cbot User Guide T G AC CG C FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Catalog # SY Part # Rev C

Transcription

1 cbot User Guide FOR RESEARCH USE ONLY ACGTACGTACGTACG TACGTACGTACGTACGTA A G T G AC CG C T AC CGT ILLUMINA PROPRIETARY Catalog # SY Part # Rev C

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3 Notice 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. ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT(S). FOR RESEARCH USE ONLY Illumina, Inc. All rights reserved. Illumina, illuminadx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iselect, CSPro, GenomeStudio, Genetic Energy, HiSeq, and HiScan are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Phusion is a trademark of Finnzymes Oy. Notice to Purchaser: Limited license (proofreading DNA polymerases). The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents (5,500,363 and 5,352,778) owned by New England Biolabs, Inc. to use this product. No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product. The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents owned by BIO-RAD Laboratories, Inc., to use this product. No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product. cbot User Guide iii

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5 Revision History v Revision History Revision history for part # : Revision Date Description of Change C May 2010 Updated software descriptions to cbot v1.1, which includes on-screen indicators for flow cell type and the option to bypass sensors. Added flow cell storage recommendation. Increased water wash volume to 12 ml and DECON to 10 ml. B March 2010 Added instructions for loading the HiSeq flow cell, and associated templates and primers. Added instructions for installing the HiSeq flow cell adapter plate. Added HiSeq Cluster Generation Kit catalog numbers and kit descriptions. Added reagent preparation instructions for Small RNA Sequencing Primer. Corrected centrifuge instructions for thawing cbot reagent plate. Added instructions for setting local date and time using the Time tab. Added monthly maintenance wash procedure. Removed safety labeling section. See the cbot Safety and Compliance Booklet for important cautions and warnings. A October 2009 Initial release cbot User Guide

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7 Table of Contents Notice iii Revision History v Table of Contents vii List of Tables ix Chapter 1 Overview Introduction Audience and Purpose cbot Components Technical Assistance Chapter 2 Getting Started Preparing Your Site Installing the cbot Configuring the cbot Changing the Adapter Plate Chapter 3 Using the Software Interface Introduction Start Screen Run Setup Screens Run Status Screen Shutting Down the cbot Remote Monitoring Overview Chapter 4 Using the cbot Introduction cbot Cluster Generation Kits Flow Cell Adapter Plates cbot Cluster Generation Workflow Preparing DNA Template for Cluster Generation Well Reagent Plate Thawing cbot Reagents cbot User Guide vii

8 viii Table of Contents Prepare Small RNA Primer Setting Up the Run Monitoring the Run Performing Post-Run Procedures Chapter 5 Maintenance and Troubleshooting Performing Periodic Maintenance Performing a Monthly Maintenance Wash Troubleshooting Appendix A Cluster Generation Chemistry Introduction Adapter Ligation and Selection Cluster Generation Appendix B Manual Settings Manual Commands Resetting the Barcode Scanner Protocol Editor Index Catalog # SY Part # Rev. C

9 List of Tables Table 1 Illumina General Contact Information Table 2 Illumina Customer Support Telephone Numbers Table 3 Shipping Container Dimensions Table 4 cbot Dimensions Table 5 Environmental Constraints Table 6 Sensor Status Icons Table 7 cbot Cluster Generation Kits Table 8 Adjustments to the Protocol for High Final DNA Concentrations Table 9 cbot Periodic Maintenance Table 10 Troubleshooting Run Problems Table 11 Protocol Editor Icons cbot User Guide ix

10 x List of Tables Catalog # SY Part # Rev. C

11 Chapter 1 Overview Topics 2 Introduction 2 Audience and Purpose 3 cbot Components 5 Technical Assistance cbot User Guide 1

12 2 CHAPTER 1 Overview Introduction The Illumina cbot Cluster Generation System provides complete automation of a complex process. With very little hands-on time and no reagent preparation, the cbot uses bridge amplification to create over 100 million DNA templates simultaneously in less than five hours. The cbot dispenses reagents from a pre-aliquoted 96-well plate, and controls reaction times, flow rates, and temperatures. The run is set up using the cbot software interface, which simplifies operation and provides a visual report of run status. An on-instrument barcode reader records the reagents and flow cell used for each experiment. Figure 1 Illumina cbot The cbot User Guide provides information about installing, operating, and maintaining the Illumina cbot. Additionally, this guide describes site requirements, instrument components, and software features. Audience and Purpose This guide is for laboratory personnel and other individuals responsible for: Operating the Illumina cbot Performing instrument maintenance Training personnel on the cbot Catalog # SY Part # Rev. C

13 cbot Components 3 cbot Components cbot Lid cbot features include an on-instrument touch screen monitor and barcode scanner. The thermal stage and reagent stage are located under the cbot lid. The waste bottle is conveniently stored in the waste bottle compartment. Touch screen monitor The touch screen monitor displays the software interface, which guides you through each step of the cluster generation process. For more information, see Using the Software Interface on page 21. Barcode scanner The on-instrument barcode scanner records the unique ID numbers of the reagent plate and flow cell used with the run. The cbot uses this information to verify the compatibility of the selected protocol and loaded reagents. Thermal stage The thermal stage holds the flow cell and controls its temperature throughout the run. For more information, see Thermal Stage on page 4. Reagent stage The reagent stage holds the 96-well reagent plate, the templates, and any custom or specialty primers. For more information, see Reagent Stage on page 4. Waste bottle compartment The waste bottle compartment holds a sensor-controlled 250 ml waste bottle that collects reagents after they have passed through the flow cell and manifold. Thermal Stage Reagent Stage Waste Bottle Compartment Barcode Scanner Touch Screen Monitor Figure 2 cbot Components cbot User Guide

14 4 CHAPTER 1 Overview Thermal Stage The thermal stage holds the flow cell and the manifold is seated over the flow cell. The flow cell clamp locks the flow cell and manifold in place. Outlet Clamp Flow Cell Clamp Flow Cell and Manifold Thermal Stage Sipper Comb Figure 3 Thermal Stage The manifold is a single-use component used to perform all chemistry steps of a cluster generation run. The inlet end of the manifold, positioned toward the front of the cbot, contains a series of sippers called the sipper comb. The sipper comb pierces the foil-sealed reagent tubes seated in the reagent plate and delivers the appropriate reagents to the flow cell. cbot sensors detect if the sipper comb is correctly snapped into place before allowing a run to proceed. The outlet end of the manifold, positioned toward the rear of the cbot, receives the liquid flowing out of the flow cell, and then transfers it to the waste container. The outlet clamp locks the outlet end in place. Reagent Stage The reagent stage holds a 96-well reagent plate, a 0.2 ml eight-tube strip containing the template, and an optional 0.2 ml eight-tube strip containing any additional primers required for applications that do not use the standard primers included in the reagent plate. The reagent plate is loaded onto the stage and locked into position by the white reagent plate lever. 96-Well Reagent Plate Reagent Plate Lever Templates Custom Primers Figure 4 cbot Reagent Stage Catalog # SY Part # Rev. C

15 Technical Assistance 5 Technical Assistance For technical assistance, contact Illumina Customer Support. Table 1 Illumina General Contact Information Illumina Website techsupport@illumina.com Table 2 Region Illumina Customer Support Telephone Numbers Contact Number North America toll-free ILMN ( ) United Kingdom toll-free Germany toll-free Netherlands toll-free France toll-free Other European time zones Other regions and locations ILMN ( ) MSDSs Material safety data sheets (MSDSs) are available on the Illumina website at Product Documentation If you require additional product documentation, you can obtain PDFs from the Illumina website. Go to When you click on a link, you will be asked to log in to icom. After you log in, you can view or save the PDF. If you do not already have an icom account, then click New User on the icom login screen and fill in your contact information. Indicate whether you wish to receive the icommunity newsletter (a quarterly newsletter with articles about, by, and for the Illumina Community), illuminotes (a monthly newsletter that provides important product updates), and announcements about upcoming user meetings. After you submit your registration information, an Illumina representative will create your account and login instructions to you. cbot User Guide

16 6 CHAPTER 1 Overview Catalog # SY Part # Rev. C

17 Chapter 2 Getting Started Topics 8 Preparing Your Site 11 Installing the cbot 15 Configuring the cbot 20 Changing the Adapter Plate cbot User Guide 7

18 8 CHAPTER 2 Getting Started Preparing Your Site This section describes the cbot physical dimensions, electrical requirements, and environmental constraints. Lab Space Reserve sufficient laboratory space to unpack your cbot. The cbot shipping container has the following dimensions: Table 3 Shipping Container Dimensions Width Height Depth Weight 52 cm (20.5 in.) 51 cm (20.25 in.) 75 cm (29.5 in.) 34 kg (75 lbs.) Reserve sufficient laboratory space and a lab bench that is capable of safely holding the weight of the cbot. The cbot has the following dimensions: Table 4 cbot Dimensions Width Height (Lid Closed) Height (Lid Open) Depth Weight 38 cm (15 in.) 39 cm (15.5 in.) 70 cm (27.5 in.) 71 cm (25.5 in.) 31 kg (68 lbs.) 70 cm (27.5 in.) 39 cm (15.5 in.) 71 cm (25.5 in.) Figure 5 cbot Dimensions, Lid Open Power Connections Place the instrument at least six inches away from the wall so that you can easily reach the power connections on the back of the cbot. Catalog # SY Part # Rev. C

