Case 1:20-cv-01644-RGA Document 1-54 Filed 12/03/20 Page 1 of 5 PageID #: 1308
`Case 1:20-cv-01644-RGA Document 1-54 Filed 12/03/20 Page 1 of 5 PageID #: 1308
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`EXHIBIT 54
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`EXHIBIT 54
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`Case 1:20-cv-01644-RGA Document 1-54 Filed 12/03/20 Page 2 of 5 PageID #: 1309
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`TruSight™ Oncology 500 ctDNA
`Enabling in-house comprehensive genomic profiling from liquid biopsy samples.
`
`Highlights
`
`• Enable comprehensive genomic profiling from blood
`Assess DNA variants across 523 genes in house, including
`bTMB and bMSI*
`
`• Unlock liquid biopsy in-house
`Leverage noninvasive blood plasma samples as an alternative,
`or complement, to limited tissue samples
`
`• Produce trustworthy results
`Realize low limits of detection with UMI-based hybrid–capture
`library preparation, high-intensity sequencing with the
`NovaSeq™ 6000 System, and advanced bioinformatics
`
`• Take advantage of an integrated workflow
`Go from cfDNA to report interpretation using the DRAGEN™
`Bio-IT Platform for variant calling in five days
`
`*
`
`bTMB and bMSI refer to plasma-based (blood) markers
`
`Introduction
`Liquid biopsy enables comprehensive analysis of circulating cell-
`free DNA (cfDNA) in plasma, providing a noninvasive approach for
`profiling solid tumors. To take advantage of liquid biopsy, it is critical
`to use a highly sensitive and specific assay capable of detecting
`somatic mutations at low frequencies. TruSight Oncology 500 ctDNA
`harnesses the power of proven Illumina next-generation sequencing
`(NGS) technology to achieve this high sensitivity and enables
`comprehensive genomic profiling of circulating tumor DNA (ctDNA)
`found in cfDNA (Figure 1, Table 1). Combining this advanced solution
`with the bioinformatics power of the DRAGEN TruSight Oncology 500
`ctDNA Analysis Software gives clinical researchers a DNA-to-report
`solution for evaluating multiple variant types across hundreds of genes
`in a single assay.
`TruSight Oncology 500 ctDNA is compatible with NovaSeq 6000 v1.5
`sequencing reagents. In addition to increases in operating efficiencies
`that result in potential price per sample reductions > 35%, these
`reagents offer an extended shelf-life of six months and improved
`Q30 scores.
`
`Table 1: TruSight Oncology 500 ctDNA at a glance
`Parameter
`Details
`
`Instrument
`Panel size
`
`Panel content
`
`NovaSeq 6000 System
`1.94 Mb DNA
`
`523 genes
`59 genes for CNVs
`23 genes for gene fusions
`MSI (> 2400 loci)
`TMB
`
`DNA input requirement
`Sample type
`Total assay time
`
`30 ng cfDNAa
`cfDNA derived from blood
`5 days from library prep to variant report
`
`Sequencing run time
`
`36 hr run, 10 hrs analysis (S2 flow cell)
`45 hr run, 22 hrs analysis (S4 flow cell)
`
`Sequence run
`
`2 × 151 bp
`
`Sample throughput
`
`Limit of detection
`
`8 samples per run (S2 flow cell)
`24 samples per run (S4 flow cell)
`48 samples per library prep kit
`
`0.5% VAF for small variants
`≥ 1.4-fold change for gene amplifications
`≤ 0.6-fold change for gene deletions
`≥ 2% tumor fraction for MSI
`
`Analytical sensitivity
`
`≥ 95% (at LOD for all variant types)
`
`Analytical specificity
`
`≥ 95%
`
`a. Recommend quantification with Agilent TapeStation or Fragment Analyzer systems
`
`The power of liquid biopsy
`Unlike a tissue biopsy that provides information from only a fraction of
`the tumor, liquid biopsy provides insights about intra- and inter-tumor
`heterogeneity throughout the body. Recent studies show that cfDNA
`analysis detected a significant number of guideline-recommended
`biomarkers and resistance alterations not found in matched tissue
`biopsies.1 In addition, a non-small cell lung cancer study revealed that
`cfDNA analyses are highly concordant with tissue-based analyses.2
`
`Indel
`
`1
`
`CNV
`
`3
`
`MSI
`
`5
`
`2
`
`SNV
`
`4
`
`6
`
`Gene fusion
`
`TMB
`
`DNA from tumors
`circulates in the blood
`
`TruSight Oncology 500 ctDNA detects
`somatic variants from 523 genes
`in circulating tumor DNA
`Figure 1: Liquid biopsy enables profiling of biomarkers for multiple variant types and multiple cancer types—Sophisticated variant calling algorithms and high
`depth of sequencing enable detection of key biomarkers in cfDNA with 0.5% limit of detection (LOD).
