MiSeq. System Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v04 Material # July 2018

Transcription

1 MiSeq System Gide Docment # v04 Material # Jly 2018 ILLUMINA PROPRIETARY

2 MiSeq System Gide This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(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, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S). ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) Illmina, Inc. All rights reserved. All trademarks are the property of Illmina, Inc. or their respective owners. For specific trademark information, see Docment # v04 Material # ii

3 Revision History Docment # Date Description of Change Material # Docment # v04 Material # Docment # v03 Material # Docment # v02 Jly 2018 May 2018 Janary 2018 Added new rn configration options for Local Rn Manager, Sample Sheets, and Manal seqencing modes. Replaced references to MiSeq Reporter with Local Rn Manager. Added Resolve Chiller Temperatre Ranges to Trobleshooting Appendix. Removed the defalt ser name and password reqired to log on to the operating system. Illmina recommends sing site-specific credentials. Added information abot the Illmina Proactive monitoring service in the Cstomize System Settings section. Added note to Reagent Compartment regarding reqired chiller temperatre range. Added back MiSeq Reporter and removed references to Local Rn Manager. Changed the name of the gide from the MiSeq Seqencing System Gide to the MiSeq System Gide. Minor text edits. Added new rn configration options for Local Rn Manager, Sample Sheets, and Manal seqencing modes. Replaced references to MiSeq Reporter with Local Rn Manager. Added Resolve Chiller Temperatre Ranges to Trobleshooting Appendix. Added new instrction for collecting instrment performance data in the Cstomize System Settings topic. Changed the name of the gide from the MiSeq System Gide to the MiSeq Seqencing System Gide. Docment # v04 Material # iii

4 MiSeq System Gide Docment # Date Description of Change Material # Docment # v01 Part # Rev. O September 2015 September 2014 Changed the name of the gide from the MiSeq System User Gide to the MiSeq System Gide. New in MCS v2.6, added BaseSpace Onsite information. Rearranged gide information to center arond tasks rather than screens. All references to the Welcome screen are changed to the Home screen. Added information that is in the MiSeq Reagent Kit Reagent Prep Gides, inclding flow cell types, reagent cartridge contents, and directions for thawing the reagent cartridge. Visit the Illmina website for all other MiSeq Reagent Kit information at spport.illmina.com/seqencing/seqencing_kits/miseq_ reagent_kit.html. Corrected the expected wash volme of a maintenance wash from ml to ml. Updated the order of workflow steps to first prepare the reagent cartridge, and then if reqired, denatre and dilte libraries. Moved trobleshooting information to Appendix A. Moved otpt folder information and flow cell tile information to Appendix B. Moved information on managing files to the Maintenance chapter. Moved the post-rn wash instrctions from the Maintenance chapter to the Seqencing chapter. The name of the Performing a Rn chapter is now Seqencing. Changed references from primary analysis to analysis by RTA software. Updated the diltion directions for the post-rn wash procedre with a template line wash from 6% NaOCl to 5% NaOCl. Added an NaOCl, 5% part nmber to the ser-spplied consmables list. Updated the following information: New in MCS v2.5, pdated post-rn wash option to inclde a template line wash Updated directions for a post-rn wash with sodim hypochlorite to the template line wash Added the expected wash volme for a post-rn washes Added VeriSeq PGS workflow information regarding additional resorces, rn options, secondary analysis options, instrment washes, and flow cell cap color. Part # Rev. N Jne 2014 Added information applicable to the VeriSeq PGS workflow. Updated rn metrics information for clster generation and density. Removed anti-virs software information. See MiSeq System Site Preparation Gide. Docment # v04 Material # iv

