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`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 1 of 36 PageID #: 1313
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`EXHIBIT 55
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`EXHIBIT 55
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`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 2 of 36 PageID #: 1314
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`TruSight Oncology 500 ctDNA
`Reference Guide
`
`Document # 1000000092559 v00
`February 2020
`For Research Use Only. Not for use in diagnostic procedures.
`
`ILLUMINA PROPRIETARY
`
`

`

`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 3 of 36 PageID #: 1315
`TruSight Oncology 500 ctDNA Reference Guide
`
`This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for
`the contractual use of its customer in connection with the use of the product(s) described herein and for no other
`purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise
`communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina
`does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third
`parties by this document.
`The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in
`order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must
`be fully read and understood prior to using such product(s).
`FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN
`MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS,
`AND DAMAGE TO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S).
`ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)
`DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).
`© 2020 Illumina, Inc. All rights reserved.
`All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information, see
`www.illumina.com/company/legal.html.
`
`Document # 1000000092559 v00
`For Research Use Only. Not for use in diagnostic procedures.
`
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`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 4 of 36 PageID #: 1316
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`Table of Contents
`
`CChapter 1 Overview
`Introduction
`cfDNA Input Recommendations
`Compatibility
`Reference Samples
`Additional Resources
`
`Chapter 2 Protocol
`Introduction
`Tips and Techniques
`Library Prep Workflow
`Enrichment Workflow
`Prepare cfDNA and Perform End Repair and A-Tailing
`Ligate Adapters
`Clean Up Ligation
`Index PCR
`Set Up First Hybridization
`Capture Targets One
`Set Up Second Hybridization
`Capture Targets Two
`Amplify Enriched Library
`Clean Up Amplified Enriched Library
`Quantify Libraries (Optional)(Acculear)
`Normalize Libraries
`Pool Libraries and Dilute to the Loading Concentration
`
`Appendix A Supporting Information
`Introduction
`Kit Contents
`Consumables and Equipment
`
`Technical Assistance
`
`1
`1
`1
`1
`1
`2
`
`3
`3
`3
`6
`7
`8
`9
`10
`11
`12
`14
`16
`17
`20
`21
`22
`23
`26
`
`27
`27
`27
`29
`
`31
`
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`Chapter 1 Overview
`
`IIntroduction
`cfDNA Input Recommendations
`Compatibility
`Reference Samples
`Additional Resources
`
`1
`1
`1
`1
`2
`
`Introduction
`The TruSight™ Oncology 500 Circulating Tumor DNA (ctDNA) protocol describes an enrichment-based
`approach to convert cell-free DNA (cfDNA) extracted from plasma samples into libraries enriched for
`cancer-related genes that can be sequenced on Illumina® sequencing systems. The TruSight Oncology
`500 ctDNA Kit enables the preparation of 48 libraries from cfDNA.
`The kit is optimized to provide high sensitivity and specificity for low-frequency somatic variants across
`523 genes. DNA biomarkers include:
` Single nucleotide variants (SNVs)
` Insertions
` Deletions
` Multinucleotide variants (MNVs)
` Gene amplifications
` Gene deletions
` Gene rearrangements
`TruSight Oncology 500 ctDNA also detects immunotherapy biomarkers for tumor mutational burden
`(TMB) and microsatellite instability (MSI).
`
`cfDNA Input Recommendations
` The TruSight Oncology 500 ctDNA Kit assay requires a minimum of 30 ng cfDNA input.
` Quantify the nucleic acids before starting the assay.
` To quantify the nucleic acids, use a capillary electrophoresis-based method, such as the Agilent
`Fragment Analyzer.
` To ensure optimal nucleic acid input, quantify the cfDNA fraction only.
` For recommendations for obtaining sufficient nucleic acid material, see the TruSight Oncology 500
`ctDNA support page on the Illumina support site.
`
`Compatibility
`For information on sequencing compatibility, see the TruSight Oncology 500 ctDNA support page on the
`Illumina support site.
`
`Reference Samples
` Use reference materials with known variant composition, such as SeraSeq ctDNA Complete
`Mutation Mix.
` Use RNase/DNase-free water as a non-template control. Do not sequence the non-template control.
