Oncomine BRCA Research Assay

Transcription

1 Oncomine BRCA Research Assay USER GUIDE For manual library preparation Catalog Number A32840 Publication Number MAN Revision B.0 For Research Use Only. Not for use in diagnostic procedures.

2 Manufacturer: Multiple Life Technologies Corporation manufacturing sites are responsible for manufacturing the products associated with the workflow covered in this guide. Corporate entity: Life Technologies Corporation Carlsbad, CA USA Toll Free in USA The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Revision history: Pub. No.MAN Revision Date Description B.0 8 June 2017 Support added for exon deletion functionality in updated Oncomine BRCA Research Assay workflows in Ion Reporter Software 5.4. A.0 12 October 2016 New user guide for the Oncomine BRCA Research Assay with detailed instructions for manual library preparation. Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses. Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Agencourt and AMPure are trademarks of Beckman Coulter, Inc. Eppendorf and Eppendorf LoBind are trademarks of Eppendorf AG. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc Thermo Fisher Scientific Inc. All rights reserved.

3 Contents CHAPTER 1 Product information... 5 Product description... 5 Kit contents and storage... 6 Ion Library Equalizer Kit... 7 Ion Xpress Barcode Adapters Kits... 7 Required materials not supplied... 8 Recommended materials and equipment (optional)... 9 Oncomine BRCA Research Assay, Chef-Ready Kit Oncomine BRCA Research Assay manual workflow CHAPTER 2 Prepare Oncomine BRCA Research Assay libraries Procedural guidelines Amplify the targets Combine the target amplification reactions Partially digest primers Ligate adapters to the amplicons and purify Combine and dilute the adapters Perform the ligation reaction Purify the library Option 1: Equalize the library Before you begin Amplify the library Wash the Equalizer Beads (if not previously performed) Add Equalizer Capture to the amplified library Add Equalizer Beads and wash Elute the equalized libraries Option 2: Quantify the library by qpcr Elute the library Quantify libraries by qpcr and calculate the dilution factor Oncomine BRCA Research Assay User Guide 3

4 Contents Option 3: Quantify the amplified libraries by Qubit Fluorometer or Agilent 2100 Bioanalyzer instrument Amplify the library Purify the amplified library Qubit Fluorometer: Quantify the library and calculate the dilution factor Agilent 2100 Bioanalyzer instrument: Quantify the library and calculate the dilution factor Combine Oncomine BRCA Research Assay barcoded libraries for sequencing on the same Ion chip Guidelines for templating and sequencing CHAPTER 3 Create a Planned Run and analyze results with an Ion Reporter workflow Configure the Torrent Browser to use your Ion Reporter account About Ion Reporter Oncomine BRCA Research workflows Upload Target Regions and Hotspots BED files into Torrent Suite Software Create a Planned Run with an Oncomine BRCA Research template Using the Ion AmpliSeq Sample ID Panel View Ion Reporter analysis results and generate a report Download results or generate a report Edit an Ion Reporter Oncomine BRCA Research workflow with a new Annotation Set Create a new Annotation Set Copy and edit a workflow to add a new Annotation Set Oncomine Knowledgebase Reporter Software APPENDIX A Tips and troubleshooting Tips Troubleshooting Library yield and quantification Other APPENDIX B Safety Chemical safety Biological hazard safety Documentation and support Customer and technical support Limited product warranty Oncomine BRCA Research Assay User Guide

5 1 Product information IMPORTANT! Before using this product, read and understand the information in the Safety appendix in this document. Product description The Oncomine BRCA Research Assay (Cat. No. A32840) provides reagents for the manual preparation from genomic DNA of up to 24 Oncomine BRCA Research Assay sample libraries targeting the BRCA1 and BRCA2 genes. Use of Ion Xpress Barcode Adaptors in library preparation enables you to combine barcoded libraries in templating reactions and sequencing runs. Up to 32 germline libraries, or up to 8 somatic libraries, can be combined and sequenced on a single Ion 318 chip, and up to 32 germline or somatic libraries can be combined and sequenced on an Ion 530 chip. The protocol requires 10 ng of DNA isolated from unfixed or formalin-fixed paraffin-embedded (FFPE) tissue per target amplification reaction (20 ng total). This guide also provides optional protocols for normalizing library concentration using the Ion Library Equalizer Kit, quantifying library concentration using qpcr, and quantifying library concentration with the Qubit Fluorometer, or the Agilent 2100 Bioanalyzer instrument. Note: The Oncomine BRCA Research Assay Chef-Ready Library Preparation Kit (Cat. No. A32841) is also available for automated library preparation (see Oncomine BRCA Research Assay, Chef-Ready Kit on page 10). The kit provides Oncomine BRCA Research Assay Pools 1 and 2 at 2X concentration pre-measured in barcoded Primer Pool tubes ready to load into an Ion AmpliSeq Chef Reagents DL8 cartridge. Oncomine BRCA Research Assay User Guide 5

6 1 Chapter 1 Product information Kit contents and storage Kit contents and storage The Oncomine BRCA Research Assay (Cat. No. A32840) is a bundle of the Oncomine BRCA Research Assay Manual Library Preparation panel and the Ion AmpliSeq Library Kit Plus, sufficient for manually preparing 24 libraries. Component Amount Storage Oncomine BRCA Research Assay Manual Library Preparation, 5X concentration (Part No. A32842) Oncomine BRCA Pool 1 (blue cap) 48 µl 30ºC to 10ºC Oncomine BRCA Pool 2 (red cap) 48 µl Ion AmpliSeq Library Kit Plus (Part. No ) 5X Ion AmpliSeq HiFi Mix (red cap) 120 µl 30ºC to 10ºC FuPa Reagent (brown cap) 48 µl Switch Solution (yellow cap) 96 µl DNA Ligase (blue cap) 48 µl 25X Library Amp Primers [1] (pink cap) 48 µl 1X Library Amp Mix (black cap) 1.2 ml Low TE (clear cap) 1 each 15 C to 30 C [2] [1] Can be used with the Ion Library Equalizer Kit, and also for library amplification if quantifying amplified library with the Qubit 3.0 Fluorometer or Agilent 2100 Bioanalyzer. [2] Can be stored at 30ºC to 10ºC. 6 Oncomine BRCA Research Assay User Guide

7 Chapter 1 Product information Ion Library Equalizer Kit 1 Ion Library Equalizer Kit The Ion Library Equalizer Kit, ordered separately, provides a streamlined method for normalizing library concentration without quantification. The Ion Library Equalizer Kit (Cat. No ) contains reagents for 96 reactions. Component Amount Storage Equalizer Primers (pink cap) 200 µl 2ºC to 8ºC Equalizer Capture (purple cap) Equalizer Elution Buffer (clear cap) 1 ml 10 ml Equalizer Beads (orange cap) 300 µl Equalizer Wash Buffer (clear cap) 35 ml Ion Xpress Barcode Adapters Kits Each kit, ordered separately, provides 16 different barcode adapters sufficient for 640 total reactions. Component Quantity Volume Storage Ion Xpress P1 Adapter (violet cap) 1 tube 320 µl Ion Xpress Barcode X (white cap) 16 tubes (1 per barcode) 20 µl each 30ºC to 10ºC The following Ion Xpress Barcode Adapters Kits are available: Ion Xpress Barcode Adapters 1 16 (Cat. No ) Ion Xpress Barcode Adapters (Cat. No ) Ion Xpress Barcode Adapters (Cat. No ) Ion Xpress Barcode Adapters (Cat. No ) Ion Xpress Barcode Adapters (Cat. No ) Ion Xpress Barcode Adapters (Cat. No ) Complete set of adapters: Ion Xpress Barcode Adapters 1 96 (Cat. No ) Oncomine BRCA Research Assay User Guide 7

8 1 Chapter 1 Product information Required materials not supplied Required materials not supplied Unless otherwise indicated, all materials are available through thermofisher.com. MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier. Item Veriti 96 well Thermal Cycler, or equivalent MicroAmp Optical 96-well Reaction Plates Source See web product pages N (with barcode) MicroAmp Adhesive Film MicroAmp Compression Pad Agencourt AMPure XP Kit DynaMag -96 Side Magnet, or other plate magnet Nuclease-free Water Absolute ethanol Pipettors, μl, and low-retention filtered pipette tips Eppendorf DNA LoBind Microcentrifuge Tubes 96-well plate centrifuge Fisher Scientific NC , NC D AM9932 MLS MLS Fisher Scientific MLS 8 Oncomine BRCA Research Assay User Guide

