Protocol 6B: The chip loading protocol has been updated according to the Thermo Fisher instructions for use.

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2 IMPORTANT NOTES AND UPDATES RUO Edition Minor textual changes throughout the document. Protocol 5A: The protocol split into two parts. Clonal amplification can be performed by either emulsion PCR (protocol 5A-1) or isothermal amplification (Protocol 5A-2). Isothermal amplification is recommended when read depth at the start of exon 3 of GC rich alleles needs to be improved. Protocol 6B: The chip loading protocol has been updated according to the Thermo Fisher instructions for use. IFU RUO Edition /01 2 OF 35

3 IFU RUO Edition /01 3 OF 35

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 8 7 Procedure 9 8 Equipment and reagents 10 9 Protocols 12 Protocol 1. HLA locus-specific amplification 12 Protocol 2A. DNA quantification with Qubit 14 Protocol 2B. Pooling of amplicons 15 Protocol 3A. Fragmentation and adapter ligation 16 Protocol 3B. Library pooling 18 Protocol 3C. DNA clean-up and size selection with 0.45x SPRI beads 18 Protocol 4. Library quantification 20 Protocol 5A-1: Clonal amplification by emulsion PCR 22 Protocol 5A-2: Clonal amplification by isothermal amplification 23 Protocol 5B: Enrichment of ISPs 26 Protocol 6A: Set barcode for template run 28 Protocol 6B: Chip loading and Ion PGM sequencing run Appendix A. contamination control Troubleshooting guide Limited license agreement 34 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 may be 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 2017 IFU RUO Edition /01 4 OF 35

5 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 Contains reagents in a volume of N µl Add liquid 2. KIT CONTENT The NGSgo workflow consists of three parts: 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 Ion Torrent consists of library preparation reagents (fragmentation, DNA end-repair, datailing, adapter ligation) for the generation of DNA libraries for downstream application in NGS. 3. NGSgo-IndX for Ion Torrent consists of a panel of barcoded adapter mixes for the barcoding of DNA libraries, for sample identification and sequencing on the Ion Torrent PGM platform. 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. For a complete overview of all reagents and equipment required for the NGSgo workflow, please see Table 1. For a full list of all available NGSgo products please visit our website. IFU RUO Edition /01 5 OF 35

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 is 24 months when stored at -20 C, and can be exposed to at least 24 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 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. NGSgo is not intended to be used for transplantation procedures where time is a critital factor. 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. GenDx cannot provide support for any problems resulting from non-adherence to this Instruction 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. IFU RUO Edition /01 6 OF 35

7 In some cases, allele imbalances can be observed for: o NGSgo HLA-DRB1: allele imbalances for DRB1*01, DRB1*04 and DRB1*14 alleles can occur in case of imbalanced amplification. o 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. o 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. For samples with a homozygous typing, which require additional verification with an independent method, there is a risk of reporting a false homozygous typing result when using SBTexcellerator as a verification assay. Samples with a homozygous typing resul should therefore be rather verified with a different GenDx assay that applies another amplification strategy, e.g. AlleleSEQR HLA or SBTessenz, or any other validated third party (amplification) typing strategy. Validation studies for NGSgo compatible with Ion Torrent have been performed on the One Touch and Ion PGM. Other Ion Torrent template preparation and sequencing platforms (e.g. Ion Chef, Proton, Ion S5), using a similar chemistry as the One Touch and Ion PGM, are compatible with NGSgo. Optimal conditions to use the output of this product (NGSgo libraries) on these Ion Torrent platforms, have to be determined by the user. 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). IFU RUO Edition /01 7 OF 35

8 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 all Ion Torrent NGS platforms (Proton, Ion PGM and Ion S5), however NGSgo is fully validated on the Ion PGM and this protocol is therefore focussed on the Ion PGM. 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 can be 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 increase the sample throughput. Library preparation with NGSgo-LibrX and NGSgo-IndX for Ion Torrent NGSgo-LibrX consists of library preparation reagents for the HLA amplicons. In the fragmentation reaction two enzymes fragment the HLA amplicons randomly. In the same reaction the DNA fragments are end-repaired and da-tailed. DNA end-repair and da-tailing are required to create compatible binding sites for the dsdna adapters that are subsequently ligated to the fragments, using ligation reagents from the NGSgo-LibrX kit and the adapters from the NGSgo-IndX kit. Adapter ligation is necessary to create compatible binding sites for the clonal amplification, barcoding of individual samples and binding sites for the sequencing primer. After adapter ligation, the individual libraries can be pooled together equivolume. The library pool is thereafter size-selected and cleaned-up, using SPRI beads. DNA fragments of ~400 bp and larger are selected and any shorter fragments or remaining enzymes and salts are removed. Finally the library concentration is determined to be able to achieve an optimal clonal amplification and chip loading. The library quantification protocol described in this IFU refers to the KAPA Library Quantification kit, which is based on qpcr using adapter-specific primers to accurately quantify the HLA library. Next-generation sequencing and data analysis The libraries are sequenced on the Ion Torrent PGM platform. The NGS data can be analysed with the software package NGSengine to determine the HLA genotype. NGSgo is compatible with Ion Torrent sequencing platforms and has been validated on the Ion Torrent PGM. IFU RUO Edition /01 8 OF 35