19 Preparing Your Site 9 Electrical Requirements Power Consumption The line voltage of the cbot is volts AC, running at 50/60 Hz. The cbot typically consumes approximately 300 watts and has a maximum of 500 watts. It should be connected to an uninterruptible power supply (UPS) to protect your cbot in the event of a power surge or loss. The cbot requires a 6 10 amp grounded, dedicated line with proper voltage ( volts AC) and an electrical ground. Electrical Standards The cbot is certified to these standards: Conforms to UL STD Certified to CSA STD C22.2 No Low Voltage Directive (Product Safety) IEC EMC Directive IEC Power Cords The cbot comes with an international standard IEC receptacle and is shipped with a region-specific power cord. Allow a maximum of 2 m (6 ft.) between the instrument AC power inlet and facility power. CAUTION Never use an extension cord to connect your cbot. Fuses Use only the manufacturer s recommended fuse specification, as described below. Littelfuse: part # HXP Time Lag (SLO BLO) Type 250 VAC, 5A 5 20 mm Do not replace any fuses. Contact Illumina Technical Support for preventative maintenance. Coin Cell Battery The coin cell battery on the instrument computer motherboard is not a user-replaceable part. The coin cell battery is not rechargeable. Under no circumstances attempt to recharge the battery. cbot User Guide

20 10 CHAPTER 2 Getting Started Environmental Constraints Consult your facilities department regarding environmental constraints before installing your cbot. Table 5 Environmental Constraints Condition Temperature Humidly Acceptable Range 22 C ±3 C Relative humidity between 20 80%, non-condensing Ventilation Maximum thermal output is approximately ~2550 Btu/h (~750 W) Catalog # SY Part # Rev. C

21 Installing the cbot 11 Installing the cbot Installing the Illumina cbot takes approximately 15 minutes. It is shipped in one box with the following contents: cbot instrument HiSeq flow cell adapter plate Power cord Waste bottle Quick setup poster User guide Exterior Components Lid Covers the thermal stage, reagent stage, and wash reservoir. Waste bottle compartment Holds the sensor-controlled waste bottle. Barcode scanner Scans barcodes of run components. Monitor Displays the cbot user interface. Power switch Turns on your instrument. Start switch Launches the cbot software. The start switch is located to the left of the waste bottle compartment. Lid Waste Bottle Compartment Monitor Barcode Scanner Figure 6 cbot Exterior Components Power Switch NOTE If your cbot was stored in a cold location, allow it to reach room temperature before proceeding. NOTE Save the packing material and shipping box for use in case you ever need to ship your cbot to Illumina for servicing. cbot User Guide

22 12 CHAPTER 2 Getting Started Unpack and Inspect 1. Snip the shipping straps wrapped around the shipping box. NOTE Do not cut the shipping tape on top of the box. The top of the shipping box is designed to lift off of the instrument. 2. Lift the top of the shipping box straight up using the cutout handles on each end. 3. Locate the power cord and waste bottle, and set them aside. NOTE Never lift the cbot from the front or rear panels, barcode scanner, or any location other than the long side panels. These panels are not designed to support the weight of the instrument. 4. With one person standing in front of the instrument and one person standing behind it, lift the instrument from the packaging by placing your hands under the long side panels. Cutouts in the foam packing mark the best positions for lifting. 5. Place the cbot on a lab bench or suitable location, positioning it so you can inspect all sides of the instrument. 6. Inspect the condition of the following components: Make sure the instrument panels are intact. Inspect the touch screen for cracks or scratches. Ensure that coolant did not leak during shipment. Check for moisture inside the box. If any of the above items are unsatisfactory, immediately contact Illumina Technical Support for further instructions. Ethernet Connection AC Power Inlet Coolant Reservoir Cap Coolant Level Figure 7 cbot Rear Panel Components Catalog # SY Part # Rev. C

23 Installing the cbot Check the coolant level through the view port on the rear panel of the instrument. The green liquid coolant level should be just below the coolant reservoir cap. Coolant Level Good Figure 8 Coolant Level Coolant Level Low If the coolant level is low, top up the coolant. Use the same coolant provided with the Illumina sequencing instrument (part # ). a. Use a wide coin or flat screwdriver to remove the reservoir cap. b. Fill the reservoir with Illumina-supplied coolant to just below the reservoir fill port. c. Replace the reservoir cap. Position and Connect 1. Remove the protective foam block from the barcode scanner. 2. Lift the cbot lid. 3. Remove the protective foam block from the reagent area. 4. Press the top-right corner of the waste compartment door, then quickly release to open the door. 5. Remove the protective foam block from the waste bottle compartment. 6. Lift the waste bottle lever and position the waste bottle under the lever, slightly tipping the mouth of the bottle to clear the spout. Waste Bottle Lever Waste Bottle Figure 9 Install the Waste Bottle cbot User Guide

24 14 CHAPTER 2 Getting Started 7. Lower the lever to secure the waste bottle into position. The spout should sit inside the mouth of the wash bottle. NOTE Always use the Illumina-supplied waste bottle. Do not use any other waste bottle in the cbot waste compartment or the electronic sensors that detect the presence and fill level of the waste bottle will not function properly. 8. Remove the protective sheet from the cbot monitor. 9. Connect the power cord to the AC power inlet on the rear panel, and then connect to facility power. Ethernet Connection AC Power Inlet Figure 10 Power Inlet and Ethernet Connection 10. If you are planning to configure your cbot for a network connection, connect a user-supplied RJ45 Ethernet cable to the Ethernet port. 11. Position your cbot on the lab bench at least cm (6 in.) away from the wall or other obstruction. Ensure it is sitting level on the bench. 12. Locate the power switch on the right side of the instrument and toggle the switch to the ON position. 13. Locate the start button to the left of the waste bottle compartment door and press to start the cbot software. Start Button Power Switch Figure 11 cbot Start Button The software initiates a start-up routine. When the start-up routine is complete, the Start screen appears on the monitor. 14. Proceed to Configuring the cbot on page 15. Catalog # SY Part # Rev. C

25 Configuring the cbot 15 Configuring the cbot You can configure the cbot using the touch screen monitor. Configuration steps include naming your instrument, determining run requirements, and selecting input types. Using a network connection, you can enable remote monitoring and alerts. Start Screen Menu 1. Touch Menu in the upper-left corner of the screen, and select Configure. The keyboard appears. Start Screen Menu Figure 12 Start Screen Menu 2. Enter the default password using the keyboard: a. Touch the Shift key to display lower case letters. b. Type the default password, admin. The default password must be entered using lower case letters. Figure 13 Keyboard c. Touch Enter. The configuration screen appears. cbot User Guide

26 16 CHAPTER 2 Getting Started Configure Run Requirements The configuration screen contains three tabs: the Run Setup tab, the Remote tab, and the Alerts tab. From the Run Setup tab, you can name your instrument, configure wash and sensor bypass options, and indicate which fields are required input before each run can begin. These settings can be modified as needed before the start of each run. Station Name Required Fields Wash and Sensor Bypass Options Figure 14 Run Setup Tab 1. Touch StationName to name your cbot. The keyboard appears. NOTE Touch the Shift key to activate lower case letters. Touch the Alt key to activate symbol keys. 2. Type the name of your instrument using the touch screen keyboard, and touch Enter. NOTE For optimal performance, Illumina recommends an instrument water wash before and after each run. 3. Touch the radial buttons to select pre-run and post-run wash options. 4. To provide the option to allow a run to proceed even if an invalid sensor reading occurs during the pre-run check, touch the checkbox Allow Sensor Bypass. This option is disabled by default. If this feature is enabled, the option appears on the pre-run check screen if the pre-run check fails due to sensor failure. You must visually confirm that the run components are loaded correctly before bypassing sensors and proceeding to the fluidics check. For more information, see Perform Pre-Run Check on page Touch the checkbox to select which fields are required input at the start of each run. Fields include user name, experiment name, reagent kit ID, flow cell ID, primer name, and template name. Catalog # SY Part # Rev. C

27 Configuring the cbot 17 Enable Remote Monitoring From the Remote tab, you can configure the system to monitor your run remotely using the remote monitoring feature. A network connection is required. 1. Touch the Remote tab. 2. Touch the checkbox next to Allow Remote Access. The IP address of the instrument appears on the screen. Figure 15 Remote Tab 3. Use your web browser from another computer to access the remote monitoring feature. For more information, see Remote Monitoring Overview on page 30. Set Up Alerts From the Alerts tab, you can configure the system to send an alert if a system issue occurs or when a run is complete. A network connection is required. 1. Touch the Alerts tab. Server Notification Events Addresses Figure 16 Alerts Tab 2. Touch SMTP Server. The keyboard appears. cbot User Guide

28 18 CHAPTER 2 Getting Started NOTE Consult your network administrator for the name of your smtp server and smtp port. 3. Using the keyboard, enter the smtp server name, and then touch Enter. 4. Touch Server Path. The keyboard appears. 5. Using the keyboard, enter the smtp server port, and then touch Enter. 6. Enter the address for each intended alert recipient: a. Touch Add to add the address of alert recipients. The keyboard appears. b. Using the keyboard, enter an address, and then touch Enter. c. Touch Add again to enter an additional address. d. To test an address, highlight the address and touch Test. 7. In the Notification Events area, touch the checkbox to identify which events trigger an alert. Set Date and Time From the Time tab, you can set your instrument to the current date and your local time. 1. Touch the Time tab. 2. Touch Edit Date/Time. The Windows Date and Time Properties dialog box appears. Figure 17 Time Tab 3. Select the appropriate time zone for your area, the current date, and the current time. 4. Touch OK to close the Windows Date and Time Properties dialog box. The changes appear on the Time Tab. Catalog # SY Part # Rev. C

29 Configuring the cbot 19 Close Configuration Screen When you have finished configuring the setup options, touch Save and Exit. Save and Exit Figure 18 Save and Exit The cbot Start screen appears. You are ready to start a cluster generation run. cbot User Guide

