted DNA library Component Volume Library pool (Protocol 4A) 10 l Nuclease-free H2O 990 l Total Volume 1000 l 78. Prepare a 1000x dilution of the pooled DNA library, as described in Table 18, and mix well. Table 18. Composition of the 1000x diluted DNA library Component Volume 100x diluted DNA library 10 l Nuclease-free H2O 90 l Total Volume 100 l 79. Prepare a 2000x, 4000x, 8000x, 16000x, and 32000x dilution series of the library, as described in Table 19. This is done by adding 10 µl of the pooled library (1000x diluted) to 10 µl of nuclease-free H 2O to generate a 2000x dilution. Mix the reaction mix by resuspending throroughly. Next, add 10 µl of the pooled library (2000x diluted) to 10 µl of H 2O to generate a 4000x dilution. Mix the reaction mix by resuspending throroughly. Proceed with this 2x dilution series to generate the 8000x, 16000x and 32000x dilutions, and make sure to resuspend between each dilution step. IFU RUO Edition /06 21 OF 28

22 Table 19. 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 Nuclease-free H2O 10 l 10 l 10 l 10 l 10 l Total Volume 20 l 20 l 20 l 20 l 20 l 80. Prepare the qpcr tubes or plate, using the 2000x, 4000x, 8000x, 16000x and 32000x dilutions of the DNA libraries, according to 20. It is recommended to make a master mix with a sufficient volume for all samples and Standards. For this, make a master mix containing qpcr Master Mix, ROX reference dye (dependent on QPCR instrument), nuclease-free H 2O, and aliquot 16 µl per reaction. Table 20. Composition of the KAPA qpcr reaction mix Components KAPA SYBR FAST qpcr Master Mix containing Primer Premix Nuclease-free H2O Diluted library DNA or DNA Standard (1-4) Total Volume Volume 12 l 4 l 4 l 20 l 81. Ensure that the qpcr tubes or plate are sealed. Collect all components in the bottom of the wells by brief centrifugation. 82. Run the KAPA cycling protocol according to Table 21 on the laboratory s qpcr instrument and analyze the data. The qpcr instrument must acquire at the SYBR Green channel. Table 21. Cycling protocol for the KAPA assay Step Time Temperature Initial denaturation 5 min 95 C Denaturation 30 sec 95 C 35 cycles Annealing, extension, data acquisition 45 sec 60 C 83. Calculate the library concentration by generating a standard curve using the DNA Standards described in Table 22. 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 22. DNA Standards in KAPA kit Standards dsdna concentration (pm) IFU RUO Edition /06 22 OF 28

23 PROTOCOL 4C. SAMPLE SHEET SET-UP 84. A sample sheet has to be created for the Illumina NGS instrument to operate the machine and to identify the samples by their index sequences. Download the sample sheet template and instruction manual from the website, on the following location: Fill in the sample sheet according the instructions in the online manual. 86. Proceed to Protocol 4D to prepare the DNA library for the Illumina run. PROTOCOL 4D. LIBRARY DENATURATION Important notes before starting In this procedure, the pooled DNA libraries are denatured with NaOH and diluted in HT-1 hybridization buffer (Illumina). The HT-1 buffer from the Illumina MiSeq reagent kit should be thawed completely. After the buffer is thawed, put the HT-1 buffer at 4 C to pre-chill before starting this protocol. The MiSeq reagent cartridge should be thawed completely prior to use. The 0.2 M NaOH solution should 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 amount of DNA to reach an optimal cluster density may be different. Optimally, this protocol should result in a cluster density of K/mm². In case the cluster density is beyond this range, ensure that the cluster passing filter is high enough to achieve sufficient coverage for HLA typing. Protocol 87. Dilute the pooled DNA library from Protocol 4A in nuclease-free H 2O to a final concentration of 4 nm. 88. Prepare a fresh solution of 0.2 M NaOH from a 1 M NaOH stock, according to Table 23, and mix well. Table 23. Composition of the 0.2 M NaOH solution Component Volume Laboratory-grade H2O 800 μl 1 M NaOH 200 μl Total Volume 1 ml 89. Add the pooled DNA library (4 nm) to the 0.2 M NaOH solution in a tube, according to Table 24. Table 24. 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 90. Vortex briefly and spin-down the DNA library solution. 91. Incubate the solution for 5 minutes at room temperature to denature the DNA into single strands. 92. Stop the reaction by adding 990 µl of pre-chilled HT-1 buffer, as described in Table 25. IFU RUO Edition /06 23 OF 28

