AmoyDx BRCA1 and BRCA2 Gene Mutation Detection Kit (Next Generation Sequencing)

Transcription

1 AmoyDx BRCA1 and BRCA2 Gene Mutation Detection Kit (Next Generation Sequencing) Instruction for Use W024I 24 tests For Illumina MiSeq Amoy Diagnostics Co., Ltd. 39 Dingshan Road, Haicang District, Xiamen , P. R. China Tel: Fax: Website: Wellkang Ltd Suite B, 29 Harley Street, London W1G 9QR United Kingdom Version: B2.0 November 2017

2 Background BRCA1 and BRCA2 gene are autosomal dominant inheritance tumor suppressor genes, involved in DNA damage repair and transcription regulation. Mutations in BRCA1 and BRCA2 gene have been linked to the development of hereditary breast cancer and ovarian cancer. Female mutation carriers, for either gene, have 60% to 80% lifetime risk of developing breast cancer and 20% to 50% of developing ovarian cancer. Male BRCA2 mutation carriers have 6% to 8% lifetime risk of developing pancreatic, hepatic, prostate and breast cancer and can pass the mutation on to their children. Patients with BRCA1 and BRCA2 mutant could benefit from PARP inhibitor (Olaparib). Intended Use The AmoyDx BRCA1 and BRCA2 Gene Mutation Detection Kit, based on Next Generation Sequencing (NGS) technology, is intended for qualitative detection of BRCA1 and BRCA2 gene mutations ( including all coding exons, exon-intron boundaries, UTRs and promoters) in extracted DNA from human blood samples. The kit is intended to be used to assess BRCA1 and BRCA2 gene status in ovarian cancer and breast cancer patients. The kit is for in vitro diagnostic use, and intended to be used by trained professionals in a laboratory environment. Principles of the Procedure The kit is developed based on the technology of multiplex PCR amplification and Next Generation Sequencing (detection system). The library construction procedure includes two steps of PCR. For the first PCR step, all targeted regions of the genes are enriched by specific primers with a tag sequence. The Tag 1 and Tag 2 are added to both ends of each amplicon (see Fig. 1). For the second step, the amplicons are further amplified by matched universal primers with the same sequences of Tag1 and Tag2. In addition, p5 and p7 adaptor sequences for MiSeq sequencing and index for massively parallel sequencing are also included in the primers (see Fig.2). Multiple samples could be tagged with various indices and be identified during simultaneously sequencing. The amplicons from the second step are purified and sequenced on a MiSeq instrument. Mutation status of BRCA1 and BRCA2 genes are analyzed by AmoyDx BRCA1/2 Analysis System. Fig.1 First step: multiplex PCR Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 1). Fig.2 Second step: Universal PCR 1/9

