MiSeq System Guide - Illumina, Inc.

1 MiSeqSystemGuideILLUMINA PROPRIETARYD ocument # 1000000061014 v00 July 2018 For Research Use Only. Not for use in diagnostic document and its contents are proprietary to illumina , Inc. and its affiliates (" illumina "), and are intended solely forthe contractual use of its customer in connection with the use of the product(s) described herein and for no otherpurpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwisecommunicated, disclosed, or reproduced in any way whatsoever without the prior written consent of illumina .

2 Illuminadoes not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any thirdparties by this instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel inorder to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must befully read and understood prior to using such product(s).FAILURE TO COMPLETELY read AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAYRESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGETO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S).

3 illumina DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE). 2018 illumina , Inc. All rights trademarks are the property of illumina , Inc. or their respective owners. For specific trademark information, # 1000000061014 v00 For Research Use Only. Not for use in diagnostic GuideTable of ContentsChapter 1 Overview1 Introduction1 Additional Resources1 Components2 MiSeq Concepts4 System Software5 Secondary Analysis Options7 Sequencing Analysis Viewer8 Required Disk Space8 MiSeq Reagent Kit Overview9 Chapter 2 Getting Started13 Start the MiSeq13 Customize System Settings13 Configure Notifications of BaseSpace Updates14 Set Email Preferences14 Set Default Folder Locations14 User-Supplied Consumables15 Chapter 3 Sequencing17 Introduction17 Run Duration17 MiSeq Workflow18 Thaw Reagent Cartridge19 Inspect

4 The Reagent Cartridge19 Denature and Dilute Libraries20 Load Sample Libraries20 Set Up a Run Using MCS20 Clean the Flow Cell21 Load the Flow Cell22 Load Reagents23 Starting the Run25 Monitor the Run26 Perform a Post-Run Wash28 Chapter 4 Maintenance33 Maintenance Frequency33 Maintenance Frequency for the VeriSeq PGS Workflow33 Perform a Maintenance Wash34 Perform a Standby Wash36 Manage Files38 Software Updates39 Shut Down the Instrument39 Document # 1000000061014 v00 For Research Use Only. Not for use in diagnostic A Troubleshooting41 Introduction41 Bundle Logs for Troubleshooting41 Perform a System Check42 Pause or Stop a Run42 Raise Reagent Cartridge Sippers Manually44 Resolve Run Setup Errors44 Resolve RFID read Failure44 Perform a Volume Test45 Measure Expected Wash Volumes46 Configure System Settings46 Appendix B Output Files and Folders49 Run Folders49 MiSeqOutput Folder Contents49 RTA Folders and Files51 Index53 Technical Assistance57 Document # 1000000061014 v00 For Research Use Only.

5 Not for use in diagnostic GuideChapter 1 OverviewIntroduction1 Additional Resources1 Components2 MiSeq Concepts4 System Software5 Secondary Analysis Options7 Sequencing Analysis Viewer8 Required Disk Space8 MiSeq Reagent Kit Overview9 IntroductionThe illumina MiSeq System combines proven sequencing by synthesis (SBS) technology with arevolutionary workflow that lets you go from DNA to analyzed data in as few as eight hours. The MiSeqintegrates cluster generation, sequencing, and data analysis on a single away automation After setting up your run, which includes loading the pre-filled reagent cartridge,buffer bottle, and flow cell, no additional hands-on time is reagent cartridge A specially designed single-use prefilled reagent cartridge provides reagentsfor cluster generation and sequencing, including paired-end sequencing reagents and indexing radio-frequency identification (RFID) tracking enables accurate consumable controls The MiSeq Control Software (MCS)

6 Interface provides controls to configure theinstrument, set up and monitor runs, and perform maintenance flow cell loading A clamping mechanism auto-positions the flow cell as it is loaded onto theinstrument. Integrated radio-frequency identification (RFID) tracking enables accurate fluidics architecture The MiSeq fluidics System enables unmatched efficiency in chemistrycycle time during analysis (RTA) Integrated analysis software performs real-time on-instrument data analysisduring the sequencing run, which includes image analysis and base calling, and saves valuabledownstream analysis secondary analysis software MiSeq Reporter software processes data from analysis by RTAto perform alignment and provides information about each sample ResourcesThe MiSeq System support pageson the illumina website provide additional resources.

7 These resourcesinclude software, training, compatible products, and the following documentation. Always check supportpages for the latest # 1000000061014 v00 For Research Use Only. Not for use in diagnostic System Site PrepGuide (document #15027615)Provides specifications for laboratory space, electrical requirements, and System Safety andCompliance Guide (document # 15027616)Provides information about instrument labeling, compliance certifications, and ExperimentManager User Guide (document # 15031335)Provides instructions for creating sample plates and sample sheets for different workflowsand library WorkflowManager User Guide (document #1000000028842)

8 Provides instructions for creating sample plates and sample sheets for use with theVeriSeq PGS Sample SheetQuick Reference Guide (document # 15028392)Provides information about adding sample sheet settings to your sample System Denatureand Dilute Libraries Guide (document # 15039740)Provides instructions for denaturing and diluting prepared sample libraries beforesequencing on the MiSeq , and preparing a PhiX control. This step applies to most Custom PrimersGuide (document #15041638)Provides instructions for preparing and loading custom primers, and editing the samplessheet for custom Reporter User Guide (document # 15042295)Provides a comprehensive overview of analysis procedures, analysis workflows, andoutput files generated by MiSeq Reporter, as well as computing requirements, off-instrument installation instructions, and troubleshooting Reporter Online HelpProvides instructions for using the MiSeq Reporter Multi SoftwareGuide (document #15053620)

9 Provides a comprehensive overview of analysis procedures, analysis workflows, and filesgenerated by BlueFuse Multi, as well as computing requirements, and troubleshootinginformation. Use this Guide with the VeriSeq PGS User Guide (document # 15044182)Provides instructions for using BaseSpace and descriptions of the graphs generated foreach analysis Onsite SystemGuide (document #15049148)Provides instructions for using the BaseSpace Onsite MiSeq comprises a touch screen monitor, a status bar, a power button with adjacent USB ports, andthree # 1000000061014 v00 For Research Use Only.

10 Not for use in diagnostic GuideAFlow cell compartment Contains the flow cell stage that houses the flow cell throughout the run. Flow cellstage motors move the stage out of the enclosed optical module for flow cell loading and returns the stagewhen the run optics compartment Contains optical components that enable imaging of the flow bar Indicates flow cell status as ready to sequence (green), processing (blue), or needs attention(orange).DTouch screen monitor Displays the control software interface for System configuration and run USB ports Facilitates the transfer of files and data to the instrument computer from the touchscreen compartment Contains reagents at proper temperatures, wash solutions, and a bottle for usedreagents.