9 Chapter 1 Product information Recommended materials and equipment (optional) 1 Recommended materials and equipment (optional) Unless otherwise indicated, all materials are available through thermofisher.com. MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier. Item Source Recommended additional equipment for qpcr Real-time PCR instrument (for example, Applied Biosystems 7900HT, 7500, StepOne, StepOnePlus, ViiA 7, QuantStudio 3/5 Systems, or QuantStudio 12K Flex Real-Time PCR System) See web product pages Recommended for gdna isolation Ion AmpliSeq Direct FFPE DNA Kit RecoverAll Total Nucleic Acid Isolation Kit A31133, A31136 AM1975 MagMAX FFPE Total Nucleic Acid Isolation Kit PureLink Genomic DNA Mini Kit K Recommended for gdna quantification TaqMan RNase P Detection Reagents Kit Qubit 3.0 Fluorometer or Qubit 2.0 Fluorometer [1] Qubit dsdna HS Assay Kit Q33216 Q32851, Q32854 Recommended for library quantification (If you are NOT using the Ion Library Equalizer Kit for library normalization, select one of the following:) Ion Library TaqMan Quantitation Kit Qubit 3.0 Fluorometer or Qubit 2.0 Fluorometer [1] Qubit dsdna HS Assay Kit Agilent 2100 Bioanalyzer Instrument Agilent High Sensitivity DNA Kit Q33216 Q32851, Q32854 Agilent G2939AA Agilent Additional material for the Ion AmpliSeq Direct FFPE DNA Kit (Optional) Uracil DNA Glycosylase (UDG) , or EN0361 [1] Supported but no longer available for purchase. Oncomine BRCA Research Assay User Guide 9

10 1 Chapter 1 Product information Oncomine BRCA Research Assay, Chef-Ready Kit Oncomine BRCA Research Assay, Chef-Ready Kit The Oncomine BRCA Research Assay, Chef-Ready Kit (Cat. No. A32841, ordered separately) provides Oncomine BRCA Research Assay Pools 1 and 2 at 2X concentration pre-measured in barcoded Primer Pool tubes ready to load into an Ion AmpliSeq Chef Reagents DL8 cartridge. In addition, the kit provides all the reagents and supplies in an Ion AmpliSeq Kit for Chef DL8 sufficient for preparing 32 libraries. See the Ion AmpliSeq Library Preparation on the Ion Chef System User Guide (Pub. No. MAN ) for detailed information on preparing Oncomine BRCA Research Assay libraries on the Ion Chef System. Component Part No. Quantity per kit Storage Oncomine BRCA Research Assay Chef-Ready Library Preparation Oncomine BRCA Tube 1 A µl 30 C to 10 C Oncomine BRCA Tube µl Ion AmpliSeq Kit for Chef DL8 Ion AmpliSeq Chef Reagents DL8 A cartridges 30 C to 10 C Ion AmpliSeq Chef Solutions DL8 A cartridges 2 C to 8 C [1] Ion AmpliSeq Chef Supplies DL8 (per insert) Ion AmpliSeq Tip Cartridge L8 PCR Frame Seal Enrichment Cartridge A box with 4 inserts 15 C to 30 C IonCode in 96 Well PCR Plates (dried) A set of 4 plates 15 C to 30 C Set includes 4 PCR plates: IonCode in 96 Well PCR Plate (red) IonCode in 96 Well PCR Plate (yellow) IonCode in 96 Well PCR Plate (green) IonCode in 96 Well PCR Plate (blue) [1] Ion AmpliSeq Chef Solutions DL8 cartridges are shipped at ambient temperature, but need to be stored at 2 C to 8 C upon arrival. 10 Oncomine BRCA Research Assay User Guide

11 Chapter 1 Product information Oncomine BRCA Research Assay manual workflow 1 Oncomine BRCA Research Assay manual workflow Isolate and quantify gdna Prepare master mix TM Oncomine BRCA Research Assay Pool 1 Pool 2 Add DNA to individual wells Amplify DNA (18 cycles for non-ffpe gdna, 21 cycles for FFPE gdna) Sample A B C D E F G H Primer pool 1 2 Combine amplification reactions in a new column Digest primers Ligate barcode adapters Purify amplicons Equalize, or quantify and dilute libraries Combine libraries (optional) Processing of 8 gdna samples and libraries at two wells per sample is shown, with Sample 1 in wells A3 and A4, Sample 2 in wells B3 and B4, etc. After target amplification in 10 µl reactions, Pool 1 and Pool 2 amplification reactions are combined into new wells in the plate. After partial digestion of primers, ligation of barcode adapters, and amplicon purification, barcoded libraries are equalized to ~100 pm concentration, or quantified and diluted to 100 pm concentration, and can be combined before template preparation. Oncomine BRCA Research Assay User Guide 11

12 2 Prepare Oncomine BRCA Research Assay libraries Procedural guidelines Amplify the targets Combine the target amplification reactions Partially digest primers Ligate adapters to the amplicons and purify Option 1: Equalize the library Option 2: Quantify the library by qpcr Option 3: Quantify the amplified libraries by Qubit Fluorometer or Agilent 2100 Bioanalyzer instrument Combine Oncomine BRCA Research Assay barcoded libraries for sequencing on the same Ion chip Guidelines for templating and sequencing Note: This chapter gives procedures for manually preparing Oncomine BRCA Research Assay libraries. For procedures for using the Oncomine BRCA Research Assay, Chef-Ready Kit to prepare libraries on the Ion Chef System, see the Ion AmpliSeq Library Preparation on the Ion Chef System User Guide (Pub. No. MAN ). Procedural guidelines Reagent preparation: Thaw components that contain enzymes such as 5X Ion AmpliSeq HiFi Mix, FuPa Reagent, and DNA Ligase on ice, and keep on ice during procedure. Thaw kit components other than enzymes, including genomic DNA and primer panels, at room temperature until no ice is present in the tubes. Vortex all reagents for 5 seconds (EXCEPT for enzymes, which should be flick-mixed) and pulse-centrifuge before use. A pulse-centrifugation is a 5-second centrifugation at maximum speed. If there is visible precipitate in the Switch Solution or the tube cap after thawing, vortex or pipet up and down at room temperature to dissolve. If using the Ion Library Equalizer Kit, minimize freeze-thawing of Equalizer Primers by aliquoting as needed for your experiments. Primers can be stored at 4 C for one year. Pipet viscous solutions slowly and ensure complete mixing by vigorous vortexing or pipetting up and down 5 times. 12 Oncomine BRCA Research Assay User Guide

13 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Procedural guidelines 2 Laboratory and PCR setup: Use good laboratory practices to minimize cross-contamination of products. If possible, perform PCR setup in an area or room that is separate from template preparation. Always change pipette tips between samples. Before starting and after use, wash the working surface with 10% bleach followed by two water rinses. Use inner wells of PCR plate if possible, and skip wells or columns to prevent cross-contamination between samples. Use a calibrated thermal cycler that is specified in Required materials not supplied on page 8. Ensure that the correct cycling protocol is being used before starting the thermal cycler. Store one 96-well cold block at 30 C to 10 C and one 96-well cold block at 2 C to 8 C before use. Use the heated lid (105 C) for all thermal cycling conditions. Pipetting recommendations: Use aerosol-barrier pipette tips. Change pipette tips between samples. Pipet viscous solutions (enzymes, beads, Switch Solution) slowly and ensure complete mixing in the MicroAmp 96-well plate. Avoid introducing air bubbles when pipetting by keeping the pipette tip at the bottom of the solution in the wells. Set pipette to the recommended volume for up and down mixing and insert tip into solution with pipette plunger depressed to avoid introducing air bubbles. Visually check tips to ensure that volumes are equivalent if using a multi-channel pipette. Touch tip to side of well and slowly dispense reagent on the side of the well to form a droplet. This practice enables you both to pipet small volumes accurately and to see that you added reagent to the well. Ensure that reagent is adequately dispensed from the pipette tip. Guidelines for DNA isolation and quantification: For each target amplification reaction, use 3,000 copies (10 ng of genomic DNA in 6 µl) from peripheral blood, cell lines, or FFPE tissue. See Required materials not supplied on page 8 for kits that are recommended for isolating DNA. We recommend the TaqMan RNase P Detection Reagents Kit (Cat. No ) for quantifying amplifiable human genomic DNA. The Qubit dsdna HS Assay Kit (Cat. No. Q32851 or Q32854) can also be used with the Qubit 2.0 or Qubit 3.0 Fluorometer. We do not recommend methods such as densitometry (for example, NanoDrop Spectrophotometers), because these methods do not discriminate between DNA and RNA and therefore are sensitive to small fragments of hydrolyzed RNA. Use of densitometry can lead to overestimation of the concentration of sample DNA, under-seeding of the target amplification reaction, low-quality libraries, and low library yields. Oncomine BRCA Research Assay User Guide 13

