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Technical brochure DEVELOP YOUR LAB, YOUR WAY The FLOW Solution: Expand your potential today

Redesign your lab with the FLOW Solution The FLOW Solution is a highly flexible modular, semi-automated data and sample workflow. It seamlessly processes various sample and tube types in a completely traceable barcoded manner from primary sample handling, NA extraction and setup to q. LIMS connectivity, middleware software management and a clever IT structure enables a smooth data transition without leaving room for manual errors. The modular instrument and assay concept allows you to truly develop your lab, your way. FLOW Flex FLOW Flex is the minimal instrument configuration necessary to be supported by the FLOW Software. It consists of a Setup Instrument, a MagNA Pure 96 and a Roche q Instrument. FLOW Flex operates with one pipetting robot used for both Primary Handling and Setup. It generates a non linear sample workflow with dual use of the Roche Liquid Handling Platform ( Setup Instrument). FLOW Classic FLOW Classic is the same instrumentation configuration as FLOW Flex with an additional pipetting robot. This could be either a Primary Handling Instrument or a Setup Instrument. In this configuration, the workflow is linear and has dedicated tasks for each pipetting robot. 2

Expand your possibilities There are two ways to grow your FLOW Solution: Install an additional FLOW Flex or FLOW Classic or add extraction and amplification systems to an existing workflow. Here are some examples for options: FLOW Fusion FLOW Fusion is a software application that allows you to run two separate FLOW installations in a coordinated manner. It allows for independent loading of samples and generates more flexible workflow options. This software tool operates both, linear and nonlinear workflows. To support this configuration a FLOW Software Additional License is necessary. FLOW Fusion and FLOW Software Additional License will be available at the launch of FLOW Software version 2.1. FLOW Ultra FLOW Ultra is a configuration which expands one FLOW installation with multiple MagNA Pure Instruments (up to 3) and q Instruments (up to 5). This option is supported in linear and nonlinear workflows and only one FLOW Software license is required. You may also combine FLOW Ultra with FLOW Fusion. 3

FLOW Primary Handling Instrument Works for different sample types Flexibly accepts primary & secondary sample tubes Pipets sample into MagNA Pure 96 Processing Cartridge Offers clot detection and a closed housing Comprehensive process surveillance GMP manufactured by Hamilton Primary Handling Instrument Purpose TypeBenchtop instrument Primary Handling for the Roche MagNA Pure 96 System Transfer of samples from common tube types into the Roche MagNA Pure 96 Processing Cartridges Dimensions 112.4 90.3 100.6 cm (W D H) Weight Run time Setup time types Supported primary/ secondary tube types Plate capacity volume Liquid handling Liquid level detection Min. pipetting volume Barcode scanning System sterility Disposables Tips 163 kg Transfer of 96 samples from sample tubes to plates in approximately 20 minutes depending on sample material Up to 15 minutes to manually place samples, tips, and plates onto loading tray Stage loading, including barcode reading is performed automatically Without pretreatment: Whole blood, serum, plasma, cell suspension (resuspended in PBS), saliva, urine With pretreatment: BAL, stool, swab eluates, CSF, sputum 15 100 mm, 15 75 mm, 12 100 mm, 12 75 mm Adapters to fit FLOW Tubes (2 ml) Can support up to 256 samples per run 1 Processing FLOW Cartridge carrier for maximum 3 MagNA Pure 96 Processing Cartridges 50 500 µl, 1,000 µl for 48 samples only One pipetting arm with 4 independently controlled air displacement channels Capacitive liquid level detection (clld) 50 µl for sample, 20 µl for internal control Barcode scanner mounted on the autoload slide for tips and tubes Barcode scanner fixed on the instrument deck for plates UV light Tip High Vol, 1 ml with Filter Plates 96 well MagNA Pure 96 Processing Cartridges, Note: Maximum volume depends on used sample volume Tubes FLOW Tubes (2ml) 4

