Devyser BRCA. Instructions for Use

Transcription

1 Devyser BRCA Art. No.: 8-A100 For in vitro Diagnostic Use Instructions for Use Page 1 of 45

2 TABLE OF CONTENTS TABLE OF CONTENTS 2 1. INTRODUCTION TO DEVYSER BRCA Intended use Background Assay principle Assay design Assay procedure DNA extraction Amplification Sequencing Data analysis 6 2. MATERIALS AND EQUIPMENT Kit configurations for Devyser BRCA Equipment and reagents required but not provided DNA extraction Reagent preparation and amplification Library purification Sequencing Software Downloads Other resources Devyser Sequence Coverage Calculator STORAGE REQUIREMENTS WARNINGS AND PRECAUTIONS PROCEDURAL LIMITATIONS SAMPLE REQUIREMENTS Clinical samples DNA extraction from whole blood sample DNA extraction from FFPE tissue sample Determination of DNA concentration Dilution of DNA from (non-ffpe) clinical samples Dilution of DNA from FFPE tissue samples PhiX control Internal system control INSTRUCTIONS FOR USE Library generation (PCR1) for Devyser BRCA 8, 24 and 96-test kits BRCA mix preparation for 8 and 24-test BRCA mix preparation 96-test Preparation and addition of DNA Thermal cycling PCR Library indexing (PCR2) Library indexing - Devyser BRCA 8 test kit (8-A100-8) PCR1 library dilution Index preparation Addition of diluted PCR1 libraries to Index strip A Library indexing - Devyser BRCA 24 test kit (8-A100-24) PCR1 library dilution Index preparation Addition of diluted PCR1 libraries to Index strip A2 22 Page 2 of 45

3 7.2.3 Library indexing - Devyser BRCA 96 test kit (8-A100-96) PCR1 library dilution Index preparation Addition of diluted PCR1 libraries to Index plate A Thermal cycling PCR Pooling and purification of libraries using the Devyser Library Clean kit (8-A204) Preparation of Wash solution Pooling Library Purification Library elution Library quantification Library dilution SEQUENCING USING MiSeq Sample sheet generation Index description Denaturation of the purified library pool Illumina sequencing SEQUENCE DATA ANALYSIS Guidelines Sequence data analysis using the SeqNext module of the SeqPilot software Sequence data analysis using Sophia DDM Sequence data analysis using Amplicon Suite Interpretation of results PERFORMANCE CHARACTERISTICS Germline sequence variants Experimental design On target reads Assay uniformity Analytical specificity Clinical sensitivity Somatic sequence variants Experimental design On target reads Assay uniformity Limit of detection (LOD) Sensitivity, specificity, and accuracy Repeatability and reproducibility VAF accuracy Batch equivalence Impact of formalin fixation Sequencing mode equivalence SYMBOLS USED ON LABELS NOTICE TO PURCHASER CONTACT INFORMATION Legal manufacturer Technical support REFERENCES ABBREVIATIONS REVISION HISTORY 43 Page 3 of 45

4 1. INTRODUCTION TO DEVYSER BRCA 1.1 Intended 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 The Devyser BRCA kit is an in vitro diagnostic product intended for the detection of sequence variants in the BRCA1 and BRCA2 genes in genomic DNA isolated from human tissue. This product is for professional use only and is not intended for diagnosis of cancer or for standalone diagnostic purposes. 1.2 Background The BRCA1 and BRCA2 genes encode tumor suppressor proteins, which play a role in DNA repair 1. Mutations in these genes may lead to complete absence of, or production of non-functional proteins. When repair mechanisms are compromised additional genetic alterations may appear. The BRCA1 and BRCA2 genes contain repetitive elements, a feature that may cause chromosomal instability, resulting in rearrangements and somatic alterations 2. Inherited (germline) and acquired (somatic) mutations in the BRCA1 and BRCA2 genes are associated with an increased risk of developing breast or ovarian cancer. Inherited BRCA mutations account for up to 10 percent of all breast cancers and 15 percent of all ovarian cancers and these tumors often develop at young age Assay principle The method employed by the Devyser BRCA kit includes multiplex PCR amplification to create a target amplicon library from each DNA sample (PCR1). The library covers the full target region in an overlapping fashion, as illustrated in figure 1. Figure 1. Multiplex target amplification In a second PCR reaction (PCR2), sequencing adapters including unique index sequences are introduced into each amplicon (figure 2), enabling pooling of up to 96 samples. The sample pool is purified using the Devyser Library Clean (Art.No.: 8-A204). The purified sample pool is sequenced using NGS chemistry and the resulting sequences are analyzed using appropriate softwares for targeted sequencing. Page 4 of 45

5 Figure 2. Introduction of adapters including unique index sequences 1.4 Assay design Devyser BRCA allows detection of SNVs, indels and quantitative detection of exon spanning CNVs, all appearing in the coding regions and adjacent exon-intron boundaries in the BRCA1 and BRCA2 genes (figure 3). Specifically, for BRCA1 the Devyser BRCA kit offers full sequence determination of all 23 coding exons of transcript NM_ and all 22 coding exons of LRG_292t1. Additionally, all adjacent exon-intron boundaries, including minimum 20 bp proximal to the 5 end and 10 bp distal to the 3 end of each exon boundary are covered. Similarly, for BRCA2 the Devyser BRCA kit offers full sequence determination of all 26 coding exons of transcript NM_ and LRG_293t1. Additionally, all adjacent exon-intron boundaries including minimum 14 bp proximal to the 5 end and 10 bp distal to the 3 end of each exon boundary are covered. Target specific primers are designed such that primer footprints are covered by an overlapping amplicon to enable detection of primer site SNVs. Figure 3. Illustration of BRCA1 and BRCA2 exons covered in Devyser BRCA Page 5 of 45