30 20 CHAPTER 2 Getting Started Changing the Adapter Plate You can cluster a Genome Analyzer flow cell v4 or a HiSeq flow cell on the cbot. Each flow cell type requires that a specific adapter plate is installed on the cbot prior to starting the run. Icons on the start page indicate which adapter plate is currently installed on the instrument. Use the following instructions to change the flow cell adapter plate for the flow cell you are using: 1. Open the cbot lid by gently lifting from the top-right corner. 2. Lift the flow cell clamp. 3. Loosen the two captive Phillips head screws securing the adapter plate. Captive Screws Adapter Plate Figure 19 Flow Cell Adapter Plate 4. Lift the existing adapter plate from the thermal stage and set aside. 5. Make sure the thermal stage is clean. If any salt build up is present, wipe the stage with a lint-free cleaning tissue slightly moistened with water. 6. Position the new adapter plate on the thermal stage, aligning the sensor arm with the corresponding slot on the right side of the thermal stage. Sensor Arm on HiSeq Adapter Plate Sensor Arm on Genome Analyzer Adapter Plate Figure 20 Sensor Arm Location 7. Tighten the two Phillips head screws to secure the adapter plate. 8. For optimal heat transfer, ensure that the adapter plate is sitting flat and the screws are tightened evenly. 9. Wipe the installed adapter plate with a lint-free cleaning tissue moistened with water, and then wipe dry with a clean tissue. Do not allow fluids to drip inside the instrument. Catalog # SY Part # Rev. C

31 Chapter 3 Using the Software Interface Topics 22 Introduction 23 Start Screen 24 Run Setup Screens 27 Run Status Screen 29 Shutting Down the cbot 30 Remote Monitoring Overview cbot User Guide 21

32 22 CHAPTER 3 Using the Software Interface Introduction The cbot software interface provides ease of use in setting up your run, confirming installation of run components, and monitoring the run as it progresses. The following software screens are used to perform a run: Start screen See Start Screen on page 23. Run Setup screens See Run Setup Screens on page 24. Run Status screen Run Status Screen on page 27. Using the cbot software interface, you can configure input requirements, wash preferences, notifications, and remote monitoring. For more information, see Configuring the cbot on page 15. Additional features include tools for customizing protocols and setting manual controls for troubleshooting. For more information, see Manual Commands on page 74 and Protocol Editor on page 79. Catalog # SY Part # Rev. C

33 Start Screen 23 Start Screen The cbot Start screen appears when the system power is on and the software is running. You can leave your cbot sitting idle on the Start screen between runs. This section describes each area of the Start screen: Menu Button The Start screen menu contains options for accessing the configuration screen, manual command settings, protocol editor, and viewing the About screen. User Name Touch User Name to enter the name of the person performing the run. You can configure the user name to be optional input or required input. For more information, see Configuring the cbot on page 15. Start Button Touch Start to proceed to the Run Setup screen. Be sure you have made any necessary changes to run configurations before proceeding. Error Messages Error messages appear if required input is missing or if the system is not responding. For example, if you touch Start without entering a user name, and user name is required input, a message appears. For more information, see Troubleshooting on page 68. Sensor Status Provides status of cbot components. Sensors read the status of the cbot lid, waste bottle, coolant system, and sipper comb. For more information, see Sensor Status Icons on page 26. Menu Button User Name Start Button Error Messages Sensor Status Figure 21 cbot Start Screen Touch Start to proceed to the Run Setup screen. cbot User Guide

34 24 CHAPTER 3 Using the Software Interface Run Setup Screens The Run Setup screens guide you through the pre-run setup steps. For detailed instructions, see Setting Up the Run on page 41. Wash Perform a pre-run wash. You can configure wash requirements on the configuration screen. For more information, see Configuring the cbot on page 15. Protocol Name your experiment and select a protocol. Reagents Record the reagent kit ID and load reagents. Flow Cell Record the flow cell ID and load the flow cell. Manifold Load the manifold and secure the sipper comb. Tube Strips Enter a templates name and load the eight-tube strip containing templates. If you use a protocol that requires additional primers, enter the primers name and load the eight-tube strip containing primers. Pre-Run Check The software automatically checks the run readiness of each component and performs a flow check. The flow check uses a set of eight integrated bubble sensors to test the fluidics before each run. Upon completion of the pre-run check, you are ready to start a cluster generation run. Run Setup Screen Features Each Run Setup screen contains instructional messages, sensor status, and input fields. If run components are missing or installed incorrectly, the cbot system provides a warning. These warnings must be corrected before you can begin the run. Required input, such as reagent ID and flow cell ID may be scanned using the barcode scanner, or you can touch the keyboard icon to activate the touch screen keyboard and type your component ID. Run Setup Steps Required Input Keyboard Input (Optional) Previous Button Next Button (Inactive) Figure 22 Run Setup Screen Catalog # SY Part # Rev. C

35 Run Setup Screens 25 Some steps require you to manually enter input, while other input is entered by the system using instrument sensors. System Input Input entered by the system is indicated by a half checkbox. On the Wash screen, for example, sensors detect if the manifold is installed or removed. Manual Input Input you enter manually is indicated by a full checkbox. On the Wash screen, for example, you must manually touch the checkbox to indicate that the reservoir is filled with water. Instructional Messages System Input Manual Input Run Summary Sensor Icons Help Screen Figure 23 Run Setup Screen Features Sensor icons at the bottom of the screen indicate when run components are properly loaded, when the waste bottle is full, or if there is a problem in the cooling system. For more information, see Sensor Status Icons on page 26. At any time during run setup, you can view the run data summary or access the Help screen. Run data summary lists the input you have entered so far. The Help screen provides detailed descriptions and instructional videos. The Next button becomes active when you have provided the required input for each run setup step. If a component is missing or the system is waiting for input, an instructional message prompts you to the next step. cbot User Guide

36 26 CHAPTER 3 Using the Software Interface Sensor Status Icons The sensor status icons indicate if a component is properly installed and ready for the run. The following table describes the various states of the system status icons. Table 6 Sensor Status Icons Component Icon Indication/Meaning cbot lid cbot lid is open. You must close the lid during operation. cbot lid is closed. Waste Bottle Waste bottle is present and ready for use. Waste bottle is full. Waste bottle is missing. Coolant Coolant is flowing and coolant level is good. Warning: Coolant is flowing, but coolant level is low. Error: Coolant is not flowing, but coolant level is good. Error: Coolant is not flowing and coolant level is low. Manifold and Sipper Comb Manifold is loaded and sipper comb is secure. Manifold is missing or sipper comb is not secure. Flow Cell Genome Analyzer flow cell adapter plate installed. HiSeq flow cell adapter plate installed. Flow cell adapter plate type unknown. Catalog # SY Part # Rev. C

37 Run Status Screen 27 Run Status Screen The Run Status screen provides an at-a-glance status of the current cluster generation run and includes the following run details: Status bar showing run progress Start date and time, end date and time, and time remaining Cluster generation protocol steps with status bar for each step Reagent currently in use Current temperature ( C) Status of the command in the current step Visual Status Bar Protocol Steps Start Date/Time End Date/Time Time Remaining Toggle Between Run Steps and Temp Graph Sensor Icons Reagent Temperature Command Time Command Status Figure 24 Run Status Screen During the run, a screensaver appears showing the run countdown time and status bar. Touch the countdown to toggle through view options: status bar only, countdown only, or both countdown and status bar. Touch the lower portion of the screensaver to return to the Run Status screen. Figure 25 Run Status Screensaver cbot User Guide

38 28 CHAPTER 3 Using the Software Interface Pausing or Aborting a Run During a run, you can pause or abort the run using buttons on the Run Status screen. Pause Completes the current command in the protocol, and then pauses the run. The run may take a few minutes before it pauses. When the run is paused, the Pause button changes to the Resume button. When the run is active, touch Pause to pause the run. When the run is paused, touch Resume to resume the run. Pause/Resume Run Abort Run Figure 26 Run Status Screen in Pause Mode Abort Run Aborts the run without the option of resuming. At this point you are prompted to unload the run components. NOTE Aborting a run is final. You cannot resume the run from this point. Touch Unload and unload run components. Catalog # SY Part # Rev. C

39 Shutting Down the cbot 29 Shutting Down the cbot It is not necessary to shut down your cbot between runs. It is designed to continue running in an idle state on the Start screen. In the case you need to shut down your cbot, you can access the command from the configuration screen. 1. Touch Menu and select Configure. The keyboard appears. 2. Enter the default password using the keyboard: a. Touch the Shift key to display lower case letters. b. Type the default password, admin. The default password must be entered using lower case letters. c. Touch Enter. The configuration screen appears. 3. From the configuration screen, touch Menu and select Shut Down Station. The cbot software shuts down. Figure 27 Shut Down Station 4. To power down your cbot, toggle the power switch on the right side panel to the OFF position. Reboot in FSE Mode The option to reboot in FSE Mode is for use by a trained Illumina Field Application Scientist or Field Service Engineer to update software or service the instrument. cbot User Guide

40 30 CHAPTER 3 Using the Software Interface Remote Monitoring Overview Remote monitoring is a feature that allows you to monitor the status of your cbot from another computer via your internal network. To take advantage of the remote monitoring feature, make sure the following requirements are met: Your cbot must be connected to a network. Allow Remote Access is selected on the Remote tab. For more information, see Enable Remote Monitoring on page 17. The next step is to go to the remote monitoring IP address listed on the Remote tab of your cbot, and add your cbot to the remote monitoring site. Add Your cbot 1. Use your web browser to navigate to the remote monitoring IP address listed on the Remote tab of the cbot configuration screen. 2. In the text box next on the remote monitoring screen, enter the IP address of your cbot. This is the same IP address listed on the Remote tab of your cbot. For more information, see Enable Remote Monitoring on page Click Add Instrument. An image that represents your instrument appears on the screen. Enter cbot IP Address Figure 28 Remote Monitoring Screen The remote monitoring screen shows each of the instruments you have added by instrument name. At a glance, this screen allows you to monitor the progression of the run and see the current state of the instrument. There are four possible states: running, paused, stopped, or error. Catalog # SY Part # Rev. C