9 7. PROCEDURE Indicated times are for preparing 24 individual libraries simultaneously Total hands-on time: hours Total time: ~28 hours NGSgo- AmpX Protocol 1 HLA locus-specific amplification Hands-on time: 60 minutes Total time: 5-7 hours Protocol 2A DNA quantification Hands-on time: 20 minutes Total time: 20 minutes Protocol 2B Pooling of amplicons (optional) Hands-on time: 10 minutes Total time: 10 minutes NGSgo- LibrX & IndX Protocol 3A Protocol 3B Protocol 3C Fragmentation and adapter ligation Hands-on time: 10 minutes Total time: 60 minutes no safe stopping point Library pooling Hands-on time: 5 minutes no safe stopping point DNA clean-up and size selection Hands-on time: 10 minutes Total time: 5 minutes Total time: 30 minutes Protocol 4 Library quantification Hands-on time: 15 minutes Total time: 1.5 hour Protocol 5A-1 Protocol 5A-2 Clonal amplification by emulsion PCR Hands-on time: 5 minutes Total time: 8 hour Clonal amplification by isothermal amplification Hands-on time: 30 minutes Total time: 1 hour Protocol 5B Enrichment of ISPs with OT2 Hands-on time: 15 minutes Total time: 1 hour Protocol 6A Protocol 6B Set barcode for template run Hands-on time: 5 min Total time: 5 min Chip loading and PGM sequencing run Hands-on time: 70 min Total time: 6 hour Ion Torrent PGM Run IFU RUO Edition /01 9 OF 35

10 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 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 (Contains amplification primers for HLA-A, B, C, DRB1, DQB1, DPB1, DPA1, DQA1, DRB3, DRB4 and DRB5) GenDx Library preparation, compatible with Ion Torrent NGS platform NGSgo-LibrX Library Preparation compatible with Ion Torrent GenDx NGSgo-IndX Adapters 4x compatible with Ion Torrent 4x GenDx NGSgo-IndX Caps 24 (purple) GenDx 24 (white) Table 2. Equipment and reagents Equipment and reagents Catalogue number Supplier General equipment and reagents Pipettes and tips (hydrophobic filters) N.A. Multiple Multichannel pipette (10 µl and 300 µl) N.A. Multiple Ice or cooling block N.A. Multiple 96-wells reactions plates N.A. Multiple Eppendorf tubes 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 IFU RUO Edition /01 10 OF 35

11 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 GenDx.com GenDx GenDx-LongRange PCR Kit (250/1000 rxn) / GenDx Agarose gel electrophoresis system N.A. Multiple Gel loading Dye, Orange (6x) B7022S New England Biolabs Quick-Load 1Kb DNA ladder N0468S; N0468L New England Biolabs Protocol 2: DNA quantification Qubit DNA BR Assay Kit (500 assays) Q32853 Thermo Fisher Qubit Fluorometer Q33216 Thermo Fisher Qubit Assay Tubes Q32856 Thermo Fisher Protocol 3: HLA library preparation NGSgo-LibrX for Ion Torrent GenDx.com GenDx NGSgo-IndX for Ion Torrent GenDx.com GenDx NGSgo-IndX caps (purple) GenDx.com GenDx Magnetic Solid Phase Reversible Immobilisation (SPRI) beads Macherey-Nagel (50 ml) Agencourt AMPure XP beads A63880 (5 ml), A63881 (60 ml), A63882 (450 ml) Macherey-Nagel or Beckman Coulter Magnet for 1.5 ml tubes (may be supplied with Ion Torrent OT2 for enrichment step, protocol 5B) N.A. Multiple 100% ethanol N.A. Multiple 10 mm Tris-HCl, 0.1% Tween20 (ph 8.0) or 0.1x TE N.A. Multiple Protocol 4: Library quantification KAPA Biosystems library quantification kit KK4827 KAPA Biosystems for Ion Torrent (including DNA Standards) Optical tubes/plates and seal (RT instrument dependent) N.A. Multiple Real-time PCR instrument N.A. Multiple Protocol 5 and 6: Ion Torrent OT2 and PGM platform reagents DNA LoBind tubes N.A. Multiple Ion PGM HiQ View OT2 kit* A29900 Thermo Fisher Ion PGM Template IA 500 kit* A24622 Thermo Fisher Ion Sphere Quality Control kit Thermo Fisher Ion PGM Enrichment beads Thermo Fisher Ion PGM Hi-Q View Sequencing kit* A30044 Thermo Fisher Ion One Touch 2 system / Ion One Touch ES system Thermo Fisher Ion Torrent PGM sequencer Thermo Fisher Ion 314 Chip kit V2 BC Thermo Fisher Ion 316 Chip kit V2 BC Thermo Fisher Ion 318 Chip kit V2 BC Thermo Fisher 1M NaOH N.A. Multiple Heatblock for 2 ml tubes N.A. Multiple NGS data analysis NGSengine (1 or multiple users, 1 or 5 years) GenDx.com GenDx * The protocols in this IFU are based on the products listed here. The NGSgo libraries are however also fully compatible with the use of the previous kit versions (the Ion PGM Template OT2 400 / HiQ, Ion PGM 400 / HiQ Sequencing kits and Ion Chip kits V2) and other equipment such as the Ion Chef, Proton and Ion S5. IFU RUO Edition /01 11 OF 35