`
`Document # 1000000092559 v00
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`TruSight Oncology 500 ctDNA Reference Guide
`
` Processing a reference sample or non-template control reduces the total number of test samples
`that can be processed.
`
`Additional Resources
`The TruSight Oncology 500 ctDNA support pages on the Illumina support website provide software,
`training resources, product compatibility information, and the following documentation. Always check
`support pages for the latest versions.
`
`Resource
`CustomProtocolSelector
`
`TruSightOncology500ctDNAChecklist
`(document#1000000107605)
`TruSightOncology500ctDNA
`Consumables&EquipmentList
`(document#1000000107604)
`IlluminaAdapterSequences(document#
`1000000002694)
`
`Description
`A tool for generating end-to-end instructions tailored to your library prep
`method, run parameters, and analysis method, with options to refine the
`level of detail.
`Provides a checklist of steps for the experienced user.
`
`Provides an interactive checklist of user-provided consumables and
`equipment.
`
`Provides adapter sequences for Illumina library prep kits.
`
`Document # 1000000092559 v00
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`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 7 of 36 PageID #: 1319
`
`Chapter 2 Protocol
`
`IIntroduction
`Tips and Techniques
`Library Prep Workflow
`Enrichment Workflow
`Prepare cfDNA and Perform End Repair and A-Tailing
`Ligate Adapters
`Clean Up Ligation
`Index PCR
`Set Up First Hybridization
`Capture Targets One
`Set Up Second Hybridization
`Capture Targets Two
`Amplify Enriched Library
`Clean Up Amplified Enriched Library
`Quantify Libraries (Optional)(Acculear)
`Normalize Libraries
`Pool Libraries and Dilute to the Loading Concentration
`
`3
`3
`6
`7
`8
`9
`10
`11
`12
`14
`16
`17
`20
`21
`22
`23
`26
`
`Introduction
`This section describes the TruSight Oncology 500 ctDNA Kit protocol.
` Review the complete sequencing workflow, from sample through analysis, to ensure compatibility of
`products and experiment parameters.
` Before proceeding, confirm kit contents and make sure that you have the required consumables and
`equipment.
` Follow the protocol in the order described using the specified parameters.
` Before beginning library preparation, record sample information, and assign each sample a unique
`index.
`
`Prepare for Pooling
`Record information about your samples before starting library prep. For compatibility information, see the
`TruSight Oncology 500 ctDNA support pages on the Illumina support website or the support pages for
`your system.
` For index adapter sequences and how to record them, see IlluminaAdapterSequences(document#
`1000000002694).
`
`Tips and Techniques
`
`Protocol Continuity
` Review tips and techniques before starting the protocol, as many critical techniques are listed only
`here and are not repeated in the protocol.
` Follow the protocol in the order described using the specified parameters.
` Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.
`
`Document # 1000000092559 v00
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`TruSight Oncology 500 ctDNA Reference Guide
`
`AAvoiding Cross-Contamination
` When adding or transferring samples, change tips between each well.
` Remove unused index adapter tubes from the working area.
` Use a unidirectional workflow when moving from pre-amp to post-amp areas.
` To prevent amplification product or probe carryover, avoid returning to the pre-amp area after
`beginning work in the post-amp area.
` When adding indexing primers, change tips between each well.
` Change gloves if gloves come in contact with indexing primers, samples, or probes.
` Clean work surfaces thoroughly before and after the procedure.
` Clean work surfaces and equipment thoroughly before and after the procedure with an
`RNase/DNase inhibiting cleaner.
` Handle and open only one index primer at a time. Recap each index tube immediately after use.
`Extra caps are provided with the kit.
`
`Sealing the Plate
` Always seal the plate before the following steps in the protocol:
` Shaking steps
` Vortexing steps
` Centrifuge steps
` Thermal cycling steps
` Apply the adhesive seal to cover the plate and seal with a rubber roller.
` Apply a new seal every time you cover a plate.
` Use Microseal 'B' adhesive seals for shaking, centrifuging, and long-term storage. The seals are
`effective at -40°C to 110°C and suitable for skirted or semiskirted PCR plates.
` If you observe droplets hanging from the inside of a sealed plate, centrifuge at 280 x g for 1 minute.