14 2 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Amplify the targets Amplify the targets IMPORTANT! Primer pools and 5X Ion AmpliSeq HiFi Mix are viscous. Pipet slowly and mix thoroughly. We recommend PCR setup on ice or a cold block. 1. For each sample, add components to 2 adjacent wells of a 96-well plate using a low volume pipettor, as described in the following table. See the plate layout example following the table. Note: If preparing multiple libraries, master mixes without DNA are recommended. Add master mixes to the plate first. IMPORTANT! Ion AmpliSeq Sample ID Panel primer pairs are included in the Oncomine BRCA Research Assay. Do not add additional Ion AmpliSeq Sample ID Panel primers to target amplification reactions. Component Volume per well (Pool 1) Volume per well (Pool 2) 5X Ion AmpliSeq HiFi Mix (red cap) Oncomine BRCA Pool 1 (blue cap) Oncomine BRCA Pool 2 (red cap) 2 µl 2 µl 2 µl 2 µl DNA, 10 ng 6 µl 6 µl Nuclease-free Water to 10 µl to 10 µl Note: Add 10 ng of control DNA to 2 wells if running a control. Oncomine TM BRCA Research Assay Pool 1 Pool 2 2 µl/well A B C D E F G H Primer Pool Note: Avoid using columns on the periphery of the plate. 14 Oncomine BRCA Research Assay User Guide

15 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Combine the target amplification reactions 2 2. Mix thoroughly by pipetting up and down, then seal the plate with MicroAmp Adhesive Film. Alternatively, the plate can be vortexed after sealing, followed by centrifugation at 100 g for 30 seconds to collect volume at the bottom of the wells. 3. Place a MicroAmp Compression Pad on the plate, load into the thermal cycler, then run the following program: Hold Stage Step Temperature Time Cycle (18 cycles for non-ffpe DNA, 21 cycles for FFPE DNA) Activate the enzyme 99 C 2 minutes Denature 99 C 15 seconds Anneal and Extend 60 C 4 minutes Hold 10 C Hold (16 hours maximum) Note: See the Ion AmpliSeq Library Kit 2.0 User Guide (Pub. No. MAN ) for guidance on cycle number if your gdna is <10 ng, or of questionable quality. STOPPING POINT Amplification products can be stored at 10 C overnight (12 16 hours) in the thermal cycler. For longer-term storage, store at 20 C. Combine the target amplification reactions 1. Centrifuge the plate at 100 g for 30 seconds in a plate centrifuge to collect contents at the bottom of the wells. 2. Carefully remove the plate seal from the plate. 3. Combine the two 10-µL target amplification reactions for each sample by transferring them to an empty well of a new column in the plate, or in a new plate. A B C D E F G H Primer Pool 1 2 Oncomine BRCA Research Assay User Guide 15

16 2 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Partially digest primers Partially digest primers Note: FuPa Reagent is viscous. Flick to mix, then pulse-centrifuge. To avoid carrying over excess enzyme, do not submerge the whole tip in the FuPa Reagent solution. Aspirate solution from the surface. 1. Add 2 µl of FuPa Reagent (brown cap) to each amplified sample. The total volume of each reaction is now ~22 µl. 2. Seal the plate with MicroAmp Adhesive Film, vortex thoroughly, then centrifuge at 100 g for 30 seconds to collect droplets. 3. Place a MicroAmp Compression Pad on the plate, load the plate in the thermal cycler, then run the following program: Temperature Time 50 C 10 minutes 55 C 10 minutes 60 C 20 minutes 10 C Hold (for up to 1 hour) 4. Centrifuge the plate at 100 g for 30 seconds before proceeding to the next step. Ligate adapters to the amplicons and purify Combine and dilute the adapters For each Ion Xpress Barcode X chosen, prepare a mix of Ion P1 Adapter and Ion Xpress Barcode X at a final dilution of 1:4 for each adapter. Store diluted adapters at 20 C. Combine the volumes indicated in the following table and use 2 µl of this barcode adapter mix in step 1 below. Scale volumes as necessary. Component Volume Ion P1 Adapter 2 µl Ion Xpress Barcode X [1] 2 µl Nuclease-free Water 4 µl Total 8 µl [1] X = barcode chosen 16 Oncomine BRCA Research Assay User Guide

17 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Ligate adapters to the amplicons and purify 2 Perform the ligation reaction IMPORTANT! When handling barcode adapters, avoid cross-contamination by changing gloves frequently and opening one tube at a time. IMPORTANT! If there is visible precipitate in the Switch Solution, vortex or pipet up and down at room temperature to dissolve. Ensure that there is no precipitate in the tube cap. 1. Centrifuge the plate at 100 g for 30 seconds in a plate centrifuge to collect contents at the bottom of the wells. 2. Carefully remove the plate seal, then add the following components in the order indicated to each well containing digested sample. After adding each component, mix by setting a pipette to 15 µl, then pipetting up and down slowly 5 times. Note: Do not make a master mix of these components. Order of addition Component Volume 1 Switch Solution (yellow cap) 4 µl 2 Ion Xpress Barcode Adaptor/P1 Adaptor mix [1] 2 µl 3 DNA Ligase (blue cap) 2 µl Total volume (including ~22 µl of digested amplicons) ~30 µl [1] Prepared in "Combine and dilute the adaptors". 3. Seal the plate with a new MicroAmp Adhesive Film, vortex thoroughly, then centrifuge at 100 g for 30 seconds to collect droplets. 4. Place a MicroAmp Compression Pad on the plate, load the plate in the thermal cycler, then run the following program: Temperature Time 22 C 30 minutes 68 C 5 minutes 72 C 5 minutes 10 C Hold (for up to 24 hours) STOPPING POINT Samples can be stored at 20 C. 5. Centrifuge the plate at 100 g for 30 seconds before proceeding to the next step. Oncomine BRCA Research Assay User Guide 17

18 2 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Ligate adapters to the amplicons and purify Purify the library IMPORTANT! Bring the AMPure XP Reagent to room temperature and vortex thoroughly to disperse the beads before use. Pipet solution slowly. Do NOT substitute a Dynabeads -based purification reagent for the AMPure XP Reagent. 1. Prepare fresh 70% ethanol: Combine 230 µl of 100% ethanol with 100 µl of Nuclease-free Water per sample. 2. Carefully remove the plate seal, then add 45 µl (1.5X sample volume) of AMPure XP Reagent to each library. Pipet up and down 5 times to mix the bead suspension thoroughly with the DNA. 3. Incubate the mixture for 5 minutes at room temperature. 4. Place the plate in a DynaMag -96 Side Magnet, then incubate for 2 minutes. Carefully remove and discard the supernatant without disturbing the pellet. 5. Add 150 µl of freshly prepared 70% ethanol, move the plate side-to-side in the two positions of the magnet to wash the beads, then remove and discard the supernatant without disturbing the pellet. 6. Repeat step 5 for a second wash. 7. Ensure that all ethanol droplets are removed from the wells. Keeping the plate in the magnet, air-dry the beads at room temperature for 5 minutes. Do not overdry. IMPORTANT! Residual ethanol droplets inhibit library amplification. Ensure that all ethanol droplets are removed from the wells. If residual ethanol is present, place the plate back on the magnet for 2 minutes and use a pipette to remove the ethanol. Proceed immediately to one of the following: Option 1: Equalize the library. Option 2: Quantify the library by qpcr. Option 3: Quantify the amplified library with the Qubit 2.0 or 3.0 Fluorometer, or with the Agilent 2100 Bioanalyzer instrument. 18 Oncomine BRCA Research Assay User Guide