12 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. It is recommended to use purified DNA that has an A260/A280 ratio of ~1.8 as determined with a Nanodrop. 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 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. Centrifuge all NGSgo-AmpX tubes containing the amplification primers (red caps) for at least one minute before opening for the first time to ensure the pellet is at the bottom of the tube. Resuspend each primer in 27 µl nuclease free H 2O (24 reaction products) or 108 µl nuclease free H 2O (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. 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 the solutions thoroughly and centrifuge briefly before use. 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. 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 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 AmpX primer (red cap) 1 μl Template DNA 1 4 μl Total Volume 25 μl IFU RUO Edition /01 12 OF 35

13 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. 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. Safe stopping point After amplification, samples can be stored at 2 8 C. 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 9. Confirm the PCR products using an appropriate detection system such as agarose gel electrophoresis. Prepare a 1% w/v agarose gel according to your laboratory protocol, and analyse 3 μl of each PCR assay. See Table 5 for approximate size of PCR products. 10. Quantify the individual PCR products according to Protocol 2A. For new users of the NGSgo workflow, it is recommended to quantify all PCR products of all samples. For more experienced users of the NGSgo workflow, the DNA concentration range can be based on a selection of amplicons, showing different band intensities on gel. Validate your amplification procedure so that the DNA concentration range is 100 to 300 ng/μl. Once the amplification procedure has been optimized and validated, it is possible to perform the DNA quantification only on a representative selection of samples. 11. Proceed to Protocol 2B for the pooling of amplicons (optional). 12. Proceed to Protocol 3A for the fragmentation and adapter ligation of the (pooled) amplicons. Table 5. Approximate size of PCR products HLA locus Expected size A, B and C 3.1 kb to 3.4 kb DRB1 3.7 kb to 4.8 kb DRB3 3.8 kb DRB4 0.4 kb (exon 2) 1.3 kb (exon 3) DRB5 4.0 kb DQA1 5.4 kb to 5.8 kb DQB1 3.7 kb to 4.1 kb DPA1 4.7 kb DPB1 5.0 kb (exon 1) 5.7 kb (exon 2-5) IFU RUO Edition /01 13 OF 35

14 PROTOCOL 2A. DNA QUANTIFICATION WITH QUBIT Important notes before starting The concentration of the HLA amplicons is determined with the Qubit 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, using the agarose gel for selecting the amplicons. The average amplicon concentration of each HLA locus is representive for the entire sample panel when similar strengths of amplicons are observed on agarose gel. Protocol 13. 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. 14. Set up two Qubit Assay Tubes for the two DNA Standards and one Qubit Assay Tube for each DNA sample. 15. 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 Volume Qubit Reagent 1 l Qubit Buffer 199 l Total Volume 200 l 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 16. Prepare the Qubit Assay Tubes according to Table Vortex all tubes for 2-3 seconds and incubate the tubes for 2 minutes at room temperature, shielded from light. 18. Insert the tubes in the Qubit Fluorometer and take readings. 19. 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 /01 14 OF 35

15 PROTOCOL 2B. POOLING OF AMPLICONS Important notes before starting Do not pool amplicons from different samples, only pool amplicons of the same sample. 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 software package NGSengine will automatically identify the sequence data of the different HLA loci per 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. Protocol 20. Combine equimolar concentrations of each amplicon of one sample in a nuclease-free tube or 96-well plate, using the molecular weight indicated in Table 8. In case you need further help with calculating the amplicon pooling strategy, please go to the GenDx website for a pooling calculation sheet. Table 8. Molecular weight (µg/pmol) per NGSgo-AmpX amplicon HLA locus Amplicon size (kb) Molecular weight (µg/pmol) A B C DRB1 3.7 to DQB1 3.7 to DPB1 5.0 (exon 1), 5.7 (exon 2-5) 7.0 DPA DQA1 5.4 to DRB DRB4 0.4 (exon 2), 1.3 (exon 3) 1.1 DRB Determine the DNA concentration of the pooled sample using the quantification measurement that was also used for the individual amplicons, described in Protocol 2A. 22. Proceed to Protocol 3A for the fragmentation and adapter ligation of the HLA amplicons, using ~250 ng of (pooled) DNA. IFU RUO Edition /01 15 OF 35

16 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), sample pooling (Protocol 3B) and clean-up (Protocol 3C) consecutively. Do not store the samples overnight at 4 C at any time point in protocol 3A, 3B and 3C. Samples can be stored overnight at 4 C after the clean-up (Protocol 3C). Prepare a volume of reaction mix at least 10% greater than required for the total number of assays to be performed. 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. Protocol 23. Set up all reactions on a cooling block (4 o C) or on ice. 24. 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. 25. Prepare an NGSgo master mix without HLA amplicon(s) as described in Table 9. HLA amplicons to be added in step 27. 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 H 2O White Variable Total Volume 32.5 l 26. Mix the master mix thoroughly, without HLA amplicons, and centrifuge briefly. 27. Dispense the appropriate master mix volume per sample into each tube or plate well. 28. Dispense the appropriate volume of (pooled) HLA amplicon(s) (250 ng) into each tube or plate well. Total volume of reaction mix plus sample should be 32.5 µl. 29. Mix the reaction mix thoroughly, and centrifuge briefly. 30. Place the reaction mix in a thermocycler, with the heated lid on, and run the program as described in Table 10. IFU RUO Edition /01 16 OF 35

