AFA Enabled Workflows From Specimen Lysis to NGS

AFA®- Enabled Workflows: From Specimen Lysis to NGS and LCMS Analysis APAC 2019 www. covaris. com

Sample Prep The current bottleneck in High Throughput Genome Analysis Proprietary 2

NGS Workflow with current non-multiplexed Library Prep Sample Prep NGS Library Prep Sequence Generation Data Analysis Proprietary 3

New multiplexed Library Prep addresses Throughput but creates new Bottleneck Sample Prep NGS Library Prep Sequence Generation Data Analysis The Bottleneck Today Proprietary 4

Adaptive-Focused Acoustics: Precisely tuned Energy for a variety of Molecular Biology Workflow Steps Tissue Rehydration • Cell Lysis • Lysate Homogenization • Disassociation of Macromolecular Complexes Contact-free Mixing of Reactions (including SPRI Clean-up) • DNA Shearing • • Proprietary 5

Adaptive Focused Acoustics (AFA) Technology Acoustic energy induces cavitation that allows biomolecules to be manipulated Thermal Control is Key Bath Sonicator Microcentrifuge Tube • • Unfocused Transducer Non-contact Probe Sonicator Microcentrifuge Tube • • Focused Waveguide Sample Contact Covaris AFA • • Focused, Non-contact Temperature regulated Proprietary 6

AFA Platforms = Automated and Reproducible Sample Prep Revolution R 230 LE 220 Rsc ME 220 M 220 E-Series S 220 8 -96 samples High throughput laboratory automation On-Deck with Liquid handler Automation - High-Throughput Covaris AFA consumable family; small and large sample volume processing, wide energy-range Covaris fully automation compatible consumables (SBS), low sample volume processing Proprietary 7

AFA-TUBE TPX = AFA Based Consumable Covaris Consumable for High Throughput Automation • • • Specially designed polymer-based consumable LE 220 Rsc and Revolution R 230 compatible PCR-tube morphology AFA treatment requires no fiber/beads RFID-enabled sample tracking, LIMS integration Compatible with most liquid handlers and on-deck processing units (SBS specifications) Proprietary 8 8

Sample Prep Bottlenecks – Not standardized or Automation Friendly Conventional Sample • • Bead Beating/Vortex Lyticases Harsh Chemicals Volume Transfer [LYSIS] • • • Phase Separation Vortex or Pipette Mixing Volume Transfer [Extraction] • • • Filter-based/vacuum manifold Pipette Mixing Volume Transfer Purification • Covaris AFA DNA Shearing Proprietary 9

Standardized, automated and simplified Workflows driven by AFA Sample [LYSIS] [Extraction] + • • • Purification DNA Shearing AFA Mild lysis buffer Small Volumes – AFA-TUBE compatible Automated Homogenization & Dissociation Contact-free mixing One-vessel Protocol • Direct to NGS Workflows • Automation Workflows, Consumables & Platforms • Standardized and Reproducible Sample Prep Proprietary 10

Cell Lysis and Extraction DNA and RNA • Lysis Buffer Choice is important • tru. POP : High Molecular Weight DNA • TLB : RNA, DNA (and protein) Proprietary 11

AFA-enhanced Lysis & Extraction of HMW DNA in the AFA-TUBE Sample • • High Molecular Weight DNA Lysis Buffer = tru. POP + tru. POP (30 -60µl) R 230 AFA Lyse and Homogenize + Magnetic Beads/ Bind Solution tru. POP facilitates AFA-enhanced cell lysis and extraction of high molecular weight DNA R 230 AFA Mix Magnetic Separation compatible with prokaryotic as well as eukaryotic cells developed for small volume use in combination with the AFA-TUBE • compatible with downstream enzymatic processing • https: //covaris. com/kits-andreagents/trupop/ Repeat Wash Buffer LE 220 Rsc 96 AFA-TUBE TPX Plate Full Automation Library-Ready DNA AFA Mix Elution Buffer AFA Mix & Shear (optional) Proprietary 12

NGS Library Workflow: Contact-free mixing during SPRI Cleanup • AFA-mediated mixing for bind, wash and elute during SPRI clean-up. • AFA-mediated mixing during enzymatic reactions, e. g. , Adapterligation. AFA Mixing Pipette Mixing https: //covaris. com/wp-content/uploads/M 020088. pdf Proprietary 13

tru. POP: Automation for a wide Variety of Sample Types / HMW DNA Extraction Workflow AFA + tru. POP Lysates are compatible with most purification workflows & fully automatable from sample to DNA in one vessel. S. cerevisiae Y. lipolytica Most conventional kits rely on multiple up-front steps to homogenize sample. Bead beating or vortexing for tough to lyse specimens complicates automation. E. coli L. monocytogenes Sample Cell count Yield Treatment Time Y. Lipolytica Culture S. Cerevisiae Culture E. coli (gram -) Culture L. monocytogenes (gram +) Culture hg. DNA whole blood 30 µl S. epidermidis (gram +) Colony ~ 3 x 107 ~ 5 x 107 ~ 4 x 108 ~1. 7 x 105 ~ 1 x 107 ~ 1. 16 µg ~ 0. 72 µg ~ 1. 98 µg ~ 1. 95 µg ~600 ng ~100 ng 60 min/plate 15 min/plate <3 min/plate 7 min/plate <1 min/strip hg. DNA-Blood Buffer Volume 30 ml 30 ml S. epidermidis Instrument LE 220 -plus LE 220 -plus ME 220 Proprietary 14