`
`Plate Transfers
` When transferring volumes between plates, transfer the specified volume from each well of a plate
`to the corresponding well of the other plate.
`
`Centrifugation
` When instructed to centrifuge the plate, centrifuge at 280 x g for 1 minute.
`
`Handling Reagents
` Tightly recap all reagent tubes immediately after use to limit evaporation and prevent contamination.
` Return reagents to the recommended storage conditions when they are no longer needed for the
`procedure.
` Stability of the TruSight Oncology 500 Kit has been evaluated and performance demonstrated for up
`to eight uses of the kit.
` Master mix preparation tables include volume overage to ensure that there is sufficient volume per
`sample.
`
`Document # 1000000092559 v00
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`TruSight Oncology 500 ctDNA Reference Guide
`
`HHandling Beads
` Do not freeze beads.
` Pipette bead suspensions slowly.
` Before use, allow the beads to come to room temperature.
` If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, and wait
`until the liquid is clear (~2 minutes).
` When washing beads:
` Use the specified magnetic stand for the plate.
` Dispense liquid so that beads on the side of the wells are wetted.
` Keep the plate on the magnetic stand until the instructions specify to remove it.
` Do not agitate the plate while it is on the magnetic stand. Do not disturb the bead pellet.
` When mixing beads with a pipette:
` Use a suitable pipette and tip size for the volume you are mixing (for example, use a P200 for
`volumes from 20 µl to 200 µl).
` Adjust the volume setting to ~50–75% of your sample volume.
` Pipette with a slow, smooth action.
` Mix beads for 1 minute to ensure homogeneity.
` Avoid aggressive pipetting, splashing, and introducing bubbles.
` Position the pipette tip above the pellet and dispense directly into the pellet to release beads
`from the well or tube.
` Make sure that the bead pellet is fully in solution. (For example, for SMB pellets, the solution
`should look dark brown and have a homogenous consistency.)
`
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`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 10 of 36 PageID #: 1322
`TruSight Oncology 500 ctDNA Reference Guide
`
`Library Prep Workflow
`The following diagram illustrates the recommended library preparation workflow using the TruSight
`Oncology 500 ctDNA Kit. Safe stopping points are marked between steps. Hands-on and total times are
`approximate and based on eight cfDNA samples.
`
`Figure 1 TruSight Oncology 500 ctDNA Kit Workflow (Part 1)
`
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`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 11 of 36 PageID #: 1323
`TruSight Oncology 500 ctDNA Reference Guide
`
`Enrichment Workflow
`The following diagram illustrates the recommended enrichment workflow using the TruSight Oncology
`500 ctDNA Kit. Safe stopping points are marked between steps. Hands-on and total times are
`approximate and based on eight cfDNA samples.
`
`Figure 2 TruSight Oncology 500 ctDNA Kit Workflow (Part 2)
`
`Document # 1000000092559 v00
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`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 12 of 36 PageID #: 1324
`TruSight Oncology 500 ctDNA Reference Guide
`
`Prepare cfDNA and Perform End Repair and A-Tailing
`This process converts the 5' and 3' overhangs on cfDNA into blunt ends using an End Repair A-Tailing
`Master Mix (ERA1).
`The 3' to 5' exonuclease activity of this mix removes the 3' overhangs and the 5' to 3' polymerase
`activity fills in the 5' overhangs. The 3' ends are A-tailed during this reaction to prevent them from
`ligating to each other during the adapter ligation reaction.
`
`CConsumables
` ERA1-A (End Repair A-tailing Enzyme Mix 1)
` ERA1-B (End Repair A-tailing Buffer 1)
` RSB (Resuspension Buffer)
` Microseal 'B' adhesive seals
` 1.7 ml microcentrifuge tube
` 96-well MIDI plate
`
`Preparation
`1 Prepare the following consumables:
`
`Reagent
`ERA1-A
`ERA1-B
`
`RSB
`
`Storage
`
`-25°C to -15°C
`-25°C to -15°C
`
`2°C to 8°C or
`-25°C to -15°C
`
`Instructions
`
`Keep on ice. Centrifuge briefly, and then pipette to mix.
`Thaw to room temperature. Centrifuge briefly, and then
`pipette to mix. If precipitates are present, warm the tube in
`your hands, and then pipette to mix until the crystals
`dissolve.