MagNA Pure 96 Instrument Offers high speed nucleic acid isolation for up to 96 samples in less than 1 hour Flexibly accepts more than 10 different sample types within one run Provides for result safety through run surveillance and tracking Consistent sample handling with excellent intra run, inter-run, and lot-to-lot precision GMP manufactured, CE-IVD registered MagNA Pure 96 Instrument Purpose Type The MagNA Pure 96 System purifies DNA, RNA, and viral nucleic acids from a wide range of starting materials using magnetic glass particle technology (MGP) Benchtop instrument Dimensions 136 81.5 100 cm (W D H) Weight Run time Racks number Plate capacity volume 235 kg Elution volume 50 to 200 µl Liquid handling Liquid pressure detection Barcode scanning System sterility Disposables Tips Plates Reagents System fluid Prefilled, ready-to-use reagents Approximately 50 to 60 minutes for 200 μl sample volumes Approximately 80 to 90 minutes for 500 μl sample volumes Drawer-like loading racks for extraction reagents 1 to 96 reactions per run 1 for MagNA Pure 96 Processing Cartridges 1 for MagNA Pure 96 Output Plates 50 to 500 μl with 96 reactions per run, 1,000 µl for 48 samples only Two robotic arms: 1. Reagent head with four individually controlled fluid channels 2. Process head with a 96-nozzle pipette head (CO-RE tip technology) Liquid pressure sensor Internal and external barcode scanner UV lights MagNA Pure 96 Filter Tips (1,000 µl) MagNA Pure 96 Processing Cartridges MagNA Pure 96 Output Plates MagNA Pure System Fluid Internal Container, for 2 L, or MagNA Pure System Fluid External Container, for 5 L MagNA Pure 96 DNA and Viral NA Small Volume Kit (CE-IVD) MagNA Pure 96 DNA and Viral NA Large Volume Kit (CE-IVD) MagNA Pure 96 Cellular RNA Large Volume Kit (For life science research only. Not for use in diagnostic procedures.) 5

FLOW Setup Instrument Data driven pipetting Highly flexible Setup 96 and 384 well plate capability Automated master mix creation GMP manufactured by Hamilton Supports FLOW Primary Handling Roche Liquid Handling Instrument* Purpose TypeBenchtop instrument Primary Handling and/or Setup Dimensions 112.4 90.3 100.6 cm (W D H) Weight180 kg Setup time Run time Up to 15 minutes Dependent on number of samples, complexity of reaction mix setup, and number of total reactions; 30-50 minutes for most common applications Plate capacity 5 positions for LightCycler 480 Multiwell Plates 3 positions for the MagNA Pure 96 Output Plates 2 positions for RT reaction plates (requires external incubation) Automated master mix preparation For ready-to-use master mixes, detection mixes, or single components Up to 3 components (enzyme, water, additional component) can be combined for master mix reactions Up to 3 components (forward primer, reverse primer, probe) can be combined for reaction mix preparation Supports up to 21 components to create one master mix Liquid handling 8 independent 1 ml pipetting channels Liquid level detection Capacitive liquid level detection (clld) Minimal pipetting volume 5 μl to empty well, 2 μl to partially filled well in 384-well, and 5 μl to empty well, 5 μl to partially filled well in 96-well Barcode scanning Barcode scanner mounted on the autoload slide for tips and tubes Barcode scanner fixed on the instrument deck for plates Disposables Tips 1 ml, 300 µl and 50 µl filter tips Plates MagNA Pure 96 Output Plates LightCycler 480 Multiwell Plates 96 wells and 384 wells (Specific plates with 4 barcodes only) Tubes FLOW Tubes (2ml) * All specs described here are relevant for Setup only. For Primary Handling specifications refer to Primary Handling Instrument specification on page 4. 6