19 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 1: Equalize the library 2 Option 1: Equalize the library IMPORTANT! We recommend the Ion Library Equalizer Kit when unamplified library concentration is consistently pm after elution in 50 µl. If sample quality or quantity is variable or unknown, we recommend using the qpcr method (see Option 2: Quantify the library by qpcr on page 22). The Ion Library Equalizer Kit (Cat. No ) provides a method for normalizing library concentration at ~100 pm without the need for special instrumentation for quantification. First amplify the Oncomine BRCA Research Assay library, then capture the library on Equalizer Beads. After elution of the equalized library, proceed directly to combining libraries and/or template preparation. Before you begin Amplify the library Warm all the reagents in the Ion Library Equalizer Kit to room temperature. Vortex and centrifuge all reagents before use. 1. Prepare library amplification mix according to the following table. Prepare a master mix for multiple reactions. Order of addition Component Volume per reaction Volume for 8 reactions [1] 1 1X Library Amp Mix (black cap) 50 µl 450 µl 2 Equalizer Primers (pink cap) 2 µl 18 µl Total 52 µl 468 µl [1] One additional reaction added as overage to compensate for pipetting error. 2. Remove the plate from the magnet (at step 7 of "Purify the library"), then add 52 µl of library amplification mix to each well containing air-dried beads. 3. Seal the plate with a new MicroAmp Adhesive Film, vortex thoroughly, then centrifuge at 100 g for 30 seconds to collect droplets. 4. Place the plate back on the magnet for 2 minutes, then carefully transfer ~50 µl of supernatant from each well to a new plate without disturbing the pellet. 5. Seal the plate with the adhesive film, place a MicroAmp Compression Pad on the plate, load the plate in the thermal cycler, then run the following program: Stage Temperature Time Hold 98 C 2 minutes Cycling (9 cycles) 98 C 15 seconds 64 C 1 minute Hold 10 C Hold (for up to 1 hour) Note: During cycling, wash the Equalizer Beads for later use (see Wash the Equalizer Beads (if not previously performed) on page 20). Oncomine BRCA Research Assay User Guide 19

20 2 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 1: Equalize the library Wash the Equalizer Beads (if not previously performed) 1. Bring the Equalizer Beads to room temperature, then mix thoroughly. Note: Beads for multiple reactions can be prepared in bulk, and stored in Equalizer Wash Buffer at 4 C for up to 12 months until use. After 12 months, rewash beads with an equal volume of Equalizer Wash Buffer. 2. For each reaction, pipet 3 µl of beads into a clean 1.5-mL tube, then add 6 µl/reaction of Equalizer Wash Buffer. For example, if you have 4 reactions, add 12 µl of beads and 24 µl of wash buffer. 3. Place the tube in a magnetic rack for 3 minutes or until the solution is clear. 4. Carefully remove the supernatant without disturbing the pellet, then discard. 5. Remove the tube from the magnet, add 6 µl per reaction of Equalizer Wash Buffer, then pipet up and down to resuspend. Add Equalizer Capture to the amplified library 1. Centrifuge the plate at 100 g for 30 seconds in a plate centrifuge to collect contents at the bottom of the wells. 2. Carefully remove the seal from the plate, then add 10 µl of Equalizer Capture to each library amplification reaction. 3. Seal the plate with a clear adhesive film, vortex thoroughly, then centrifuge to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times before sealing the plate. 4. Incubate at room temperature for 5 minutes. Add Equalizer Beads and wash 1. Mix the washed Equalizer Beads by gentle vortexing or pipetting up and down. 2. Add 6 µl of washed beads to each plate well containing the captured library. 3. Set the pipette volume to 40 µl, then pipet the mixture up and down at least 5 times to mix thoroughly. 4. Incubate at room temperature for 5 minutes. Briefly centrifuge the plate to collect all the liquid to the bottom of the plate wells. 5. Place the plate in the magnet, then incubate for 2 minutes or until the solution is clear. 6. Carefully remove the supernatant without disturbing the pellet. Note: Check for droplets on the sides of the plate wells. If droplets are observed, seal the plate, then gently tap the plate on a hard, flat surface, or briefly centrifuge to collect all the liquid to the bottom of the plate wells. Note: Save the supernatant for repeat analysis if needed. 7. Add 150 µl of Equalizer Wash Buffer to each reaction. 20 Oncomine BRCA Research Assay User Guide

21 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 1: Equalize the library 2 8. Move the plate side-to-side in the two positions of the magnet to wash the beads. Note: If your magnet does not have two positions for shifting the beads, remove the plate from the magnet and gently pipet up and down 5 times (with the pipettor set to at least half the total volume), then return the plate to the magnet and incubate for 2 minutes or until the solution clears. 9. With the plate still in the magnet, carefully remove, then discard the supernatant without disturbing the pellet. 10. Repeat the bead wash as described in steps 7 9. Note: Ensure that as much wash buffer as possible is removed without disturbing the pellet. Elute the equalized libraries 1. Remove the plate from the magnet, then add 100 µl of Equalizer Elution Buffer to each pellet. 2. Seal the plate with MicroAmp Clear Adhesive Film, vortex thoroughly, then centrifuge to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times before sealing the plate. Note: Centrifuge with sufficient force to collect droplets, but not pellet beads. If beads are pelleted, vortex again and centrifuge more gently. 3. Elute the libraries by incubating in a thermal cycler at 32 C for 5 minutes. 4. Place the plate in the magnet, then incubate at room temperature for 5 minutes or until the solution is clear. The supernatant contains the equalized library at ~100 pm, which can be stored with beads for up to 1 month at 4 8 C. Proceed to Combine Oncomine BRCA Research Assay barcoded libraries for sequencing on the same Ion chip on page 28 and template preparation, or store libraries. Store libraries at 4 8 C for up to 1 month, or at 30 C to 10 C for longerterm storage. Oncomine BRCA Research Assay User Guide 21

22 2 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 2: Quantify the library by qpcr Option 2: Quantify the library by qpcr Elute the library, then determine the concentration by qpcr with the Ion Library TaqMan Quantitation Kit (Cat. No ). Libraries that have not undergone a second round of amplification typically have yields of pm. However, yield is not indicative of library quality. After quantification, determine the dilution factor that results in a concentration of ~100 pm, which is suitable for template preparation using an Ion template kit. Elute the library 1. Remove the plate with purified libraries from the plate magnet, then add 50 μl of Low TE to the pellet to disperse the beads. 2. Seal the plate with MicroAmp Adhesive Film, vortex thoroughly, then briefly centrifuge to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times before sealing the plate. 3. Incubate at room temperature for at least 2 minutes. 4. Place the plate on the magnet for at least 2 minutes. 5. Prepare a 100-fold dilution for quantification. Remove 2 µl of supernatant, containing the library, then combine with 198 µl of Nuclease-free Water. Quantify libraries by qpcr and calculate the dilution factor Determine the concentration of each Oncomine BRCA Research Assay library by qpcr with the Ion Library TaqMan Quantitation Kit using the following steps. Analyze each sample, standard, and negative control in duplicate 20-µL reactions. 1. Prepare three 10-fold serial dilutions of the E. coli DH10B Ion Control Library (~68 pm; from the Ion Library TaqMan Quantitation Kit) at 6.8 pm, 0.68 pm, and pm. Mark these tubes as standards, then use these concentrations in the qpcr instrument software. 2. Prepare reaction mixtures. For each sample library, control, and standard, combine 20 µl of 2X TaqMan MasterMix and 2 µl of 20X Ion TaqMan Assay, then mix thoroughly. Dispense 11-µL aliquots into the wells of a PCR plate. 3. Add 9 µl of the diluted (1:100) Oncomine BRCA Research Assay library or 9 µl of each control dilution to each well (two wells per sample as noted before), for a total reaction volume of 20 µl. 4. Program your real-time instrument as follows: a. Enter the concentrations of the control library standards. b. Select ROX Reference Dye as the passive reference dye. c. Select a reaction volume of 20 µl. d. Select FAM dye/mgb as the TaqMan probe reporter/quencher. 22 Oncomine BRCA Research Assay User Guide

23 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 2: Quantify the library by qpcr 2 e. The Ion Library TaqMan qpcr Mix can be used on various instruments, as listed in the following table. The fast cycling program was developed using the StepOnePlus System in Fast mode. Real-time PCR System Reaction plate Run mode Stage Temperature Time 7500 Fast 96-well Fast 7900 HT 7900 HT Fast 96-well Fast Hold (UDG incubation) Hold (polymerase activation) 50 C 2 minutes 95 C 20 seconds Fast ViiA well Standard 95 C 1 second QuantStudio 3 or 5 Cycle (40 cycles) StepOne StepOnePlus 48-/96-well Fast 60 C 20 seconds HT 7900 HT Fast ViiA 7 QuantStudio 3 or 5 96-well Standard Standard Hold (UDG incubation) Hold (polymerase activation) Cycle (40 cycles) 50 C 2 minutes 95 C 2 minutes 95 C 15 seconds 60 C 1 minute 5. Following qpcr, calculate the concentration of each undiluted Oncomine BRCA Research Assay library by multiplying the concentration determined with qpcr by Based on the calculated library concentration, determine the dilution factor that result in a concentration for each library of ~100 pm. For example: The undiluted library concentration is 300 pm. The dilution factor is 300 pm/100 pm = 3. Therefore, 10 µl of library mixed with 20 µl of Low TE (1:3 dilution) yields approximately 100 pm. 7. Dilute the libraries to ~100 pm with Low TE. Note: A library that yields less than 100 pm can be rescued with library amplification. Combine 25 µl of unamplified library with 71 µl of 1X Library Amp Mix and 4 µl of 25X Library Amp Primers from the Ion AmpliSeq Library Kit Plus. Perform 5 10 library amplification cycles (see step 4 of Amplify the library on page 19 or Amplify the library on page 24 for cycling conditions). Proceed to Combine Oncomine BRCA Research Assay barcoded libraries for sequencing on the same Ion chip on page 28 and template preparation, or store libraries at 4 8 C for up to 1 month, or at 20 C for longer-term storage. Oncomine BRCA Research Assay User Guide 23