`
`Cell-free DNA is
`extracted from plasma
`
`For Research Use Only. Not for use in diagnostic procedures.
`
`1170-2019-006-D | 1
`
`Prepare Library | Sequence | Analyze Data
`
`PioM
`
`PioM
`
`

`

`Case 1:20-cv-01644-RGA Document 1-54 Filed 12/03/20 Page 3 of 5 PageID #: 1310
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`Specimen
`
`Library prep
`and enrichment
`
`Sequence
`
`Variant report
`
`Interpretation
`and reporting
`
`DRAGEN™
`
`cfDNA extracted from plasma
`
`TruSight Oncology 500
`ctDNA Kit
`
` NextSeq 6000 System
`
`DRAGEN TruSight Oncology 500
`ctDNA Analysis Software on the
`DRAGEN Bio-IT Platform
`
`Powered by PierianDx Clinical
`Genomics Workspace
`
`Figure 2: TruSight Oncology 500 ctDNA assay workflow—The TruSight Oncology 500 ctDNA assay integrates into current lab workflows, going from cfDNA to a
`variant report in five days.
`
`Turnaround time = 5 days
`
`A foundation of comprehensive content
`
`Content for TruSight Oncology 500 ctDNA was designed with
`recognized authorities in the oncology community and includes
`current and emerging biomarkers with comprehensive coverage of
`genes involved in key guidelines and clinical trials for multiple tumor
`types. The panel probe design captures both known and novel gene
`fusions and includes 523 genes for detecting variants likely to play a
`role in tumorigenesis. Biomarkers comprise single-nucleotide variants
`(SNVs), insertions/deletions (indels), copy-number variants (CNVs),
`gene fusions, and complex immuno-oncology genomic signatures,
`such as microsatellite instability (MSI) and tumor mutational burden
`(TMB) (Table 2). For a complete list of genes, visit www.illumina.com/
`products/by-type/clinical-research-products/trusight-oncology-500-
`ctdna.html.
`
`Table 2: Examples of variants detected using TruSight
`Oncology 500 ctDNA
`Variant type
`
`Relevant examples
`
`SNVs and indels
`
`Gene fusions
`
`EGFR, POLE, TMPRSS2, BRAF
`
`ALK, ROS1, NTRK, RET
`
`CNVs
`
`MSI
`
`TMB
`
`HER2
`
`MSI-Score
`
`TMB-Score
`
`Proven technology for detecting low-level
`biomarkers
`Using proven Illumina sequencing by synthesis (SBS) chemistry,
`TruSight Oncology 500 ctDNA enables comprehensive genomic
`profiling from just 30 ng cfDNA, making it an ideal alternative for
`use when tissue testing is not available. Library preparation takes
`advantage of target enrichment chemistry, using biotinylated probes
`and streptavidin-coated magnetic beads to enrich for selected targets
`from DNA-based libraries. Targeted hybridization–capture enrichment
`chemistry uses probes that are large enough to impart high binding
`specificity, but still allow hybridization to targets containing small
`mutations. This approach reduces sample dropouts in the presence of
`both natural allelic variations and sequence artifacts.
`
`Because ctDNA represents a small fraction of cfDNA, powerful
`methods are required to separate signal from noise. Library
`preparation incorporates unique molecular identifiers (UMIs) that
`enable ultra-low frequency variant identification.3 TruSight Oncology
`500 ctDNA libraries are sequenced on the NovaSeq 6000 System
`at high depth (400M reads per sample at ~ 35,000×) to enhance
`sensitivity. The result is the ability to detect mutations at 0.5% variant
`allele frequency (VAF) for small variants, with 95% analytical sensitivity
`and > 99.995% analytical specificity (Table 3).