17 Table 10. Cycling protocol for the NGSgo fragmentation, 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 fragmentation reactions at 4-15 C, immediately continue with the proceeding protocol: 31. Prepare the NGSgo master mix for adapter ligation in a tube on ice, as described in Table 11. Table 11. Composition of the NGSgo master mix for adapter ligation Component Cap colour Volume (n=1) NGSgo-LibrX Ligase Mix Red 7.5 μl NGSgo-LibrX Ligation Enhancer Red 0.5 μl Total Volume 8 μl 32. Briefly vortex the NGSgo master mix, followed by a quick spin to collect all liquid from the sides of the tube. 33. Add 8 μl of the master mix for adapter ligation to the 32.5 μl of the da-tailed DNA fragments from step 29, as described in Table Vortex and centrifuge the NGSgo-IndX tubes thoroughly before opening, finishing with a centrifugation step for at least 30 seconds. Add 1.25 μl of barcoded adapter to the individual samples, as shown in Table 12. Use a different adapter for each sample. Table 12. Composition of the reaction mix for adapter ligation Component Volume Adapter ligation master mix (Table 11) 8 μl NGSgo-IndX AD-Ion-X 1.25 μl da-tailed DNA fragments (Protocol 3A) 32.5 μl Total Volume μl 35. Briefly vortex the reaction mix, followed by a quick spin to collect all liquid from the sides of the tube. 36. Incubate at 20 C for 15 minutes in a thermal cycler. This is not a safe stopping point. Do not store the adapter ligation reactions at 4-15 C, but immediately proceed to Protocol 3B. 37. Briefly spin the tubes or plate and proceed to Protocol 3B for the pooling of the libraries. IFU RUO Edition /01 17 OF 35

18 PROTOCOL 3B. LIBRARY POOLING Important notes before starting Libraries pooled at equal volumes is possible, since difference in concentration will be minimal. The protocol below describes how to pool libraries at equal volumes. To determine which Ion Torrent PGM chip is most suitable, the expected data output can be calculated for any loci/sample combination. In general we recommend to use a maximum of 6 samples (5 loci) for the 314 chip, 24 samples (5 loci) for the 316 chip and 24 samples (11 loci) for the 318 chip. Protocol 38. Vortex and spin down the individual DNA libraries. 39. Pool the DNA libraries by adding 8 µl of each library into one tube. 40. Vortex the pooled DNA libraries, followed by a quick spin to collect all liquid from the sides of the tube. This is not a safe stopping point. Do not store the library pool at 4-15 C, but immediately proceed to Protocol 3C. 41. Proceed to Protocol 3C for the clean-up and size selection of the DNA samples using magnetic SPRI beads. PROTOCOL 3C. 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. A magnetic stand for 1.5 ml Eppendorf Tubes should be used for this protocol, which is delivered together with the One Touch ES system. The same stand is also used for the enrichment beads purification in Protocol 5B. Protocol 42. Set up all reactions at room temperature. 43. Vortex the magnetic SPRI beads thoroughly to resuspend the beads. 44. Add the amount of resuspended beads as indicated in Table 13 to the library pool from Protocol 3B, resulting in a 0.45x beads:dna ratio. IFU RUO Edition /01 18 OF 35

19 Table 13. Composition of the reaction mix for size selection Total number of samples Total volume library pool Total volume beads to be added 6 48 μl 21.6 μl μl 43.2 μl μl 72 μl μl 86.4 μl N N x 8 μl N x 8 x 0.45 μl 45. Mix well by vortexing or pipetting up and down at least 10 times, and incubate for 5 minutes at room temperature. 46. Quickly spin the tube and place it on the magnetic stand to separate beads from supernatant. 47. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant by pipetting. Be careful not to disturb the beads that contain DNA targets. Caution: do not discard beads. 48. Add 1 ml of freshly prepared 80% ethanol to the tube while in the magnetic stand. 49. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. 50. Repeat step [48] and step [49] twice, for a total of three washes. 51. Remove all remaining ethanol with a small pipette without touching the bead pellet, leaving the tube on the magnetic stand. 52. 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 clonal amplification step. If necessary spin down the tube and collect the remaining liquid, while the tube is on the magnetic stand. 53. Remove the tubes from the magnetic stand and elute the DNA target from the beads by adding 50 μl of elution buffer (10 mm Tris-HCl, 0.1% Tween20, ph 8.0) or 0.1x TE. 54. Mix well by vortexing thoroughly for seconds, and ensure the beads are in solution. 55. Quickly spin the tube and incubate for two minutes at room temperature. 56. Place the tube on the magnetic stand. After the solution is clear (about 5 minutes), transfer 46 μl to a new tube. Make sure not to transfer any SPRI beads. 57. Optional: It is recommended to confirm 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 5 μl of the library. 58. Proceed to Protocol 4 for the library quantification. Safe stopping point After clean-up, samples can be stored overnight at 2-8 C. IFU RUO Edition /01 19 OF 35