24 2 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 3: Quantify the amplified libraries by Qubit Fluorometer or Agilent 2100 Bioanalyzer instrument Option 3: Quantify the amplified libraries by Qubit Fluorometer or Agilent 2100 Bioanalyzer instrument Oncomine BRCA Research Assay libraries must be amplified before quantification to enrich amplifiable material and obtain sufficient material for accurate quantification. Amplify the library using 1X Library Amp Mix, then purify. Quantify the library using the Qubit 2.0 or 3.0 Fluorometer, or the Agilent 2100 Bioanalyzer instrument. Amplified libraries typically have yields of 2,000 10,000 pm. Yield is not indicative of library quality. After quantification, determine the dilution factor that results in a concentration of ~100 pm, which is appropriate for template preparation using an Ion template kit. Alternatively, the Ion Library TaqMan Quantitation Kit can be used to quantify amplified libraries. Amplify the library 1. Remove the plate with purified libraries from the plate magnet, then add 50 μl of 1X Library Amp Mix and 2 μl of 25X Library Amp Primers to each bead pellet. Note: The 1X Library Amp Mix and 25X Library Amp Primers and can be combined before addition. 2. Seal the plate with MicroAmp Adhesive Film, vortex thoroughly, then centrifuge briefly to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times before sealing the plate. 3. Place the plate back on the magnet for at least 2 minutes, then carefully transfer ~50 µl of supernatant from each well to a new well or a new plate without disturbing the pellet. 4. Seal the plate with MicroAmp Adhesive Film, place a MicroAmp Compression Pad on the plate, load in the thermal cycler, then run the following program: Stage Temperature Time Hold 98 C 2 minutes 5 cycles 98 C 15 seconds 64 C 1 minute Hold 10 C Hold STOPPING POINT Samples can be stored at 20 C. Purify the amplified library Perform a two-round purification process with the Agencourt AMPure XP Reagent: First round at 0.5X bead-to-sample-volume ratio: High molecular-weight DNA is bound to beads, while amplicons and primers remain in solution. Save the supernatant. Second round at 1.2X bead-to-original-sample-volume ratio: Amplicons are bound to beads, and primers remain in solution. Save the bead pellet, and elute the amplicons from the beads. 24 Oncomine BRCA Research Assay User Guide

25 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 3: Quantify the amplified libraries by Qubit Fluorometer or Agilent 2100 Bioanalyzer instrument 2 IMPORTANT! Bring Agencourt AMPure XP Reagent to room temperature and vortex thoroughly to disperse the beads before use. Pipet the solution slowly. Use freshly prepared 70% ethanol for the next steps. Combine 230 µl of ethanol with 100 µl of Nuclease-free Water per sample. Do NOT substitute a Dynabeads -based purification reagent for the Agencourt Agencourt AMPure XP Reagent. First-round purification 1. Tap the plate gently on a hard flat surface, or centrifuge briefly to collect the contents at the bottom of the wells, then remove the plate seal. 2. Add 25 μl (0.5X sample volume) of Agencourt AMPure XP Reagent to each plate well containing ~50 µl of sample. Pipet up and down 5 times to mix the bead suspension with the DNA thoroughly. 3. Incubate the mixture for 5 minutes at room temperature. 4. Place the plate in a magnet such as the DynaMag Side Magnet for at least 5 minutes or until the solution is clear. 5. Carefully transfer the supernatant from each well to a new well of the 96-well PCR plate without disturbing the pellet. IMPORTANT! The supernatant contains the desired amplicons. Do not discard! Second-round purification 1. To the supernatant from step 4 above, add 60 μl (1.2X original sample volume) of Agencourt AMPure XP Reagent. Pipet up and down 5 times to mix the bead suspension with the DNA thoroughly. 2. Incubate the mixture for 5 minutes at room temperature. 3. Place the plate in the magnet for 3 minutes or until the solution is clear. Carefully remove, then discard the supernatant without disturbing the pellet. IMPORTANT! The amplicons are bound to the beads. Save the bead pellet. 4. Add 150 μl of freshly prepared 70% ethanol to each well, then move the plate side to side in the magnet to wash the beads. Remove and discard the supernatant without disturbing the pellet. Note: If your magnet does not have two positions for shifting the beads, remove the plate from the magnet and gently pipet up and down five times (with the pipettor set at 100 µl), then return the plate to the magnet and incubate for 2 minutes or until the solution clears. 5. Repeat step 4 for a second wash. 6. Ensure that all ethanol droplets are removed from the wells. Keeping the plate in the magnet, air-dry the beads at room temperature for 2 5 minutes. Do not overdry. Oncomine BRCA Research Assay User Guide 25

26 2 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 3: Quantify the amplified libraries by Qubit Fluorometer or Agilent 2100 Bioanalyzer instrument 7. Remove the plate from the magnet, then add 50 μl of Low TE to the pellet to disperse the beads. 8. Seal the plate with MicroAmp Adhesive Film, vortex thoroughly, then centrifuge to collect droplets. Alternatively, mix by setting a pipettor to 40 µl, then pipet the mixture up and down at least 5 times before sealing the plate. 9. Incubate at room temperature for at least 2 minutes. 10. Place the plate in the magnet for at least 2 minutes, then analyze an aliquot of the supernatant as described in: "Qubit Fluorometer: Quantify the library and calculate the dilution factor" or "Agilent 2100 Bioanalyzer instrument: Quantify the library and calculate the dilution factor". IMPORTANT! The supernatant contains the desired amplicons. Do not discard! Qubit Fluorometer: Quantify the library and calculate the dilution factor Analyze 10 µl of each amplified library using the Qubit 2.0 or 3.0 Fluorometer and the Qubit dsdna HS Assay Kit. Amplified libraries typically have concentrations of ng/ml. Libraries below 300 ng/ml can still provide good quality sequences. For more information, see the Qubit dsdna HS Assay Kits User Guide (Pub. No. MAN ). 1. Determine the amplified library concentration: a. Make a 1:200 working dilution of Qubit dsdna HS reagent using the Qubit dsdna HS Buffer. b. Combine 10 µl of the amplified Ion AmpliSeq library with 190 µl of dye reagent, mix well, then incubate for at least 2 minutes. c. Prepare each Qubit standard as directed in the user guide. d. Measure the concentration on the Qubit 2.0 or Qubit 3.0 Fluorometer. e. Calculate the concentration of the undiluted library by multiplying by Based on the calculated library concentration, determine the dilution that results in a concentration of ~100 pm. Note: The Oncomine BRCA Research Assay panel has an average amplicon size of approximately 140 bp, giving a library with average MW = 140 bp 650 Da/bp = 91,000 Da. A 100 pm concentration is equivalent to approximately 9 ng/ml: 100 pmole/l 91,000 pg/pmole = pg/l = 9,100 pg/ml = 9.1 ng/ml For example, The library concentration is 450 ng/ml. The dilution factor is 450 ng/ml divided by 9 ng/ml = 50. Therefore, 10 µl of library mixed with 490 µl of Low TE (1:50 dilution) yields 9 ng/ml (~100 pm). 3. Dilute library to ~100 pm with Low TE. 26 Oncomine BRCA Research Assay User Guide