5 MiSeq System Gide Docment # Date Description of Change Part # Rev. M Part # Rev. L Part # Rev. K Part # Rev. J Part # Rev. H Janary 2014 October 2013 Agst 2013 Agst 2013 March 2013 Updated to change introdced in MCS v2.4: Added bndle logs featre for sending trobleshooting files. Added the reboot of system software as a pre-rn step. Added microcentrifge tbes to the list of ser-spplied consmables. Eliminated MiSeq Software as a separate chapter and distribted chapter contents throghot the gide. Removed information abot folders for cstom recipes. Removed information abot recommended clster density ranges for MiSeq reagent kits. Removed details abot MiSeq reagent kits and added an overview of reagent kit featres. For detailed information, see the reagent prep docmentation for the kit yo are sing. Added content to the trademarks notice. Corrected formatting errors. Added rn descriptions for MCS v2.3 and MiSeq Reagent Kit v3. Updated the following information: Reagent kit and version compatibility to inclde MiSeq Reagent Kit v3 Cstom Recipes folder description to inclde a v3 sbfolder Changed clster density range for v2; added range for v3 Otpt path for image files Corrected flow cell bar codes for nano flow cells (D) and micro flow cells (G). Removed information abot MiSeq Reagent Kit, inclding contents and flow cell types. For more information, see MiSeq Reagent Preparation Gide (docment # ). Added section titled MiSeq Concepts that introdces the analysis workflow, manifest file, and sample sheet. Removed information abot FASTQ file generation, manifest file formats, analysis workflow details, and sample sheet details. For information abot these topics, see the MiSeq Reporter User Gide, part # , or the MiSeq Sample Sheet Qick Reference Gide, part # Removed instrctions for preparing cstom primers. For more information, see Using Cstom Primers on the MiSeq, part # Docment # v04 Material # v

6 MiSeq System Gide Docment # Date Description of Change Part # Rev. G Part # Rev. F Part # Rev. E Janary 2013 November 2012 October 2012 Removed instrctions for denatring and dilting DNA libraries and preparing an Illmina PhiX control. See Preparing DNA Libraries for Seqencing on the MiSeq, part # Updated instrment wash instrctions to add 25 ml 10% Tween 20 to 475 ml laboratory-grade water, instead of 500 ml laboratory-grade water. Added the following new information: Added kit descriptions for new MiSeq reagent kits: MiSeq Reagent Nano Kit and MiSeq Reagent Micro Kit Added overview of flow cell types Added description of Enrichment analysis workflow Updated the following information: New in MCS v2.1, pdated Perform Wash screen to add a post-rn wash option and command to raise sippers Updated version compatibility table to inclde nano and micro kit dependencies Updated version compatibility information to inclde new reagent kits Updated the following information: Corrected PhiX control preparation instrctions and expected clster density of prepared PhiX control to K/mm² Noted that the procedre for denatring and dilting libraries, Preparing Yor Libraries, does not apply to Nextera XT libraries as well as TrSeq Amplicon libraries Changed pgrade name from MiSeq Expansion Pack to MiSeq hardware pgrade Add the MiSeq Reporter User Gide to Additional Resorces list. Docment # v04 Material # vi

7 MiSeq System Gide Docment # Date Description of Change Part # Rev. D Jly 2012 Updated software descriptions to MCS v2.0. Added the following new information: Added a section titled What's New in MCS to describe new software featres, interface changes, and workflow changes Added catalog nmber and description of the MiSeq Reagent Kit v2, 500 Cycles Added Version Compatibility and Reqirements section Added description of MiSeq Expansion Pack, which is reqired for 14-tile dal-srface flow cell imaging Added description of dal-srface flow cell tile nmbering Added the PCR Amplicon analysis workflow for Nextera XT libraries Added the se of 10% Tween 20 in wash procedres and expected wash volmes Added the reagent cartridge version to the RFID read failre procedre Updated the following information: Changed reagent acronyms for IMF, CMF, and AMX to v2 reagent names IMS, CMS, and AMS, respectively Changed the PhiX concentration from 8 pm to 12.5 pm Changed the maximm recommended NaOH concentration to 1 mm in final soltion Noted that a maintenance wash is reqired to remove the instrment from standby mode and begin the setp steps for a sbseqent rn Removed Sample Sheet Parameters section and sample sheet setp step in the workflow; Illmina recommends creating the sample sheet prior to sample preparation (See the MiSeq Sample Sheet Qick Reference Gide, part # and the Illmina Experiment Manager User Gide, part # ). Docment # v04 Material # vii