6 1.5 Assay procedure DNA extraction The Devyser BRCA kit has been validated using QIAamp DNA Blood Mini Kit (Qiagen, cat.# 51104/51106) and QIAsymphony DSP DNA Midi Kit (Qiagen, cat.#937255) for extraction of genomic DNA from human whole blood and QIAamp DNA FFPE Tissue Kit (Qiagen, cat.# 56404) and High Pure FFPET DNA Isolation Kit (Roche, cat.# ) for extraction of genomic DNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. Detailed information on sample requirements can be found in chapter Amplification The Devyser BRCA kit has been validated using the Veriti Thermal Cycler (Thermo Fisher Scientific). Detailed information on using the Veriti Thermal Cycler can be found in sections and Sequencing The Devyser BRCA kit has been validated using the MiSeq NGS system (Illumina). Detailed information on using the MiSeq NGS system can be found in sections and Data analysis The Devyser BRCA kit has been validated for data analysis and variant calling using the SeqNext module of the SeqPilot software (JSI Medical Systems GmbH), the Sophia DDM platform (Sophia Genetics) and Amplicon Suite (SmartSeq s.r.l). Further information on data analysis software can be found in sections 2.3 and 9. Page 6 of 45

7 2. MATERIALS AND EQUIPMENT 2.1 Kit configurations for Devyser BRCA The Devyser BRCA kit for Illumina sequencing is available in three configurations according to tables 1 to 3 below. Table 1. Devyser BRCA 8 test configuration (8-A100-8) Component Art.No. Number/kit Cap color Storage condition BRCA mix 4-A244 1 Blue Below -18 C Start 4-A243 1 Purple Below -18 C Dilution buffer 4-A245 1 White -25 C to +8 C Index mix 4-A247 1 Red Below -18 C Index strip A1-1 - Below -18 C Index buffer 4-A258 1 Green -25 C to +8 C Sealer S Ambient Table 2. Devyser BRCA 24 test configuration (8-A100-24) Component Art.No. Number/kit Cap color Storage condition BRCA mix 4-A244 3 Blue Below -18 C Start 4-A243 3 Purple Below -18 C Dilution buffer 4-A245 3 White -25 C to +8 C Index mix 4-A247 3 Red Below -18 C Index strip A2-1 - Below -18 C Index buffer 4-A258 3 Green -25 C to +8 C Sealer S Ambient Table 3. Devyser BRCA 96 test configuration (8-A100-96) Component Art.No. Number/kit Cap color Storage condition BRCA mix, 48 test 4-A273 2 Blue Below -18 C Start, 48 test 4-A274 2 Purple Below -18 C Dilution buffer, 96 test 4-A275 1 White -25 C to +8 C Index mix, 48 test 4-A276 2 Red Below -18 C Index plate A3-1 - Below -18 C Index buffer, 96 test 4-A277 1 Green -25 C to +8 C Sealer L Ambient Page 7 of 45

8 2.2 Equipment and reagents required but not provided DNA extraction DNA extraction reagents described in 1.5.1, and according to manufacturer s instructions for use If alternative DNA extraction methods and sample materials are used, a thorough evaluation of the performance together with the Devyser BRCA kit should be performed Micropipette or multipipette with aerosol barrier tips Disposable protective gloves (powder free) Reagent preparation and amplification Consumables for the thermal cycler Micropipette with aerosol barrier tips or dispenser with displacement tips Disposable protective gloves (powder free) Veriti Thermal Cycler with MicroAmp 96-Well Tray/Retainer Set (Thermo Fisher Scientific) If an alternative thermal cycler is used, a thorough evaluation of its performance together with the Devyser BRCA kit should be performed. It is of high importance that the following ramp rates are applied: heating 1,6 C/s, cooling 1,6 C/s Library purification Required kit: Devyser Library Clean (8-A204) Table 4. Devyser Library Clean (8-A204) Component Art.No. Number/kit Cap color Storage condition Clean 4-A255 1 Orange +2 to +8 C Wash 4-A256 1 Yellow +2 to +8 C Dilution buffer 4-A245 1 White +2 to +8 C Magnetic rack for test tubes (DynaMag -2 Magnet, Thermo Fisher Scientific or equivalent) Ethanol (96 %) Qubit Fluorometer (Thermo Fisher Scientific, cat.# Q33216) Qubit dsdna HS Assay Kit (Thermo Fisher Scientific, cat.# Q32851/Q32854) Reaction tubes Micropipette or multipipette with aerosol barrier tips Disposable protective gloves (powder free) Page 8 of 45

9 2.2.4 Sequencing Illumina MiSeq Illumina Miseq reagent Kits v2: Miseq Reagent Nano Kit v2 (300-cycles), cat.# MS ) Miseq Reagent Nano Kit v2 (500-cycles), cat.# MS ) Miseq Reagent Micro Kit v2 (300-cycles), cat.# MS ) Miseq Reagent Kit v2 (300-cycles), cat.# MS ) Miseq Reagent Kit v2 (500-cycles), cat.# MS ) Illumina PhiX control v3 (cat.# FC ) Other user-supplied consumables needed for sequencing, according to Illumina's sequencing guide Micropipette or multipipette with aerosol barrier tips Disposable protective gloves (powder free) NOTE All equipment should be validated, calibrated and maintained regularly 2.3 Software SeqPilot v4.3.1 software including the SeqNext module (JSI Medical Systems GmbH). If using a different version of the software, please consult the manufacturer Sophia DDM (Sophia Genetics) pipeline for Devyser BRCA. Contact Devyser or Sophia Genetics to create a Sophia DDM account Amplicon Suite (SmartSeq s.r.l) pipeline for BRCA.Contact Devyser for information Page 9 of 45

10 2.4 Downloads Supplementary information and files can be downloaded from the Devyser website: using the download code printed on the kit label. See table 5 for details. Table 5. Download files Download Illumina double index MiSeq IEM files SeqNext guide for Devyser BRCA MiSeq sample sheet guide Devyser BRCA SeqNext sge files Devyser BRCA BED files Description Index sequence information Devyser setting files for sample sheet generation: Generating a Devyser sample sheet for MiSeq.doc DEVYSER double Index MiSeq.txt DevyserGenerateFASTQ.txt DevyserGenerateFASTQ.jpg SeqNext guide for Devyser BRCA analysis Guide for generating a Devyser sample sheet for MiSeq Devyser settings files (.sge files) for BRCA data analysis with SeqNext using genome reference Hg19/GRCh37 or Hg38/GRCh38. The.sge file includes information about the regions of interest (ROI) and settings for read alignments and variant calling. BED file detailing amplicon positions according to Hg19/GRCh37 and Hg38/GRCh Other resources Devyser Sequence Coverage Calculator In order to ensure sufficient read coverage of each sample when using the Devyser BRCA kit, and for pooling multiple sequencing libraries with different sample types and coverage needs, please consult our sequencing coverage calculator and guide at Download update service Sign up for the download update service to receive notifications via whenever there is a new version of the downloads available. Visit to sign up Page 10 of 45