`Bring to room temperature. Vortex before use.
`If stored at -25°C to -15°C, thaw at room temperature and
`vortex before use.
`
`2 Bring cfDNA to room temperature.
`3 See cfDNAInputRecommendations on page 1 to quantify samples.
`4 Use RSB to prepare a minimum of 30 ng of each purified cfDNA sample in a final volume of 50 µl
`(0.6 ng/µl).
`5 Pipette mix or vortex cfDNA and then centrifuge briefly.
`6
`Label the MIDI plate LP (Library Preparation).
`7 Preheat two Hybex incubators with MIDI heat block inserts as follows.
` Preheat the first incubator to 30°C.
` Preheat the second incubator to 72°C.
`Transfer 50 µl of each cfDNA sample to corresponding wells of a new 96-well MIDI plate.
`8
`9 Prepare an ice bucket.
`
`Document # 1000000092559 v00
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`TruSight Oncology 500 ctDNA Reference Guide
`
`Procedure
`1 Combine the appropriate volumes from the table below in a microcentrifuge tube to prepare ERA1
`Master Mix. Please note that master mix volumes include overage.
`
`Master Mix Component
`ERA1-B
`ERA1-A
`
`3 Samples (µl)
`26
`10
`
`8 Samples (µl)
`69
`27
`
`16 Samples (µl)
`138
`54
`
`24 Samples (µl)
`207
`81
`
` Prepare for a minimum of 3 libraries.
` Discard any remaining master mix after use.
`2 Pipette 10 times to mix, and then place ERA1 Master Mix on ice.
`3 Add 10 µl ERA1 Master Mix to each sample in the LP MIDI plate.
`4 Apply Microseal ‘B’ to the LP MIDI plate and shake the LP MIDI plate at 1800 rpm for 1 minute.
`5
`Incubate at 30°C for 30 minutes.
`6
`Immediately transfer to another incubator at 72°C and incubate for 20 minutes.
`7 Place the LP MIDI plate on ice for 5 minutes.
`
`Ligate Adapters
`UMI1 adapters that contain unique molecular indexes are ligated to cfDNA fragments. This process
`ligates adapters to the ends of cfDNA fragments.
`
`Consumables
` ALB1 (Adapter Ligation Buffer 1)
` LIG3 (DNA Ligase 3)
` STL (Stop Ligation Buffer)
` UMI1 (UMI Adapters v1)
` Microseal 'B' adhesive seals
`
`About Reagents
` ALB1 is highly viscous. Pipette slowly to avoid forming bubbles.
`
`Preparation
`1 Prepare the following consumables:
`
`Item
`ALB1
`
`LIG3
`
`STL
`
`UMI1
`
`Storage
`-25°C to -15°C
`
`-25°C to -15°C
`
`-25°C to -15°C
`
`-25°C to -15°C
`
`Instructions
`Thaw to room temperature. Vortex ≥ 10 seconds to
`resuspend. Centrifuge briefly.
`Keep on ice.
`Centrifuge briefly, and then pipette to mix.
`Thaw to room temperature. Vortex ≥ 10 seconds to
`resuspend. Centrifuge briefly.
`Thaw to room temperature. Vortex ≥ 10 seconds to
`resuspend. Centrifuge briefly.
`
`Document # 1000000092559 v00
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`TruSight Oncology 500 ctDNA Reference Guide
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`Procedure
`1 Add 60 µl ALB1 to each well.
`2 Add 5 µl LIG3 to each well.
`3 Add 10 µl UMI1 to each well.
`4 Apply Microseal ‘B’ to the LP MIDI plate and shake the LP MIDI plate at 1800 rpm for 1 minute.
`5
`Incubate at room temperature for 30 minutes.
`6 Add 5 µl STL to each well.
`7 Apply Microseal ‘B’ to the LP MIDI plate and shake the LP MIDI plate at 1800 rpm for 1 minute.
`
`Clean Up Ligation
`This process uses SPB to purify the cfDNA fragments and remove unwanted products, such as
`unligated adapters.