8 MiSeq System Gide Docment # Date Description of Change Part # Rev. C April 2012 Updated software descriptions to MCS v1.2 Added the following new procedres and sections: BaseSpace overview, Using Cstom Primers, Generating FASTQ files, Trobleshooting Flow Rate Error, Performing a Volme Test, Performing a Maintenance Wash, and Idling the Instrment, which incldes a standby wash. Updated the following information: Updated name of Amplicon workflow to Cstom Amplicon; pdated name of DenovoAssembly workflow to Assembly; added GenerateFASTQ workflow Added descriptions of rn folders and files; pdated rn folder naming; added otpt file size Listed genome folder as reqired for amplicon seqencing in Sample Sheet Parameters Added instrctions for dilting NaOH to denatre libraries Updated Resolving RFID Read Failre to inclde MiSeq Self-Service instrctions Listed files and folders sed for trobleshooting rn performance Part # Rev. B Part # Rev. A December 2011 September 2011 Updated software descriptions to MCS v1.1 Added information abot anti-virs protection Updated the following information: Instrctions to resolve RFID failre Preparing libraries Changed to 0.2 N NaOH Rn folder naming convention Reqired disk space and storage capacity Rn setp steps Added more information to Setting Up the Sample Sheet Rn setp steps Added note to dispose of remaining PR2 Analysis dration Added when analysis exceeds two hors Analysis Inpt Reqirements Listed manifest files as reqired for TrSeq Cstom Amplicon libraries Corrected HT1 tbe size in MiSeq Reagent Kit Contents Changed icom references to MyIllmina Initial release Docment # v04 Material # viii

9 Table of Contents Chapter 1 Overview 1 Introdction 1 Additional Resorces 1 Components 2 MiSeq Concepts 4 System Software 5 Secondary Analysis Options 6 Seqencing Analysis Viewer 8 Reqired Disk Space 8 MiSeq Reagent Kit Overview 8 Chapter 2 Getting Started 11 Start the MiSeq 11 Cstomize System Settings 11 Configre Notifications of BaseSpace Updates 12 Set Preferences 12 Set Defalt Folder Locations 12 User-Spplied Consmables 13 Chapter 3 Seqencing 15 Introdction 15 Rn Dration 15 MiSeq Workflow 17 Thaw Reagent Cartridge 18 Inspect the Reagent Cartridge 19 Denatre and Dilte Libraries 19 Load Sample Libraries 19 Set Up a Rn Using MCS 20 Clean the Flow Cell 21 Load the Flow Cell 23 Load Reagents 24 Start the Rn 26 Monitor the Rn 27 Perform a Post-Rn Wash 29 Chapter 4 Maintenance 33 Maintenance Freqency 33 Maintenance Freqency for the VeriSeq PGS Workflow 33 Perform a Maintenance Wash 34 Perform a Standby Wash 36 Manage Files 38 Software Updates 39 Sht Down the Instrment 39 Docment # v04 Material # ix

10 MiSeq System Gide Appendix A Trobleshooting 41 Introdction 41 Bndle Logs for Trobleshooting 41 Perform a System Check 42 Pase or Stop a Rn 42 Raise Reagent Cartridge Sippers Manally 44 Resolve Rn Setp Errors 44 Resolve RFID Read Failre 44 Perform a Volme Test 45 Measre Expected Wash Volmes 46 Resolve Reagent Chiller Temperatre Errors 46 Resolve Local Rn Manager Analysis Errors 46 Configre System Settings 46 Appendix B Otpt Files and Folders 47 Rn Folders 47 MiSeqOtpt Folder Contents 47 RTA Folders and Files 49 Index 51 Technical Assistance 55 Docment # v04 Material # x