24 Table 25. Composition of the denatured DNA library solution (20 pm) in HT-1 buffer Component Volume Denatured DNA library solution (2 nm) 10 μl Pre-chilled HT-1 buffer 990 μl Total Volume 1000 μl 93. Invert several times and spin-down the DNA library solution. The concentration of the denatured library is 20 pm. 94. If necessary, dilute the denatured library further in pre-chilled HT-1 buffer to the desired concentration between 6 and 20 pm, using Table 26. 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 26. Library dilutions for MiSeq Final Concentration 6 pm 8 pm 10 pm 12 pm 15 pm 17 pm 20 pm 20 pm denatured DNA 180 μl 240 μl 300 μl 360 μl 450 μl 510 μl 600 μl Pre-chilled HT-1 buffer 420 μl 420 μl 300 μl 240 μl 150 μl 90 μl 0 μl 95. Invert several times and spin-down the DNA library solution. 96. Keep the reaction mix on ice until ready to proceed to the MiSeq. 97. Load 600 μl of the final denatured library to to the MiSeq reagent cartridge. 98. Start the MiSeq according to the manufacturer s instructions, using the sample sheet from Protocol 4C. 10. 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 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. Use of fresh nuclease-free H 2O as provided in the kit 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 H 2O. Avoid contamination of indices: Do not interchange the caps of the NGSgo-IndX tubes. Caps can be reused, as long as caps are not interchanged. Adhere to the safe stopping points in the protocol to ensure optimal library yield. IFU RUO Edition /06 24 OF 28

25 11. TROUBLESHOOTING GUIDE For all your questions and remarks regarding the NGSgo workflow IFU please contact our technical support, Additional information such as frequently asked question about the NGSgo workflow and product information and updates can be found on our website, Amplification: No, weak, or unusual PCR product Locus-specific amplicons are visualized on an agarose gel prior to library preparation. In case of amplification issues, insufficient amount of amplicon might be generated for achieving sufficient amount of library with NGSgo. Consequently, the read depth may be too low. Sufficient read depth is necessary for reliable HLA typing. Quality parameters for read depth should be set and validated to determine the acceptance criteria for typing analysis. a) LongRange PCR Enzyme Mix was not added to the amplification mix or not mixed properly when added. Repeat amplification paying attention to the addition and mixing of LongRange PCR Enzyme Mix with the amplification mix. b) NGSgo-AmpX primers not dissolved properly Ensure that NGSgo-AmpX primers are resuspended sufficiently in nuclease-free H2O. Repeat the centrifugation step to spin all primers to the bottom. Primer concentration can be verified by Nanodrop system. c) Cycling conditions not optimal When using a fast thermal cycler, reduce the ramp rate to d) Weak PCR products below recommended concentration e) Poor-quality or degraded genomic DNA 1 C/s. Evaluate the quality of gdna and the concentration of gdna used. Do not exceed the recommended gdna concentration ( ng) or volume (1 4 μl) to be used in PCR. If the obtained amplicon is weak and below the recommended concentration of 50 ng/µl, the amplicon can still be sequenced. Acceptable sequence and typing may be achievable as long as the coverage is sufficient. Run genomic DNA on an 1% agarose gel to evaluate quality. Purified DNA should have an A260/A280 ratio between 1.7 and 1.9. f) Genomic DNA from buccal swabs Robustness of amplification was not validated for buccal swabs. In case sufficient gdna concentrations with high qualities are obtained, reliable amplification can be performed. g) No HLA-DRB3/4/5 PCR product Generation of DRB3/4/5 amplicons is haplotype-specific. To determine whether the absence/presence of a DRB3, DRB4 and/or DRB5 amplification is true or false, the DRB1 typing can be used to confirm its appearance. For instance: the presence of a DRB4 amplicon can be confirmed in case DRB1 is typed for DRB1*04/07/09 alleles. h) Two PCR products visible after amplification of HLA-DRB1 or HLA- DQB1 i) Two PCR products visible after amplification of HLA-DPB1 or HLA- DRB4 j) One PCR product visible after amplification of HLA-DRB4 k) PCR product with excessive aspecific background bands In a heterozygous sample, two bands may appear for the HLA- DRB1 or HLA-DQB1 PCR products due to length polymorphism in intron regions. DPB1 and DRB4 amplification are both duplex PCRs which yield two PCR products. Amplification of HLA-DRB4 normally results in two PCR products (exon 2 = 0.4 kb, exon 3 = 1.3 kb), except for DRB4*03:01N, which does not possess exon 2. In case of a very weak exon 3, repeat the DRB4 amplification using a 63 C annealing temperature instead of 65 C or continue sequencing and limit the typing analysis to exon 2 only. Ensure that a hot-start has been applied before initiation of the PCR. Evaluate the quality of gdna and the concentration of gdna used. Do not exceed the recommended gdna concentration to be used in PCR. IFU RUO Edition /06 25 OF 28