3 Table 1 Kit Contents Content Main Ingredient Quantity BRCA1/2 PCR1 Reaction Mix 8-tube strip (Primer, PCR Buffer, Mg 2+, dntps) 24 strips BRCA1/2 DNA Polymerase Taq DNA Polymerase 50 µl/tube 1 BRCA1/2 PCR2 Reaction Mix PCR Buffer, Mg 2+, dntps, Taq DNA Polymerase 18 µl/tube 24 BR-p7-01 Primer 5 µl/tube 1 BR-p7-02 Primer 5 µl/tube 1 BR-p7-03 Primer 5 µl/tube 1 BR-p7-04 Primer 5 µl/tube 1 BR-p7-05 Primer 5 µl/tube 1 BR-p7-06 Primer 5 µl/tube 1 BR-p7-07 Primer 5 µl/tube 1 BR-p7-08 Primer 5 µl/tube 1 BR-p7-09 Primer 5 µl/tube 1 BR-p7-10 Primer 5 µl/tube 1 BR-p7-11 Primer 5 µl/tube 1 BR-p7-12 Primer 5 µl/tube 1 BR-p5-01 Primer 5 µl/tube 1 BR-p5-02 Primer 5 µl/tube 1 BR-p5-03 Primer 5 µl/tube 1 BR-p5-04 Primer 5 µl/tube 1 BR-p5-05 Primer 5 µl/tube 1 BR-p5-06 Primer 5 µl/tube 1 BR-p5-07 Primer 5 µl/tube 1 BR-p5-08 Primer 5 µl/tube 1 BRCA1/2 Positive Control DNA 50 µl/tube 1 One BRCA1/2 PCR 1 Reaction Mix strip and one BRCA1/2 PCR 2 Reaction Mix tube is employed for each sample (see Table 2). Table 2 Detailed Information of Reaction Mix Composition Tube No. Detection Reagent Volume BRCA1/2 PCR1 Reaction Mix 1 BRCA1/2 PCR1 Reaction Mix 1 10 µl 2 BRCA1/2 PCR1 Reaction Mix 2 10 µl 3 BRCA1/2 PCR1 Reaction Mix 3 10 µl 4 BRCA1/2 PCR1 Reaction Mix 4 10 µl 5 BRCA1/2 PCR1 Reaction Mix 5 10 µl 6 BRCA1/2 PCR1 Reaction Mix 6 10 µl 7 BRCA1/2 PCR1 Reaction Mix 7 10 µl 8 BRCA1/2 PCR1 Reaction Mix 8 10 µl BRCA1/2 PCR 2 BRCA1/2 PCR2 Reaction Mix 18 µl 2/9

4 Reaction Mix Note: The tube No. of PCR strips are marked on the side as follows: Storage and Stability The kit requires shipment on frozen ice packs. All contents of the kit should be stored immediately upon receipt at -20±5 and protected from light. The shelf-life of the kit is six months. The recommend maximum freeze-thaw cycle is five cycles. Additional Reagents and Equipment Required but Not Supplied 1) Thermal cycler (e.g. ABI2720, ABI9700.) 2) Illumina MiSeq Sequencing System. 3) Fluorometer and DNA quantitation kit (fluorometric based), we recommend use Qubit dsdna HS Assay Kit from Thermo Fisher Scientific, Cat. No. Q32851 or ) DNA purification kit, we recommend use Agencourt AMPure XP Kit from Beckman Coulter Genomics, Cat. No. A63880, A63881 or A ) Capillary electrophoresis analyzer kit, we recommend use Agilent 2100 High Sensitivity DNA Kit from Agilent Technologies, Cat. No ) Illumina MiSeq Reagent Nano Kit V2, Cat. No. MS or Illumina MiSeq Reagent Kit V2, Cat. No. MS ) Illumina PhiX Control V3, Cat. No. FC ) Magnet, we recommend use DynaMag-2 Magnet from Thermo Fisher Scientific, Cat. No D. 9) 80% ethanol, freshly prepared. 10) Sterile, nuclease-free H 2 O. 11) Sterile, nuclease-free tubes. 12) Dedicated pipettes and filtered pipette tips for handling DNA. Precautions and Handling Requirements For in vitro diagnostic use. Precautions Please read the instruction carefully and become familiar with all components of the kit prior to use, and strictly follow the instruction during operation. Please check the compatible real-time PCR instruments prior to use. DO NOT use the kit or any kit component after their expiry date. DO NOT use any other reagents from different lots in the tests. DO NOT use any other reagent in the other test kits. Safety Information Handle all specimens and components of the kit as potentially infectious material using safe laboratory procedures. Only trained professionals can use this kit. Please wear suitable lab coat and disposable gloves while handling the reagents. Avoid skin, eyes and mucous membranes contact with the chemicals. In case of contact, flush with water immediately. DO NOT pipet by mouth. 3/9