27 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Option 3: Quantify the amplified libraries by Qubit Fluorometer or Agilent 2100 Bioanalyzer instrument 2 Proceed to Combine Oncomine BRCA Research Assay barcoded libraries for sequencing on the same Ion chip on page 28 and template preparation, or store libraries at 4 8 C for up to 1 month, or at 20 C for longer-term storage. Agilent 2100 Bioanalyzer instrument: Quantify the library and calculate the dilution factor Analyze 1 µl of amplified library on the Agilent 2100 Bioanalyzer instrument with the Agilent High Sensitivity DNA Kit (Cat. No ). Amplicon libraries should have multiple peaks in the bp size range. Amplified libraries typically have concentrations of ,000 pm. Yield is not indicative of library quality, and libraries below 1,000 pm can still provide good quality sequences. If the library concentration is over 20,000 pm, dilute the library 1:10 and repeat the quantification to obtain a more accurate measurement. 1. Determine the molar concentration of the amplified library using the Bioanalyzer software. Ensure that the upper and lower marker peaks are identified and assigned correctly. Follow the manufacturer s instructions to perform a region analysis (smear analysis). Briefly: a. Select the Data icon in the Contexts panel, then view the electropherogram of the sample to be quantified. b. Select the Region Table tab below, then create a region spanning the desired amplicon peaks. Correct the baseline if needed. c. The molarity is automatically calculated and displayed in the table in pmol/l (pm). 2. Based on the calculated library concentration, determine the dilution that results in a concentration of ~100 pm. For example: The library concentration is 3,000 pm. The dilution factor is 3,000 pm/100 pm = 30. Therefore, 10 µl of library mixed with 290 µl of Low TE (1:30 dilution) yields approximately 100 pm. 3. Dilute library to ~100 pm with Low TE. Proceed to Combine Oncomine BRCA Research Assay barcoded libraries for sequencing on the same Ion chip on page 28 and template preparation, or store libraries at 4 8 C for up to 1 month, or at 20 C for longer term storage. Oncomine BRCA Research Assay User Guide 27

28 AX Chapter 2 Prepare Oncomine BRCA Research Assay libraries Combine Oncomine BRCA Research Assay barcoded libraries for sequencing on the same Ion chip Combine Oncomine BRCA Research Assay barcoded libraries for sequencing on the same Ion chip Multiple Oncomine BRCA Research Assay libraries can be sequenced on a single Ion 318 v2 BC or Ion 530 Chip by combining equal volumes of each barcoded library before template preparation. Recommendations for the maximum number of libraries sequenced per chip are given in the following table: Ion sequencing chip Germline libraries sequenced per chip Somatic libraries sequenced per chip Ion 318 v2 BC Chip 32 8 Ion 530 Chip Oncomine BRCA Research Assay libraries after amplication and barcoding Combine equal volumes of barcoded libraries after equalization or quantification Combined library Prepare template Sequence on one Ion 318 v2 BC or Ion 530 Chip ion 318 ion torrent 530 DAAG02858 In this example, a barcoded library is generated from each of 8 samples using the two Oncomine BRCA Research Assay primer pools. After equalizing, or quantifying and diluting to 100 pm, equal volumes of the 8 barcoded libraries are combined before template preparation for sequencing on one Ion 318 v2 BC or Ion 530 Chip. 28 Oncomine BRCA Research Assay User Guide

29 Chapter 2 Prepare Oncomine BRCA Research Assay libraries Guidelines for templating and sequencing 2 Guidelines for templating and sequencing Proceed to template preparation and sequencing using the following kits appropriate to your instrument setup. See the appropriate user guide or guides listed in the table for more information. Ion PGM Hi Q View OT2 Kit and Ion PGM Hi Q View Sequencing Kit Kit Cat. No. User Guide A29900 A30044 Ion PGM Hi Q View OT2 Kit User Guide (Pub. No. MAN ) Ion PGM Hi Q View Sequencing Kit User Guide (Pub. No. MAN ) Ion PGM Hi Q View Chef Kit A29902 Ion PGM Hi Q View Chef Kits User Guide (Pub. No. MAN ) Ion 520 & Ion 530 Kit Chef A27757 A30010 Ion 520 & Ion 530 Kit Chef User Guide (Pub. No. MAN ) See Chapter 3, Create a Planned Run and analyze results with an Ion Reporter workflow for detailed instructions for using an Ion Reporter BRCA Research Assay workflow to analyze your results. Oncomine BRCA Research Assay User Guide 29

30 3 Create a Planned Run and analyze results with an Ion Reporter workflow Configure the Torrent Browser to use your Ion Reporter account About Ion Reporter Oncomine BRCA Research workflows Upload Target Regions and Hotspots BED files into Torrent Suite Software Create a Planned Run with an Oncomine BRCA Research template Using the Ion AmpliSeq Sample ID Panel View Ion Reporter analysis results and generate a report Download results or generate a report Edit an Ion Reporter Oncomine BRCA Research workflow with a new Annotation Set Oncomine Knowledgebase Reporter Software Note: We recommend that you update to the latest available versions of Torrent Suite and Ion Reporter Software to enable the data analysis and reporting options described in this chapter. Torrent Suite Software 5.2 is required to access the premade Planned Run templates. We recommend that you update to Ion Reporter Software 5.4 to access new Ion Reporter workflows [1] and enable exon deletion analysis. [1] Torrent Suite Software and Ion Reporter Software are for Research Use Only. Not for use in diagnostic procedures. 30 Oncomine BRCA Research Assay User Guide

31 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Configure the Torrent Browser to use your Ion Reporter account 3 Configure the Torrent Browser to use your Ion Reporter account 1. In the Torrent Browser, click Ion Reporter Configure in the Settings dropdown list. 2. Click Add Account, then select the appropriate Ion Reporter format from the dropdown list. Oncomine BRCA Research Assay User Guide 31

32 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Configure the Torrent Browser to use your Ion Reporter account 3. Enter Display Name, Username and Password for your IR account, then click Get Versions. 4. Select Ion Reporter 5.4 for Version, then click 3Add. 32 Oncomine BRCA Research Assay User Guide

33 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow About Ion Reporter Oncomine BRCA Research workflows 3 About Ion Reporter Oncomine BRCA Research workflows Somatic and germline Ion Reporter Oncomine BRCA Research workflows are available for the Ion PGM and Ion S5 /Ion S5 XL sequencing platforms. The w3.0 versions of the workflows are enabled to detect BRCA1 and BRCA2 exon deletions and duplications through inclusion of a Variability Correction Information Baseline (VCIB) CNV baseline (Oncomine BRCA DNA Baseline v1.0 (5.4)). Ion Reporter Oncomine BRCA Research workflow Enabled for exon deletion detection Sequencing platform Oncomine BRCA Research Somatic w2.1 - DNA - Single Sample No Ion PGM Oncomine BRCA Research Germline w2.1 - DNA - Single Sample Oncomine BRCA Research Somatic w3.0 - DNA - Single Sample Yes Oncomine BRCA Research Germline w3.0 - DNA - Single Sample Oncomine BRCA Research Somatic w2.1 - DNA - Single Sample No Ion S5/S5 XL Oncomine BRCA Research Germline w2.1 - DNA - Single Sample Oncomine BRCA Research Somatic w3.0 - DNA - Single Sample Yes Oncomine BRCA Research Germline w3.0 - DNA - Single Sample Note: The w2.1 workflows are visible to previous users of Ion Reporter 5.2 only. New Ion Reporter 5.4 users can only access the w3.0 workflows. Results with w2.1 workflows in Ion Reporter 5.4 are the same as in 5.2, except that exon deletion analysis is not functional. IMPORTANT! To create a custom Ion Reporter Oncomine BRCA Research workflow, copy an existing Oncomine BRCA Research workflow and edit it. You cannot create a workflow with full functionality starting from the Create workflow button on the Ion Reporter homepage. The Oncomine Variant Annotator (OVAT) v2.2 Plugin functionality is highly dependent on the panel used for the assay. Do not enable the OVAT plugin when copying or editing a non-oncomine annotation workflow. Oncomine BRCA Research Assay User Guide 33

34 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Upload Target Regions and Hotspots BED files into Torrent Suite Software Upload Target Regions and Hotspots BED files into Torrent Suite Software 1. Upload the following BED files, required for your Oncomine BRCA Research Ion Reporter workflow and provided in Ion Reporter Software 5.4, into Torrent Suite Software: Oncomine_BRCA_Research_Assay designed.bed Oncomine_BRCA_Research_Assay hotspots.blist.318.bed Oncomine_BRCA_Research_Assay hotspots.blist.530.bed 2. In the Torrent Browser, select References from the Settings dropdown list. 3. Select Target Regions, then click Add Target Regions. 4. Click Select File, then navigate to the Oncomine_BRCA_Research_Assay designed.bed file on your Torrent Server. Click Upload Target Regions File. 34 Oncomine BRCA Research Assay User Guide