41 31 Chapter 4 Using the cbot Topics 32 Introduction 32 cbot Cluster Generation Kits 33 Flow Cell Adapter Plates 34 cbot Cluster Generation Workflow Well Reagent Plate 35 Preparing DNA Template for Cluster Generation 39 Thawing cbot Reagents 41 Setting Up the Run 58 Monitoring the Run 59 Performing Post-Run Procedures cbot User Guide

42 32 CHAPTER 4 Using the cbot Introduction This chapter lists the kits required for cluster generation on the cbot, and explains how to properly thaw the cbot reagent plate, load run components, and perform a cluster generation run. If you are planning to alter the protocol in any way, you must do so before setting up the run. For more information, see Protocol Editor on page 79. cbot Cluster Generation Kits cbot cluster generation kits are shipped on dry ice. When you receive your kit, store kit components at temperatures specified in Table 7. Single-read kits and paired-end kits are available for the Genome Analyzer and the HiSeq sequencing system. Each kit contains the required components for performing one cluster generation run on the cbot. Table 7 cbot Cluster Generation Kits Cluster Generation Kit Kit Components Storage Requirements Genome Analyzer Single-Read Cluster Generation Kit Catalog # GD Single-read Genome Analyzer Flow Cell v4 2 to 8 C cbot manifold for Flow Cell v4 Room temperature Single-read 96-well reagent plate (For more information, see 96-Well Reagent Plate on page 38) HT1 (Hybridization Buffer) for diluting templates and primers Small RNA Sequencing Primer (optional) -15 to -25 C -15 to -25 C -15 to -25 C Genome Analyzer Paired-End Cluster Generation Kit Catalog # PE Paired-End Genome Analyzer Flow Cell v4 2 to 8 C cbot manifold for Flow Cell v4 Room temperature Paired-end 96-well reagent plate (For more information, see 96-Well Reagent Plate on page 38) HT1 (Hybridization Buffer) for diluting templates and primers Paired-End Reagent Kit (used with sequencing protocols on the Genome Analyzer) (For more information, see the Genome Analyzer User Guide) -15 to -25 C -15 to -25 C -15 to -25 C Catalog # SY Part # Rev. C

43 Flow Cell Adapter Plates 33 Table 7 cbot Cluster Generation Kits (Continued) Cluster Generation Kit Kit Components Storage Requirements HiSeq Single-Read Cluster Generation Kit Catalog # GD Single-read HiSeq flow cell 2 to 8 C cbot manifold for HiSeq flow cell Room temperature Single-read 96-well reagent plate (For more information, see 96-Well Reagent Plate on page 38) HT1 (Hybridization Buffer) for diluting templates and primers Small RNA Sequencing Primer (optional) HiSeq Accessories Kit (contains gaskets, bottle caps, and ICR instrument bottle used with sequencing protocols on the HiSeq) (For more information, see the HiSeq 2000 User Guide) -15 to -25 C -15 to -25 C -15 to -25 C Room temperature HiSeq Paired-End Cluster Generation Kit Catalog # PE Paired-end HiSeq flow cell 2 to 8 C cbot manifold for HiSeq flow cell Room temperature Paired-end 96-well reagent plate (For more information, see 96-Well Reagent Plate on page 38) HT1 (Hybridization Buffer) for diluting templates and primers Read 2 Cluster Resynthesis Kit (used with sequencing protocols on the HiSeq) (For more information, see the HiSeq 2000 User Guide) HiSeq Accessories Kit (contains gaskets, bottle caps, and ICR instrument bottle used with sequencing protocols on the HiSeq). (For more information, see the HiSeq 2000 User Guide) -15 to -25 C -15 to -25 C -15 to -25 C Room temperature For troubleshooting purposes, a cbot Rehybridization Kit (part # ) is available through Illumina Technical Support or your Field Application Scientist (FAS). This kit is used on the cbot with protocols titled Repeat_Hyb_v<#> or Repeat_TubeStripHyb_v<#> included with the cbot software. Flow Cell Adapter Plates The HiSeq flow cell and Genome Analyzer flow cell require specific adapter plates. Adapter plates are provided with the instrument. If you do not have the appropriate adapter plate for the flow cell you are using, contact Illumina Technical Support for ordering information. cbot User Guide

44 34 CHAPTER 4 Using the cbot cbot Cluster Generation Workflow A cluster generation run on the cbot requires very little preparation. Cluster generation reagents are provided in an easy to use 96-well plate containing pre-mixed reagents. After thawing and vortexing, the reagents are ready for use on the instrument. For more information, see Thawing cbot Reagents on page 39. To prepare for a run, you first need to select a protocol and then load four run components. A run requires the flow cell, one single-use manifold, the cbot reagent plate, and your templates. Additional primers may also be loaded on the cbot should you wish to customize your experiment. Following run preparation, a cluster generation run on the cbot takes about four hours to complete. Prepare DNA Template Thaw & Mix Reagents Perform a Pre-Run Wash Select a Protocol & Load Run Components Perform a Pre-Run Check & Start the Run Unload Run Components Perform a Post-Run Wash Confirm Reagent Delivery Figure 29 Cluster Generation Workflow Catalog # SY Part # Rev. C

45 Preparing DNA Template for Cluster Generation 35 Preparing DNA Template for Cluster Generation This section explains how to prepare your DNA template for cluster generation. An alternate template preparation method is describe in the qpcr Quantitation Protocol, part # (Appendix B). There are two steps involved in preparing the DNA template. The first step is to denature with 2 N NaOH, and then dilute DNA into hybridization buffer. Consumables Illumina-Supplied HT1 (Hybridization Buffer) User-Supplied 2 N NaOH Tris-Cl 10 mm, ph ml eight-tube strip DNA Template DNA Template Storage Illumina recommends storing prepared DNA template at a concentration of 10 nm. For single-read libraries, 10 nm is ~1.9 ng/μl for 290 bp fragments including adapter length. For paired-end libraries, 10 nm is ~2.0 ng/μl for 310 bp fragments including adapter length. Adjust the concentration for your prepared DNA samples to 10 nm using Tris-Cl 10 mm, ph 8.5. For long-term storage of DNA samples at a concentration of 10 nm, add Tween 20 to the sample to a final concentration of 0.1% Tween. This helps to prevent adsorption of the template to plastic tubes upon repeated freezethaw cycles, which would decrease the cluster numbers over time. DNA Concentration The flow cell has eight parallel lanes for processing up to eight different DNA samples. The first time you process a sample, it is useful to try a concentration range to optimize the number of clusters formed. If the DNA concentration is too low, too few clusters will be generated and the sequencing yield will be low. If the DNA concentration is too high, the clusters will be too dense and can overlap, complicating sequencing data analysis. Procedure Denature DNA Template Use the following instructions to denature the template DNA with 2 N NaOH to a final DNA concentration of 1 nm. This is suitable for performing the hybridization step on the cbot at a DNA concentration up to 8 pm. If you require a higher DNA concentration, see Denaturing High Concentrations of DNA on page 36 for suggested adjustments. cbot User Guide

46 36 CHAPTER 4 Using the cbot CAUTION Excess NaOH concentrations (greater than 800 μm) in diluted samples inhibits the formation of clusters, an effect which occurs if you add more than 8 μl of the NaOH denaturated DNA sample to 1 ml of hybridization buffer. 1. Combine template DNA, Tris-Cl, and 2 N NaOH in the following volumes: 10 nm Template DNA (2 μl) Tris-Cl 10 mm, ph 8.5 (17 μl) 2 N NaOH (1 μl) The total volume should be 20 μl (template final concentration 1 nm). 2. Vortex briefly to mix the template solution. 3. Pulse centrifuge the solution. 4. Incubate for five minutes at room temperature to denature the template into single strands. 5. Place the template on ice until you are ready to proceed to final dilution. Denaturing High Concentrations of DNA Use the following table only if you require a DNA concentration higher than 4 pm; otherwise, follow the protocol Denature DNA Template on page 35. Table 8 Adjustments to the Protocol for High Final DNA Concentrations Desired Final DNA Concentration in 1 ml Template DNA (10 nm) Tris-Cl 10 mm, ph N NaOH Concentration of Denatured Template DNA 4 8 pm 2 μl 17 μl 1 μl 1.0 nm 8 12 pm 3 μl 16 μl 1 μl 1.5 nm pm 4 μl 15 μl 1 μl 2.0 nm pm 5 μl 14 μl 1 μl 2.5 nm pm 6 μl 13 μl 1 μl 3.0 nm Dilute Denatured DNA Use the following instructions to dilute the denatured DNA with pre-chilled HT1 to a total volume of 1,000 μl. Illumina recommends that you perform a titration of your DNA template to determine a good density of clusters. 1. Remove HT1 (Hybridization Buffer) from -15 to -25 C storage and thaw at 2 to 8 C overnight or in a beaker of room temperature deionized water. 2. To reach the desired final concentration for the hybridization step, dilute denatured DNA as follows: Part # Rev. C