5 Decontamination and Disposal The kit contains positive control; strictly distinguish the positive control from other reagents to avoid contamination which may cause false positive. PCR amplification is extremely sensitive to cross-contamination. The flow of tubes, racks, pipets and other materials used should be from pre-amplification to post-amplification, and never backwards. Gloves should be worn and changed frequently when handling samples and reagents to prevent contamination. Using separate, dedicated pipettes and filtered pipette tips when handling samples and reagents to prevent exogenous nucleic acid contamination to the reagents. All disposable materials are for one time use. DO NOT reuse. The unused reagents, used kit, and waste must be disposed of properly. Cleaning After the experiment, wipe down the work area, spray down the pipettes and equipment with 75% ethanol or 10% hypochlorous acid solution. Instrument Setup For ABI2720 used in PCR1 amplification, it s essential to remove the adaptor to allow the PCR tubes direct contact with metallic well of the thermal block to ensure the amplification performance. Specimen Preparation Sample DNA should be extracted from whole blood collected with EDTA anticoagulant tube, avoid using heparin anticoagulant tube. The collected blood samples should be handled and stored following proper procedures. Selection of high quality DNA extraction reagents is essential for the kit. We recommend use Qubit 2.0 for concentration measurement of extracted DNA. Make sure the DNA concentration is more than 5 ng/μl, and total DNA should be more than 450 ng. The extracted DNA should be used for DNA library preparation immediately, if not, it should be stored at below -20 for no more than 6 months. Assay Procedure Note: It is recommended to include a BRCA1/2 Positive Control (PC) and a No-template Control (ddh 2 O, NTC) in the process of library preparation, sequencing and data analysis. 1. PCR1 Reagent Preparation: 1) Take out BRCA1/2 PCR1 Reaction Mix strips from refrigerator (-20 ), thaw them at room temperature. When the reagents completely thawed, centrifuge the strips briefly to collect the contents at the bottom of the tube. 2) Take out BRCA1/2 DNA Polymerase when used, and centrifuge it briefly prior to use. 2. PCR1 Amplification: 1) Transfer 90 μl ddh 2 O (NTC) into a new centrifuge tube. 2) DNA dilution: dilute the sample DNA to 5 ng/μl with ddh 2 O. The concentration of sample DNA should be quantified with DNA quantitative kit, and we recommend use of Qubit dsdna HS Assay Kit from Thermo Fisher Scientific. Transfer 90 μl of diluted DNA into a new centrifuge tube. 3) Transfer 9 μl BRCA1/2 Positive Control into 81 μl ddh 2 O within a new centrifuge tube, mix them thoroughly, and then centrifuge briefly. 4) Add 1.8 μl BRCA1/2 DNA Polymerase into the following solutions respectively: 90 µl No-template Control (ddh 2 O, NTC), 90 µl diluted sample DNA, and 90 µl diluted BRCA1/2 Positive Control (PC). Mix each solution thoroughly and centrifuge briefly. 5) Take out the 8-tube BRCA1/2 PCR1 Reaction Mix strips, and gently uncover the caps prior to use. Dispense above mixed solutions to the BRCA1/2 PCR1 Reaction Mix strips, one strip for one sample, and 10 µl for each tube of the strip. 4/9