11 3. STORAGE REQUIREMENTS Store the Devyser BRCA kit below -18 C or the individual kit components as specified on the label (see also tables 1 to 3 in 2.1) Store the components of the Devyser Library Clean kit at +2 to +8 C Do not use components beyond the kit lot expiration date If handled, reclosed and stored properly, kit components will remain stable until the expiration date of the kit or according to in use stability specified in the IFU (chapter 7) Frozen kit components should be thawed in a refrigerator or at room temperature before use Avoid repeated freezing-thawing Page 11 of 45

12 4. WARNINGS AND PRECAUTIONS Use of this product should be limited to personnel trained in PCR, NGS techniques and NGS data analysis The procedure should be performed according to this IFU Deviations from the IFU will compromise the kit performance Modifications of software settings 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 damaged reagent vials Frozen components should be completely thawed in a refrigerator or at room temperature before use Use, storage and disposal of kit components and samples, 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 It is recommended 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 workflow 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 12 of 45

13 5. PROCEDURAL LIMITATIONS Sequence variants present in other genes than the BRCA1 and BRCA2 genes will not be detected using Devyser BRCA This assay has not been validated for diagnosis of cancer or any other disease Results obtained with the Devyser BRCA kit can only be directly applied to the tissue or specific sample material tested Rare primer site sequence alterations may affect the function of individual PCR primers used in the Devyser BRCA kit The following parameters might affect the overall performance and in particular the CNV analysis Quality and concentration of the clinical sample DNA Deviations from the protocol The number of control samples used in the analysis The type of control samples used Sequencing depth Bioinformatic pipeline The result of a chemical reaction known as deamination is prevalent in FFPE DNA samples. Transitional C:G>T:A SNVs are the most frequent sequence artifacts arising from deamination of cytosine in FFPE DNA It is recommended that only samples that have been processed and sequenced together and with the same kit lot should be analysed together for a CNV analysis The Devyser BRCA kit has not been validated for detection of somatic CNVs present in clinical samples The bioinformatic characterization of CNVs is based on different computational strategies. None of the strategies can guarantee correct detection of all CNVs Patients who have undergone a previous allogenic bone marrow transplant should not be tested with the Devyser BRCA kit CNV detection using SeqNext has not been validated Analysis of somatic sequence variants using Sophia DDM has not been validated Page 13 of 45

14 6. SAMPLE REQUIREMENTS DNA concentration, integrity and purity are important parameters for successful testing using the Devyser BRCA kit. DNA should be free from contaminating proteins, salts and other PCR inhibitors, e.g. residual ethanol from DNA extraction procedures. Low quality DNA may result in amplification failure and/or increased background signals. 6.1 Clinical samples The Devyser BRCA kit has been validated using human genomic DNA extracted from whole blood and FFPE tissue samples DNA extraction from whole blood sample Results are consistently obtained for the Devyser BRCA kit with DNA extracted from human whole blood using QIAamp DNA Blood Mini Kit (Qiagen, cat.# /51106) and QIAsymphony DSP DNA Midi Kit (Qiagen, cat.#937255) Follow the protocol of the extraction kit starting with 200 µl fresh whole blood and elute in 200 µl elution buffer DNA extraction from FFPE tissue sample Results are consistently obtained for the Devyser BRCA kit with DNA extracted from FFPE tissue samples using QIAamp DNA FFPE Tissue Kit (Qiagen, cat.# 56404) and High Pure FFPET DNA Isolation Kit (Roche, cat.# ) Follow the protocol of the extraction kit starting with at least one section (15-20 μm) of the FFPE tissue sample and elute in 30 μl elution buffer The performance of the Devyser BRCA kit with DNA extracted from FFPE tissue samples is dependent on the DNA quality and therefore it is recommended to assess the integrity of the extracted DNA prior to analysis Determination of DNA concentration High quality DNA is important for accurate and reproducible determination of DNA concentration All DNA concentrations referred to in this IFU were determined using the Qubit Fluorometer and the Qubit dsdna HS Assay Kit The DNA concentration determined for a DNA sample may differ between Qubit systems and between the Qubit system and other techniques. It is important to verify that the technique used for determination of DNA concentration correlates to the actual results obtained with the Devyser BRCA kit A spectrophotometric or fluorometric analysis of the DNA extracted from FFPE tissue samples may not give reliable results for the assessment of the amount of DNA accessible for PCR amplification. DNA cross linking and fragmentation effects during formalin fixation may limit purification and amplification efficiency. RNA remaining after DNA extraction may also Page 14 of 45

15 effect spectrophotometric analysis. When using DNA extracted from FFPE tissue samples a pre-evaluation of the DNA quality and integrity is recommended Dilution of DNA from (non-ffpe) clinical samples For optimal performance it is recommended to adjust the concentration of the extracted DNA to 2 ng/µl (see 7.1.3) Dilution of DNA from FFPE tissue samples For optimal performance it is recommended to adjust the concentration of the extracted DNA to 2-10 ng/µl (see 7.1.3) NOTE The use of high quality DNA with carefully determined concentration enables direct pooling of equal volumes from each clinical sample library prior to purification and quantification of the library pool (see 7.3) 6.2 PhiX control Include PhiX control v3 library DNA (Illumina cat.# FC ) in each sequencing run to ensure that the sequencing pool has the required diversity for high quality sequencing (see 8.3). 6.3 Internal system control We recommend to perform regular internal system control of all equipment and software used in this procedure. Samples with pre-characterized BRCA1 and BRCA2 gene sequence variants (in-house developed or externally sourced) are suitable as system controls. Page 15 of 45