26 l) No template control has band Repeat amplification making fresh PCR mix. m) PCR product of aberrant size Repeat amplification. n) Aspecific DRB3/4/5 amplification In rare occasions, aspecific DRB1 amplicons are generated during DRB3/4/5 amplification. In case DRB3/4/5 and DRB1 are in the same amplicon pool, this does not affect the typing of either locus when using NGSengine for data analysis. In case DRB3/4/5 is not pooled together with DRB1, an additional correct heterozygous DRB1 typing may be generated from the DRB3/4/5 reads. In case of a homozygous DRB1 typing, additional DRB1 typing confirmation is required. False homozygous typing or locus dropout In case of imbalanced amplification, one of the two alleles is underrepresented in the data. If the read depth is too low or out of balance for one of the two alleles, this may result in a false homozygous typing. Allele ratios should always be inspected and verified for typing analysis. a) False homozygous typing due to imbalanced allele ratios Inspect the allele ratios in the NGS typing software. In case of imbalanced amplification, the second allele may be present below the detection limit. By adjusting the allele threshold settings in NGSengine the second allele can be detected. b) Imbalanced DRB1 alleles DRB1*01, DRB1*04 and DRB1*14 alleles can be underrepresented in the data. Manually lower the allele ratio threshold in the software settings. If this does not work, repeat the DRB1 amplification, following the recommendations for amplification as described above. c) Imbalanced DRB3/4/5 alleles DRB3/4/5 amplicons should be pooled at equimolar levels. Use NGSengine v2.1. or higher for optimized DRB3/4/5 analysis d) Locus is not identified in locus pool Amplicons should be pooled at equimolar levels. In case of underrepresentation of one of the amplicons, the locus may not be automatically detected and analysed by the software. NGSengine settings can be adjusted by manually assigning one or more loci to a sample. Restrict the spread of amplicon input concentration variation between loci and multiplexed samples. Amplicon and/or library contamination or mix-up Amplicon and/or library contamination or mix-up, prior to indexing, can result in a wrong typing result and should be avoided in all circumstances. Extra care should be taken when pooling the amplicons and when processing multiple libraries in the NGSgo workflow. Always confirm the NGS-based HLA typing with a second independent HLA-typing typing method, as required by EFI and ASHI standards. a) Suspected amplicon and/or library contamination or mixup. Contamination and/or mixup can be detected by inspecting the HLA typing result, the allele ratios and noise levels in the NGS data. Reads are unusually short or large Take the safe stopping points in the library preparation procedure into account. It is recommended to verify the library fragment sizes of the final libraries before proceeding to NGS. Overfragmented libraries may have a lower library concentration and shorter DNA inserts, resulting in insufficient read depth and suboptimal phasing. Underfragmented libraries may show overrepresentation of reads aligning to the 3 and 5 ends of the amplicon, whereas the clinically relevant middle part of the amplicon may be underrepresented. a) Fragmentation/End Prep enzymes were not added to the master mix or not mixed properly when added. Repeat library preparation paying attention to the addition and mixing of the amplicon(s), Fragmentase and End Prep enzymes and buffers. b) Overfragmentation Repeat library preparation, taking care that the preparation of the fragmentation is performed on ice. Take the safe stopping points in the library preparation procedure into account. c) Underfragmentation Re-quantify the amplicon pool and ensure that the amplicon input is within 50 ng to 1000 ng. Repeat library preparation. d) Incorrect size selection with SPRI beads Ensure that the correct DNA:bead ratios are applied, as described in the IFU. Ensure that the SPRI beads are at room temperature and mixed to a homogenous solution when used. IFU RUO Edition /06 26 OF 28