20 PROTOCOL 4. LIBRARY QUANTIFICATION Important notes before starting Ensure that all components of the Ion Torrent library quantification kit, including the standards, are completely thawed and thoroughly mixed prior to use. 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 clonal amplification and chip loading. When using a qpcr instrument that requires a reference dye, please add 0.4 µl of 50x ROX to each reaction. See instrument compatibility table from KAPA Biosystems for further details. Protocol 59. For first time use of a new kit: Prepare the qpcr/primer mix by adding 1 ml of Ion Torrent Primer Premix (10X) to 5 ml bottle of KAPA SYBR FAST qpcr Master Mix (2X) and mix well. 60. Prepare a 100x dilution of the pooled DNA library, as described in Table 14, 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 14. Composition of the 100x diluted DNA library Component Volume Pooled barcoded DNA libraries (Protocol 4A) 5 l Nuclease-free H 2O 495 l Total Volume 500 l 61. Prepare a 1000x dilution of the pooled DNA library, as described in Table 15, and mix well. Table 15. Component 100x diluted DNA library Nuclease-free H 2O Total Volume Composition of the 1000x diluted DNA library Volume 10 l 90 l 100 l 62. Prepare a 2000x, 4000x, 8000x and 16000x dilutions of the DNA library, as described in Figure 1. Figure 1. How to prepare DNA library dilutions IFU RUO Edition /01 20 OF 35

21 63. Prepare the qpcr tubes or plate, using the 1000x, 2000x, 4000x, 8000x, and 16000x dilutions of the DNA libraries, according to Table 16. Table 16. Composition of the KAPA qpcr reaction mix Components Volume KAPA SYBR FAST qpcr Master Mix containing Primer Premix 12 l Nuclease-free H 2O 4 l Diluted library DNA or DNA Standard (1-6) 4 l Total Volume 20 l 64. Ensure that the qpcr tubes or plate are sealed. Collect all components in the bottom of the wells by brief centrifugation. 65. Run the KAPA cycling protocol according to Table 17 on the laboratory s qpcr instrument and analyze the data. The qpcr instrument must acquire at the SYBR Green channel. Table 17. 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 acquisition 60 C 45 sec 35 cycles 66. Calculate the library concentration by generating a standard curve using the DNA Standards described in Table 18. And making use of the conversion factor of as described below. Multiply the concentration of the diluted libraries with the dilution factor to calculate the intermediate 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 18. DNA concentration standards. Standards dsdna concentration (pm) Proceed with the clonal amplification depending on the template preparation: - Protocol 5A-1: Emulsion PCR using the Ion One Touch - Protocol 5A-2: Isothermal amplification - When using the Ion Chef please refer to the Thermo Fisher protocol and continue with Protocol 6A thereafter. IFU RUO Edition /01 21 OF 35

22 PROTOCOL 5A-1. CLONAL AMPLIFICATION BY EMULSION PCR Important notes before starting This protocol is for the Ion One Touch. Let the Ion PGM HiQ Reagent Mix reach ambient temperatures prior to use. Make sure that the Ion OneTouch 2 instrument is ready for use. Start the run on the Ion OneTouch 2 Instrument within ± 15 minutes after preparing the amplification solution. This protocol is a short version of the official protocol from Thermo Fisher for the Ion PGM HiQ View OT2 kit and only meant as a guide. For full instructions, please visit the website It is strongly advised to use the official protocol containing full instructions upon first time use and obligatory to do so when another Ion PGM Template kit is applied. Protocol 68. When using the PGM dilute the library to such extend that a concentration of 8 pm in a volume of at least 25 μl is obtained. For Proton and Ion S5 the input is significantly different and needs to be validated first. The concentrations given in step [70] are a guideline for the PGM. Optimal conditions can deviate from instrument to instrument. Optimization of the DNA library concentration to get the optimal clonal amplification and chip loading may be required. Based on the chip loading, the library concentration in the next NGS run can be adjusted if necessary. 69. Dispense 150 l Ion OneTouch Breaking Solution into each of the recovery tubes. 70. Install recovery tubes and amplification plate. 71. To the tube containing Ion PGM HiQ View Reagent Mix (violet cap), add the following components in the designated order according to Table 19: Table 19. Clonal amplification solution Order Reagent Cap Colour Volume 1 Nuclease free water - 25 l 2 Ion PGM HiQ Enzyme Mix Brown 50 l 3 Diluted library (8 pm) - 25 l Total 900 l Table 20. Clonal amplification solution 72. Vortex the Ion PGM HiQ View Ion Sphere Particles (ISPs) thoroughly to resuspend the beads. 73. Add 100 μl of the Ion PGM HiQ View ISPs to the clonal amplification solution. 74. Fill the Ion PGM One Touch Plus Reaction Filter Assembly through the sample port with the clonal amplification solution (1000 μl total). 75. Add 850 μl of Ion PGM Reaction Oil to the Filter Assembly. 76. Add another 850 μl of Ion PGM Reaction Oil to the Filter Assembly. 77. In a fluent motion turn the filter assembly upside down and load it into the OneTouch 2 instrument and follow the on-screen instructions to start the run. The run will take approximately 8 hours. IFU RUO Edition /01 22 OF 35

23 It is advisable to start the clonal amplication at the end of the day and to let it run overnight, in order to continue the next day. 78. At the end of the run of the Ion One Touch 2 instrument, follow the instructions on the screen that will prompt to centrifuge the sample. 79. Immediately after the centrifuge stops, touch Open Lid and remove the recovery router and both recovery tubes. 80. Remove all but 100 μl of recovery solution from both recovery tubes using the same pipette tip. 81. With a new tip, but using the same tip for both tubes, resuspend the ISPs by pipetting up and down at least 10 times. 82. Add 500 μl wash solution to each recovery tube. 83. Combine both solutions containing the ISPs into a new 1.5 ml LoBind tube. 84. Centrifuge the ISPs for 2.5 minutes at 15,500x g 85. Remove all but 100 μl of the wash solution from the tube and resuspend the ISPs by pipetting the beads up and down 10 times. 86. Continue with protocol 5B. If necessary the templates ISPs can be stored at 4 o C for up to 16 hours. IFU RUO Edition /01 23 OF 35