16 7. INSTRUCTIONS FOR USE Figure 4. Schematic overview of the Devyser BRCA library preparation procedure The Devyser BRCA library preparation procedure consists of the following steps: PCR1 (7.1) A combined BRCA1 and BRCA2 amplicon library is generated in one multiplex PCR reaction for each clinical sample. PCR2 (7.2) Index addition to the PCR1 library is performed in PCR2 to allow pooling of multiple clinical sample libraries for sequencing. Purification (7.3) Unique clinical sample libraries generated in PCR2 are pooled and purified in a single tube. The purified library pool is analyzed by NGS. Each step ( ) is followed by a suitable stopping point where the procedure can be paused and restarted within 30 days. NOTE This kit is available in three test configurations; 8, 24, and 96. Each Start and BRCA mix tube in the 8 and 24 test configuration is sufficient for 8 reactions, and each Start and BRCA mix tube in the 96 test configuration is sufficient for 48 reactions. Determine the required number of each component tube before starting Page 16 of 45

17 7.1 Library generation (PCR1) for Devyser BRCA 8, 24 and 96-test kits Figure 5. Schematic overview of steps to BRCA mix preparation for 8 and 24-test Calculate the number of Start and BRCA mix tubes required. Each tube is sufficient for 8 reactions. Required kit components: Start (4-A243), BRCA mix (4-A244) A. Ensure that the Start and the BRCA mix are completely thawed before use B. Vortex the Start tube(s) briefly C. Briefly centrifuge the Start and BRCA mix tube(s) to collect the content D. Add 50 µl of Start to the BRCA mix tube(s) to obtain an activated BRCA mix E. Vortex the activated BRCA mix tube(s) and then centrifuge it briefly to collect the content F. Dispense 10 µl of the activated BRCA mix into separate PCR reaction tubes and cap the tubes G. Store the dispensed BRCA mix at +2 to +8 C and continue to H. Any remaining activated BRCA mix can be stored in a freezer below -18 C for 6 weeks Page 17 of 45

18 7.1.2 BRCA mix preparation 96-test Calculate the number of Start, 48 test and BRCA mix, 48 test tubes required. Each tube is sufficient for 48 reactions. Required kit components: Start, 48 test (4-A274), BRCA mix, 48 test (4-A273) A. Ensure that the Start, 48 test and the BRCA mix, 48 test are completely thawed before use B. Vortex the Start, 48 test tube(s) briefly C. Briefly centrifuge the Start, 48 test and BRCA mix, 48 test tube(s) to collect the content D. Add 270 µl of Start to the BRCA mix, 48 test tube to obtain an activated BRCA mix, 48 test E. Vortex the activated BRCA mix, 48 test tube(s) and then centrifuge it briefly to collect the content F. Dispense 10 µl of the activated BRCA mix, 48 test into separate PCR reaction tubes or separate wells in a plate and cap the tubes or seal the plate G. Store the dispensed BRCA mix, 48 test at +2 to +8 C and continue to H. Any remaining activated BRCA mix, 48 test can be stored in a freezer below -18 C for 6 weeks Preparation and addition of DNA Required kit component: Dilution buffer (4-A245) or Dilution buffer, 96 test (4-A275) A. Determine the DNA concentration of each DNA sample (see 6.1.3) B. Ensure that the Dilution buffer is completely thawed before use C. Dilute the DNA samples to a final concentration of 2 ng/µl (non-ffpe) or 2-10 ng/µl (FFPE) using the provided Dilution buffer D. Add 5 µl of diluted DNA from each clinical sample to the separate PCR reaction tubes or the separate wells in the plate containing activated BRCA mix (from or 7.1.2) E. Mix by pipetting F. Cap the tubes or seal the plate and centrifuge briefly to collect the content G. Continue to Page 18 of 45

19 7.1.4 Thermal cycling PCR1 Figure 6. Thermal profile PCR1 A. Program the thermal cycler according to the PCR1 thermal profile in figure 6 B. Set the ramp rates to heating 1,6 C/s and cooling 1,6 C/s C. Set the reaction volume to 15 µl D. Place the tubes or the plate in the thermal cycler E. Start the amplification (duration approximately 1 hr 45 min) F. Following amplification, centrifuge briefly if necessary to collect the content and continue to 7.2 The PCR1 library can be stored in a freezer below -18 C for 30 days. SUITABLE STOPPING POINT NOTE It is of high importance that the following ramp rates are applied: heating 1,6 C/s, cooling 1,6 C/s NOTE To program the correct ramp rate for the Veriti Thermal Cycler: In the "Tools Menu" select "Convert a Method". In the next select "9700 Max Mode" and then enter the PCR profile as outlined in NOTE If using tubes/strips in a Veriti Thermal Cycler they should first be placed in the MicroAmp 96-Well Tray/Retainer Set for Veriti Systems Page 19 of 45

20 7.2 Library indexing (PCR2) For the Devyser BRCA 8 test kit proceed to For the Devyser BRCA 24 test kit proceed to For the Devyser BRCA 96 test kit proceed to Library indexing - Devyser BRCA 8 test kit (8-A100-8) Figure 7. Schematic overview of steps to PCR1 library dilution Required kit component: Index buffer (4-A258) A. Ensure that the Index buffer is completely thawed B. For each PCR1 library to be diluted, dispense 198 µl Index buffer to a new tube C. Add 2 µl of each PCR1 library to the separate tubes containing 198 µl Index buffer. Make sure no liquid remains in the tip by pipetting repeatedly in the Index buffer D. Mix the diluted PCR1 libraries thoroughly by pipetting (using a pipetting volume of at least 100 µl) E. Continue to Page 20 of 45