`
`CConsumables
` RSB (Resuspension Buffer)
` SPB (Sample Purification Beads)
` Freshly prepared 80% ethanol (EtOH)
` Microseal 'B' adhesive seals
` 96-well PCR plate
`
`About Reagents
` Aspirate and dispense SPB slowly due to the viscosity of the suspension.
`
`Preparation
`1 Prepare the following consumables.
`
`Item
`RSB
`
`SPB
`
`Storage
`2°C to 8°C or
`-25°C to -15°C
`
`2°C to 8°C
`
`Instructions
`Bring to room temperature. Vortex before use.
`If stored at -25°C to -15°C, thaw at room temperature
`and vortex before use.
`Bring to room temperature for at least 30 minutes. Vortex
`for 1 minute before using.
`
`Label a new 96-well PCR plate LS (Library Samples).
`2
`3 Prepare fresh 80% EtOH.
`
`Procedure
`
`Bind
`1 Vortex SPB for 1 minute to resuspend the beads.
`2 Add 112 µl SPB to each well of the LP MIDI plate.
`
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`TruSight Oncology 500 ctDNA Reference Guide
`
`3 Apply Microseal ‘B’ to the LP MIDI plate and shake the LP MIDI plate at 1800 rpm for 1 minute.
`4
`Incubate at room temperature for 5 minutes.
`
`Wash
`1 Place the LP MIDI plate on the magnetic stand for 10 minutes.
`2 Remove and discard all supernatant from each well.
`3 Wash beads as follows.
`
`a Keep on magnetic stand and add 200 µl fresh 80% ethanol to each well.
`b Wait 30 seconds.
`c Remove and discard all supernatant from each well.
`
`4 Wash beads a ssecond time.
`5 Use a P20 pipette with fine tips to remove residual supernatant from each well.
`
`Elute
`1 Remove from the magnetic stand.
`2 Add 27.5 µl RSB to each well.
`3 Apply Microseal ‘B’ to the LP MIDI plate and shake the LP MIDI plate at 1800 rpm for 1 minute.
`4
`Incubate at room temperature for 2 minutes.
`5 Place on a magnetic stand for 2 minutes.
`6
`Transfer 25 µl of each eluate from the LP MIDI plate to the corresponding well of the LS PCR plate.
`
`Index PCR
`In this step, library fragments are amplified using primers that add index sequences for sample
`multiplexing. The resulting product contains the complete library of cfDNA fragments flanked by index
`sequences and adapters required for cluster generation.
`
`Consumables
` EPM (Enhanced PCR Mix)
` UPxx (Unique Index Primer)
` Microseal 'B' adhesive seals
`
`WARNING
`This set of reagents contains potentially hazardous chemicals. Personal injury can occur
`through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment,
`including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle
`used reagents as chemical waste and discard in accordance with applicable regional, national,
`and local laws and regulations. For additional environmental, health, and safety information, see the
`SDS at support.illumina.com/sds.html.
`
`Document # 1000000092559 v00
`For Research Use Only. Not for use in diagnostic procedures.
`
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`TruSight Oncology 500 ctDNA Reference Guide
`
`Preparation
`1 Prepare the following consumables:
`
`Reagent
`EPM
`UPxx
`
`Storage
`-25°C to -15°C
`-25°C to -15°C
`
`Instructions
`Thaw on ice. Vortex to resuspend. Centrifuge briefly.
`Thaw to room temperature. Vortex to resuspend. Centrifuge briefly.
`
`3
`
`2 Assign one UPxx index primer per library (xx = index primer number).
`When sequencing multiple libraries on a single flow cell, assign a different indexing primer to each
`sample library. Record sample layout orientation and the indexes for each sample library.
`In the post-amp area, save the following I-PCR program on the thermal cycler:
` Choose the preheat lid option and set to 100°C
` Set the reaction volume to 50 µl
` 98°C for 30 seconds
` 15 cycles of:
` 98°C for 10 seconds
` 60°C for 30 seconds
` 72°C for 30 seconds
` 72°C for 5 minutes
` Hold at 10°C
`
`Procedure
`1 Add 5 µl of the assigned indexing primer (UPxx) to each well of the LS PCR plate. Apply a new tube
`cap to the indexing primer tube with a cap provided in your kit.
`2 Add 20 µl EPM to each well.