11 Chapter 1 Overview Introdction 1 Additional Resorces 1 Components 2 MiSeq Concepts 4 System Software 5 Secondary Analysis Options 6 Seqencing Analysis Viewer 8 Reqired Disk Space 8 MiSeq Reagent Kit Overview 8 Introdction The Illmina MiSeq system combines proven seqencing by synthesis (SBS) technology with a revoltionary workflow that lets yo go from DNA to analyzed data in as few as eight hors. The MiSeq integrates clster generation, seqencing, and data analysis on a single instrment. Featres Walk away atomation After setting p yor rn, which incldes loading the pre-filled reagent cartridge, bffer bottle, and flow cell, no additional hands-on time is reqired. Prefilled reagent cartridge A specially designed single-se prefilled reagent cartridge provides reagents for clster generation and seqencing, inclding paired-end seqencing reagents and indexing reagents. Integrated radio-freqency identification (RFID) tracking enables accrate consmable tracking. Interface controls The MiSeq Control Software (MCS) interface provides controls to configre the instrment, set p and monitor rns, and perform maintenance procedres. Convenient flow cell loading A clamping mechanism ato-positions the flow cell as it is loaded onto the instrment. Integrated radio-freqency identification (RFID) tracking enables accrate consmable tracking. Innovative flidics architectre The MiSeq flidics system enables nmatched efficiency in chemistry cycle time dring seqencing. Real-time analysis (RTA) Integrated analysis software performs real-time on-instrment data analysis dring the seqencing rn, which incldes image analysis and base calling, and saves valable downstream analysis time. Local Rn Manager Integrated secondary analysis software processes data from analysis by RTA to perform alignment and provides information abot each sample analyzed. Additional Resorces The MiSeq system spport pages on the Illmina website provide additional resorces. These resorces inclde software, training, compatible prodcts, and the following docmentation. Always check spport pages for the latest versions. Docment # v04 Material #

12 MiSeq System Gide Resorce MiSeq System Site Prep Gide (docment # ) MiSeq System Safety and Compliance Gide (docment # ) Illmina Experiment Manager User Gide (docment # ) BleFse Workflow Manager User Gide (docment # ) MiSeq Sample Sheet Qick Reference Gide (docment # ) MiSeq System Denatre and Dilte Libraries Gide (docment # ) MiSeq Cstom Primers Gide (docment # ) Local Rn Manager Software Gide (docment # ) BleFse Mlti Software Gide (docment # ) BaseSpace User Gide (docment # ) BaseSpace Onsite System Gide (docment # ) Description Provides specifications for laboratory space, electrical reqirements, and environmental considerations. Provides information abot instrment labeling, compliance certifications, and safety considerations. Provides instrctions for creating sample plates and sample sheets for different workflows and library types. Provides instrctions for creating sample plates and sample sheets for se with the VeriSeq PGS workflow. Provides information abot adding sample sheet settings to yor sample sheet. Provides instrctions for denatring and dilting prepared sample libraries before seqencing on the MiSeq, and preparing a PhiX control. This step applies to most library types. Provides instrctions for preparing and loading cstom primers, and editing the samples sheet for cstom primers. Provides an overview of the Local Rn Manager software, instrctions for sing software featres, and instrctions for installing analysis modles on the instrment compter. Provides a comprehensive overview of analysis procedres, analysis workflows, and files generated by BleFse Mlti, as well as compting reqirements, and trobleshooting information. Use this gide with the VeriSeq PGS workflow. Provides instrctions for sing BaseSpace and descriptions of the graphs generated for each analysis workflow. Provides instrctions for sing the BaseSpace Onsite System. Components The MiSeq comprises a toch screen monitor, a stats bar, a power btton with adjacent USB ports, and three compartments. Docment # v04 Material #

13 MiSeq System Gide A B C D E F Flow cell compartment Contains the flow cell stage that hoses the flow cell throghot the rn. Flow cell stage motors move the stage ot of the enclosed optical modle for flow cell loading and retrns the stage when the rn begins. Enclosed optics compartment Contains optical components that enable imaging of the flow cell. Stats bar Indicates flow cell stats as ready to seqence (green), processing (ble), or needs attention (orange). Toch screen monitor Displays the control software interface for system configration and rn setp. External USB ports Facilitates the transfer of files and data to the instrment compter from the toch screen monitor. Reagent compartment Contains reagents at proper temperatres, wash soltions, and a bottle for sed reagents. A magnetic latch secres the reagent compartment door. The MiSeq interface gides yo throgh the rn setp steps sing the toch screen monitor. Loading rn components reqires access to the reagent compartment and the flow cell compartment. Flow Cell Compartment The flow cell compartment contains the flow cell stage, thermal station, and flidics connections to the flow cell. The flow cell stage holds the flow cell and the flow cell clamp secres and positions the flow cell. When the flow cell clamp closes, two pins near the clamp hinge ato-position the flow cell. The thermal station, located beneath the flow cell stage, controls changes in flow cell temperatre reqired for clster generation and seqencing. Figre 1 Flow Cell Compartment A B C D E Flow Cell Stage Flow Cell Compartment Door Flow Cell Clamp Flow Cell Flow Cell Clamp Release Btton Reagent Compartment The reagent compartment contains the reagent chiller, and positions for the wash bffer (PR2) bottle and the waste bottle. To maintain a consistent temperatre, open and close the reagent chiller only when instrcted. NOTE The reqired temperatre range of the reagent chiller is 2 C to 11 C. Docment # v04 Material #