47 Preparing DNA Template for Cluster Generation 37 Required Final Concentration 5 pm 6 pm 7 pm 8 pm 1.0 nm Denatured DNA 5 μl 6 μl 7 μl 8 μl Pre-chilled HT1 995 μl 994 μl 993 μl 992 μl NOTE Cluster generation on the cbot results in a higher cluster density than the same DNA library on the Cluster Station. 3. Invert several times to mix the template solution. 4. Pulse centrifuge the solution. NOTE The Multiplexing Sequencing Primers and PhiX Control Kit contains a PhiX174 library amplified using PCR Primer Index 3. Illumina recommends using this control library on lane 4 of the flow cell. 5. Label the tubes of an eight-tube strip Dispense 120 μl of the control library into tube 4 of the eight-tube strip. This will place the control sample in lane 4 on the flow cell. 7. Add 120 μl of diluted, denatured sample DNA template into the remaining tubes of an eight-tube strip. Take careful note of which template goes into which numbered tube. 8. Record each sample position and concentration on the lab tracking form. 9. Set aside on ice until you are ready to load it onto the instrument. cbot User Guide

48 38 CHAPTER 4 Using the cbot 96-Well Reagent Plate To ensure proper performance, it is very important to promptly store the cbot reagents at -15 to -25 C until you are ready to use them. The 96-well reagent plate contains 11 rows of foil-sealed eight-tube strips filled with cluster generation reagents. Row 12 of the reagent plate is empty. Row 1 Row 11 Figure 30 cbot 96-Well Reagent Plate Each reagent tube strip is labeled with the reagent name followed by a number. The number indicates the row it occupies on the reagent plate. Should any tube strips become dislodged from the reagent plate holder, use the row number on the label to return the tube strip to the appropriate position on the plate. Reagent Plate Contents Row 1 HT1 (Hybridization Buffer) Row 2 HFE (1X Phusion Master Mix, Finnzymes Oy) Row 3 APM1 (AMX1 Premix) Row 4 AMX1 (Amplification Mix) Row 5 AT1 (100% Formamide) Row 6 AMX1 (Amplification Mix) Row 7 HT2 (Wash Buffer) Row 8 Single-Read Kit: LS1 (Linearization Solution) Row 8 Paired-End Kit: LMX1 (Linearization Mix) Row 9 BMX (Blocking Mix) Row 10 HP5 (0.1 N NaOH) Row 11 HP1 (Sequencing Primer) Row 12 Empty NOTE HP1 (Sequencing Primer) is compatible with all applications except small RNA and experiments that require a custom primer for Read 1. For more information, see Prepare Small RNA Primer on page 40. WARNING This set of reagents contain an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. For more information, see the MSDS for this kit, at Catalog # SY Part # Rev. C

49 Thawing cbot Reagents 39 Thawing cbot Reagents Use the following instructions to thaw cbot reagents. Consumables Best Practices Illumina-Supplied 96-well reagent plate from a cbot Cluster Generation Kit Wear a fresh pair of gloves when setting up a run. Hold the 96-well reagent plate by the plate base to avoid dislodging any reagent tubes. Always check that the tubes are securely seated in the reagent plate before and after vortexing or inverting reagents. Loose reagent tubes can damage the cbot manifold and stop the run. NOTE The clear plastic lid on the reagent plate protects the foil seals from being damaged or punctured during thawing. Remove the protective lid only when necessary, such as when you are checking that tubes are securely seated. Procedure Reagents take a minimum of 60 minutes to thaw using a water bath. Alternatively, you can thaw the reagents at 2 to 8 C overnight or approximately 16 hours. 1. Remove the reagent plate from -15 to -25 C storage. 2. Press down on each row of tubes in the reagent plate to ensure that they are securely seated in the plate. 3. Place the reagent plate in an ice bucket or water bath containing only enough room temperature tap water to submerge the reagent plate base. To prevent contamination, do not allow the water to touch the foil seals. Allow the reagents to thaw in the water bath for at least 60 minutes. NOTE Reagent tubes in the center of the plate take longer to thaw. Inspect the reagents to ensure they have completely thawed. 4. Remove the thawed reagent plate from the water bath and gently pat dry with paper towels. 5. Hold the reagent plate by the base and place your other hand on top of the tubes, then invert the reagent plate five times to mix the thawed reagents. 6. Hold the reagent plate by the base and place your other hand on top of the tubes, then vortex the plate for approximately 10 seconds to dislodge any trapped air bubbles. 7. Tap the reagent plate on a hard surface 5 10 times to collect any reagent droplets to the bottom of the tubes. Alternatively, pulse centrifuge the reagent plate to collect reagent droplets. 8. Proceed to setting up the run. Do not allow reagents to sit at room temperature for an extended period of time. cbot User Guide

50 40 CHAPTER 4 Using the cbot Prepare Small RNA Primer If you are preparing a single-read flow cell with small RNA or gene expression libraries, you need to prepare Small RNA Sequencing Primer prior to your run. The prepared primer is loaded onto the cbot in the eight-tube strip holder labeled Primers at the front of the reagent stage. For more information, see Load Primers on page 53. Consumables Illumina-Supplied Small RNA Sequencing Primer (provided in the cbot Single-Read Cluster Generation Kit) HT1 (Hybridization Buffer) Procedure 1. Remove HT1 (Hybridization Buffer) from -15 to -25 C storage and thaw at 2 to 8 C overnight or in a beaker of room temperature water. 2. Thaw the tube containing 10 μl Small RNA Sequencing Primer at room temperature. This will take only a few minutes. 3. When the Small RNA Sequencing Primer is thawed, vortex for a few seconds and briefly centrifuge. 4. Add 5 μl of Small RNA Sequencing Primer to 995 μl of HT1. 5. Vortex for a few seconds, and then briefly centrifuge. 6. Label the tubes of an eight-tube strip Aliquot 120 μl of the diluted sequencing primer into each tube of the eight-tube strip. 8. Proceed directly to Setting Up the Run on page 41. When prompted, load the eight-tube strip containing primer on the cbot as described in Load Primers on page 53. Catalog # SY Part # Rev. C

51 Setting Up the Run 41 Setting Up the Run This section describes how to set up a run and load run components on the cbot. You may configure some steps to be optional, such as user name or experiment name, for example. For more information, see Configure Run Requirements on page 16. Enter User Name 1. Touch User Name. The keyboard appears. 2. Using the keyboard, type your name and then touch Enter. 3. Touch Start to proceed to the pre-run wash. Figure 31 Start Screen Perform a Wash The cbot software checks the system to ensure that the flow cell and manifold have been removed before beginning a wash. 1. Confirm that the checkbox next to Manifold Removed is selected. If it is not selected, you must remove the manifold before proceeding. For more information, see Unload Run Components on page 59. If the pre-run wash has been configured as optional, you can touch Skip to bypass the pre-run wash. Figure 32 Perform a Wash cbot User Guide

52 42 CHAPTER 4 Using the cbot 2. Raise the cbot lid by gently lifting from the top-right corner. 3. Fill the wash reservoir, located behind the thermal stage, with approximately 12 ml deionized water. Wash Reservoir Figure 33 Fill the Wash Reservoir 4. Close the cbot lid. 5. Touch the checkbox on the screen to indicate that water is present. The Wash button becomes active. 6. Touch Wash. After the wash is finished, the Wash Reservoir Dry button becomes active. 7. Blot out any excess water remaining in the wash reservoir with a low-lint wipe, taking care not to rub the outlet ports as this can cause fibers to clog the holes. Catalog # SY Part # Rev. C

53 Setting Up the Run 43 Figure 34 Dry the Wash Reservoir 8. Touch the checkbox on the screen to indicate that the wash reservoir is dry. The Next button becomes active. 9. Touch Next to proceed to selecting the protocol. Select a Protocol 1. Touch Experiment Name. The keyboard appears. 2. Using the touch screen keyboard, type your experiment name and then touch Enter. NOTE cbot cluster generation protocols perform a complete cluster generation run, including steps for amplification, linearization, blocking, and primer hybridization. 3. Select the appropriate protocol for your experiment from the list of protocols. Touch the scroll bar to scroll through available protocols. Figure 35 Select a Protocol 4. Touch Next to proceed to the reagents step. cbot User Guide

54 44 CHAPTER 4 Using the cbot Load Reagents 1. Remove the clear plastic lid from the cbot reagent plate. 2. Gently press down on the thawed and vortexed tubes in the reagent plate to make sure they are securely seated in the plate. 3. Touch Scan Reagent ID to activate the barcode scanner. Alternatively, you can enter the reagent ID using the keyboard. Touch the keyboard icon to activate the keyboard option. Keyboard Option Figure 36 Load Reagents 4. Hold the reagent plate level with the barcode label facing the instrument, and move the reagent plate into the light path of the barcode scanner. Figure 37 Scan Reagent ID You will hear a beep when the scanner has successful read the barcode. The reagent ID appears on the screen. 5. Raise the cbot lid. 6. With the reagent plate in one hand, use the other hand to pull the spring-loaded reagent plate lever towards you to release the clamp. 7. Place the reagent plate onto the reagent stage, positioned with row 1 facing towards the front of the instrument directly behind the eight-tube strip holder. Ensure the beveled corner of the plate is positioned in the front-right corner. Catalog # SY Part # Rev. C

55 Setting Up the Run 45 Reagent Lever Figure 38 Position the Reagent Plate Row 1 Figure 39 Position of Row 1 8. Push the reagent plate lever back to the secure the reagents in place. 9. Touch the checkbox on the screen to indicate that the reagent plate is loaded. Figure 40 Reagent Plate Loaded 10. Touch Next to proceed to loading the flow cell. cbot User Guide