6 6) Centrifuge the PCR tubes briefly to collect the reagents at the bottom of tubes and avoid generating bubbles. Note: This centrifugation step is essential for proper mixing of the reagents. 7) Place the 8-tube strips into the thermal cycler. A recommended plate layout is shown in Table 3. Table 3 Suggested PCR Plate Layout Well A B J K L 1 Sample1 Sample2 Sample10 PC NTC 2 Sample1 Sample2 Sample10 PC NTC 3 Sample1 Sample2 Sample10 PC NTC 4 Sample1 Sample2 Sample10 PC NTC 5 Sample1 Sample2 Sample10 PC NTC 6 Sample1 Sample2 Sample10 PC NTC 7 Sample1 Sample2 Sample10 PC NTC 8 Sample1 Sample2 Sample10 PC NTC 8) Carry out the PCR using the cycling condition in Table 4. Table 4 PCR1 Cycling Parameters Stage Temperature Time Cycles Initial denaturation 94 C 2 minutes 1 Denaturation 94 C 45 seconds Annealing 60 C 45 seconds 20 Extension 68 C 2 minutes Final extension 72 C 10 minutes 1 Stop reaction 10 C <12 h 1 Note: If not proceed to the next procedure when PCR1 amplification finished, the PCR tubes should be stored at -15 C~-25 C for no more than 2 days. 3. PCR1 Products Mixture Preparation: 1) Take out the amplified BRCA1/2 PCR1 Reaction Mix strips, mix the reagents thoroughly by vortex and then centrifuge them briefly. 2) Gently uncover the caps. Transfer 4 μl of PCR1 product from every tube of the strip into a new centrifuge tube to generate 32 μl of PCR1 products mixture. Mix them thoroughly and then centrifuge briefly. 4. PCR1 Clean-up: Purify the PCR1 products mixture with Agencourt AMPure XP Kit. 1) Incubate the AMPure XP magnetic beads at room temperature for at least 30 minutes. Mix the magnetic beads thoroughly to re-suspend any magnetic particles that may have settled. 2) Add 35 μl of AMPure XP magnetic beads to each above PCR1 product mixture, and mix them thoroughly by pipetting up and down for more than 20 times. The color of the mixture should appear homogenous after mixing. Incubate the mixture for 5 minutes at room temperature to allow the DNA binding to the magnetic beads. 3) Place the tubes on a magnet for 3 minutes to separate the beads from the solution or wait for the solution to clear. Gently remove and discard the supernatant while the tube is on the magnet. Do not touch the magnetic beads. 4) Keep the tubes on the magnet, add 500 μl of fresh 80% ethanol to each tube, slowly rotate the tube about 2 turns and incubate it for at least 30 seconds at room temperature. Remove and discard the supernatant while the tube is on the magnet. Be sure to 5/9

7 remove all the ethanol completely. 5) Repeat the above step (4). ADx-NBR01 6) Uncover the tubes on the magnet, and incubate them for 1~3 minutes at room temperature to make sure no ethanol residual. Do not over dry the beads (avoid the surface of beads appearing cracked) that may result in loss of DNA. 7) Remove the tubes from the magnet, add 30 μl of ddh 2 O to each tube and mix them by pipetting up and down for no less than 20 times. Incubate the mixture for 3 minutes at room temperature. 8) Place the tube on the magnet to separate the beads from the solution, wait for the solution to clear. Transfer the eluate to a new tube, the resulting purified PCR1 products are used for PCR2 amplification. Be sure the beads are not carried over into the new tube. Note: If not proceed to the next procedures when PCR1 clean-up finished, the purified PCR1 products tubes could be remained at 4 C for no more than 4 hours or stored at -15 C~-25 C for no more than 3 days. 5. PCR2 Reagent Preparation: 1) Take out BRCA1/2 PCR2 Reaction Mix tubes and the index primers (BR-p7 and BR-p5) from the refrigerator, and thaw them at room temperature. When the reagents completely thawed, centrifuge it briefly to collect the contents at the bottom of the tube. 2) Place the BRCA1/2 PCR2 Reaction Mix tubes on ice. Note: If there are multiple samples, do use different indices in the same run to distinguish various samples. It s recommended to select highly diversified set of BR-p5 and BR-p7 indices to keep sequencing balance and get high quality sequencing data. A list of indices sequences are shown in Table 5. Index in Kit Table 5 Indices Sequences Index Sequence Corresponding Index in Nextera XT V2 Set A BR-p7-01 TAAGGCGA N701 BR-p7-02 CGTACTAG N702 BR-p7-03 AGGCAGAA N703 BR-p7-04 TCCTGAGC N704 BR-p7-05 GGACTCCT N705 BR-p7-06 TAGGCATG N706 BR-p7-07 CTCTCTAC N707 BR-p7-08 CGAGGCTG N710 BR-p7-09 AAGAGGCA N711 BR-p7-10 GTAGAGGA N712 BR-p7-11 GCTCATGA N714 BR-p7-12 ATCTCAGG N715 BR-p5-01 CTCTCTAT S502 BR-p5-02 TATCCTCT S503 BR-p5-03 GTAAGGAG S505 BR-p5-04 ACTGCATA S506 BR-p5-05 AAGGAGTA S507 BR-p5-06 CTAAGCCT S508 BR-p5-07 CGTCTAAT S510 BR-p5-08 TCTCTCCG S511 6/9