21 Index preparation Required kit components: Index mix (4-A247), Index strip A1 A. Ensure that the Index mix is completely thawed before use B. Vortex and then briefly centrifuge the Index mix tube to collect the content C. Carefully remove the transport seal of Index strip A1. Note! Do not reuse the transport seal D. Add 20 µl of Index mix to each of the 8 wells of Index strip A1. Note! Tips must be changed between each individual well E. Continue to Addition of diluted PCR1 libraries to Index strip A1 Required kit component: Sealer S A. Add 5 µl of each diluted PCR1 library (from ) to separate tubes in Index strip A1 (prepared in ) B. Mix thoroughly by pipetting to dissolve the coloured reagent pellets, using a pipetting volume of at least 10 µl. Note! Make sure that the coloured reagent pellets are completely dissolved before proceeding to the next step. Avoid bubbles C. Cut a piece of Sealer S to completely cover Index strip A1 D. Carefully seal Index strip A1 and make sure that all wells are covered E. Centrifuge briefly to collect the content F. Continue to Library indexing - Devyser BRCA 24 test kit (8-A100-24) Figure 8. Schematic overview of s to Page 21 of 45

22 PCR1 library dilution Required kit component: Index buffer (4-A258) A. Ensure that the Index buffer is completely thawed before use B. For each PCR1 library to be diluted, dispense 198 µl Index buffer to a new tube C. Add 2 µl of each PCR1 library to the separate dilution tubes containing 198 µl Index buffer. Make sure no liquid remains in the tip by pipetting repeatedly in the Index buffer D. Mix the diluted PCR1 libraries thoroughly by pipetting (using a pipetting volume of at least 100µL) E. Continue to Index preparation Required kit components: Index mix (4-A247), Index strip A2 A. Ensure that the Index mix is completely thawed before use B. Vortex and then briefly centrifuge the Index mix tube to collect the content C. Carefully remove the transport seal of Index strip A2. Note! Do not reuse the transport seal D. Add 20 µl of Index mix to each of the wells to be used in Index strip A2. Note! Tips must be changed between each individual well E. Continue to Addition of diluted PCR1 libraries to Index strip A2 Required kit component: Sealer S A. Add 5 µl of each diluted PCR1 library pool (from ) to separate wells in Index strip A2 (prepared in ) B. Mix thoroughly by pipetting to dissolve the coloured reagent pellets, using a pipetting volume of at least 10 µl. Note! Make sure that the coloured reagent pellets are completely dissolved before proceeding to the next step. Avoid bubbles C. Cut a piece of Sealer S to completely cover Index strip A2 D. Carefully seal Index strip A2 and make sure that all wells are covered E. Centrifuge briefly to collect the content F. Continue to Page 22 of 45

23 7.2.3 Library indexing - Devyser BRCA 96 test kit (8-A100-96) Figure 9. Schematic overview of s to PCR1 library dilution Required kit component: Index buffer, 96 test (4-A277) A. Ensure that the Index buffer, 96 test is completely thawed before use B. For each PCR1 library to be diluted, dispense 198 µl Index buffer, 96 test to a new tube C. Add 2 µl of each PCR1 library to the separate dilution tubes containing 198 µl Index buffer, 96 test. Make sure no liquid remains in the tip by pipetting repeatedly in the Index buffer D. Mix the diluted PCR1 libraries thoroughly by pipetting (using a pipetting volume of at least 100 µl) E. Continue to Index preparation Required kit components: Index mix, 48 test (4-A276), Index plate A3 A. Ensure that the Index mix, 48 test is completely thawed before use B. Vortex and then briefly centrifuge the Index mix tube to collect the content C. Carefully remove the transport seal of Index plate A3. Note! Do not reuse the transport seal D. Add 20 µl of Index mix, 48 test to each of the wells to be used in Index plate A3. Note! Tips must be changed between each individual well E. Continue to Page 23 of 45

24 Addition of diluted PCR1 libraries to Index plate A3 Required kit component: Sealer L A. Add 5 µl of each diluted PCR1 library pool (from ) to separate tubes in Index plate A3 (prepared in ) B. Mix thoroughly by pipetting to dissolve the coloured reagent pellets, using a pipetting volume of at least 10 µl. Note! Make sure that the coloured reagent pellets are completely dissolved before proceeding to the next step. Avoid bubbles C. Use Sealer L to completely cover Index plate A3, or cut a piece of Sealer L to cover wells in use. D. Carefully seal Index plate A3 and make sure that wells in use are covered E. Centrifuge briefly to collect the content F. Continue to Thermal cycling PCR2 Figure 10. Thermal profile PCR2 A. Program the thermal cycler according to the PCR2 thermal profile in figure 10 B. Set the ramp rates to heating 1,6 C/s and cooling 1,6 C/s C. Set the reaction volume to 25 µl D. Place the tubes or the plate in the thermal cycler E. Start the amplification (duration approximately 1 hr 55 min) F. If proceeding with sequencing the same day, prepare sequencing reagents (see note in 7.3) G. Following amplification, centrifuge briefly if necessary to collect the content and continue to 7.3 PCR2 libraries can be stored in a freezer below -18 C for 30 days. SUITABLE STOPPING POINT Page 24 of 45

25 NOTE It is of high importance that the following ramp rates are applied: heating 1,6 C/s, cooling 1,6 C/s NOTE To program the correct ramp rate for the Veriti Thermal Cycler: In the "Tools Menu" select "Convert a Method". In the next select "9700 Max Mode" and then enter the PCR profile as outlined in section NOTE If using tubes/strips in a Veriti Thermal Cycler they should first be placed in the MicroAmp 96-Well Tray/Retainer Set for Veriti Systems 7.3 Pooling and purification of libraries using the Devyser Library Clean kit (8-A204) NOTE Defrost the MiSeq reagent cartridge well in advance prior to sequencing according to the procedure described in the current "MiSeq System Guide 10 Figure 11. Schematic overview of to Page 25 of 45

