Devyser Index Plate A. Instructions for Use
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1 Devyser Index Plate A Art. No.: 8-A200 Instructions for Use Page 1 of 17
2 TABLE OF CONTENTS TABLE OF CONTENTS 2 1. INTRODUCTION TO DEVYSER INDEX PLATE A Intended use Kit configuration Devyser Index Plate A Equipment and reagents required but not provided Storage requirements Instructions for use 3 2. WARNINGS AND PRECAUTIONS 4 3. PROCEDURAL LIMITATIONS 5 4. INSTRUCTIONS FOR USE Index preparation using the Devyser Index Plate A (8-A200) PCR1 library dilution Addition of diluted PCR1 libraries to index Plate A 8 5. SEQUENCING USING MiSeq Flowcell selection Preparation for sequencing Sample sheet generation Index description Sequencing SYMBOLS USED ON LABELS CONTACT INFORMATION Legal manufacturer Technical support REFERENCES ABBREVIATIONS REVISION HISTORY 17 Page 2 of 17
3 1. INTRODUCTION TO DEVYSER INDEX PLATE A Instructions for use (IFU) update service Sign up for the IFU update service to receive notifications via whenever there is a new version of the IFU available. Visit to sign up 1.1 Intended use The Devyser Index Plate A is intended for use as an accessory only in conjunction with the following products: Devyser BRCA,CE-IVD (8-A100) Devyser CFTR, CE-IVD (8-A101) Devyser BRCA, RUO (8-A102) Devyser CFTR, RUO (8-A103) 1.2 Kit configuration Devyser Index Plate A The Devyser Index Plate A includes predispensed Illumina double index primer combinations for molecular barcoding of up to 96 different samples simultaneously. Each index primer combination is predispensed into individual wells of the 96-well plate as outlined in Table 4. Table 1. Devyser Index Plate A configuration (8-A200) Storage Product Component Number/kit Cap color condition Index Plate A3 1 - Below -18 C Devyser Index Plate Sealer L 2 - Ambient 1.3 Equipment and reagents required but not provided Index mix (art. #: 4-A247) from any of the products outlined in 1.1 Index buffer (art. #: 4-A258) from any of the products outlined in 1.1 Consumables for the thermal cycler Micropipette with aerosol barrier tips or dispenser with displacement tips Disposable protective gloves (powder free) Veriti Thermal Cycler (Thermo Fisher) Specific instructions for use for the individual products outlined in Storage requirements Store the individual components of the Devyser Index Plate A as outlined in Table 1 (section 1.2). 1.5 Instructions for use Consult specific instructions for use for the individual products outlined in 1.1 and Table 2. Page 3 of 17
4 2. WARNINGS AND PRECAUTIONS Use of this product should be limited to personnel trained in PCR and NGS techniques Deviations from the IFU will compromise the kit performance Wear powder free disposable gloves, laboratory coat and eye protection when handling clinical samples and kit reagents Do not pool reagents with different kit lot numbers or different vials of the same lot Do not use opened or damaged reagent vials Frozen components should be thawed in a refrigerator or at room temperature before use Handling of kit components and samples, their use, storage and disposal should be in accordance with the procedures defined by national biohazard safety guidelines and in accordance with country, federal, state and local regulations Avoid microbial contamination of reagents when removing aliquots from reagent vials The use of sterile disposable aerosol barrier pipette tips is recommended We recommend using different sets of pipettes for the initial addition of DNA samples and for diluting and handling samples after PCR amplification Highly concentrated amplicons produced during PCR amplification must be handled with care to avoid contamination in the laboratory environment The work flow in the laboratory should proceed in a unidirectional manner, beginning in the reagent preparation area, moving to the DNA extraction area, then to the amplification area and finally to the sequencing area Supplies and equipment should be dedicated to each activity and not used for other activities or moved between areas. Gloves should be changed between activities Page 4 of 17