8 6. PCR2 Amplification: 1) Add 5 μl purified PCR1 products, 1 μl of selected BR-p7 and 1 μl of selected BR-p5 into BRCA1/2 PCR2 Reaction Mix tubes. Seal the PCR tubes. Note: Do not label different samples in the same run with the same pair of BR-p7 and BR-p5. 2) Centrifuge the tubes for several seconds to collect the reagents at the bottom of the tube. This centrifugation step is essential for proper mixing of the reagents. 3) Place the PCR tubes into the thermal cycler. Carry out the PCR procedure using the cycling condition in Table 6. Table 6 PCR2 Cycling Parameters Stage Temperature Time Cycles Initial Denaturation 94 C 2 minutes 1 Denaturation 94 C 45 seconds Annealing 60 C 45 seconds 9 Extension 68 C 2 minutes Final Extension 72 C 10 minutes 1 Stop Reaction 10 C < 12 h 1 Note: If not proceed to the next procedures when PCR2 amplification finished, the PCR tubes should be stored at -15 C~-25 C for no more than 1 day. 7. PCR2 Clean-up Preparation: 1) Take out the amplified BRCA1/2 PCR2 Reaction Mix tubes, mix them by vortex and then centrifuge them briefly. 2) Transfer 25 μl of each PCR2 product into a new 1.5 ml centrifuge tube, and centrifuge them briefly. 8. PCR2 Clean-up: Purify the PCR2 products mixture with Agencourt AMPure XP Kit. 1) Incubate the AMPure XP magnetic beads at room temperature for at least 30 minutes. Mix the magnetic beads thoroughly to re-suspend any magnetic particles that may have settled. 2) Add 21.3 μl of AMPure XP magnetic beads to each above PCR2 product, and mix them thoroughly by pipetting up and down for more than 20 times. The color of the mixture should appear homogenous after mixing. Incubate the mixture for 5 minutes at room temperature to allow the DNA to bind to the magnetic beads. 3) Place the tubes on a magnet for 3 minutes to separate the beads from the solution or wait for the solution to clear. Gently remove and discard the supernatant while the tube is on the magnet. Do not touch the magnetic beads. 4) Keep the tubes on the magnet, add 500 μl of fresh 80% ethanol to each tube, slowly rotate the tube about 2 turns and incubate it for at least 30 seconds at room temperature. Remove and discard the supernatant while the tube is on the magnet. Be sure to remove all the ethanol completely. 5) Repeat the above step (4). 6) Uncover the tubes on the magnet, incubate for 1~3 minutes at room temperature to allow the ethanol completely evaporation. Do not over dry the beads (avoid the surface of beads appearing cracked) that may result in loss of DNA. 7) Remove the tubes from the magnet, add 35 μl of ddh2o to each tube and mix them by pipetting up and down for no less than 20 times. Incubate the mixture for 3 minutes at room temperature. 8) Place the tube on the magnet to separate the beads from the solution, wait for the solution to clear. Transfer the eluate to a new tube, and the resulting purified PCR2 products are used as DNA library for further sequencing. Be sure the beads are not carried over into the new tube. Note: If not proceed to the next procedure when PCR2 clean-up finished, the purified PCR2 products tubes could be remained at 4 C for no more than 4 hours or stored at -15 C~-25 C. 7/9