Whole Blood Nucleic Acid: From Sample to HMW DNA or RNA in the same Plate “ 96 tru. XTRAC Blood DNA” QIAamp 96 DNA Blood (Qiagen) Blood DNA Isolation 96 Well (Norgen) Number of Reagents 6 6 6 Reagent Volumes (96 samples) 6 ml - 20 ml 2 ml - 50 ml 2 ml - 60 ml Reagent Reservoirs 1 2 2 Lysate Mixing Step AFA Vortexing Vortex in Single Tube Centrifuge No Yes No Magnet Yes No Yes Lysate Transfer Step No Yes Incubator Yes (56 C) Yes (70 C) No Pipette Racks (a' 96) 2 4 2 Hands off Automation Yes No No Proprietary 15

https: //covaris. com/wp-content/uploads/ABRF 2019 -Poster_HMW-Extraction-Whole-Blood. pdf Proprietary 16

DNA Shearing during SPRI Clean-up: Normalize your DNA before Library Prep Sample + tru. POP (30 -60µl) AFA Lyse and Homogenize + Magnetic Beads/ Bind Solution AFA Mix Magnetic Separation Wash Buffer Pulsing Repeats/ Iterations Av. Mode SD %CV 10 591 29. 3 5 30 313 9. 3 3 60 182 10. 6 5. 8 LE 220 Rsc 96 AFA-TUBE TPX Plate Full Automation Library-Ready DNA Repeat AFA Mix Elution Buffer AFA Mix & Shear (optional) Proprietary 17

AFA-enhanced Lysis & Extraction of High Quality total RNA in the AFA-TUBE Sample • RNA Lysis Buffer = TLB (Tissue Lysis Buffer) TLB (30 -60µl) AFA Lyse and Homogenize + Magnetic Beads/ Bind Solution • TLB facilitates AFA-enhanced cell lysis and extraction of total RNA AFA Mix Magnetic Separation • compatible with prokaryotic as well as eukaryotic cells • developed for small volume use in combination with the AFA-TUBE • purification workflow essentially follows the one of HMW DNA extraction Repeat Wash Buffer LE 220 Rsc 96 AFA-TUBE TPX Plate Full Automation High Quality RNA AFA Mix Elution Buffer AFA Mix & Shear (optional) Proprietary 18

RNA from Bacteria: E. coli Cell Lysis & total RNA Extraction AFA-TUBE-TPX/LE 220 -Plus; bath temperature 12°C; n x 2 second pulsed AFA-treatment. Input 1 -2 x 108 cells from liquid culture (LB) in stationary phase Lysis Buffer: TLB Purification: tru. XTRAC FFPE RNA purification reagents (Covaris) 10. 00 100. 00 90. 00 80. 00 70. 00 60. 00 50. 00 40. 00 30. 00 20. 00 10. 00 BB 2 BB 3 No DNase RIN No AFA DV 200 (%) RIN Score RNA Quality 0. 00 DV 200 Proprietary 19

Lysis of Human Leukemia Cells and Extraction of High Quality total RNA 1 x 105 cells concentrated by brief centrifugation. After media was removed, TLB (30µl) was added and lysis was induced by AFA. • Yield: 1. 4 (+/-0. 2) µg • DV 200 >95% • RIN >9 Next: Determine amount of fulllength (5’ m. G capped). Proprietary 20

Total RNA extracted and purified from human Blood collected into PAXgene BCT AFA –enhanced Lysis Classical Vortex Lysis 191 [3. 3%] 192 [1. 9%] CV (%) 3. 3 1. 9 RIN Score 7. 8 5. 7 CV (%) 10. 3 17. 5 DV 200 84 73 CV (%) 10. 4 7. 0 Q-Ratio* (200/71) 0. 67 0. 56 Yield (ng by Qubit) [CV] * As described in: Kashofer et al. (2013) PLo. S ONE 8(7): e 70714. Proprietary 21

Cell Lysis and Co-Extraction of RNA and Protein Lysis Buffer Choice is important • TLB : RNA & Protein • Extract from FFPE Extract from fresh/frozen Cells (e. g. , FNA) • • Proprietary 22

Total RNA and Protein extracted and from FFPE Tissue • Protocol developed for Broad Institute: LMD tissue sections from deparaffinized FFPE slides. • Essentially a modified FFPE (t. NA) workflow. • TLB without Proteinase K (protein!) as lysis buffer. • RNA is extracted and purified in the supernatant. • Protein is extracted from remaining tissue pellet. Proprietary 23

AFA-Enhanced Workflows: Eliminate Bottlenecks Sample Prep direct to NGS & PCR No purification required (tru. POP) Cells/Tissue Microbiome Bacteria tru. XTRAC Extraction & Purification FFPE, cf. DNA, Ch. IP AFA Blood FFPE Sputum Yeast Liquid Biopsy Agnostic Workflows Wide Range of Samples/Species Adaptable Lysis Conditions High Throughput Automation No bead-beating, one-vessel lysis, extraction and purification Proprietary 24

So, what does this all mean? • Covaris R&D is focusing on Biomolecule Extraction (RNA/DNA & Protein) • AFA-TUBE Configuration • Small Sample size; small lysis volume • Lysis, extraction and purification in ONE AFA-TUBE • Purified DNA and RNA are ready for NGS, PCR… • Protein Extraction pursued in Collaboration (focus on Tryptic Digest LC-MS applications) • Co-extraction of RNA/DNA, RNA/Protein (future DNA/RNA/Protein) Proprietary 25