5 3. PROCEDURAL LIMITATIONS The Devyser Index Plate A should only be used as an accessory in conjunction with the products outlined in section 1.1. This IFU describes an alternate procedure for molecular barcoding of samples analysed using any of the products outlined in section 1.1. The molecular barcoding procedure is referred to as PCR2 in the individual user manuals. The procedure outlined in section 4 of this IFU replaces section 7.4 "Index preparation" in the individual IFUs for the products outlined in section 1.1. The procedure outlined in section 5 of this IFU replaces section 8 "Preparation for sequencing" in the individual IFUs for the products outlined in section 1.1. Page 5 of 17
6 4. INSTRUCTIONS FOR USE Schematic overview of the Devyser NGS library preparation procedure. The Devyser Index Plate A allows an alternate selection of molecular barcodes/indexes used during PCR2 of Devysers' NGS library kits as outlined in Table 2. The Devyser Index Plate A can be used if other Illumina double Index combinations are to be used than those already included in the kits outlined below: Table 2. Devyser NGS library kits compatible for use with the Devyser Index Plate A. NGS library kit name Product art. #. IFU section replaced Devyser BRCA,CE-IVD 8-A100 Complete section 7.4 Devyser CFTR, CE-IVD 8-A101 and 8 Devyser BRCA, RUO 8-A102 Complete section 7.4 Devyser CFTR, RUO 8-A103 and 8 Page 6 of 17
7 4.1 Index preparation using the Devyser Index Plate A (8-A200) Required kit components: Index mix (from the NGS library kit), Index Plate Calculate the number of Index mix tubes required. Each tube is sufficient for 8 reactions. A. Prepare the appropriate number of strips to be used from Index Plate A by removing them from the plate. B. Ensure that the Index mix is completely thawed before use. C. Vortex and then briefly centrifuge each Index mix tube to collect the content. D. Remove the transport seal from Index Plate A. Do not reuse the transport seal. E. Add 20 µl Index mix to each of the required number of wells in Index Plate A or strips removed from the plate. Tips must be changed between each individual well. F. Mix by pipetting to dissolve the coloured reagent pellets. G. Temporarily cover the strip(s) or plate. 4.2 PCR1 library dilution Required kit component: Index buffer (from the NGS library kit) A. Ensure that each Index buffer is completely thawed before use. B. Vortex and then briefly centrifuge each Index buffer tube. C. Prepare one new empty tube for each clinical sample. D. Dispense 198 µl Index buffer into each of the separate tubes. E. Add 2 µl PCR1 library from each clinical sample (from step 7.3 in the IFU of the individual NGS library kit) to the separate dilution tubes. F. Mix thoroughly by pipetting (using a pipetting volume of at least 100 µl). Page 7 of 17
8 4.3 Addition of diluted PCR1 libraries to index Plate A Required kit component: Sealer L A. Add 5 µl of each diluted PCR1 library from 4.2 to separate tubes in the strips removed from Index Plate A (prepared in 4.1). B. Mix by pipetting. Make sure that the coloured reagent pellets are completely dissolved before proceeding to the next step. C. Cut a piece of Sealer L to completely cover the strip(s) from step B. D. Carefully seal the strip(s) and make sure that all wells are covered. E. Centrifuge briefly to collect the content. F. Continue to step 7.5 in the IFU of the individual NGS library kit. Page 8 of 17
9 5. SEQUENCING USING MiSeq 5.1 Flowcell selection In order to ensure sufficient read coverage of each amplicon generated, it is important to avoid overloading the MiSeq flowcell. We recommend the use of a maximum number of samples as indicated in table 3. Table 3. Flowcell selection Illumina reagent Kit Number of samples per flowcell MiSeq Reagent Nano Kit v2 (300 cycles) up to 16 samples MiSeq Reagent Nano Kit v2 (500 cycles) up to 32 samples in PE run MiSeq Reagent Kit Micro Kit v2 (300 cycles) up to 48 MiSeq Reagent Kit Micro Kit v2 (500 cycles) up to 96 samples in PE run MiSeq Reagent Kit v2 (300 cycles) up to 96 MiSeq Reagent Kit v2 (500 cycles) up to 96 samples in PE run 5.2 Preparation for sequencing Sample sheet generation Generate a sample sheet for each run by using the effective version of the Illumina Experiment Manager (IEM) software. The IEM software is installed on the MiSeq computer and can also be downloaded from the Illumina website to any personal computer. Download and import the Devyser settings files for sample sheet generation to the correct location on your computer or the MiSeq computer. The procedure is described in the document "Generating a Devyser sample sheet for MiSeq ". All files are included in the zip-bundle "MiSeq IEM files" (see section 2.5 for download instructions in the IFU of the respective Devyser kit listed in section 1.1 of this manual). Open the IEM software and execute the following steps to generate a sample sheet for a single read 300 cycle run: A. Select Create Sample Sheet B. Select MiSeq and then click Next C. Select Other and DevyserFASTQ Only and then click Next D. Enter the information as outlined in steps E - J E. Reagent Cartridge Barcode: Enter the barcode of the reagent cartridge F. Library Prep Kit: Select Devyser Double Index G. Index reads:: Select 2 H. Read Type: Select Single Read I. Cycles Read 1: Select 301 Page 9 of 17