56 46 CHAPTER 4 Using the cbot Load Flow Cell CAUTION Lane orientation for the HiSeq flow cell is opposite of the Genome Analyzer flow cell. The Genome Analyzer flow cell lane orientation is lane 1 8, left to right, while the HiSeq flow cell lane orientation is lane 8 1, left to right. Use the following instructions to correctly load the flow cell. 1. Lift the flow cell clamp. 2. Wash the adapter plate on the thermal stage with a small amount of deionized water, and wipe dry with a lint-free cleaning tissue. Do not allow fluids to drip inside the instrument. 3. Remove the flow cell from the storage tube using plastic forceps or metal forceps with tips wrapped tightly in parafilm. NOTE If you plan to store the flow cell after completing cluster generation, make sure you retain the storage tube and storage buffer. 4. Holding the flow cell by the edges, rinse the flow cell with deionized water. 5. Gently dry the flow cell with a lint-free cleaning tissue using a sweeping motion. Repeat until the flow cell is completely clean and dry. 6. Touch Scan Flow Cell ID to activate the barcode scanner. Alternatively, you can enter the reagent ID using the keyboard. Touch the keyboard icon to activate the keyboard option. Keyboard Option Figure 41 Load the Flow Cell 7. Hold the flow cell close to the scanner tray with the barcode positioned toward the instrument. The white background of the barcode scanner tray is necessary for successful scanning. 8. Slowly slide the flow cell into the light path of the barcode scanner so the entire barcode crosses the scan light at the same time. Catalog # SY Part # Rev. C

57 Setting Up the Run 47 Figure 42 Scan Flow Cell ID You will hear a beep when the scanner has successfully read the barcode. The flow cell ID appears on the screen. Instructions for proper flow cell orientation (Genome Analyzer shown) Figure 43 On-Screen Loading Instructions 9. Do one of the following depending on the flow cell you are using: Genome Analyzer Place the flow cell on the thermal stage with the holes facing upward, lane 1 on the left side, and lane 8 and the barcode on the right side. If your flow cell has black masking, the barcode may not be visible. Lane 8 Barcode on Right Side Figure 44 Position Genome Analyzer Flow Cell cbot User Guide

58 48 CHAPTER 4 Using the cbot HiSeq Place the flow cell on the thermal stage with holes facing upward, lane 1 on the right side, and lane 8 and the barcode on the left side. With holes facing up, the barcode on the HiSeq flow cell is facing down and not visible when the flow cell is positioned correctly. NOTE When the HiSeq flow cell is loaded onto the cbot, the lanes are oriented lane 8 to lane 1 from left to right. You must load your samples and additional primers in the same orientation. For more information, see Load Templates on page 51 and Load Primers on page 53. Lane 8 Barcode on Left Side (shown for reference only) Figure 45 Position HiSeq Flow Cell 10. Touch the checkbox to indicate that you have loaded the flow cell. 11. Touch Next to proceed to loading the manifold. Load Manifold Make sure that you use the manifold provided in the same cluster generation kit as the flow cell. Manifolds are specific to the size of the flow cell. 1. Remove the manifold from the packaging and inspect the sippers on the sipper comb. Ensure the sippers are straight and have not been bent or damaged. Ensure the black rubber gaskets are evenly seated. 2. Position the manifold over the flow cell with the sipper comb pointing toward the front of the cbot. Catalog # SY Part # Rev. C

59 Setting Up the Run 49 Figure 46 Position the Manifold 3. Align the manifold with the guide pins on the thermal stage, and set the manifold into place on top of the flow cell. 4. Wiggle the manifold to ensure it is evenly seated over the flow cell. The manifold must be evenly seated to form a tight seal. 5. Touch the checkbox to indicate that the manifold is seated. Figure 47 Load the Manifold 6. Close the flow cell clamp to lock the manifold in position, ensuring that the bracket snaps securely under the white clip. 7. Touch the checkbox to indicate that the flow cell clamp is closed. cbot User Guide

60 50 CHAPTER 4 Using the cbot Flow Cell Clamp 8. Connect the outlet end of the manifold to the outlet port in the wash reservoir, and ensure it is evenly seated. Outlet Clamp Outlet Port Figure 48 Secure Outlet End 9. Snap the outlet clamp closed to secure the outlet end of the manifold. 10. Touch the checkbox to indicate that you have connected the manifold to the outlet port and the rear clamp is secured. 11. Align the sipper comb with the two metal guide pins on the front edge of the thermal stage. 12. Snap the sipper comb into place using the plastic tabs on either side of the sipper comb. Catalog # SY Part # Rev. C

61 Setting Up the Run 51 Metal Guide Pins Figure 49 Secure the Sipper Comb When the sipper comb is secure, the system checks the final checkbox and the Next button is activated. 13. Ensure that the sipper comb sippers are straight and perpendicular to the reagent plate. 14. Touch Next to proceed to loading the tube strips. Load Templates CAUTION Lane orientation for the HiSeq flow cell is opposite of the Genome Analyzer flow cell. The Genome Analyzer flow cell lane orientation is lane 1 8, left to right, while the HiSeq flow cell lane orientation is lane 8 1, left to right. Use the following instructions to correctly load the eight-tube strip containing templates for the flow cell you are using. 1. Touch Enter Template Name. The keyboard appears. 2. Using the keyboard, type the template ID and then touch Enter. cbot User Guide

62 52 CHAPTER 4 Using the cbot Figure 50 Load Tube Strips Reagent Plate Templates Primers Figure 51 Load Templates and Primers 3. Load the eight-tube strip containing the template into the second row of the tube strip holder in one of the following orientations depending on the flow cell you are using: Genome Analyzer Position the eight-tube strip containing the template into the tube strip holder with the tube labeled #1 on the left side and the tube labeled #8 on the right side. Tube #1 Tube #8 Figure 52 Tube Strip Orientation for Genome Analyzer Flow Cell Catalog # SY Part # Rev. C

63 Setting Up the Run 53 HiSeq Position the eight-tube strip containing the template into the tube strip holder with the tube labeled #8 on the left side and the tube labeled #1 on the right side. This orientation aligns with the lanes on the HiSeq flow cell when correctly loaded onto the cbot. Tube #8 Tube #1 Figure 53 Tube Strip Orientation for HiSeq Flow Cell 4. Touch the checkbox to indicate that you have loaded templates. 5. If you are using additional primers, proceed to Load Primers. Otherwise, close the cbot lid. The Next button becomes active. Figure 54 Close cbot Lid to Continue Load Primers The option to load the primer strip tube appears if the protocol you have selected uses custom or specialty primers. Use the following steps to load custom or specialty primers. Otherwise, close the cbot lid and touch Next to proceed to the pre-run check. CAUTION Lane orientation for the HiSeq flow cell is opposite of the Genome Analyzer flow cell. The Genome Analyzer flow cell lane orientation is lane 1 8, left to right, while the HiSeq flow cell lane orientation is lane 8 1, left to right. Use the following instructions to correctly load the eight-tube strip containing primers for the flow cell you are using. 1. Touch Enter Primer Name. The keyboard appears. 2. Using the keyboard, type the primer ID and then touch Enter. cbot User Guide

64 54 CHAPTER 4 Using the cbot 3. Load the eight-tube strip containing primers into the first row of the tube strip holder in one of the following orientations depending on the flow cell you are using: Genome Analyzer Position the eight-tube strip containing primers into the tube strip holder with the tube labeled #1 on the left side and the tube labeled #8 on the right side. Tube #1 Tube #8 Figure 55 Tube Strip Orientation for Genome Analyzer Flow Cell HiSeq Position the eight-tube strip containing primers into the tube strip holder with the tube labeled #8 on the left side and the tube labeled #1 on the right side. This orientation aligns with the lanes on the HiSeq flow cell when correctly loaded onto the cbot. Tube #8 Tube #1 Figure 56 Tube Strip Orientation for HiSeq Flow Cell 4. Touch the checkbox to indicate that you have loaded additional primers. Catalog # SY Part # Rev. C

65 Setting Up the Run 55 Figure 57 Close cbot Lid to Continue 5. Close the cbot lid. The Next button becomes active. 6. Touch Next to proceed. The system automatically performs a pre-run check. Perform Pre-Run Check The pre-run check reads the instrument sensors to detect the correct installation of run components, and then performs a flow check using bubble sensors to detect air in the lines. The pre-run check takes approximately three minutes. Successful Pre-Run Check The Start button becomes active if the pre-run check is successful. Figure 58 Perform Pre-Run Check 1. Touch Start. The Run Status screen opens and the run begins. 2. Proceed to Monitoring the Run on page 58. cbot User Guide

66 56 CHAPTER 4 Using the cbot Pre-Run Check Run Component Errors Figure 59 Pre-Run Check Failure, Run Component Errors If the pre-run check fails due to errors related to run components, perform the following steps: 1. Check any run component with an indicated error to ensure it is present and loaded correctly. 2. Touch Rerun Check to repeat the sensor check. 3. If the check continues to fail and your system is configured to allow sensor bypass, do the following: a. Visually inspect run components to ensure corrected orientation. b. Touch Bypass Sensor Check and Rerun Check. The system proceeds to the flow check. If the Bypass Sensor Check option is not visible, your system is not configured to allow sensor bypass. For more information, see Configure Run Requirements on page 16. Pre-Run Flow Check Failure Figure 60 Pre-Run Check Failure, Flow Check Fails Catalog # SY Part # Rev. C

67 Setting Up the Run 57 If the flow check fails, perform the following steps: 1. Ensure the flow cell is positioned correctly with the ports facing upward and the barcode on the right side. 2. Inspect the manifold to ensure it is evenly seated over the flow cell, and the flow cell clamp is closed. 3. Ensure the outlet end of the manifold is evenly seated in the outlet port and the clamp is closed. 4. Ensure row #1 of the reagent plate is fully thawed and contains reagent. 5. Ensure the sippers are straight and that the foil-sealed tubes in row #1 of the reagent plate are pierced. NOTE You can rerun the check up to three times before you risk running out of reagent. 6. Touch Rerun Check to repeat the check. For more information, see Troubleshooting on page If the flow check fails repeatedly, the option to bypass the flow check becomes active. Touch Bypass Flow Check to proceed with the run. Figure 61 Bypass Flow Check NOTE If you choose to bypass the flow check, Illumina does not guarantee that the failed lanes will cluster successfully. 8. After the run, check reagent delivery from all tubes. For more information, see Confirm Reagent Delivery on page 62. cbot User Guide