24 PROTOCOL 5A-2. CLONAL AMPLIFICATION BY ISOTHERMAL AMPLIFICATION Important notes before starting Before starting, set a heat block to 40 o C and fill the wells half-way with water. This protocol is a short version of the official protocol from Thermo Fisher for the Ion PGM Template IA 500 kit and only meant as a guide. For full instructions, please visit the website It is strongly advised to use the official protocol containing full instructions upon first time use and obligatory to do so when another Ion PGM Template kit is applied. Protocol 87. Dilute the library to such extend that a concentration of 45 pm in a volume of at least 5 μl is obtained. The concentrations given in step [80] are a guideline for the PGM. Optimal conditions can deviate from instrument to instrument. Optimization of the DNA library concentration to get the optimal clonal amplification and chip loading may be required. Based on the chip loading, the library concentration in the next NGS run can be adjusted if necessary. 88. Vortex the Ion PGM Template IA ISPs thoroughly to resuspend the beads. 89. Prepare the Templating Solution in a 2 ml LoBind tube on ice as indicated in Table 20. Table 20. Templating Solution Order Reagent Cap colour Volume 1 Ion PGM Template IA ISP Dilution Buffer White 128 l 2 Ion PGM Template IA Primer Mix L Blue 8 l 3 Ion PGM Template IA ISPs Orange 21 l 4 Diluted library (45 pm) l Total l Table 21. Clonal amplification solution 90. Vortex the Templating Solution for 2 seconds at maximum speed, pulse-centrifuge and return the tube to ice till further use. 91. Invert the Ion PGM Template IA Rehydration buffer (white cap) three times to mix and add 720 μl to the Ion PGM Template IA Pellet. 92. Vortex the rehydrated pellet for 2 seconds at maximum speed, pulse-centrifuge and place the tube on ice. 93. Transfer the rehydrated pellet to the Templating Solution on ice. Make sure to transfer the complete rehydrated pellet (the solution is viscous). 94. Vortex the mixture for 2 seconds at maximum speed, pulse-centrifuge and place the tube on ice till further use. 95. Invert the Ion PGM Template IA Start Solution (white cap) three times to mix and add 300 μl to the Templating Solution containing the rehydrated pellet. IFU RUO Edition /01 24 OF 35

25 96. Vortex the mixture ten times for 1 second at maximum speed, invert the tube and again vortex the mixture ten times for 1 second at maximum speed. Pulse-centrifuge the mixture and place the tube on ice till a heat block has reached 40 o C (fill the wells half-way with water before heating). 97. Start the isothermal amplification by gently placing the tube in a 40 o C heat block. Make sure the tube is immersed in water. 98. Incubate the reaction for 25 minutes at 40 o C. 99. Stop the reaction by removing the tube from the heat block and adding 650 μl of Ion PGM Template IA Stop Solution Vortex the tube thoroughly and centrifuge for 3 minutes at 7500 g Discard the supernatant except for 100 μl without disturbing the pellet Add 1 ml of Ion PGM Template IA Recovery Solution and resuspend the pellet. Followed by another addition of 700 μl of Recovery Solution Vortex thoroughly and leave to incubate for 5 minutes while vortexing every minute for 5 seconds Centrifuge for 3 minutes at g Immediately remove and discard all supernatant without disturbing the ISP pellet. Remove any bubbles prior to removing the bulk of the liquid to avoid frothing in subsequent steps Add 100 μl of the Ion PGM Template IA Wash Solution Resuspend the ISPs by vortexing for 4 seconds at maximum speed and pipetting the suspension up and down 4 times Continue with protocol 5B. If necessary the templates ISPs can be stored at 4 o C for up to one week. IFU RUO Edition /01 25 OF 35

26 PROTOCOL 5B. ENRICHMENT OF ISPs Important notes before starting Make sure the Ion One Touch ES system is ready for use. Well 1 of the eight-well strip is the position with the square-shaped tab. This protocol is a short version of the official protocol from Thermo Fisher for the Ion PGM HiQ View OT2 kit / Ion PGM Template IA 500 kit and only meant as a guide. For full instructions, please visit the website It is strongly advised to use the official protocol containing full instructions upon first time use and obligatory to do so when another Ion PGM Template kit is applied. Protocol Recover the template positive ISPs 109. Transfer the 100 μl with templated ISPs from Protocol 5A-1 or Protocol 5A-2 to well 1 of the eight-well strip When Protocol 5A-1 has been used; save 2 μl of the recovered ISPs for the Quality Control. The percentage of DNA covered ISPs can then be measured using Qubit (according to the protocol supplied with the Ion Sphere Quality Control kit and on Do note that the percentage of DNA covered ISPs should ideally be between 3-15%. If this is not the case you can expect data of reduced quality after the sequencing run. Do contact us at support@gendx.com if you need further advice Vortex the tube containing the enrichment beads for 30 seconds to thoroughly resuspend the beads Transfer 13 μl of the beads to a new 1.5 ml LoBind tube Place the tube on a magnet for 2 minutes, then carefully remove and discard the supernatant Remove the tube from the magnetic stand and add 130 μl of MyOne TM bead wash solution Vortex the tube for 30 seconds followed by a quick spin and transfer the 130 μl of beads to well 2 of the eight-well strip Prepare the Ion One Touch ES system. Prepare fresh melt-off solution by combining the components as described in Table 21. Table 21. Melt-off solution Order Reagent Volume 1 Tween Solution 280 μl 2 1M NaOH 40 μl Total 320 μl 117. Transfer 300 μl of the melt-off solution to well 7 of the eight-well strip Fill the wells 3, 4 and 5 from the eight-well strip with 300 μl wash solution Load a new tip in the Tip Arm of the Ion One Touch ES system. IFU RUO Edition /01 26 OF 35