`
`Table 3: Detection of low-level variants with high accuracy
`Variant type
`Analytical sensitivitya Analytical specificityb
`
`Small variants (≥ 0.5% VAF)
`
`Gene amplifications (≥ 1.4-
`fold change)
`
`Gene deletions (≤ 0.6-fold
`change)
`
`Gene fusions (0.5%)
`
`MSI high detection (≥ at 2%
`tumor fraction)
`
`≥ 95%
`
`≥ 95%
`
`≥ 95%
`
`≥ 95%
`
`≥ 95%
`
`≥ 99.995%
`
`≥ 95%
`
`≥ 95%
`
`≥ 95%
`
`≥ 95%
`
`a. Analytical sensitivity is defined as percent detection at the stated variant level
`b. Analytical specificity is defined as the ability to detect a known negative
`
`Accurate and accelerated analysis
`
`DRAGEN TruSight Oncology 500 ctDNA Analysis Software uses
`accelerated, fully integrated bioinformatics algorithms to overcome
`cfDNA challenges and ensure optimal assay performance. The
`software performs sequence alignment, error correction by collapsing
`the sequence, then variant calling based on the raw data. Duplicated
`reads and sequencing errors are removed without losing signal
`for low-frequency variants while yielding high-sensitivity variant
`calling results.
`
`Unlike qualitative results from PCR-based assays, DRAGEN TruSight
`Oncology 500 ctDNA Analysis Software provides a quantitative MSI
`score derived from > 2400 homopolymer MSI marker sites. For TMB
`analysis, the DRAGEN software optimizes sensitivity by measuring
`both nonsynonymous and synonymous SNVs and indels. After variant
`calling and error correction, the accuracy of TMB measurement
`is further enhanced by filtering germline variants, low-confidence
`variants, and variants associated with clonal hematopoiesis of
`indeterminate potential.
`
`2 | 1170-2019-006-D
`
`For Research Use Only. Not for use in diagnostic procedures.
`
`Prepare Library | Sequence | Analyze Data
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`

`

`Case 1:20-cv-01644-RGA Document 1-54 Filed 12/03/20 Page 4 of 5 PageID #: 1311
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`100%
`
`80%
`
`60%
`
`40%
`
`20%
`
`0%"
`
`Detection rate
`
`G1202R
`
`F1174L
`
`T790 M
`
`DRAGEN TruSight Oncology 500 ctDNA Analysis Software runs on
`a local Illumina DRAGEN Server v3.* This ultra-rapid platform offers
`enhanced hardware and software that reduce data analysis time by
`~85%, or from nine days to ~20 hours (Table 4).
`
`PierianDx Clinical Genomics Workspace completes tertiary analysis.
`Simply upload variant report files directly into the Clinical Genomics
`Workspace cloud. Clinical Genomics Workspace performs variant
`annotation and filtering for smooth interpretation and reporting. From
`thousands of variants in the genome, Clinical Genomics Workspace
`filters and prioritizes biologically relevant variants for the final
`automated, customizable genomic report. The entire workflow, from
`cfDNA to consolidated variant reporting, takes only five days (Figure 2).
`
`Extensive validation delivers accurate and
`highly reproducible results
`
`To demonstrate the high-quality results achieved with TruSight
`Oncology 500 ctDNA, Illumina performed various studies evaluating
`the ability to call SNVs, CNVs, gene fusions, TMB, and MSI (Figures 3
`and 4, Tables 5 and 6).
`
`Table 4: Time required for data analysis is reduced with the
`onsite DRAGEN Server v3
`
`Data analysis step
`
`Non-DRAGEN
`solutiona
`
`BCL conversion
`
`Alignment + collapsing
`+ realignment
`
`Gene fusion calling
`
`Variant calling
`
`Total time
`
`6 hours
`
`170 hours
`
`10 hours
`
`24 hours
`
`~9 days
`
`TruSight Oncology
`500 ctDNA DRAGEN
`Analysis Software
`
`1 hour
`
`11 hours
`
`2 hours
`
`8 hours
`
`~20 hours
`(~85% reduction)
`
`a. Single node (128G memory, 24 cores CPU), nonparallelized pipeline for 24 samples
`using an S4 flow cell
`
`* A cloud-based DRAGEN TruSight Oncology 500 ctDNA Analysis Software solution is
`coming soon.
`
`ALK
`
`EGFR
`
`KRAS
`
`1.00%
`
`0.50%
`
`0.25%
`
`0.10%
`
`Figure 3: Small variant detection at low VAF—Samples with known VAF for
`each variant were diluted to values ranging from 0.10-1.00% VAF. Five replicates
`of each sample were analyzed with TruSight Oncology 500 ctDNA using 30 ng of
`commercial reference control DNA.