14 MiSeq System Gide Dring the rn, the reagent chiller holds a single-se reagent cartridge. Dring the instrment wash, the reagent chiller holds the wash tray. The software atomatically lowers sippers into each reservoir of the reagent cartridge at the appropriate time dring a rn depending on the process being performed. To the right of the reagent chiller are form-fitted slots for the PR2 bottle and the waste bottle. The sipper handle locks the bottles in place and lowers the appropriate sipper into each bottle. Reagents are pmped throgh the sippers and flidics lines, and then to the flow cell. Reagent waste is delivered to the waste bottle throghot the process. Figre 2 Reagent Compartment Components A B C D E Reagent Chiller Sipper Handle (shown in raised position) PR2 Bottle Waste Bottle Reagent Cartridge MiSeq Concepts The following concepts and terms are common to the rn setp steps on the MiSeq. Concept Analysis Workflow Manifest Reference Genome Rn Folder Sample Sheet Description A secondary analysis procedre is performed by Local Rn Manager. The analysis workflow for each rn is specified in the sample sheet or by the selected modle. The file that specifies a reference genome and targeted reference regions to be sed in the alignment step. For workflows that reqire a manifest, the manifest file is specified in the sample sheet and copied to the manifest folder designated in MCS. Manifest files can also be loaded in Local Rn Manager for se dring secondary analysis. A FASTA format file that contains the genome seqences sed dring analysis. For most analysis workflows, the reference genome file is specified in the sample sheet. The folder strctre poplated by RTA software (MiSeqOtpt folder) or the folder poplated by Local Rn Manager (MiSeqAnalysis). For more information, see Rn Folders on page 47. A comma-separated vales file (*.csv) that contains information sed to set p and analyze a seqencing rn, inclding a list of samples and their index seqences. The sample sheet can be provided dring the rn setp steps on the MiSeq. After the rn begins, the sample sheet is renamed to SampleSheet.csv and copied to the rn folders: MiSeqTemp, MiSeqOtpt, and MiSeqAnalysis. Docment # v04 Material #

15 MiSeq System Gide For more information on analysis workflows and manifest file formats, see the Local Rn Manager Software Gide (docment # ). For more information on sample sheets, see the MiSeq Sample Sheet Qick Reference Gide (docment # ). System Software The instrment software site incldes integrated applications that perform seqencing rns, on-instrment analysis, and related fnctions. MiSeq Control Software (MCS) Controls instrment operation. The MiSeq Control Software (MCS) interface gides yo throgh the steps to load the flow cell and reagents before beginning the rn. An overview of qality statistics appears as the rn progresses. Dring the rn, MCS operates the flow cell stage, dispenses reagents, controls flow cell temperatres, and captres images of clsters on the flow cell. MCS performs the rn according to parameters specified in the Local Rn Manager software. Real-time analysis (RTA) software Performs image analysis and base calling, and assigns a qality score to each base for each cycle. Images are temporarily stored in the rn folder for processing by RTA, and then atomatically deleted when analysis by RTA is complete. Local Rn Manager An on-instrment integrated soltion sed to create a rn, monitor stats, analyze seqencing data, and view reslts. Local Rn Manager also tracks sample information and controls ser permissions. The software rns as a Windows service on the instrment compter and is viewed throgh a web browser. For more information see Local Rn Manager Software on page 7. Optional software sed off-instrment incldes the Seqencing Analysis Viewer (SAV). For more information, see Seqencing Analysis Viewer on page 8. Stats Icons A stats icon on the control software interface indicates a change in conditions dring rn setp or dring the rn. A nmber on the icon indicates the nmber of conditions for a stats. When a rn stats changes, the icon blinks to alert yo. Select the icon to view a description of the condition. Select Acknowledge to clear the message, and then Close to close the dialog box. Filter the types of messages that appear in the stats window by selecting the icons along the top margin of the window. Selecting an icon toggles the condition to show or hide. Stats Icon Stats Name Stats OK Description No change. System is normal. Attention Important information. Action is recommended. Warning Warnings do not stop a rn. However, some warnings reqire action before proceeding. Error Errors sally stop a rn and generally reqire action before proceeding with the rn. Docment # v04 Material #