27 120. Add 10 μl of neutralization solution to a new 0.2 ml PCR tube and insert the opened tube into the hole in the base of the Tip Holder Ensure that all the components are present in the 8-well strip (as indicated in Figure 2) and that the strip is orientated correctly and placed at the far right of the slot. Check that the neutralization solution is present and the tube is opened. Figure 2. Lay out of the 8 well strip, indicating the contents of each well Press the Start/Stop button to start the run. The enrichment takes ~35 minutes Immediately when the enrichment is finished, mix your sample by inverting the tube 5 times and store your sample at 4 o C. Your sample can subsequently be stored at 4 o C up to 24 hours before continuing with protocol 6A. IFU RUO Edition /01 27 OF 35

28 PROTOCOL 6A. SET BARCODE FOR TEMPLATE RUN Important notes before starting When using NGSgo-IndX for Ion Torrent for the first time you have to import the barcode sequences into your Torrent Browser. Once you have generated a template this can be used in following runs. For full instructions on the use of Torrent Browser please refer to the Torrent Suite software documentation. Additionally we have more detailed instructions available on and upon request. Protocol 124. Go to the download section of GenDx.com to obtain the GenDx Barcode kit file for NGSgo-IndX. Save this file to a location so you know where to find it. More extensive instructions on the installation of this file are also available on the same webpage Log into your Torrent Browser account and move to the references section On the left hand side you will see Barcodes click on this followed by clicking on the Add new DNA Barcodes button in the right top corner A new pop-up window will open where you can select the GenDx Barcode kit file. Select the correct file and click on Upload and Save When creating a new template for your run (sample sheet) you will now be able to select the GenDx Barcode set at the Kits tab For further detailed information on how to set-up a template in Torrent Browser we would like to refer to the Torrent Suite software documentation. Additionally we have more detailed instructions available upon request. IFU RUO Edition /01 28 OF 35

29 PROTOCOL 6B. CHIP LOADING AND ION PGM SEQUENCING RUN Important notes before starting Select the correct chip based upon the capacity that you would need (see Protocol 3B for details). This protocol is a short version of the official protocol from Thermo Fisher for the use of a 316 chip in combination with a weighted chip bucket and only meant as a guide. For full instructions, please visit the website It is strongly advised to use the official protocol containing full instructions upon first time use and obligatory to do so when another chip (314 or 318) or chip bucket is applied. Do note that the final length of reads at the end of the sequencing run strongly depends on the Torrent Browser settings. By default read lengths of bp can be achieved. If longer reads are required the trimming options in Torrent Browser can be adjusted leading to an average bp read length. Full instructions are available at GenDx upon request. Protocol (316 chip only) Clean and initialize the Ion PGM sequencer 130. Start the Ion PGM sequencer Follow the on-screen instructions for a clean with water. Using the assisted option that will take you through the program step by step Follow the on-screen instructions for initialization. Using the assisted option that will take you through the program step by step Follow the on-screen instructions for performing a run, until the Ion PGM sequencer asks you to load the chip with your sample. When prompted add the run template from Torrent Browser and perform the Chip Check, using the empty chip that you will load with your sample afterwards. Anneal the sequencing primer (316 chip only) 134. Vortex the Control ISPs and add 5 μl to the entire volume of enriched, template positive ISPs Mix the contents of the tube by vortexing for 15 seconds. Centrifuge for 2 minutes at 15,500x g Carefully remove the supernatant without disturbing the pellet, leaving 15 μl in the tube (visually compare to 15 μl of liquid in a separate tube) Add 12 μl of the sequencing primer and confirm that the total volume is 27 μl (add Annealing buffer if necessary) Vortex the solution followed by a quick spin. Incubate in a thermal cycler at 95 C for 2 minutes and 37 C for 2 minutes. After incubation the reaction can be kept at room temperature. IFU RUO Edition /01 29 OF 35

30 Bind sequencing polymerase to the ISPs (316 chip only) 139. Add 3 μl of Ion PGM Sequencing Polymerase to the ISPs, for a total final volume of 30 μl Pipet the reaction up and down to mix and incubate at room temperature for 5 minutes. Load the chip and start sequencing run (316 chip only) 141. Tilt the chip 45 degrees so that the loading port is the lower port. Insert a pipette tip in the loading port and remove as much liquid as possible Place the chip upside-down in the bucket with the tab pointing in and centrifuge the chip in the MiniFuge for 5 seconds. Remove the chip from the bucket and wipe off any liquid from the bucket Place the chip back in the chip bucket oriented correctly Load the total 30 μl sample from step 115 on the chip by inserting the pipette tip in the loading port and dialing down at a rate of 1 μl per second. To avoid introducing bubbles, leave a small amount (~0.5 μl) of sample in the pipette tip Make sure that the chip tab is pointing in and centrifuge for 30 seconds Flip the chip so that the chip tab is pointing out and centrifuge for 30 seconds Tilt the chip at a 45-degree angle and slowly remove about 25 μl of liquid. Without removing the pipette slowly add the liquid back on the chip. Finally, slowly remove as much liquid as possible from the chip and discard liquid Turn the chip upside-down in the bucket and with the tab pointing out perform a quick 5 second spin. Remove and discard any additional liquid. If some liquid remains on the chip, lightly and rapidly tap the point of the chip against the benchtop a few times and remove any collected liquid Immediately transfer the chip to the Ion PGM sequencer for sequencing When the chip is loaded on the machine follow the additional on-screen instructions of the Ion PGM sequencer and start the sequencing run. IFU RUO Edition /01 30 OF 35