9 9. DNA Library Quality Control: 1) Take out the DNA libraries (purified PCR2 products), mix them thoroughly by vortex and then centrifuge them briefly. 2) Quantify the DNA library with the fluorometric based assay such as Qubit dsdna HS Assay Kit. The concentration of the qualified library should be > 0.5 ng/μl or > 2 nm. 3) Meanwhile, qualify the DNA library with bio-analyzer and related reagents such as the Agilent 2100 HS DNA Kit. The fragment size distribution should be around 500 bp and there are no obvious fragments with size of 150 ~250 bp for qualified libraries. 4) Calculate the concentration of each library in nm with the following formula : 10. DNA Library Sequencing: Library conc [nm] = Select proper sequencing reagents according to Table 7. Library conc [ng/μl] Mean DNA fragment size [bp] Table 7 Suggested Assays Assay Illumina MiSeq Reagent Nano Kit v2, 500 Cycles (Catalog # MS ) Illumina MiSeq Reagent Kit v2, 500 Cycles (Catalog # MS ) Sample Size 15 samples > 15 samples 1) Take out the DNA library (purified PCR2 products), mix them thoroughly by vortex and then centrifuge them briefly. 2) Dilute the DNA libraries to 2 nm with HT1. Pipet 5 μl of each diluted sample library to generate a 2 nm library mixture. 3) Denature the 2 nm library mixture and PhiX Control v3 (Cat. No. FC ) according to manufacturer s instruction of Illimina Miseq Sequencing System, and then dilute the denatured library mixture and PhiX Control v3 to the required concertation (it is recommended to start with 8~10 pm). 4) Mix 570 μl of denatured library mixture and 30 μl of denatured PhiX control library (with 5% PhiX control spike-in) and load the mixture onto the MiSeq reagent cartridge. 5) Carry out a run as described in the MiSeq System User Guide. 11. Data Analysis: When sequencing finished, use AmoyDx BRCA1/2 Analysis System to analyze the sequencing data and detect the mutation status of BRCA1 and BRCA2 genes. Result Interpretation 1) If any fragment around 500 bp detected in NTC library, the data must be discarded as there is contamination. 2) The concentration of the BRCA1/2 Positive Control library should be > 0.5 ng/μl and mutations should be correctly detected. 3) Analyze the DNA library: i. If the concentration of DNA library is < 0.5 ng/μl or < 2 nm, the quality of the DNA sample may be not good or not quantified accurately, or there is a PCR amplification failure. In this case, the experiments should be repeated. ii. If single base depth >100, the ratio of the allele inconsistent with the human genome reference sequence (Hg19) > 15% and the recorded frequency <1% in 1000Genomes ( determine the mutation as positive. Performance Characteristics 1) Sensitivity: The kit can detect as low as 25% mutant DNA in a background of 75% normal DNA with 50ng DNA amount. 2) Coverage: The target regions can be uniformly covered, with a coverage of 100%. 3) Accuracy: Accuracy of the kit was validated by testing 8 positive reference controls and 8 negative reference controls, with a detection concordance rate of 100% 4) Precision: Precision of the kit was validated by performing a certain mutant positive reference control for 10 repeats with all the 8/9

10 results as expected. Limitations 1) The kit is to be used only by personnel specially trained in the techniques of PCR and NGS. 2) The results can be used to assist clinical diagnosis, combining with other clinical and laboratory findings. 3) The kit has been only validated for use with human blood sample. 4) The kit can only be used for qualitative detection of BRCA1/2 mutation, not intended for measuring therapeutic efficacy. 5) Reliable results are dependent on proper sample processing, transport, and storage. 6) The kit is based on multi-amplification, DNA degradation may affect the kit performance, therefore the kit is not applicable for DNA sample of which the main segments are <500 bp. Symbols 1) Authorized Representative in the European Community 2) In Vitro Diagnostic Medical Device 3) Manufacturer 4) Catalogue Number 5) Batch Code 6) Use By 7) Contains Sufficient for <n> Tests 8) Temperature Limitation 9) Consult Instructions For Use 10) Keep Dry 11) This Way Up 12) Fragile, Handle With Care 9/9