35 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Upload Target Regions and Hotspots BED files into Torrent Suite Software 3 5. Select Hotspots from the list, then click Add Hotspots. Note: There are no hotspots for the Oncomine BRCA Research Assay panel - the positions are better characterized as blacklist positions. 6. Click Select File, then navigate to the Oncomine_BRCA_Research_Assay hotspots.blist.bed file on your drive appropriate to the 318 or 530 workflow you are using. Click Upload Hotspots File. Oncomine BRCA Research Assay User Guide 35

36 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Create a Planned Run with an Oncomine BRCA Research template Create a Planned Run with an Oncomine BRCA Research template 1. Sing in to the Torrent Server via the Torrent Browser, then navigate to Plan4Plan Template Run, Click Inherited Disease, or Oncology - Solid Tumor in the Applications list. 2. Select the applicable Oncomine BRCA Planned Run template: Oncomine BRCA Research for S5 Oncomine BRCA Research for PGM Alternatively, click Plan Run from the Settings of the template dropdown list to the right 3. The Planned Run wizard opens to the Plan step. Enter or select the following: a. Run Plan name b. Appropriate BED files from the Target and Hotspots Regions dropdown lists (if not selected) c. Sample Tube Label and Chip ID (if using the Ion Chef Instrument) d. The number of barcodes used in your sample e. Sample information 36 Oncomine BRCA Research Assay User Guide

37 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Create a Planned Run with an Oncomine BRCA Research template 3 Note: Assign unique, descriptive names to your samples to ensure that results for multiple samples are not combined in the run report. Oncomine BRCA Research Assay User Guide 37

38 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Create a Planned Run with an Oncomine BRCA Research template 4. Click the Ion Reporter step, select your Ion Reporter account, select an appropriate Oncomine BRCA Research workflow from the Existing Workflow list, then click Next >. 38 Oncomine BRCA Research Assay User Guide

39 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Create a Planned Run with an Oncomine BRCA Research template 3 5. In the Kits step, confirm settings and select Ion AmpliSeq Sample Id panel from the Control Sequence dropdown list. Example of Kits page selections for the Ion S5 System. 6. In the Plugins step, a. Select the sampleid plugin from the plugins list. Oncomine BRCA Research Assay User Guide 39

40 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Create a Planned Run with an Oncomine BRCA Research template b. Select variantcaller from the plugins list, click Configure, then select Chip Type and sample type. For Parameter Settings, select Custom, then navigate to the matching json file. Click Save Changes. Note: json file names: json file name oncomine_brca_somatic_318_w2.1- somatic_lowstringency_pgm_parameters.json oncomine_brca_germline_318_w2.1- germline_lowstringency_pgm_parameters.json oncomine_brca_somatic_318_w3.0- somatic_lowstringency_pgm_parameters.json oncomine_brca_germline_318_w3.0- germline_lowstringency_pgm_parameters.json oncomine_brca_somatic_530_w2.1- somatic_lowstringency_parameters.json oncomine_brca_germline_530_w2.1- germline_lowstringency_parameters.json oncomine_brca_somatic_530_w3.0- somatic_lowstringency_parameters.json oncomine_brca_germline_530_w3.0- germline_lowstringency_parameters.json Sequencing platform Ion PGM Ion S5/S5 XL 40 Oncomine BRCA Research Assay User Guide

41 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Create a Planned Run with an Oncomine BRCA Research template 3 c. Select coverageanalysis from the plugins list, click Configure, click the Sample Tracking checkbox, then click Save Changes. Note: Checking Sample Tracking enables the analysis to produce a statistic for reads mapped to Sample ID targets so that the level of off-target reads is accurately represented in the Coverage Analysis Report. d. Click Next on the lower right corner of the Plugins page to proceed to the next page. 7. Enter optional information in the Projects step, then return to the Plan step. Click Plan Run to complete the Planned Run. Your run is listed as pending in the Planned Runs List. 8. After completion of the sequencing run, navigate to your run in the Torrent Browser Data4Completed Runs & Reports list to view your results. Go to the Ion Reporter home page and click View Analyses, or the Analyses tab to view your Ion Reporter analysis. Oncomine BRCA Research Assay User Guide 41

42 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Using the Ion AmpliSeq Sample ID Panel Using the Ion AmpliSeq Sample ID Panel The Oncomine BRCA Research Assay includes the nine primer pairs that make up the Ion AmpliSeq Sample ID Panel, so do not add additional Sample ID primers to target amplification reactions. If you have not done so already, use the following procedure to configure a Planned Run or Planned Run template and view the Sample ID Report following a run. Note: The Sample ID Panel may be used to match a tumor and normal sample. However, copy number variations in the tumor sample may distort the allele balance in the fingerprint. IMPORTANT! Adding additional Ion AmpliSeq Sample ID Panel primer pairs can interfere with target amplification reactions. Do not add additional primers. 1. When creating a new Planned Run in the Torrent Browser, select Ion AmpliSeq Sample ID panel from the Control Sequence (optional): dropdown menu on the Kits page. 2. When creating a new Planned Run in the Torrent Browser, select the sampleid plugin on the Plugins page. Selection of the plugin automatically generates a Sample ID Report after the run. 3. On the Plugins page, select the coverageanalysis plugin checkbox, then click Configure. In the configuration dialog, select the Sample Tracking checkbox. This enables the analysis to produce a statistic for reads mapped to Sample ID targets so that the level of off-target reads is accurately represented in the Coverage Analysis Report. Note: If Sample Tracking is not selected, Sample ID reads are counted as offtarget reads. 42 Oncomine BRCA Research Assay User Guide

43 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Using the Ion AmpliSeq Sample ID Panel 3 4. Following sequencing, select the Data tab in the Torrent Browser and select Completed Runs and Results. Open the report for your run and scroll down to the Plugin Summary section, where you find the sampleid plugin results. Oncomine BRCA Research Assay User Guide 43

44 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report View Ion Reporter analysis results and generate a report This section describes how to use Ion Reporter Software to view analysis results, sort and classify variants, and generate a report. See the next section for information on how to view exon deletion results. The Oncomine BRCA Research Assay used with Ion Reporter Software 5.4 enables detection and visualization of whole exon and multiple exon deletion in BRCA1 and BRCA2 genes in somatic and germline samples with high sensitivity. The following steps describe how to use the software to visualize results and generate variant reports. 1. On the Ion Reporter home page, click View analyses, then navigate to your analysis on the Analyses page and select it. 44 Oncomine BRCA Research Assay User Guide

45 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report 3 2. On the Analysis Results page, view a summary of called variants and their genotypic and functional properties, and sort and select variants of interest by clicking the checkboxes at far left. Variant types and descriptions are listed in the following table. Description of variant types CNV Type CNV Subtype Description BigDel BigDup GeneCNV NOCALL REF Deletion of at least one exon Duplication of at least one exon Whole BRCA1/BRCA2 gene deletion or duplication Read count differs from baseline by noninteger amount; evidence for a BigDel or BigDup call is weak Read count matches reference baseline SNV Single nucleotide substitution MNP Multiple nucleotide polymorphism at adjacent nucleotide positions INDEL Single or multiple nucleotide insertion or deletion Oncomine BRCA Research Assay User Guide 45

46 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report Note: Scroll to the right using the scroll bar on the bottom to view additional columns. You can customize the columns that appear in the Analyses and Analysis Results tables by clicking Select Columns... in the Preferences dropdown list in the upper right corner. Select or deselect columns in the Available Columns list, then click Apply. 46 Oncomine BRCA Research Assay User Guide

47 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report 3 3. Click the Summary link to view a summary of called variants. You can classify variants by selecting a classification from the dropdown list, if desired. Oncomine BRCA Research Assay User Guide 47

48 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report 4. Click the Pharmacogenomics link to view the ClinVar column. Clicking the ClinVar link (boxed) for a selected variant takes you to an NCBI ClinVar variantspecific home page where information regarding the ClinVar variant annotation is maintained. Note: Other functional annotations in the Functional tab can also be used to classify, sort, and filter variants. Sample ID results for a sample is displayed at the top of the Analysis Results page, and on the Generate Report page in the Sample Information section. 48 Oncomine BRCA Research Assay User Guide

49 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report 3 5. On the ClinVar variant home page, click the Variation Report link for the variant (boxed) to go to a Clinical assertion and Summary evidence page, where additional information about a selected variant can be viewed. Oncomine BRCA Research Assay User Guide 49

50 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report The following are examples of the type of information available in the ClinVar Clinical assertions, Summary evidence, and Supporting observations tabs: Clinical assertions tab 50 Oncomine BRCA Research Assay User Guide

51 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report 3 Summary evidence tab Supporting observations tab Oncomine BRCA Research Assay User Guide 51