Roche q Instrument Speeds through 40 cycles in 40 60 minutes Well established industry standard with thousands of placements worldwide Top notch in: Temperature accuracy Sensitivity Connection of up to five 96-well or 384-well Roche q Instruments GMP manufactured Roche q Instrument Purpose Type q reaction in 96 or 384 plate formats Benchtop instrument Dimensions 57.4 58.8 49.7 cm (W D H) Weight Run time Reactions per run Probe /assay format Multiplexing capability 55 kg < 40 min. for 40 cycles in 384-well plate format 40 cycles, 2-step protocols: < 40 min. (384) 40 cycles, 2-step protocols: < 60 min. (96) Up to 96 or 384 reactions per run (384 well block available for LightCycler 480 only) Intercalating dyes and Hydrolysis probes 6x Cq uniformity SD < 0.2 Temperature accuracy ± 0.2 C of target temperature FLOW Reaction volumes 7 20 μl (384)/10 100 μl (96) Analysis type Disposables Absolute quantification based on 2nd derivative maximum method Plates LightCycler 480 Multiwell Plates 96/384 wells (Specific plates with 4 barcodes only) 7

FLOW Software: Connect your lab, your way Applications and performance data 1. Qualitative Detection of multiple targets FLOW Software Application Qualitative and Quantitative Analysis Run time used Approximately 4 hours depending on number and type of sample materials used, complexity of setup, and used profile Throughput Up to 256 sample extractions per run (with 3 MagNA Pure 96 Instruments) Assay flexibility Detect a multitude of targets per sample and per plate; use one or multiple plates for separate run profiles Configurations FLOW Classic, FLOW Flex, FLOW Fusion and FLOW Ultra (Check page 2 and 3 for details) Hands-on time 20 30 sec. per sample for the complete FLOW Solution Experimental Goal: Detect 3 different targets and internal control with quadruplex. Daily maintenance and cleaning activities approximately 15min. (for all instruments) FLOW Software The FLOW Solution is used for many applications. Here is an example of a multiplex for 3 targets and an internal control. anages the entire workflow M Manages information exchange between all modules Communicates with LIS system: LIMS download of sample information LIMS download of test requests Generates work lists Reports test results back to LIMS Experimental Set Up: A multiplex assay with 3 different target genes and one internal control was designed. Negative process and positive controls were used to validate the result. FLOW setup Separate setup for all benchtop instruments and FLOW control unit possible (Multi room concept) Overlapping runs hen purification of first order is finished, a new order can be started approximately every 1.5 hours. W This results in approximately 4 overlapping FLOW runs per day for FLOW Classic and 3 for FLOW Flex. Retests Yes, from samples and eluates Reflex tests Yes, from already tested eluates Materials Used: Primary sample material was suspended in S.T.A.R buffer. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the Pathogen Universal 200 protocol. q was performed with the RNA Process Control Kit which includes the LightCycler Multiplex RNA Master. Results and Data: Positive samples (Red curves, yellow circles) were found for all targets. Controls are in green. For each target positive samples were detected, showing the FLOW Solution can be run in a multiplex set up without crosstalk (Fig.2). Run schedule for FLOW Classic Complete one FLOW process in approximately 4 hours. When the purification of the first run is finished, a new workflow can be started (approximately every 1.5 hours), resulting in 4 overlapping FLOW runs* per day (see Fig.1). 4 runs on the FLOW Classic Solution every 8 hours Setup 1 Setup 2 Setup 8:00 9:00 10:00 11:00 12:00 13:00 WORKING DAY 3 Setup 14:00 15:00 4 16:00 Fig.2: FLOW Software display for Target 1,2,3 and internal control. Graphs shows target gene samples (red) controls (green). Fig.1: Run schedule for FLOW Classic. * A run depends on number and type of sample materials used, complexity of setup, and profile. 9