26 7.3.1 Preparation of Wash solution Required kit: Devyser Library Clean (8-A204) Required kit component: Wash (4-A256) A. Prepare the Wash solution by adding 1500 μl of 96 % ethanol to the Wash tube B. Mix thoroughly by vortexing C. Tick the box on the Wash tube label to indicate that ethanol was added D. Note! The Wash solution should be stored at +2 to +8 C and used within 3 months from day of activation E. Continue to Pooling A. To obtain a library pool volume of at least 40 μl for subsequent purification, pool 5 μl from each of the PCR2 libraries (from 7.2.4) into a single tube B. Mix thoroughly by vortexing and then briefly centrifuge the library pool to collect the content C. Transfer 40 µl of the library pool into a new tube suitable for placing on a magnetic rack D. Continue to NOTE If less than 8 libraries are pooled, add equal volumes of each PCR2 library to obtain a library pool volume of 40 µl. If the total pooled volume is less than 40 µl, use equal volumes of the pooled PCR2 libraries and Clean (see 7.3.3). For elution, use equal volume of Dilution buffer as the library pool volume (see 7.3.4). However, do not use less than 25 µl of Dilution buffer for elution NOTE If highly divergent clinical samples are processed together (e.g. FFPE and blood samples or clinical samples processed with different Devyser library kits), only clinical samples of the same category should be pooled prior to library purification Page 26 of 45

27 7.3.3 Library Purification Required kit: Devyser Library Clean (8-A204) Required components: Clean (4-A255), Wash (4-A256) A. Briefly centrifuge the Clean tube to collect the content B. Firmly tap the Clean tube. Make sure that the bead pellet is re-suspended and that the content is homogenous. If necessary, briefly vortex the tube but avoid extensive vortexing C. Add 40 µl re-suspended Clean to the library pool from and mix by pipetting. See note in for use of alternative volumes of library pool and Clean D. Incubate the tube at room temperature for 3 minutes E. Place the tube onto a magnetic rack until all beads are pelleted and the solution has become clear F. While keeping the tube on the magnetic rack, carefully remove and discard the solution. Note! It is important to avoid touching the bead pellet during this step (see figure 12) G. Add 150 µl of prepared Wash solution (from 7.3.1) to the tube without removing it from the magnetic rack H. Slightly lift and rotate the tube two half circles to wash the beads I. Place the tube onto the magnetic rack to pellet the beads J. Carefully remove as much Wash solution as possible by pipetting from the bottom of the tube. Note! It is important to avoid touching the bead pellet and the walls of the tube during this step (see figure 12) K. Leave the lid open until all remaining Wash solution has evaporated and the bead pellet has change from being luster to lustereless, approximatly 3-5 minutes, while remaining on the magnetic rack. Important! See note below L. Remove the tube from the magnetic rack and continue to NOTE It is important that all Wash solution has evaporated and that the pellet is dry before continuing. The pellet appearance should change from being luster to lusterless and the color should change slightly to a lighter nuance when dry. If Wash solution remains, briefly centrifuge the tube to collect all remaining Wash solution, pellet the beads using the magnetic rack, remove the residual Wash solution and air dry the pellet again Figure 12. Bead pellet Page 27 of 45

28 7.3.4 Library elution Required kit: Devyser Library Clean (8-A204) Required kit component: Dilution buffer (4-A245) A. Briefly centrifuge the Dilution buffer to collect the content B. Add 40 µl Dilution buffer to the tube from and re-suspend the pellet by pipetting and/or tapping the tube. If necessary collect the liquid by a very brief centrifugation C. Place the tube onto the magnetic rack until all beads are pelleted D. While keeping the tube on the magnetic rack, transfer the cleared supernatant, containing the purified library pool, to a new tube E. Continue to Library quantification Required kit: Qubit dsdna HS Assay Kit (see 2.2.3) Quantify the library as described below and consult the current manual for Qubit dsdna HS Assay Kits for details A. Prepare Qubit ds DNA HS working solution by diluting Qubit DNA HS reagent 1:200 in Qubit ds DNA HS buffer B. Mix thoroughly by vortexing C. Mix 190 µl Qubit ds DNA HS working solution with 10 µl of Qubit standard 1 D. Mix 190 µl Qubit ds DNA HS working solution with 10 µl of Qubit standard 2 E. Mix 190 µl Qubit ds DNA HS working solution with 10 µl of the purified library pool from F. Briefly vortex, centrifuge and incubate each tube for 2 minutes at room temperature G. Measure the concentration (ng/µl) of the purified library pool on a Qubit Fluorometer H. Continue to Library dilution Required kit: Devyser Library Clean (8-A204) Required kit component: Dilution buffer (4-A245) A. Dilute the purified library pool from to a final concentration of ng/µl (corresponding to approximately 2 nm) using the Dilution buffer B. Measure the concentration (ng/µl) of the diluted library pool to confirm the concentration by repeating E to G in C. Proceed to sequencing using the Illumina MiSeq according to chapter 8 The purified library pool can be stored in a freezer at below -18 C for 30 days. SUITABLE STOPPING POINT Page 28 of 45

29 8. SEQUENCING USING MiSeq To ensure sufficient read coverage of each sample when using the Devyser BRCA kit, and for pooling multiple sequencing libraries with different sample types and coverage needs, please consult our sequencing coverage calculator at The Devyser BRCA libraries can be sequenced in paired-end mode (2x151 bp or 2x251 bp) or single read mode (1x301 bp). Ensure the software pipeline to be used is compatible with the selected sequencing mode. 8.1 Sample sheet generation Generate a sample sheet for each run in the Illumina Experiment Manager (IEM) software by using the Devyser Guide "Generating a Devyser Sample Sheet for MiSeq " available at and the Illumina document # : "Illumina Experiment Manager Software Guide" Index description The Illumina double index introduced during PCR2 are listed in tables 6 to 8. Detailed information about the index combinations and index sequences can be found online (see 2.4 for details). Table 6. Illumina double index used in Index strip A1 Index 1-8 Index1: N701 Index2: N501 1 Index2: N502 2 Index2: N503 3 Index2: N504 4 Index2: N505 5 Index2: N506 6 Index2: N507 7 Index2: N508 8 Table 7. Illumina double index used in Index strip A2 Index 1-8 Index 9-16 Index Index1: N701 Index1: N702 Index1: N703 Index2: N Index2: N Index2: N Index2: N Index2: N Index2: N Index2: N Index2: N Page 29 of 45