31 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 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. IFU RUO Edition /01 31 OF 35

32 11. TROUBLESHOOTING GUIDE Little or no PCR product LongRange PCR Enzyme Mix was not added to the amplification mix or not mixed properly when added. Cycling conditions not optimal. DNA concentration not optimal. Poor-quality or degraded genomic DNA. Unusual PCR products Two PCR products visible after amplification of HLA-DRB1 or HLA- DQB1 locus. HLA-DRB1 or HLA-DQB1 amplicons of different samples have different sizes. Weak amplification of HLA-DRB4 exon 3 SPRI bead elution difficult to perform Beads keep clumping together. Beads keep stucking to tube. Unusual KAPA data output Library pool concentration higher/lower than usual. The different dilution series do not match. ISP coverage outside 3-15% The Quality Control gives an ISP coverage of less than 3%. The Quality Control gives an ISP coverage of more than 15%. Variation between template kits from Life Technologies. Unusual data output Torrent Browser High percentage of chip loading (above 80%). High percentage of polyclonal ISPs (above 45%). Repeat amplification paying attention to the addition and mixing of LongRange PCR Enzyme Mix with the amplification mix. When using a fast thermal cycler, reduce the ramp rate to 1 o C/s. Re-quantify the DNA and adjust to at least 50 ng/µl (100 ng/µl is optimal). If the sample concentration is below the recommended range and little or no amplification product is visible, sequence the sample anyway. Acceptable sequence and typing may still be achievable. Run genomic DNA on a 1% agarose gel to evaluate quality. It is recommended to use purified DNA that has an A260/A280 ratio of ~1.8. In a heterozygous sample, 2 bands may appear for the HLA-DRB1 or HLA-DQB1 PCR products due to length polymorphism in intron regions of the HLA-DRB1 or HLA-DQB1 gene. Due to length polymorphisms in intron regions of the HLA-DRB1 or HLA-DQB1 gene, amplicons may vary in size in different samples. Use 63 o C annealing temperature instead of 65 o C as described in Table 3, page 14. Acceptable amplification level should still be obtained. Reduce the waiting time for evaporation of ethanol just before the elution step to 2-3 minutes. Any remaining ethanol can also be removed with a small pipette, so less needs to be removed by evaporation. Use LoBind tubes in the SPRI bead step. Run the library pool on a 1% agarose gel to evaluate size and quality. A smear of DNA of different sizes should be observed from ~400 bp to ~1000 bp. If two out of three dilution series do not differ significantly, you can use these to determine the concentration. Otherwise repeat the KAPA measurement to be sure of the correct result. Proceeding would result in good quality reads, however the number of reads will be less. It is recommend to repeat the clonal amplification with a higher DNA input. Proceeding would result in high chip loading, low quality reads and therefore a reduced number of usable reads. Repeat the clonal amplification with a lower DNA input. When observed it is recommended to optimize your DNA input for every lot to reach a percentage of covered ISP between 3-15%. This results in low quality reads and therefore a reduced number of usable reads. Repeat the chip loading with a diluted sample or repeat the clonal amplification with a lower DNA input. Repeat the chip loading with a diluted sample or repeat the clonal amplification with a lower DNA input. IFU RUO Edition /01 32 OF 35

33 Unusual data output NGSengine Low mappability (below 60%). Low average read length (below 200 bp). Low median read depth (below 100 reads/position). Low read depth at start of exon 3. Low quality of reads, most likely in combination with a high ISP coverage and/or chip loading. Repeat the clonal amplification and sequencing steps using a lower DNA input. Switch off the automatic trimming option in Torrent Browser (full instructions available from GenDx). If the ISP coverage and chip loading were low, repeat the clonal amplification and sequencing steps with a higher DNA input. If the ISP coverage and chip loading were high, the number of reads is reduced due to low quality, repeat the clonal amplification and sequencing steps with a lower DNA input. The alleles typed have a high GC content in this region, making it difficult to be amplified during clonal amplification using Protocol 5A-1. Isothermal amplification as decribed in protocol 5A-2 improved the read depth for the GC rich regions. Increase the amount of amplicon for this locus in the amplicon pool that is used as input for the library preparation. For all other questions and remarks regarding this NGS workflow for HLA typing IFU we 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, IFU RUO Edition /01 33 OF 35

34 12. 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, re-furbished, or resold. 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 trademarks of Genome Diagnostics. LongRange PCR kit (QIAGEN), Qubit (Life Technologies), Ion Torrent equipment (Life Technologies), KAPA (KAPA Biosystems), Fragmentase (NEB). All other trademarks are the property of their respective owners, more info IFU RUO Edition /01 34 OF 35

35 IFU RUO Edition /01 35 OF 35

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. IFU RUO Edition /01 36 OF 35