52 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report 6. To view the exon deletion results, return to the Analysis Results page and click Visualize at the upper right. The BRCA Report on the Analysis Visualization page graphically shows the read counts of BRCA1 and BRCA2 exons normalized to the Oncomine BRCA DNA Baseline. Click Download Results to download a.png file of the graphic and a.tsv variant summary file. 52 Oncomine BRCA Research Assay User Guide

53 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow View Ion Reporter analysis results and generate a report 3 7. Click the IRGV tab to display the IRGV view of exon deletions or duplications on chromosomes 13 and Click to open an IRGV Details tab in your browser 2 Zoom in and zoom out controls 3 Click to view chromosome 4 Scrollable alignment panel 5 Confidence filter 6 MAPD filter 7 Click to access more download options 8 Oncomine BRCA Research Assay designed BED file alignment Note: The MAPD (Median of the Absolute values of all Pairwise Differences) metric is an estimate of coverage variability between adjacent amplicons, and is used in CNV scoring. The default threshold is 0.5, so sample results with a MAPD above this value should be viewed with lower confidence. For further information, see Ion Reporter Help. Oncomine BRCA Research Assay User Guide 53

54 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Download results or generate a report Download results or generate a report 1. After selecting variants of interest on the Ion Reporter Analysis Results page, you can report your results in two ways: Download a.vcf (variant call format) file, or a.tsv (tab separated value) file of complete or filtered results from the Download dropdown list, which you can then customize to suit your reporting needs. Click Generate Report to produce a customizable IR report. Note: These actions can also be performed in the Analysis Visualization pages. 54 Oncomine BRCA Research Assay User Guide

55 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Download results or generate a report 3 Clicking Generate Report takes you to the Configuration page where you can customize your report. Complete the required field and add any other information. 2. Scroll down the Generate Report page to customize your report with the annotations that you want to include. 3. After making any additional comments and signing-off on the report, click Next -> in the lower right corner. Oncomine BRCA Research Assay User Guide 55

56 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Download results or generate a report 4. On the Preview page, review the report, then click Lock & Publish to finalize your report. After generating the report, download it immediately. Note: Only one report can be generated per analysis. Results require reanalysis to generate a second report. To capture all the information from the Ion Reporter annotation columns, generate a.tsv file using the Download dropdown list, then customize accordingly. Example of an IR report 56 Oncomine BRCA Research Assay User Guide

57 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Edit an Ion Reporter Oncomine BRCA Research workflow with a new Annotation Set 3 Edit an Ion Reporter Oncomine BRCA Research workflow with a new Annotation Set We recommend that you update the ClinVar annotation source in the Oncomine BRCA Research Ion Reporter workflow Annotation Sets to the latest available version. This section describes how to create a new Annotation Set with an updated ClinVar annotation, then add it to a copy of an existing Oncomine BRCA Research workflow. See Ion Reporter Help for more information on how to update and customize Ion Reporter workflows. Create a new Annotation Set 1. Sign in to Ion Reporter Software, then click Workflows4 Presets. 2. On the Workflow Presets page, select Annotation Sets from the dropdown list, then click the Annotation Set that you want to update. Take note of the names of the annotations in the Details panel at right. You will add these annotation sources to the new set in the next steps. Oncomine BRCA Research Assay User Guide 57

58 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Edit an Ion Reporter Oncomine BRCA Research workflow with a new Annotation Set 3. Select Annotation Set from the Create Preset dropdown list. 4. In the Create Annotation Set dialog, enter a new name for your Annotation Set, then select annotation sources from the dropdown list one at a time. 58 Oncomine BRCA Research Assay User Guide

59 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Edit an Ion Reporter Oncomine BRCA Research workflow with a new Annotation Set 3 5. Select the Source Version of the annotation that you want to include in your Annotation set, then click Use. In this case, select the Source Version to replace the Source Version in the Annotation Set. Repeat the selection process to populate the remaining annotations in the Annotation Set, referring to the list of annotations in the original Annotation Set. 6. When you have finished entering all the annotation sources, click Save. Oncomine BRCA Research Assay User Guide 59

60 3 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Edit an Ion Reporter Oncomine BRCA Research workflow with a new Annotation Set Copy and edit a workflow to add a new Annotation Set 1. Click the Workflows tab, then select the Oncomine BRCA Research workflow that you want to copy and edit. Select Copy from the Actions dropdown list. 2. Click the Annotation step in the Edit Workflow wizard, then select the new annotation set that you created from the Annotation Set dropdown list. 60 Oncomine BRCA Research Assay User Guide

61 Chapter 3 Create a Planned Run and analyze results with an Ion Reporter workflow Oncomine Knowledgebase Reporter Software 3 3. Make any additional changes, or confirm settings in the other steps. In the Confirm step, enter a workflow name, then click Save Workflow. The new workflow with an updated Annotation Set is listed on the Workflows page. Oncomine Knowledgebase Reporter Software You can also use Oncomine Knowledgebase Reporter Software to analyze your Oncomine BRCA Research Assay sequencing data, and manage and report results. To access the Thermo Fisher Cloud version of Oncomine Knowledgebase Reporter Software, go to: Contact your local Thermo Fisher Scientific representative if you have any questions regarding Oncomine Knowledgebase Reporter Software. Oncomine BRCA Research Assay User Guide 61

62 A Tips and troubleshooting Tips Arrange samples in columns on the plate for easier pipetting with multichannel pipettes during purification with the DynaMag Side Magnet. Plate seals can be firmly applied using the applicator in the MicroAmp Optical Adhesive Film Kit. You can remove plate seals with much less effort when the plate is hot. Try removing seals right after taking the plate out of the thermal cycler. When transfer to a new plate is specified, you can transfer solutions to a clean well in the same plate instead, if desired. 62 Oncomine BRCA Research Assay User Guide

63 Appendix A Tips and troubleshooting Troubleshooting A Troubleshooting Library yield and quantification Library yield is low Observation Possible cause Recommended action Equalizer beads were unwashed. Residual salt was present after wash. Be sure to wash Equalizer Beads before use. Carefully remove all of the Equalizer Wash Soln before elution. Input DNA was mis-quantified. Re-quantify input DNA using the TaqMan RNase P Detection Reagents Kit, or Qubit 2.0 or Qubit 3.0 Fluorometer. Input DNA was less than 10 ng. PCR, digestion, or ligation was inefficient. AMPure XP Beads were overdried. Residual ethanol inhibited library amplification. Add more DNA or increase target amplification cycles. Ensure proper dispensing and mixing of viscous components at each step. Do not dry the AMPure XP Beads more than 5 minutes. If the beads appear to be cracked, incubate the library amplification mix with the beads for 5 minutes, mix well with a pipettor until completely resuspended before placing the tube on the magnet to pellet the beads. Carefully remove all ethanol drops, using an additional centrifugation and removal step, if necessary. Library yield is high Input DNA was mis-quantified. Re-quantify input DNA using the TaqMan RNase P Detection Reagents Kit, or Qubit 2.0 or Qubit 3.0 Fluorometer; quantify RNA with Qubit 2.0 or Qubit 3.0 Fluorometer. Input DNA was more than 10 ng. Add less DNA/RNA or decrease target amplification cycles. Oncomine BRCA Research Assay User Guide 63

64 A Appendix A Tips and troubleshooting Troubleshooting Other Observation Possible cause Recommended action Number of on-target reads is lower than expected Percentage of polyclonal ISPs is high Percentage of low quality ISPs is high Unknown causes. Library was over-seeded. Other causes. Library was under-seeded. Other causes. Increase the number of target amplification cycles by two, or increase the anneal/extend temperature of the target amplification reaction from 60 C to 62 C for the first two cycles of the target amplification reaction. Decrease amount of library added to the template preparation reaction by 50%. Check an appropriate Ion PGM Hi Q View template preparation guide for more information. Double the volume of library used in template preparation. Use a fresh dilution of library prepared in an Eppendorf LoBind Tube. Check an appropriate Ion PGM Hi Q View template preparation guide for more information. 64 Oncomine BRCA Research Assay User Guide

65 B Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device. Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the Documentation and Support section in this document. Oncomine BRCA Research Assay User Guide 65

66 B Appendix B Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the Documentation and Support section in this document. Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. Handle chemical wastes in a fume hood. Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) After emptying a waste container, seal it with the cap provided. Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. 66 Oncomine BRCA Research Assay User Guide

67 Appendix B Safety Biological hazard safety B Biological hazard safety WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment. U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) , Revised December 2009; found at: World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/ ; found at: Oncomine BRCA Research Assay User Guide 67