`
`Summary
`
`TruSight Oncology 500 ctDNA is an NGS-based, multiplex assay
`that analyzes hundreds of cancer-related biomarkers from plasma
`simultaneously. Assay content is aligned with current guidelines and
`clinical trials, with the ability to detect multiple variant types from
`523 genes implicated in various tumor types, without requiring multiple
`samples for iterative testing. TruSight Oncology 500 ctDNA also
`provides assessment of immuno-oncology and emerging biomarkers
`(TMB, MSI, NTRK, and ROS1). Taking advantage of extensive genomic
`content, industry- leading sequencing technology, and enhanced
`software, TruSight Oncology 500 ctDNA provides an integrated
`solution for accelerating clinical research projects, in your own
`laboratory with minimal operational and analysis complexity.
`
`B.
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.08
`
`(Jensen-Shannon Distance Sum)
`
`MSI score
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Sample
`
`A.
`
`160
`
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`Mutations per Mb
`
`A B C D
`
`A B C D A B C D
`
`A B C D A B C D A B C D
`
`1
`
`2
`
`Operator
`
`3
`
`4
`Sample
`Operator A B
`
`5
`
`6
`
`Figure 4: Reproducible TMB and MSI measurement—(A) TMB was evaluated in six different plasma samples across four operators in replicate. (B) MSI was
`evaluated in three nucleosomal prepped cell lines with known MSI-high status (1-3) and three cfDNA samples from low prevalence MSI-high tumors (4-6) across two
`different operators (green, blue)..
`
`For Research Use Only. Not for use in diagnostic procedures.
`
`1170-2019-006-D | 3
`
`
`
`
`
`
`
`S752_I759 delE746_A750 delL858R L747_P753>S
`
`G12C
`
`G12D
`
`Q61H
`
`Q61R
`
`Prepare Library | Sequence | Analyze Data
`
`

`

`Case 1:20-cv-01644-RGA Document 1-54 Filed 12/03/20 Page 5 of 5 PageID #: 1312
`
`Table 5: Sensitive detection of CNVs
`Expected
`Observed
`fold-change
`mean
`
`Gene
`
`Amplifications
`
`AKT2
`
`BRAF
`
`BRCA2
`
`CCND3
`
`CDK6
`
`FGF14
`
`FGF3
`
`FGF4
`
`1.4
`
`1.5
`
`1.8
`
`1.5
`
`1.5
`
`1.3
`
`2.1
`
`1.4
`
`1.4
`
`1.5
`
`1.5
`
`1.4
`
`1.5
`
`1.5
`
`1.6
`
`1.2
`
`Learn more
`
`Standard
`deviation
`
`Detection
`rate
`
`To learn more about TruSight Oncology 500 ctDNA, visit
`www.illumina.com/tso500-ctDNA
`
`To more about the DRAGEN Bio-IT Platform, visit
`www.illumina.com/DRAGEN
`
`0.02
`
`0.01
`
`0.01
`
`0.01
`
`0.01
`
`0.01
`
`0.01
`
`0.01
`
`0.01
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`FGFR2
`
`MET
`
`MYC
`
`Deletions
`
`BRCA1
`
`BRCA2
`
`AR
`
`1.3
`
`1.4
`
`1.7
`
`0.8
`
`0.8
`
`0.7
`
`1.5
`
`1.5
`
`1.8
`
`0.8
`
`0.8
`
`0.8
`
`0.02
`
`0.02
`
`0.01
`
`0.01
`
`0.01
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`Samples with known fold-changes for gene amplifications and deletions were evaluated
`using TruSight Oncology 500 ctDNA with 30 ng of cfDNA input. Five replicates of each
`sample were analyzed.
`
`Table 6: Gene fusion detection at low VAF
`Expected
`Observed
`VAF
`VAF
`
`Gene fusion
`
`Standard
`deviation
`
`Detection
`rate
`
`References
`1. Parikh AR, Leshchiner I, Elagina L, et al. Liquid versus tissue biopsy for
`detecting acquired resistance and tumor heterogeneity in gastrointestinal
`cancers. Nat Med. 2019;25(9):1415-1421.