30 Table 8. Illumina double index used in Index plate A3 Index 1-8 Index 9-16 Index Index Index Index Index Index Index Index Index Index Index2: N501 Index2: N502 Index2: N503 Index2: N504 Index2: N505 Index2: N506 Index2: N507 Index2: N508 Index1: N701 Index1: N702 Index1: N703 Index1: N704 Index1: N705 Index1: N706 Index1: N707 Index1: N708 Index1: N709 Index1: N710 Index1: N Index1: N Denaturation of the purified library pool A. Prepare 20 pm PhiX, HT1 and a fresh dilution of 0.2 N NaOH according to the current version of the Illumina document # : "Denature and Dilute Libraries Guide" 8 B. Combine 5 µl purified library pool from with 5 µl 0.2 N NaOH C. Briefly vortex, centrifuge and incubate for 5 minutes at room temperature D. Add 1410 μl prechilled HT1 to dilute the denatured library pool E. To obtain a sequencing mix, add 9 µl 20 pm denaturated PhiX control DNA. The added PhiX will represent approximately 1 % of the total number of reads from the sequencing run F. Repeateadly invert and then vortex the tube to mix and briefly centrifuge to collect the content 8.4 Illumina sequencing A. Prepare the sequencing run according to the current version of the Illumina document # : "MiSeq System Guide" 10 B. For loading the reagent cartridge, transfer 600 µl of the sequencing mix from 8.3 to the sample well in the reagent cartridge C. Load the desired flow cell and execute the sequencing run D. After completion of the sequencing run, locate the generated sequencing data files (FASTQ) and move them to the correct location for analysis, see 2.3 and 9 Page 30 of 45

31 9. SEQUENCE DATA ANALYSIS 9.1 Guidelines See references 4-8 in chapter Sequence data analysis using the SeqNext module of the SeqPilot software Transfer the sequencing data files (FASTQ) to an appropriate location and start the analysis in SeqNext according to manufacturer s instructions for use. Consult the SeqNext guide for Devyser BRCA (see 2.4) for details on downloading settings and performing analysis and data interpretation. 9.3 Sequence data analysis using Sophia DDM Upload the sequencing data files (FASTQ) and start the analysis in Sophia DDM according to manufacturer s instructions for use. 9.4 Sequence data analysis using Amplicon Suite Upload the sequencing data files (FASTQ) and start the analysis in Amplicon Suite according to manufacturer s instructions for use. 9.5 Interpretation of results For detection of sequence variants of germline origin, ensure that each amplicon has at least 100x coverage. For detection of sequence variants of somatic origin, ensure that each amplicon has at least 1000x coverage for detection of VAF down to 5%. An expert should perform technical validation of the results and interpret variants in a clinical context. Page 31 of 45

32 10. PERFORMANCE CHARACTERISTICS The performance of the Devyser BRCA kit for the detection of sequence variants of germline and somatic origin using the Illumina MiSeq was evaluated according to the defined conditions of use outlined in this IFU. The target region was defined as the BRCA1 (NM_007300) and BRCA2 (NM_000059) coding regions +/- 2 bp Germline sequence variants Experimental design A total of 49 samples were included in the study. All samples were previously characterized to carry a BRCA1 or BRCA2 sequence variant. The sequence variants included indels, SNVs and CNVs. Three sequencing libraries were prepared in parallel for each sample, resulting in a total of 147 sample libraries. Two Illumina MiSeq Reagent Kit v2 (300 cycles) flowcells were used and samples were sequenced in single read mode, 1x301 bp. A coverage cut-off of 100 reads per amplicon was employed. Three different software were used to analyse the generated sequence data SeqNext 4.3, JSI Medical Systems GmbH Sophia DDM, Sophia Genetics Amplicon Suite, SmartSeq s.r.l. All five (5) samples containing a CNV were excluded from SeqNext analysis On target reads The number of quality filtered sequencing reads, mapped and aligned against the target region, were counted and compared to the total number of reads. Table 9. On target reads SeqNext 4.3 Sophia DDM AmpliconSuite On target reads 98,1% (96.8% - 98,7%) 97,9% (97,5% - 98,5%)* 96.6% (91.4% %) *The on target determined by Sophia DDM is 99,6% (98,9% - 99,9%) Assay uniformity The total number of amplicons exhibiting at least 20% of the mean amplicon coverage within each sample were calculated. Table 10. Assay uniformity SeqNext 4.3 Sophia DDM AmpliconSuite Assay uniformity 99,99% 99,99% 99.99% Page 32 of 45

33 Analytical specificity The obtained consensus sequence for forward and reverse reads were compared for 3 randomly chosen amplicons in 3 different clinical samples in each of 3 independent data sets. The analysed sequences displayed complete correlation of sequences with each other, supporting excellent analytical specificity and inter-run reproducibility. Analytical specificity: > 99% Inter-run reproducibility: > 99% Clinical sensitivity The ability of the software to correctly identify the previously characterized sequence variants was evaluated. All five (5) samples containing a CNV were excluded from SeqNext analysis. Table 11. Clinical sensitivity SeqNext 4.3 Sophia DDM Amplicon Suite True positive calls 132/ / /147 Clinical sensitivity > 99% > 99% > 99% 10.2 Somatic sequence variants Experimental design A total of 86 sample libraries from 41 unique samples were processed and sequenced at three different sites in order to validate the ability of Devyser BRCA to detect SNVs and indels down to 5% variant allele frequency (VAF) in genomic DNA derived from formalin fixed paraffin embedded (FFPE) samples. Sequencing libraries from a proficiency panel (PP) consisting of seven (7) samples were prepared and sequenced at all three sites. The PP consisted of the following samples: Three (3) samples prepared by mixing the FFPE derived samples GM24149, GM24143, and GM2463 (Horizon Discovery), referred to as PP Mixes, containing 13 mutations with theoretical VAF between 5% and 95% Three (3) samples with known sequence variants and various levels of degradation. The respective samples are classified as mildly (HD798), moderately (HD799) or severely (HD803) formalin compromised (Horizon Discovery) One (1) sample (HD753) containing sequence variants, including one with VAF 5,6% in a homopolymer position (Horizon Discovery) Samples were sequenced in single read mode (1x301 bp) or paired end mode (2x151 bp or 2x251 bp) on the MiSeq instrument using MiSeq Reagent Kit v2 flowcells. On average, 1.5 M reads or reads-pairs per sample (min 0.77M, max 2.4M) were produced. Page 33 of 45