27 Low library yield Suboptimal fragmentation, end-repair, da-tailing, adapter ligation, indexing PCR, or SPRI bead cleanup can result in a lower library yield. When the library pool yield is >4 nm, as determined by KAPA, the libraries can be sequenced. Repeat library preparation when the final library pool is <4 nm. a) Fragmentation/End Repair/ Ligase/Ligation Enhancer/HiFi PCR enzymes or buffer were not added to the mix or not mixed properly when added. b) Adapter, indices or DNA fragments were not added to the mix or not mixed properly when added. Suboptimal library preparation can result in a lower library yield. Repeat library preparation when the final library pool is <4 nm. Pay attention to the addition and mixing of all components. Suboptimal library preparation can result in a lower library yield. Repeat library preparation when the final library pool is <4 nm. Pay attention to the addition and mixing of all components. c) Cycling conditions not optimal Check that the temperatures and cycling conditions are correct for: 1) fragmentation/end-repair/da-tailing, 2) adapter ligation, 3) indexing PCR d) Ethanol not completely removed after air-drying of the SPRI beads e) SPRI beads are clumping together during elution f) Different SPRI bead volumes per sample g) Accidentally took up beads during washing and/or elution steps. Any remaining ethanol should be removed with a 10 µl pipette. Air-dry the beads for 3-5 minutes before elution. Ensure that the beads have been air-dried for 3-5 minutes. Do not over-dry the beads. Bead solution should be vortexed before use to ensure a homogenous bead solution. The DNA/bead solution can be pipetted back in the tube. Place the tube on the magnet for ~5 minutes until the solution is clear. Repeat washing or elution procedure. h) Low library yield after clean-up Ensure that the beads are at room temperature and mixed to a homogenous solution when used and that the clean-up procedure is performed at room temperature. Unusual library quantification data output a) Library concentration lower/higher than usual b) Library quantification results are not reproducible c) KAPA library quantification results are negative, while the gel shows good quality libraries PCR product and library storage a) Storage of NGSgo-AmpX PCR products b) Storage of intermediate NGSgo libraries Library concentration is expected to range between ~ nm when using ~250 ng NGSgo-AmpX amplicon (pool) input. When the library concentration deviates, it is recommended to run a fraction of the library pool on a 1% agarose gel to evaluate fragment size, yield and quality. A smear of DNA fragments ranging between bp is expected. Perform duplicate or triplicate measurements on a library dilution series. When the replicate measurements are deviating, repeat the quantification. Library may not be functional due to absence of index primers and/or adapter sequences. Repeat KAPA assay to verify library functionality and concentration. If negative, repeat library preparation. PCR products can be stored for up to 3 months at 4 C to 8 C. If the storage temperature and/or duration exceeds this range, the integrity of PCR products can be checked on an agarose gel prior to proceeding to the library preparation. PCR products do not require a cleanup (e.g. Exo/SAP) for storage. Libraries cannot be stored after the fragmentation or adapter ligation step (Protocol 3A). Libraries can safely be stored overnight after the SPRI bead clean-up steps (at 4 C to 8 C) or after the indexing PCR (at 4 C to 15 C). c) Storage of final NGSgo libraries Final (undenatured) libraries can be stored up to 4 months at 4 C to 8 C prior to sequencing. It is recommended to do a KAPA qpcr-based library quantification method (KAPA) prior to sequencing, to ensure library functionality and accurate library concentration measurement after storage. Use freshly denatured libraries for the sequencing. IFU RUO Edition /06 27 OF 28

28 Next-generation sequencing and data analysis a) Cluster density lower/higher than usual Validate your library concentration to obtain a cluster density between k/mm 2. In case of lower/higher cluster densities, the data may still be used for HLA typing, taking the quality measure provided by the Illumina sequence into account (i.e. cluster passing filter, Q30 score etc.). Ensure that the output is high enough for obtaining a sufficient read depth. b) Low cluster passing filters Ensure that the correct DNA library concentration was loaded on the flow cell. Optimize the DNA library concentration to get the optimal cluster density. It is recommended to add PhiX DNA for increased sequence diversity when sequencing less than 12 multiplexed libraries. See Illumina for PhiX protocol. c) Low NGS output/low read depth Ensure that the selected flow cell (Nano, Micro or Standard) has sufficient capacity for the number of samples and amplicons selected. The capacity of the flow cell and the obtained cluster density should be high enough to generate enough reads for achieving a read depth that is acceptable for all samples and loci tested. d) Variable read depths between loci within one sample or between multiplexed samples. Check the DNA quality and amplicon quality. Validate your procedure such that amplicon yield is reproducible for all samples. Apply equimolar pooling to achieve balance read depths for all samples and loci in the NGS run. Restrict the spread of amplicon input concentration variation between loci and multiplexed samples. e) Error in sample sheet Provide a new correct sample sheet to the sequencer and repeat the analysis and export of NGS data from the sequencer. 12. LIMITED LICENSE AGREEMENT Use of this product signifies the agreement of any purchaser or user of the NGSgo workflow for HLA typing and NGSgo reagents with the following terms: The NGSgo kit and its components may be used solely in accordance with the IFU NGSgo workflow for HLA typing, and only with components described in the IFU. 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 NGSgo IFU and additional protocols available at Other than expressly stated licenses, 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, re-furbished, or resold. 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. 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. Qubit (Life Technologies), MiSeq (IIlumina), KAPA (KAPA Biosystems). All other trademarks are the property of their respective owners, more information IFU RUO Edition /06 28 OF 28

29 ORDERING INFORMATION products are supported either directly or by your local distributor or reseller. Please contact your local distributor or Customer Support team at or for any product information or quote request. Alexander Numan Building Yalelaan CM Utrecht, the Netherlands Phone: +31 (0) Fax: +31 (0) www: , all rights reserved. IFU RUO Edition /06 29 OF 28