`2. Leighl NB, Page RD, Raymond VM, et al. Clinical Utility of Comprehensive
`Cell-free DNA Analysis to Identify Genomic Biomarkers in Patients with
`Newly Diagnosed Metastatic Non-small Cell Lung Cancer. Clin Cancer Res.
`2019;25(15):4691-4700.
`Illumina (2017) TruSight Oncology UMI Reagents. www.illumina.com/
`content/dam/illumina-marketing/documents/products/datasheets/trusight-
`oncology-umi-reagents-datasheet-1000000050425.pdf. Accessed May 4,
`2020.
`
`3.
`
`Ordering information
`
`Product
`
`TruSight Oncology 500 ctDNA Kit
`
`TruSight Oncology 500 ctDNA Kit plus
`PierianDx Interpretation Report
`
`NovaSeq Reagent Kits
`
`Quantity
`
`Catalog no.
`
`48 samples/
`16 indexes
`
`48 samples/
`16 indexes
`
`20039252
`
`20043410
`
`100%
`
`NovaSeq 6000 S2 Reagent Kit v1.5
`
`300 cycles
`
`20028314
`
`FGFR2-
`COL14A1
`
`NPM1-ALK
`
`FGFR3-
`BAIAP2L1
`
`NPM1-ALK
`
`EML4-ALK
`
`CCDC6-RET
`
`FGFR2-
`COL14A1
`
`4.1%
`
`3.4%
`
`3.4%
`
`2.4%
`
`1.7%
`
`1.0%
`
`0.9%
`
`4.2%
`
`0.7%
`
`0.7%
`
`0.4%
`
`0.5%
`
`0.7%
`
`0.4%
`
`0.5%
`
`0.2%
`
`0.2%
`
`0.1%
`
`0.1%
`
`0.1%
`
`0.1%
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`100%
`
`NovaSeq Xp 4-Lane Kit v1.5
`
`On-premise variant reporting
`
`DRAGEN TruSight Oncology 500 ctDNA
`Analysis Software, Level 1
`
`DRAGEN TruSight Oncology 500 ctDNA
`Analysis Software, Level 2
`
`DRAGEN TruSight Oncology 500 ctDNA
`Analysis Software, Level 3
`
`1-year license
`
`20042100
`
`1-year license
`
`20042101
`
`1-year license
`
`20042102
`
`NovaSeq 6000 S4 Reagent Kit v1.5
`
`300 cycles
`
`1 kit
`
`20028312
`
`20043131
`
`EML4-ALK
`
`NCOA4-RET
`
`EML4-ALK
`
`NPM1-ALK
`
`CCDC6-RET
`
`0.7%
`
`0.5%
`
`0.5%
`
`0.5%
`
`0.2%
`
`0.2%
`
`0.1%
`
`0.8%
`
`0.1%
`
`0.2%
`
`0.1%
`
`0.0%
`
`0.2%
`
`0.0%
`
`0.1%
`
`100%
`
`100%
`
`100%
`
`100%
`
`Samples with known gene fusion allele frequencies ranging from ~0.5-4% were evaluated.
`Five replicates of each sample were analyzed using TruSight Oncology 500 ctDNA with
`30 ng cfDNA input.
`Gene fusion directionality reported based on known expression. Consult the TruSight
`Oncology 500 ctDNA Local App User Guide for more information on DNA-based fusion
`directionality.
`
`DRAGEN TruSight Oncology 500 ctDNA
`Analysis Software, Level 4
`
`DRAGEN TruSight Oncology 500 ctDNA
`Analysis Software, Level 5
`
`DRAGEN TruSight Oncology 500 ctDNA
`Analysis Software, Level 6
`
`DRAGEN TruSight Oncology 500 ctDNA
`Analysis Software, Level 7
`
`DRAGEN TruSight Oncology 500 ctDNA
`Analysis Software, Level 8
`
`1-year license
`
`20042103
`
`1-year license
`
`20042104
`
`1-year license
`
`20042105
`
`1-year license
`
`20042106
`
`1-year license
`
`20042107
`
`Illumina DRAGEN Server v3
`
`1 server
`
`20040619
`
`Illumina DRAGEN Server Advance
`Exchange Plan
`
`Illumina DRAGEN Server Installation
`
`20032797
`
`20031995
`
`Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
`© 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. 1170-2019-006-D QB9370
`
`For Research Use Only. Not for use in diagnostic procedures.
`
`1170-2019-006-D | 4
`
`Prepare Library | Sequence | Analyze Data
`
`