16 MiSeq System Gide Activity Indicators An activity indicator icon displays the activity the instrment is crrently performing. Figre 3 Activity Indicators From left to right, the activity indicators represent the following activities: Moving the Y-stage Moving the Z-stage Activating electronics fnctionality Using the camera Pmping throgh the flidics system Sensor Indicators Sensor indicators, which appear at the base of each interface screen, represent the stats of instrment components. Figre 4 Sensor Indicators From left to right, the sensor indicators represent the following components: Flow cell compartment door in the closed or open positions Temperatre of the reagent chiller in C Temperatre of the flow cell in C Stats of BaseSpace connection (not connected shown) Secondary Analysis Options MiSeq seqencing data can be analyzed on the instrment compter sing Local Rn Manager, on a networked server sing BaseSpace Onsite, or on the clod sing BaseSpace. These applications prodce information abot alignment, variants, and contig assemblies for each genome reqested and for each sample of a mltisample rn. If performing the VeriSeq PGS workflow, se BleFse Mlti software for analysis. BaseSpace and BaseSpace Onsite Overview BaseSpace is the Illmina clod compting environment. BaseSpace Onsite provides a compting environment on a dedicated server, complete with rn setp tools and analysis options. Log in to BaseSpace or BaseSpace Onsite when yo set p the seqencing rn. When sing BaseSpace or BaseSpace Onsite, yo have the additional option to store rn data locally. For more information, see Cstomize System Settings on page 11. Docment # v04 Material #

17 MiSeq System Gide When yo begin yor seqencing rn, the icon changes to indicate that the MiSeq is connected to BaseSpace or BaseSpace Onsite and data files are being transferred to the specified location. Figre 5 Connected to BaseSpace Icon Figre 6 Connected to BaseSpace Onsite Icon Using BaseSpace, data files are encrypted in transit, decrypted dring analysis, and encrypted again when stored. Using BaseSpace Onsite, data files are encrypted in transit, decrypted dring analysis, and can be optionally encrypted again when stored. BaseSpace and BaseSpace Onsite atomatically disconnect from the MiSeq at the end of the rn or as soon as all RTA analysis files have finished transfer. If the internet connection is interrpted, analysis files contine to pload after the connection is restored from the point when the interrption occrred. As soon as the last base call file is ploaded to BaseSpace or BaseSpace Onsite, secondary analysis of yor data begins. The same analysis workflows are spported on BaseSpace and BaseSpace Onsite as with oninstrment analysis sing Local Rn Manager. Yo can connect to BaseSpace at basespace.illmina.com. Log in sing yor MyIllmina accont login. For more information on BaseSpace, see the BaseSpace User Gide (docment # ) and the BaseSpace spport pages on the Illmina website. For more information on BaseSpace Onsite, see the BaseSpace Onsite System Gide (docment # ) and the BaseSpace OnSite spport pages on the Illmina website. Local Rn Manager Software Local Rn Manager software is an on-instrment integrated soltion for creating a rn, monitoring stats, analyzing data, and viewing reslts. The software integrates with MCS and processes base calls generated dring initial analysis. Local Rn Manager performs secondary analysis atomatically pon completion of a seqencing rn. Local Rn Manager is sed to record sample information dring library preparation and ensres positive sample tracking throghot the process, prodcing information abot each sample. In addition, Local Rn Manager can control ser athentication if enabled, granting varios access level permissions to sers. Permissions are saved in a database file, which the MiSeq references. Local Rn Manager can also monitor the seqencing rn. For more information, see the Local Rn Manager Software Gide (docment # ). Docment # v04 Material #