`3 Apply Microseal ‘B’ to the LS PCR plate and shake the LS PCR plate at 1800 rpm for 1 minute.
`4
`Transfer the plate to the post-amp area to prevent amplification product carryover.
`5 Briefly centrifuge at 280 × g.
`6 Place on the preprogrammed thermal cycler and run the I-PCR program.
`7 Relabel the plate ALS (Amplified Library Samples).
`8 Centrifuge briefly.
`
`SAFE STOPPING POINT
`If you are stopping, make sure that the ALS PCR plate is sealed well, and store at -25°C to -15°C for up
`to 30 days.
`
`Set Up First Hybridization
`During this process, a pool of oligos specific to the 523 genes targeted by TruSight Oncology 500
`ctDNA hybridize to DNA libraries prepared in IndexPCR on page 11. Enrichment of targeted regions
`requires two hybridization steps. In this step, oligos hybridize to the DNA libraries overnight (8–24
`hours).
`
`Document # 1000000092559 v00
`For Research Use Only. Not for use in diagnostic procedures.
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`TruSight Oncology 500 ctDNA Reference Guide
`
`CConsumables
` OPD2 (Oncology Probes DNA 2)
` TCA1 (Target Capture Additives 1)
` TCB1 (Target Capture Buffer 1)
` 96-well PCR plate
` Microseal 'B' adhesive seals
`
`WARNING
`This set of reagents contains potentially hazardous chemicals. Personal injury can occur
`through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment,
`including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle
`used reagents as chemical waste and discard in accordance with applicable regional, national,
`and local laws and regulations. For additional environmental, health, and safety information, see the
`SDS at support.illumina.com/sds.html.
`
`Preparation
`1 Prepare the following consumables:
`
`Reagent
`OPD2
`
`TCA1
`
`TCB1
`
`Storage
`-25°C to -15°C
`
`-25°C to -15°C
`
`2°C to 8°C
`
`Instructions
`Thaw to room temperature. Vortex to resuspend.
`Centrifuge briefly.
`Thaw to room temperature. Vortex to resuspend.
`Centrifuge briefly.
`Thaw to room temperature. Centrifuge briefly, then
`pipette to mix. Inspect for precipitates. If present, warm
`the tube in your hands, then pipette to mix until the
`crystals are dissolved.
`
`2
`
`If the ALS PCR plate was stored at -25°C to -15°C, prepare it as follows.
`
`a Thaw at room temperature.
`b Centrifuge at 280 × g for 1 minute.
`c Pipette to mix and centrifuge.
`
`Label a new 96-well PCR plate HYB1 (Hybridization 1).
`3
`4 Save the following HYB1 program on the thermal cycler:
` Choose the preheat lid option and set to 100°C
` Set the reaction volume to 50 µl
` 95°C for 10 minutes
` 85°C for 2.5 minutes
` 75°C for 2.5 minutes
` 65°C for 2.5 minutes
` Hold at 57°C
`
`Procedure
`1
`Transfer 20 µl of each library to the HYB1 PCR plate.
`2 Add 15 µl TCB1 to each well.
`
`Document # 1000000092559 v00
`For Research Use Only. Not for use in diagnostic procedures.
`
`13
`
`

`

`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 18 of 36 PageID #: 1330
`TruSight Oncology 500 ctDNA Reference Guide
`
`3 Add 10 µl TCA1 to each well.
`4 Add 5 µl OPD2 to each well.
`5 Apply Microseal ‘B’ to the HYB1 PCR plate and shake the HYB1 PCR plate at 1800 rpm for 1
`minute.
`6 Place on the preprogrammed thermal cycler and run the HYB1 program. Hybridize for 8—24 hours
`(overnight) at 57°C.
`
`Capture Targets One
`This step uses SMB (Streptavidin Magnetic Beads) to capture probes hybridized to the targeted library
`DNA regions of interest. Two heated washes using EEW remove nonspecific DNA binding from the
`beads. The enriched library is then eluted from the beads and prepared for a second round of
`hybridization.