68 58 CHAPTER 4 Using the cbot Monitoring the Run The Run Status screen allows you to monitor the run in progress. For more information, see Run Status Screen on page 27. During the run, watch for error messages, either on the screen or in the form of alerts. For more information, see Set Up Alerts on page 17. Figure 62 Run Status Screen Allow approximately four hours for the run to complete. After the run is complete, the cbot holds the flow cell at 20 C. The flow cell may remain on the instrument at this temperature overnight. If you need to store the flow cell, store it in storage buffer in the flow cell tube at 4 C. The flow cell is stable after primer hybridization for up to ten days when properly stored at 4 C in storage buffer in the flow cell tube. Run Data Report The run data report provides a summary of the run in progress. You can view the report at any time during the run or at the end of the run. Touch Menu and then select Run Data. Catalog # SY Part # Rev. C

69 Performing Post-Run Procedures 59 Performing Post-Run Procedures Post-run procedures confirm that the run successfully completed. Post-run procedures include viewing the run data report, unloading run components, performing an instrument wash, and checking reagent delivery. View the Run Data Report Unload Run Components At the end of the run, the run data report automatically appears to alert you that the run is complete. The run data report lists the following information: Protocol name Flow cell ID Reagent ID Template name Start time and finish time 1. When the run is complete, touch Unload to proceed. Unload Figure 63 Run Complete, Unload Components 2. Raise the cbot lid by gently lifting from the top-right corner. Figure 64 Unload Manifold cbot User Guide

70 60 CHAPTER 4 Using the cbot 3. Release the outlet clamp securing the outlet end of the manifold. 4. Disconnect the outlet end of the manifold from the outlet port in the wash reservoir. 5. Release the flow cell clamp. 6. Remove the sipper comb from the metal guide pins using the plastic tabs on either side of the sipper comb. When the sipper comb is removed, the system selects the checkbox indicating that the manifold is removed. Figure 65 Manifold Removed 7. Remove the manifold from the cbot. 8. Carefully lift the flow cell from the thermal stage. 9. Pull the reagent plate lever toward you to release the reagent plate. 10. Remove the reagent plate from the reagent stage. Set aside until you are ready to check reagent delivery. 11. Remove the eight-tube strip containing the templates. Set aside until you are ready to confirm reagent delivery. 12. If applicable, remove the eight-tube strip containing additional primers. Set aside until you are ready to confirm reagent delivery. 13. Touch the checkbox to indicate that you have unloaded the reagents, templates, and primers. The Wash button becomes active when all components have been removed. a. If a component is not removed and an error appears, remove the component and touch Rerun Check. b. If the component has been removed and the error persists, select Bypass Sensor Check to proceed to the post-run wash. If the Bypass Sensor Check option is not visible, your system is not configured to allow sensor bypass. For more information, see Configure Run Requirements on page Touch Wash to proceed to the post-run wash. If you have configured the post-run wash as optional, you can touch Exit to bypass the wash. Catalog # SY Part # Rev. C

71 Performing Post-Run Procedures 61 Flow Cell Storage Perform a Post- Run Wash If you need to store the flow cell, store it in storage buffer in the flow cell tube at 4 C. The flow cell is stable after primer hybridization for up to ten days when properly stored at 4 C in storage buffer in the flow cell tube. 1. Wash the plate on the thermal stage with deionized water to remove any salt residue, and dry with a lint-free cleaning tissue. 2. Fill the wash reservoir with approximately 12 ml deionized water. You must have a sufficient volume of water to prevent air from entering the lines. Wash Reservoir Figure 66 Fill the Wash Reservoir 3. Close the cbot lid. 4. Touch the checkbox on the screen to indicate that water is present. The Wash button becomes active. Wash Figure 67 Perform a Post-Run Wash 5. Touch Wash. 6. When the wash is complete, blot out any excess water remaining in the wash reservoir, taking care not to rub the outlet ports as this can cause fibers to clog the holes. 7. Touch the checkbox on the screen to indicated that the wash reservoir is dry. The Exit button becomes active. 8. Touch Exit. The Start screen appears. Your cbot is ready for another run. cbot User Guide

72 62 CHAPTER 4 Using the cbot Confirm Reagent Delivery 1. Visually inspect the foil-sealed tops of each tube strip and confirm that each reagent tube seal was pierced by the sipper comb. 2. Remove each tube strip from the reagent plate base: a. Hold the reagent plate base firmly with two hands. b. With your finger tips under the reagent plate base, gently press upward on the center tubes of the tube strip, releasing the tube strip from the base. c. Lift the tube strip out of the base. 3. Visually inspect each tube to confirm that reagent delivery was successful from all tubes. Successful delivery is indicated by an approximately equal remaining volume in each tube. NOTE Very small differences in delivery per lane are normal and do not affect performance. Figure 68 Successful Reagent Delivery Figure 69 Unsuccessful Reagent Delivery 4. If reagent delivery was not successful from all tubes and the foil-sealed tops of each tube is pierced, take a picture of the tube strip and contact Illumina Technical Support. 5. Inspect the eight-tube strips containing templates. 6. If you used custom primers with your run, inspect the eight-tube strips containing primers. Catalog # SY Part # Rev. C

73 Chapter 5 Maintenance and Troubleshooting Topics 64 Performing Periodic Maintenance 65 Performing a Monthly Maintenance Wash 68 Troubleshooting cbot User Guide 63

74 64 CHAPTER 5 Maintenance and Troubleshooting Performing Periodic Maintenance The cbot is easy to maintain. Perform the basic maintenance steps described in this section to ensure optimal performance. Table 9 cbot Periodic Maintenance Maintenance Frequency Description Instrument wash At least one water wash between every run and if the instrument is idle for more than a day. Always perform an instrument wash after each run to clear salts and enzymes from the instrument hardware and to prevent clogs. If the instrument has been idle for more than 24 hours, a pre-run wash is recommended. For more information, see Perform a Wash on page 41. Empty waste bottle Between every run. To ensure that your run is not interrupted, empty the waste bottle between runs. Clean surfaces Once a week. Use deionized water and a lint-free cleaning tissue to clean the surface of the thermal stage and the reagent stage. Clean the surface of the template and primer tube strip holders. Clean barcode scanner window Once a week. Use deionized water and a lint-free cleaning tissue to clean the barcode scanner window. Maintenance Wash Once a month. Use 5% DECON (or 100 mm NaOH) to remove traces of reagents from internal cbot components and inhibit the growth of microorganisms. For more information, see Performing a Monthly Maintenance Wash on page 65. Check coolant level Every three months. Ensure that the green coolant is visible through the coolant window on the rear panel of the instrument. If necessary, use a mirror to view the coolant window. If the coolant is low, use a wide coin or standard screwdriver to remove the coolant reservoir cap, and fill the reservoir to just below the reservoir cap. Use only Illumina-supplied coolant (part # ). If you need additional coolant, contact your Illumina Field Application Scientist or Field Service Engineer. Catalog # SY Part # Rev. C

75 Performing a Monthly Maintenance Wash 65 Performing a Monthly Maintenance Wash Perform a monthly maintenance using 5% DECON to remove traces of reagents from internal cbot components and inhibit microbial growth. In regions where DECON is not available, 100 mm NaOH may be substituted. The maintenance wash requires approximately ten minutes of hands-on time and consists of four wash steps: 1. A water wash. 2. A 5% DECON wash. 3. A water wash to rinse the system. 4. A second water wash to thoroughly rinse the system. Consumables Procedure User-Supplied Deionized water 5% DECON or 100 mm NaOH Low-lint lab tissues Perform a Water Wash 1. Confirm that all run components are removed before proceeding. For more information, see Unload Run Components on page From the Start screen, touch Menu and select Manual Commands. The Manual Commands screen opens. 3. Touch Commands to open the Commands tab. Figure 70 Manual Commands, Commands Tab cbot User Guide

76 66 CHAPTER 5 Maintenance and Troubleshooting 4. Fill the wash reservoir with approximately 12 ml deionized water. Wash Reservoir Figure 71 Fill the Wash Reservoir 5. Touch Wash on the Commands tab. 6. After the wash is finished, blot out any excess water remaining in the wash reservoir with a low-lint wipe, taking care not to rub the outlet ports as this can cause fibers to clog the holes. Figure 72 Dry the Wash Reservoir 7. Proceed to the DECON or NaOH wash. Perform a DECON (or NaOH) Wash 1. Fill the wash reservoir with 10 ml of 5% DECON or 100 mm NaOH. 2. Touch Wash. CAUTION DECON is a highly alkaline wash solution. Be sure to wear gloves when blotting 5% DECON to protect your skin. Do not allow DECON to sit in the wash reservoir for long periods of time as the high ph may corrode the metal pins over repeated, extended contact. Catalog # SY Part # Rev. C