18 MiSeq System Gide Seqencing Dring Analysis The MiSeq system compting resorces are dedicated to either seqencing or analysis. With Local Rn Manager, if a new seqencing rn is started on the MiSeq before secondary analysis of an earlier rn is complete, a confirmation dialog box appears. After confirming yo want the new seqencing rn to start, secondary analysis of the earlier rn is stopped ntil the new rn completes seqencing. After the new rn completes seqencing, secondary analysis of the earlier rn atomatically starts again from the beginning. Seqencing Analysis Viewer Yo can monitor yor rn in greater detail withot interfering with the rn sing the Illmina Seqencing Analysis Viewer (SAV). Yor MiSeq mst be networked to view primary analysis reslts with SAV. SAV allows yo to review metrics dring a rn as metrics are generated, and later after a rn has completed. Install SAV onto a compter independent of the MiSeq with access to the same network connected to the instrment. After lanching the software, browse to the otpt folder for yor rn. After template generation, SAV provides metrics generated by RTA and organizes the metrics into plots, graphs, and tables. NOTE SAV is niversal to Illmina seqencing systems, most of which se an 8-lane flow cell. Some views inclde drop-down lists showing lanes 1 8. Becase the MiSeq flow cell has a single lane, select All or Lane 1. For more information, see the Seqencing Analysis Viewer User Gide (docment # ). Reqired Disk Space The integrated instrment compter has approximately 550 GB of storage capacity. Before starting a rn, the software checks available disk space. If there is not enogh disk space for the rn, a message indicating how mch disk space is reqired appears. If prompted to make disk space available, move or delete older rn folders as appropriate. For more information, see Manage Files on page 38. After clearing adeqate disk space, select Restart Check. MiSeq Reagent Kit Overview The MiSeq Reagent Kit is a single-se reagent kit reqired to perform a seqencing rn. It is available in different types and sizes. Each type of MiSeq Reagent Kit incldes a kit-specific flow cell type and all reagents reqired for performing a rn. The flow cell, PR2 bottle, and reagent cartridge provided in the kit se radio-freqency identification (RFID) for accrate consmable tracking and compatibility. Always se the reagent cartridge associated with yor flow cell type. If the reagent cartridge is not compatible, a message appears dring rn setp that prompts yo to load a compatible reagent cartridge. For a description of available reagent kits, visit the MiSeq Reagent Kits prodct page on the Illmina website. Flow Cell The MiSeq flow cell is a single-se glass-based sbstrate where clsters are generated and the seqencing reaction is performed. Docment # v04 Material #

19 MiSeq System Gide Reagents enter the flow cell throgh the inlet port, pass throgh the single-lane imaging area, and then exit the flow cell throgh the otlet port. Waste exiting the flow cell is delivered to the waste bottle. Libraries are loaded onto the reagent cartridge before setting p the rn, and then atomatically transferred to the flow cell after the rn begins. Figre 7 MiSeq Flow Cell A B C Otlet Port Imaging Area Inlet Port Flow Cell Cap Color The cap color of the flow cell container indicates the flow cell type: Flow Cell Standard Flow Cell PGS Flow Cell Micro Flow Cell Nano Flow Cell Flow Cell Cap Color Clear Green Yellow Reagent Cartridge Overview The MiSeq reagent cartridge is a single-se consmable consisting of foil-sealed reservoirs prefilled with clstering and seqencing reagents sfficient for seqencing one flow cell. Each reservoir on the cartridge is nmbered. Sample libraries are loaded onto the cartridge in position 17, which is labeled Load Samples. WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. Docment # v04 Material #

20 MiSeq System Gide Reserved Reservoirs Figre 8 Reagent Cartridge with Nmbered Reservoirs Table 1 Reagent Cartridge Reservoirs Position Reagent Name Description 8 LDR Denatration Reagent (contains formamide) 17 Reserved Load Sample (Reserved for sample libraries) 18 Reserved Reserved for cstom Read 1 primer [Optional] 19 Reserved Reserved for cstom Index Read primer [Optional] 20 Reserved Reserved for cstom Read 2 primer [Optional] NOTE For more information abot sing cstom primers on the MiSeq reagent cartridge, see the MiSeq Cstom Primers Gide (docment # ). Docment # v04 Material #