2. Quantitative Detection of a single target Quantification of targets is usually done with a standard curve generated on the level only. In the following experiment a standard curve was generated by extracting each standard individually in triplicates. With this approach the standard deviation of the whole workflow is measured rather than on the level only. Experimental Goal: Show quantification efficiency of the FLOW Solution. Materials used: Negative samples: K3-EDTA negative pooled human plasma. Positive samples: K3-EDTA negative pooled human plasma spiked with pcp- plasmid in the concentrations given in table 1. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the Pathogen Universal 200 protocol. q was performed with the LightCycler 480 Probes Master. Fig.3: Standard curve generated by the FLOW Software from the data shown in Graph 1. 3. Inter- and intra-run reproducibility Experiment Setup: All standards were used in triplicates with separate extraction on each; a pool was split to 5 samples (Tab.2). s together with standards were run on FLOW Classic. Results & Conclusion: A standard curve profile with a low error (0,0147) and a high efficiency (1,988) (Fig.3, Graph 1) was generated. Standard deviations (mean 0,11) were very low, which documents an excellent precision of the whole process (Tab.1). Separate extracted samples could be quantified at a late Cq with high reproducibility. In summary FLOW offers a reliable way to quantify samples. Graph 1: Standard curve as displayed in the Roche q instrument with 7, 10-fold dilutions. Target Standards Std Dev Mean Min Max Delta Target pcp-1 pcp-1 2,00E+04 0,37 33,69 33,36 34,09 0,73 28,77 28,77 0,10 30,22 30,14 30,32 0,18 Extraction Replicates 1 2,00E+05 2 28,93 29,01 2,00E+06 0,05 26,63 26,58 26,66 0,08 3 28,57 28,44 2,00E+07 0,02 23,16 23,14 23,18 0,04 4 28,80 28,86 2,00E+08 0,13 19,86 19,77 20,01 0,24 5 28,70 28,78 2,00E+09 0,04 16,33 16,30 16,37 0,07 2,00E+10 0,06 13,32 13,25 13,38 0,13 Tab.1: Cq values for the standard curve displayed in Fig.2 and Graph 1 generated with FLOW Classic. Standards are shown in copies/ml. Each standard was measured in separate isolated triplicates. 10 PSU Replicates Tab.2: s quantified with the standard curve in Fig.2. Rows are pipetting replicates on the PSU, while columns are replicate samples on a complete FLOW run. Fig.4: First run with one positive control in A1 and all the rest negative. Fig.5: Second run with controls in A1 (pos) and B1 (neg). Fig.6: Third run with controls in A1 (pos) and B1 (neg). Fig.7: Forth run with one positive control in A1 and all the rest negative. Consistent and reliable results are key for each workflow application. In this series of experiments, the reproducibility of data generated by the FLOW Solution was evaluated. Results: Negative samples stayed negative (Fig.4 and Fig.7). No cross-contamination was found on either intra- or interrun data. Experiment Goal: Check for intra- and inter-run reproducibility data for positive and negative results. Experimental Set Up: The following experiments were performed with FLOW Flex. Four consecutive runs on the same instruments were setup to check the reproducibility performance of the FLOW Solution. The first run was done with 95 negative samples and one positive control. Two follow up runs with a checkerboard setup were accomplished with 48 negative and 48 positive samples. Finally, the first run was repeated. Materials Used: Negative samples: K3-EDTA negative pooled human plasma. Positive samples: K3-EDTA negative pooled human plasma spiked with pcp-plasmid. Extraction was done with the MagNA Pure 96 DNA Viral NA Small Volume Kit, using the 200µl universal pathogen protocol. q was performed with the LightCycler 480 Probes Master. Positive samples showed a very high reproducibility with a standard deviation of 0,20 and 0,18 respectively (Fig.5 and Fig.6). The run to run difference was 0,04 Cq on the mean (Tab.3). Plate ID N Std Dev Mean Min Max 5120646 48 0,18 17,88 17,44 18,19 5120647 48 0,20 17,92 17,57 18,29 All 96 0,19 17,9 17,44 18,29 Tab.3: Positve results of the 2 checkerboard runs from Fig.5 and Fig.6. 11

www.flow.roche.com Published by Roche Molecular Systems, Inc. 4300 Hacienda Drive Pleasanton, CA 94588 USA 2015 Roche Molecular Systems Inc. The FLOW Solution is not available in all territories due to different national regulations. This document is not intended for use in the USA. For general laboratory use. LIGHTCYCLER and MAGNA PURE are registered trademarks of Roche. All other product names and trademarks are the property of their respective owners.