77 Performing a Monthly Maintenance Wash After the wash is finished, blot out any excess 5% DECON remaining in the wash reservoir with a low-lint wipe, taking care not to rub the outlet ports as this can cause fibers to clog the holes. 4. Immediately proceed to the water wash to prevent DECON from drying and clogging the wash reservoir holes. Perform a Water Wash (First Rinse) 1. Fill the wash reservoir with approximately 12 ml deionized water. 2. Touch Wash. 3. After the wash is finished, blot out any excess water remaining in the wash reservoir with a low-lint wipe, taking care not to rub the outlet ports as this can cause fibers to clog the holes. 4. Proceed to the final water wash to remove all remaining traces of the 5% DECON from the wash reservoir and internal cbot components. Perform a Water Wash (Final Rinse) 1. Fill the wash reservoir with approximately 12 ml of clean, deionized water. 2. Touch Wash. 3. After the wash is finished, blot out any excess water remaining in the wash reservoir with a low-lint wipe, taking care not to rub the outlet ports as this can cause fibers to clog the holes. 4. Close the cbot lid and empty the waste bottle. The cbot is ready for the next cluster generation run. cbot User Guide

78 68 CHAPTER 5 Maintenance and Troubleshooting Troubleshooting Table 10 Troubleshooting Run Problems Use the following table to troubleshoot possible problems encountered during a cluster generation run. Problem Possible Cause Action Flow check failure Flow cell is installed upside down. Check positioning of the flow cell. Ensure ports are facing upward. Temperature out of range Coolant is flowing and coolant level is low Coolant is not flowing and coolant level is low Coolant is not flowing and coolant level is not low Poor seal between the manifold and the flow cell. Poor seal between the outlet end of the manifold and the outlet port. Sippers did not pierce reagent tubes in row 1. Reagents are still frozen. Problem with manifold flow. Potential Peltier or Peltier control board failure. Coolant has slowly evaporated to a low level. Coolant level may be too low to generate flow. Potential coolant pump failure. Ensure the manifold is evenly seated over the flow cell. Ensure the outlet end of the manifold is evenly seated in the outlet port. Ensure the sippers are straight and the reagent plate is positioned with row 1 facing towards the front of the instrument, directly behind the eighttube strip holder. Ensure the clear plastic lid has been removed from the reagent plate. Ensure reagents are completely thawed. Remove the manifold and ensure the black rubber gaskets are evenly seated in the clear plastic. If one lane consistently fails, the manifold may be clogged and require a replacement. Alternatively, you can bypass the flow check. However, the lanes indicated in the on-screen message may not cluster successfully. See Pre-Run Flow Check Failure on page 56 for more information. Contact Illumina Technical Support. Add Illumina-supplied coolant (part # ) to the coolant reservoir. Add Illumina-supplied coolant (part # ) to the coolant reservoir. Contact Illumina Technical Support. System is in a locked state Potential software error. Contact Illumina Technical Support. Catalog # SY Part # Rev. C

79 Appendix A Cluster Generation Chemistry Topics 70 Introduction 70 Adapter Ligation and Selection 71 Cluster Generation cbot User Guide 69

80 70 APPENDIX A Cluster Generation Chemistry Introduction During cluster generation, template molecules are attached to a flow cell and then amplified locally to form clonal clusters. The flow cells containing the clusters can then be sequenced on an Illumina sequencing instrument. This section gives an overview of the chemistry of cluster generation. Adapter Ligation and Selection Before cluster generation can occur, the template fragments need to contain the right 5 and 3 adapters, and have the proper length. This is done during sample preparation using the following steps: 1. In most protocols, samples are fragmented to get proper sized fragments. 2. Samples that consist of RNA are reverse transcribed to get a DNA template. 3. Two different Y-shaped adapters are ligated to the DNA fragments (Figure 73). 4. The fragments are PCR-amplified with primers annealing to the Y-shaped adapters. The Y-shaped adapters have been devised to generate fragments containing two non-homologous 5 and 3 ends (Figure 73). Figure 73 Non-Homologous 3 and 5 Ends Ligation of Y-shaped Adapters and PCR 5. Fragments are size-fractionated on a gel. The sample is now ready for cluster generation. Catalog # SY Part # Rev. C

81 Cluster Generation 71 Cluster Generation Cluster generation is performed on the cbot using the following steps: 1. The DNA fragment library is diluted, denatured and introduced into the lanes of the flow cell. The flow cell is a silica slide with eight lengthwise lanes containing a dense lawn of oligonucleotides. 2. The templates are captured by the oligonucleotides attached to the surface of the flow cell. Figure 74 Capture and Extension of Template 3. Templates bound to the primers are 3 -extended producing covalentlyattached discrete single molecules. 4. The double-stranded molecule is denatured, and the original template is washed away. 5. Free ends of the bound templates hybridize to adjacent lawn primers to form U-shaped bridges. Figure 75 Bridge Formation 6. The DNA bridge is copied from the primer to create a double-stranded DNA bridge. 7. The resulting dsdna is denatured, hybridized to lawn-primers to form new bridges and extended again. cbot User Guide

82 72 APPENDIX A Cluster Generation Chemistry Figure 76 Further Amplification of Template 8. This process of iso-thermal bridge amplification is repeated 35 times to create a dense cluster of over 2000 molecules. Figure 77 Clonal Cluster 9. The reverse strands in the cluster are removed by cleavage at the reverse strand-specific lawn primers, leaving a cluster with forward strands only. 10. The free 3 -OH ends are blocked to prevent non-specific priming. 11. Sequencing primers are hybridized to the free ends of the DNA templates. The flow cell with the synthesized clonal clusters is now ready to be sequenced on the Illumina sequencing instrument. Catalog # SY Part # Rev. C

83 Appendix B Manual Settings Topics 74 Manual Commands 77 Resetting the Barcode Scanner 79 Protocol Editor cbot User Guide 73

84 74 APPENDIX B Manual Settings Manual Commands Manual commands are used for troubleshooting purposes and confirm that the instrument is responding. You can access the Manual Commands screen from the Start screen menu. Touch Menu and select Manual Commands. The Manual Commands screen opens. Manual Commands Figure 78 Start Screen Menu The Manual Commands screen contains four tabs: Commands, Temp, Pump, and General. Commands Tab From the Commands tab, you can initialize the system, prime reagents through the system, or return the reagent stage to the home position. If the run is stopped unexpectedly, use this feature to release the reagent plate from the sipper comb. Use the Commands tab to perform a monthly maintenance wash. For more information, seeperforming a Monthly Maintenance Wash on page 65. Figure 79 Manual Commands, Commands Tab Catalog # SY Part # Rev. C

85 Manual Commands 75 Temp Tab From the Temp tab, you can confirm that the Peltier heater is performing correctly. If you change the temperature setting to other than 20 C, you must manually return the setting to 20 C before leaving the Manual Commands screen. Figure 80 Manual Commands, Temp Tab WARNING Never set the temperature lower than 20 C. Condensation may occur and damage the instrument. Pump Tab From the Pump tab, you can manually pump reagents to confirm reagent delivery and troubleshoot fluidics lines. For most purposes, a flow rate of 500 μl/minute is appropriate. Figure 81 Manual Commands, Pump Tab cbot User Guide

86 76 APPENDIX B Manual Settings General Tab From the General tab, you can confirm that the barcode scanner is responding. Use the settings on this tab to reset the scanner to the default configuration. For more information, see Resetting the Barcode Scanner on page 77. Figure 82 Manual Commands, General Tab Catalog # SY Part # Rev. C

87 Resetting the Barcode Scanner 77 Resetting the Barcode Scanner The cbot barcode scanner is ready for use when you receive your instrument. However, in the event that the barcode scanner is reset to an incorrect configuration, use the following instructions to restore it to the default configuration. 1. From the Manual Commands screen, touch General. 2. Touch Turn On to turn on the barcode scanner. Turn on Barcode Scanner Figure 83 Turn On Barcode Scanner 3. Scan the following barcodes in the order shown: Figure 84 Reset Barcode #1 Figure 85 Reset Barcode #2 Figure 86 Reset Barcode #3 Figure 87 Reset Barcode #4 cbot User Guide

88 78 APPENDIX B Manual Settings Figure 88 Reset Barcode #5 Figure 89 Reset Barcode #6 \ Figure 90 Reset Barcode #7 Figure 91 Reset Barcode #8 Figure 92 Reset Barcode #9 Figure 93 Reset Barcode #10 Figure 94 Reset Barcode #11 Catalog # SY Part # Rev. C

89 Protocol Editor 79 Protocol Editor You can edit protocols according to your needs using the Protocol Editor. For example, you may want to repeat steps in a protocol, or change the number of amplification cycles in the chemistry section. Each protocol consists of two main sections: Chemistry section Contains instructions for pumping reagents, temperature changes, and wait durations. The chemistry section of the protocol appears in upper portion of the Protocol Editor screen. Protocol section Contains a series of steps made up of chemistry definitions. The protocol section appears in the lower portion of the Protocol Editor screen. If you edit an existing protocol, remember to rename your protocol. 1. From the Start screen, touch Menu and select Protocol Editor. The Protocol Editor opens. 2. From the Protocol Editor, touch Menu and select one of the following commands from the menu: Select Open to open an existing protocol. Select Load from Library to load an existing chemistry definition or protocol step stored in the cbot library. Select New Chemistry Definition or New Protocol Step to create a new definition or step and store it in the cbot library. Figure 95 Protocol Editor Menu cbot User Guide

90 80 APPENDIX B Manual Settings Chemistry Section Expanded Chemistry Steps Protocol Section Figure 96 Protocol Editor, Expanded Steps 3. Use the down arrow to the left of the step to expand the commands in the step or the up arrow to collapse the commands (Figure 96). 4. To edit a step in a chemistry definition, highlight the step. Selections appear in the right-hand panel to change the pump, temperature ramp, or wait commands. 5. To edit a step in the protocol, highlight the step. Selections appear in the right-hand panel to change to the number of cycles for the selected chemistry definition. 6. Use the Protocol Editor icons to the right of the step name to rearrange, delete, or copy steps and commands. See Protocol Editor Icons on page 81 for more information. Figure 97 Protocol Editor, Editing Panel Catalog # SY Part # Rev. C