`
`CConsumables
` EE2 (Enrichment Elution 2)
` EEW (Enhanced Enrichment Wash)
` ET2 (Elute Target Buffer 2)
` HP3 (2 N NaOH)
` SMB (Streptavidin Magnetic Beads)
` 96-well MIDI plate
` 96-well PCR plate
` Microseal 'B' adhesive seals
`
`WARNING
`This set of reagents contains potentially hazardous chemicals. Personal injury can occur
`through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment,
`including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle
`used reagents as chemical waste and discard in accordance with applicable regional, national,
`and local laws and regulations. For additional environmental, health, and safety information, see the
`SDS at support.illumina.com/sds.html.
`
`About Reagents
` Make sure to use SSMB and not SPB for this procedure.
`
`Document # 1000000092559 v00
`For Research Use Only. Not for use in diagnostic procedures.
`
`14
`
`

`

`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 19 of 36 PageID #: 1331
`TruSight Oncology 500 ctDNA Reference Guide
`
`Preparation
`1 Prepare the following consumables:
`
`Reagent
`EE2
`
`EEW
`
`ET2
`
`HP3
`
`SMB
`
`Storage
`-25°C to -15°C
`
`-25°C to -15°C
`
`2°C to 8°C
`
`2°C to 8°C
`
`2°C to 8°C
`
`Instructions
`Thaw to room temperature. Vortex to resuspend.
`Centrifuge briefly.
`Thaw to room temperature. Vortex for 1 minute to
`resuspend.
`Bring to room temperature. Vortex to resuspend.
`Centrifuge briefly.
`Bring to room temperature. Vortex to resuspend.
`Centrifuge briefly.
`Bring to room temperature for 30 minutes.
`If the bead pellet is present, pipette up and down to
`release the pellet, and then vortex to resuspend.
`
`2 Preheat a Hybex incubator with MIDI heat block insert to 57°C.
`3
`Label a new 96-well MIDI plate CAP1 (Capture 1).
`4
`Label a new 96-well PCR plate ELU1 (Elution 1).
`
`Procedure
`
`Bind
`1 Remove the HYB1 PCR plate from the thermal cycler.
`2 Vortex SMB for 1 minute to resuspend the beads.
`3 Add 150 µl SMB to each well of the CAP1 MIDI plate.
`4
`Transfer 50 µl of each library from the HYB1 PCR plate to the corresponding well of the CAP1 MIDI
`plate.
`5 Apply Microseal ‘B’ to the CAP1 MIDI plate and shake the CAP1 MIDI plate at 1800 rpm for 1
`minute.
`Incubate in a Hybex incubator at 57°C for 25 minutes.
`6
`7 Place on a magnetic stand for 2 minutes.
`8 Use a pipette to remove and discard the supernatant.
`
`Wash
`1 Wash beads as follows.
`
`a Remove the CAP1 MIDI plate from the magnetic stand.
`b Add 200 µl EEW to each well.
`c Apply Microseal ‘B’ to the CAP1 MIDI plate and shake the CAP1 MIDI plate at 1800 rpm for 2
`minutes.
`d If the bead pellet is still present, remove the Microseal and pipette to mix. Make sure that all
`beads are resuspended, and then apply a new Microseal 'B'.
`e Incubate in a Hybex incubator at 57°C for 10 minutes.
`
`Document # 1000000092559 v00
`For Research Use Only. Not for use in diagnostic procedures.
`
`15
`
`

`

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`TruSight Oncology 500 ctDNA Reference Guide
`
`f Place on a magnetic stand for 2 minutes.
`g While on the magnetic stand, use a pipette to remove and discard all supernatant from each
`well.
`
`2 Repeat step 1 to wash beads a ssecond time.
`3 Use a P20 pipette with fine tips to remove any residual supernatant from each well.
`
`Elute
`1 Combine the following volumes in a microcentrifuge tube to prepare the EE2+HP3 Elution Mix:
`
`Elution Mix Component
`EE2
`HP3
`
`3 Libraries (µl)
`95
`5
`
`8 Libraries (µl)
`171
`9
`
`16 Libraries (µl)
`342
`18
`
`24 Libraries (µl)
`513
`27
`
` Prepare for a minimum of 3 libraries.
` Discard any remaining elution mix after use.
`2 Vortex briefly to mix.
`3 Remove the CAP1 MIDI plate from the magnetic stand.
`4 Carefully add 17 µl EE2+HP3 Elution Mix to each sample pellet.