10 J. Use Adapter trimming: check the box and then click Next K. Click Add blank rows until the desired number of sample rows have been generated L. Add sample names in the Sample ID column M. Choose the desired indexes (N701-N712) in the drop-down menu for Index 1. See description of index content in section N. Choose the desired indexes (N501-N508 ) in the drop-down menu for Index 2. See description of index content in section O. Click Finish and save the Sample Sheet. P. If necessary, transfer the sample sheet to the correct location on the Illumina MiSeq (as defined in the Illumina "MiSeq System User Guide 1 ) If a 500 cycle single read run (501 cycles) or a paired end run (2x151, 2x201 or 2x251 cycles) is started, replace the information given in H - I accordingly. If many samples are sequenced, we recommend that a Sample Plate is created in a 96-well format in the IEM. Detailed information on how to generate sample plates is available in the Illumina guide "Illumina Experiment Manager User Guide (Document ) Index description The Illumina double index are introduced during PCR2 described in Table 4 below. Page 10 of 17
11 Table 4. Devyser Index Plate A configuration (8-A200) Index Plate A i7 TAAGGCGA CGTACTAG AGGCAGAA TCCTGAGC GGACTCCT TAGGCATG CTCTCTAC CAGAGAGG GCTACGCT CGAGGCTG AAGAGGCA GTAGAGGA N701 N702 N703 N704 N705 N706 N707 N708 N709 N710 N711 N712 i5 Index 1-8 Index 9-16 Index Index Index Index Index Index Index Index Index Index TAGATCGC N CTCTCTAT N TATCCTCT N AGAGTAGA N GTAAGGAG N ACTGCATA N AAGGAGTA N CTAAGCCT N Table 4 illustrates the Illumina dual index combinations used in Devyser Index Plate A. Twelve different primers containing Index 1 (i7) sequences (N701-N712) are combined with eight different primers containing Index 2 (i5) sequences (N501-N508), resulting in 96 different combinations. Page 11 of 17
12 5.2.3 Sequencing The sequencing library is processed using reagent kits containing all required reagents for sequencing on the MiSeq system. Follow the effective version of the Illumina guide "Preparing Libraries for Sequencing on the MiSeq 3 to denature and dilute the 2 nm sequencing library from 7.10 in the IFU of the respective Devyser kit. For optimal sequencing performance it is recommended to further dilute the denatured 10 pm library to a final concentration of 7 pm. A. Mix 420 µl of the denatured sequencing library (10 pm) with 180 µl Hyb Buffer (Illumina REF ) B. Add PhiX control DNA to a final concentration of 1 % in the library to obtain the final sequencing mix (according to the procedure described in the MiSeq manual 3 ) C. Add the final sequencing mix to the sample well in the reagent cartridge D. Load the desired flow cell and execute the sequencing run After completion of the sequencing run, locate the generated run data files (FASTQ) and move them to the correct location for analysis, as described in section 9 in the IFU of the respective Devyser kit. Page 12 of 17
13 6. SYMBOLS USED ON LABELS Lot or batch number Expiry date Number of tests Store below temperature shown Consult instructions for use Catalogue number Manufacturer Page 13 of 17
14 7. CONTACT INFORMATION 7.1 Legal manufacturer Devyser AB Instrumentvägen 19 SE Hägersten SWEDEN Phone: Homepage: Technical support Phone: Page 14 of 17
15 8. REFERENCES 1 Illumina guide for performing a MiSeq run, _ system_user_guide_ html 2 Illumina Experiment Manager User Guide (Document ) 3 Illumina guide for preparing libraries for sequencing, downloads/prepare_libraries_for _sequencing_miseq _ html Page 15 of 17
16 9. ABBREVIATIONS IFU instruction for use IVD in vitro diagnostic RUO Research Use Only NGS next generation sequencing PCR polymerase chain reaction Page 16 of 17
17 10. REVISION HISTORY Version New Page 17 of 17
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