34 Two different software were used to analyse the generated sequence data: SeqNext 4.3, JSI Medical Systems GmbH Amplicon Suite, SmartSeq s.r.l On target reads The number of quality filtered sequencing reads, mapped and aligned against the target region, were counted and compared to the total number of reads. Table 12. On target reads SeqNext 4.3 Amplicon Suite On target reads 89.3% (79.7% %) 93.8% (77.3% %) Assay uniformity The total number of amplicons exhibiting at least 20% of the mean amplicon coverage within each sample were calculated. Table 13. Assay uniformity SeqNext 4.3 AmpliconSuite Assay uniformity 99.1% 98.9% Limit of detection (LOD) The LOD is the lowest VAF that can be distinguished from the identified background noise. LOD was determined as the mean of quantified noise plus three standard deviations 12. In order to determine VAF and noise, alignment was performed with SeqNext and the BAM format was exported and analyzed. Pysamstats ( was used to extract the consensus sequence for each base in the target region. Only bases with quality score higher than 30 and positions with coverage >1000 were included. LOD was determined for each base in the target region. The maximum and mean calculated LOD of all the positions was defined as LOD max and LOD mean respectively. For homopolymer regions with consecutive repetitions above five nucleotides (HP6-8), the LOD was calculated separately. Table 14. Limit of detection LOD max LOD mean Target region excluding HP 6 1.2% 0.4% HP6 2.3% 1.1% HP7 5.0% 2.6% HP8 9.0% 8.5% Sensitivity, specificity, and accuracy 84 of the 86 sequenced samples displayed a total of 560 known sequence variants that were used to calculate sensitivity, specificity and accuracy. A coverage of 50 reads and a reported VAF of at Page 34 of 45

35 least 5% was required for a variant to be reported as a positive call with the following exceptions: Mutations in homopolymers were classified as positive calls only if the VAF was above the LOD mean reported in Table 14 Mutations with an expected VAF of 5% were classified as a positive call if the reported VAF was 3% to compensate for the possible deviation between theoretical and actual VAF in the PP Mixes and for intrinsic variation affecting the VAF determination Sensitivity. specificity and accuracy were calculated and presented in table 15. where: True positive (TP) is the number of variants called using Devyser BRCA and confirmed by a reference method True negative (TN) is the number of variants not called using Devyser BRCA nor by a reference method False positive (FP) is the number of variants called using Devyser BRCA but not confirmed by a reference method False negative (FN) is the number of variants not called by Devyser BRCA kit but called by a reference method. Table 15. Sensitivity, specificity and accuracy SeqNext 4.3 Amplicon Suite TP TN FP 7* 6* FN 0 0 Sensitivity TP/(TP+FN) >99.9% >99.9% Specificity TN/(TN+FP) 99.9% 99.9% Accuracy (TP+TN)/(TP+TN+FP+FN) 99.9% 99.9% *All FP displayed VAF %. Six (6) FP are called identically by the two software. One variant is called at 5,0% by JSI and therefore classified as a FP while the same variant is called at 4.8% by Amplicon Suite and therefore classified as TN Repeatability and reproducibility Repeatability describes the ability to reproduce the same analysis multiple times at the same site. Reproducibility describes the ability to reproduce the same analysis multiple times at different sites. The three PP mixes were processed and sequenced in triplicates at one site and used to measure repeatability of called bases. All bases in each of the three samples showed full concordance using both software. Reproducibility: >99.9% The seven proficiency panel samples were processed and sequenced at three different sites and used to measure the reproducibility of called bases. For samples sequenced multiple times at the same site, the replicate showing the lowest concordance rate was reported. Page 35 of 45

36 For both software, 8 discordant bases out of the 7x18911 analyzed bases were identified. All 8 were found in the severely compromised FFPE sample HD803 and reported as discordant since the reported VAF between the replicates was on both side of the threshold of 5% (reported VAF 4-6%). In addition, one discordant base in one of the PP Mixes was identified at 4% VAF with SeqNext 4.3 only. Repeatability: >99,9% VAF accuracy The three PP mixes were processed and sequenced in triplicates at one site and used to determine the accuracy of VAF determination. For each theoretical VAF, the mean of the determined VAF was calculated and the maximum deviation between theoretical and determined VAF were assessed. Table 16. VAF accuracy JSI - SeqPilot SmartSeq Amplicon Suite Theoretical VAF (%) Mean Determined VAF (%) Max Deviation from Theoretical VAF (%) Mean Determined VAF (%) Max Deviation from Theoretical VAF (%) Batch equivalence The seven PP samples were processed and sequenced twice at one site using two different batches of Devyser BRCA. Concordant and discordant bases were computed for each sequenced base position. All bases in each of the seven samples showed full concordance between batches using both software Impact of formalin fixation The impact of formalin fixation on data quality was evaluated using the samples with various level of degradation; mildly (HD798), moderately (HD799) and severely (HD803) formalin compromised (Horizon Discovery). Sequencing data for the three samples was down-sampled to 1M read pairs (2M reads) and aligned using SeqNext 4.3. The coverage of each base position was determined. Page 36 of 45

37 Table 17. Sequence coverage for samples of various quality HD number % of bases at coverage 1000x HD798 >99.9% HD799 >99.9% HD % Figure 13. Impact of formalin fixation The percentage of bases (y-axis) sequenced at a given coverage (x-axis) for samples with various level of degradation. 1000x coverage is marked with a dotted line Sequencing mode equivalence Sequencing libraries of the three PP Mixes were processed and sequenced using three different sequencing modes: 1x301 bp, 2x151 bp and 2x251 bp. All bases in the target region displayed complete concordance using the three different sequencing modes. All 13 mutations were called and reported at similar VAF. Devyser BRCA displayed complete equivalence using the sequencing modes 1x301 bp, 2x151 bp and 2x251 bp for MiSeq. Page 37 of 45

38 11. SYMBOLS USED ON LABELS Lot or batch number Expiry date Number of tests Store below temperature shown Temperature limit Consult instructions for use Catalogue number Manufacturer In vitro diagnostic device Page 38 of 45