`5 Apply Microseal ‘B’ to the CAP1 MIDI plate and shake the CAP1 MIDI plate at 1800 rpm for 1
`minute.
`6 Place on a magnetic stand for 2 minutes.
`7 Carefully transfer 15 µl eluate from each well of the CAP1 MIDI plate to the ELU1 PCR plate.
`8 Add 5 µl ET2 to each eluate in the ELU1 PCR plate.
`9 Apply Microseal ‘B’ to the ELU1 PCR plate and shake the ELU1 PCR plate at 1800 rpm for 1
`minute.
`
`Set Up Second Hybridization
`This step binds targeted regions of the enriched DNA libraries with capture probes a second time. The
`second hybridization ensures high specificity of the captured regions. To ensure optimal enrichment of
`libraries, perform the second hybridization step for 1.5–4 hours.
`
`Consumables
` OPD2 (Oncology Probes DNA 2)
` TCA1 (Target Capture Additives 1)
` TCB1 (Target Capture Buffer 1)
` Microseal 'B' adhesive seals
`
`Document # 1000000092559 v00
`For Research Use Only. Not for use in diagnostic procedures.
`
`16
`
`

`

`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 21 of 36 PageID #: 1333
`TruSight Oncology 500 ctDNA Reference Guide
`
`WARNING
`TThis set of reagents contains potentially hazardous chemicals. Personal injury can occur
`through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment,
`including eye protection, gloves, and laboratory coat appropriate for risk of exposure. Handle
`used reagents as chemical waste and discard in accordance with applicable regional, national,
`and local laws and regulations. For additional environmental, health, and safety information, see the
`SDS at support.illumina.com/sds.html.
`
`Preparation
`1 Prepare the following consumables:
`
`Reagent
`OPD2
`
`TCA1
`
`TCB1
`
`Storage
`-25°C to -15°C
`
`-25°C to -15°C
`
`2°C to 8°C
`
`Instructions
`Thaw to room temperature. Vortex to resuspend.
`Centrifuge briefly.
`Thaw to room temperature. Vortex to resuspend.
`Centrifuge briefly.
`Thaw to room temperature. Centrifuge briefly, then
`pipette to mix. Inspect for precipitates. If precipitates are
`present, warm the tube in your hands, and then pipette to
`mix until the crystals are dissolved.
`
`2 Save the following HYB2 program on the thermal cycler:
` Choose the preheat lid option and set to 100°C
` Set the reaction volume to 50 µl
` 95°C for 10 minutes
` 85°C for 2.5 minutes
` 75°C for 2.5 minutes
` 65°C for 2.5 minutes
` Hold at 57°C
`
`Procedure
`1 Add 15 µl TCB1 to each well of the ELU1 PCR plate.
`2 Add 10 µl TCA1 to each well.
`3 Add 5 µl OPD2 to each well.
`4 Apply Microseal ‘B’ and shake the ELU1 PCR plate at 1800 rpm for 1 minute.
`5 Place on the preprogrammed thermal cycler and run the HYB2 program. Hybridize at 57°C for 1.5–4
`hours.
`
`Capture Targets Two
`This step uses SMB (Streptavidin Magnetic Beads) to capture probes hybridized to the targeted regions
`of interest. RSB is used to rinse the captured libraries and remove nonspecific binding from the beads.
`The enriched library is then eluted from the beads and prepared for sequencing.
`
`Consumables
` EE2 (Enrichment Elution 2)
`
`Document # 1000000092559 v00
`For Research Use Only. Not for use in diagnostic procedures.
`
`17
`
`

`

`Case 1:20-cv-01644-RGA Document 1-55 Filed 12/03/20 Page 22 of 36 PageID #: 1334
`TruSight Oncology 500 ctDNA Reference Guide
`
` ET2 (Elute Target Buffer 2)
` HP3 (2 N NaOH)
` RSB (Resuspension Buffer)
` SMB (Streptavidin Magnetic Beads)
` 96-well MIDI plate
` 96-well PCR plate
` Microseal 'B' adhesive seals
`
`WARNING
`TThis set of reagents contains potentially hazardous chemicals. Personal injury can occur
`through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment,
`including eye protection, gloves, a