TS Tumor Panel (15 Genes) Overview Technical training TS Tumor Panel (15 Genes) Overview Targets relevant regions in 15 cancer-associated genes Content created by NCCN, ESMO, KOLs, and leading pharma companies Enhanced Sample-to-Data Detailed QC steps and simplified pre-defined variant report Workflow optimized for Oncology customers Low FFPE input (20ng) and quick TAT (~36 hours) Sensitive, High-Confidence Variant Detection Identifies somatic mutations at 5% variant allele frequency with >500x coverage Gastric

TruSight Tumor Panel (15 Genes) Sample-To-Data Workflow Solution Technical training TruSight Tumor Panel (15 Genes) Sample-To-Data Workflow Solution FFPE specimens Sample Library Prep Sequencing Analysis Tissue Guidelines Extraction Methods DNA Quant Guidelines FFPE optimized Low DNA-input Multiplex PCR Library QC Guidelines MiSeq V3 Leading Sequencers Sequencing QC MSR Pre-Defined Report Variant Studio 3rd Party Annotation Text underlined is additional QC/analysis that is included in TST 15. These are things that were not included for previous kits. Extends beyond library preparation to enable a more complete solution For Research Use Only. Not for use in diagnostic procedures.

TruSight Tumor Panel (15 Genes) Sample-To-Data Turn Around Time Technical training TruSight Tumor Panel (15 Genes) Sample-To-Data Turn Around Time DAY 1 DAY 2 DAY 3 LIBRARY PREP 7 hours SEQUENCING 27 hours ANALYSIS 1 hour DNA to Analysis in less than 3 days

TruSight Tumor Panel (15 Genes) Content List Key Benefits Gene content: created with industry guidance (NCCN, CAP), leading KOLs, and pharma Relevance: Selected genes have the most analytic relevance for translational tumor research Focused: Small panel ensures quick and comprehensive sample-to-data turnaround time Gene Associated Cancers Target AKT1 Breast E17K BRAF Melanoma*, Colon* Lung VK600E/K/R/M EGFR Lung* Exon 19, 20, 21(L858R), G719A/X, S7681, T790M, L861Q ERBB2 Breast, Lung A775_G776insYVMA FOXL2 Ovary C134W GNA11 Melanoma Q209L GNAQ KIT Gastric, Melanoma* Exons 8, 9, 10, 11, 13, 14, 17, 18 KRAS Colon*, Gastric, Lung* Codons 12, 13, 59, 61, 117, 146 MET Lung*, Colon, Gastric Focal Amplification NRAS Colon* PDGFRA Gastric, Melanoma Exons 12, 14, 18 PIK3CA Lung, Breast, Prostate Exons 9, 20 RET M918T TP53 Lung, Melanoma, Ovary, Colon Full CDS *Indicates in NCCN guideline for cancer type

TruSight Tumor Panel (15 Genes) Recommended QC Technical training TruSight Tumor Panel (15 Genes) Recommended QC STEP QC Method QC METRIC FFPE Tissue Sectioning of tissue with microtome ≥ 140 mm2 (non-melanoma) ≥ 20% tumor DNA Extraction Input concentration fluorometry ≥ 2 ng/µL Library Prep Final concentration by fluorometry ≥ 20 ng/µL final yield Final fragment size on gel (except NTC) ~350bp Sequencing Review of analysis report >93.5% bases >500X

TS Tumor Panel (15 Genes) Library Prep Technical training TS Tumor Panel (15 Genes) Library Prep Mix A Mix B 5 Steps! Multiplex PCR Index PCR Clean Up Validate Sequence

TS Tumor Panel (15 Genes) Multiplex PCR Chemistry Technical training TS Tumor Panel (15 Genes) Multiplex PCR Chemistry Two libraries per sample: Pool A & Pool B

TruSight Tumor Panel (15 Genes) Analysis and Reporting Technical training TruSight Tumor Panel (15 Genes) Analysis and Reporting Illumina Experiment Manager v1.10 Adds TruSight Tumor Panel (15 Genes) workflow and library prep IEM v1.10 will be released upon TST 15 FCS MiSeq Software v2.6 MSR TruSight Tumor Panel (15 Genes) Workflow Alignment and variant calling for TST 15. Creates pre-defined filtered report for easy data interpretation. MCS/MSR 2.6 will be released upon TST 15 FCS VariantStudio TruSight Tumor 15 customers receive license for VariantStudio Enables customized filtering of variants and reporting if pre-defined report is not sufficient

Technical training TruSight Tumor Panel (15 Genes) Pre-defined report makes variant calling easy Streamlined variant filtering and classification Easy to use automated analysis

TruSight Tumor Panel (15 Genes) Data Specifications Technical training TruSight Tumor Panel (15 Genes) Data Specifications PARAMETER DETAIL DNA input requirements 20 ng Panel size 44 kb Amplicon size ~150–175 bp Throughput 8 Samples per MiSeq v3 run Variant frequency 5% Coverage > 93.5% bases covered at > 500×

TruSight Tumor Panel (15 Genes) FFPE input Technical training TruSight Tumor Panel (15 Genes) FFPE input Library concentration vs. ΔCq. ΔCq is a metric used to assess the quality of FFPE DNA. Higher values reflect poorer quality DNA. A library yield of 20ng/ul is recommended for TruSight Tumor Panel (15 Genes).

TruSight Tumor Panel (15 Genes) TST 15 vs TST 26 variant calling Technical training TruSight Tumor Panel (15 Genes) TST 15 vs TST 26 variant calling Gene Coordinate Variant Protein TST 26 TST 15 Smp1 Rep1 MixA Rep2 MixB PDGFRA 55141055 A>G/G 567P 99.6 99.84 99.82 99.73 99.54 EGFR,EGFR-AS1 55249063 G>A/A 99.64 99.93 99.98 TP53 7578469 C>C/A 154G/V 14.4 18.6 20.33 7579472 G>C/C 72P/R 99.52 99.65 99.76 99.77 99.74 100% of variants detected by TruSight Tumor (26 Genes) were detected by TruSight Tumor (15 Genes) and reported at similar frequencies.

TST (15 Genes) – Kit Configuration Technical training TST (15 Genes) – Kit Configuration Library Prep Kit Content TruSight Tumor Kit (15 Genes) TruSight Tumor Panel (15 Genes) 24 Samples, 48 libraries 3 Boxes Includes Library Prep reagents, 24 index combinations, and purification beads Cat # OP-101-1001 Includes 1 Library Prep AND 3 MiSeq Sequencing Kits Cat# OP-101-1002 1 Library Prep Kit ONLY