21 Chapter 2 Getting Started Start the MiSeq 11 Cstomize System Settings 11 Configre Notifications of BaseSpace Updates 12 Set Preferences 12 Set Defalt Folder Locations 12 User-Spplied Consmables 13 Start the MiSeq 1 Switch the toggle power switch on the back of the instrment to the (on) position. NOTE For best performance, leave the instrment on continosly. However, if the instrment mst be trned off, see Sht Down the Instrment on page 39. Wait a minimm of 60 seconds before trning the power switch back to the ON position Figre 9 Power Switch Location 2 Wait for the system to load, and then log on to the operating system. If necessary, conslt yor facility administrator for the ser name and password. When the operating system is loaded, the MiSeq Control Software (MCS) lanches and initializes the system atomatically. 3 For Local Rn Manager, if ser management is enabled, log in sing yor Local Rn Manager ser name and password, and select Next. Cstomize System Settings 1 From the Home screen, select Rn Options. 2 Select the Rn Settings tab. 3 Select Post Rn Wash or Maintenance Wash. An instrment wash is reqired after each rn. The software reqires that a wash is performed before setting p a sbseqent rn. The Post-Rn Wash Option specifies the type of wash that is performed by defalt. A post-rn wash takes approximately 30 mintes. A maintenance wash takes approximately 1 hor. Docment # v04 Material #

22 MiSeq System Gide 4 [Optional] Select Start rn after pre-rn check. Do not prompt for confirmation. to start the rn atomatically. 5 In the BaseSpace Settings tab, enter the address of the Clod Server or Onsite Server location. The Onsite Server setting is reqired if yo se BaseSpace Onsite. 6 Select Send Instrment Performance Data to Illmina to aid technical spport to enable the Illmina Proactive monitoring service. The name of the setting in the software interface might be different from the name in this gide, depending on the version of MCS in se. With this setting trned on, instrment performance data are sent to Illmina. This data helps Illmina trobleshoot more easily and detect potential failres, enabling proactive maintenance and maximizing instrment ptime. For more information on the benefits of this service, see Illmina Proactive Technical Note (docment # ). This service: Does not send seqencing data. Reqires that the instrment be connected to a network with internet access. Is trned on by defalt. To opt ot of this service, disable the Send Instrment Performance Data to Illmina to aid technical spport setting. Configre Notifications of BaseSpace Updates 1 From the Home screen, select Manage Instrment. 2 Select Software Update. 3 Select Atomatically check for new software pdates on BaseSpace. Set Preferences MiSeq can be configred to send an notification when RTA analysis is complete, when on-instrment secondary analysis is complete, or if a critical MiSeq software error occrs. 1 From the Home screen, select Rn Options. 2 Select the Notifications tab. 3 Enter the following information: Local SMTP server address Use the on-screen keyboard to enter the local SMTP server address. If necessary, contact the facility administrator for this information. Sender address Use the on-screen keyboard to enter the sender address. This address can be yor address or a different address specified for sending notifications. Recipient addresses Use the on-screen keyboard to enter the addresses of each recipient to receive notifications. Separate each address with a comma. Select Test to send a test to notification recipients. Notify via when Select the checkbox for each of the rn events that trigger a notification. Set Defalt Folder Locations Folders can be on a local network or on the instrment compter. 1 From the Home screen, select Rn Options. 2 Select the Folder Settings tab. 3 Enter defalt locations for the following folders. Docment # v04 Material #

23 MiSeq System Gide Otpt Folder Sets the defalt location for analysis otpt files. Change the defalt otpt folder to a network location for sharing, and long-term storage. For more information, see Rn Folders on page 47. Recipe Folder Sets the defalt location for recipes. Recipes are XML files that the software ses to perform the seqencing rn. A recipe is created at the start of the rn based on parameters in the sample sheet, and then the recipe is copied to the otpt folder. Sample Sheet Folder Sets the defalt location for sample sheets. Sample sheets are created before library preparation and contain parameters for the rn. Manifest Folder Manifest files are reqired for some library types. See the sample prep docmentation for yor sample prep kit, as well as the Sample Sheet Qick Reference Gide (docment # ). User-Spplied Consmables Make sre that the following ser-spplied consmables are available before beginning a rn. Consmable Spplier Prpose Stock 1.0 N NaOH, moleclar biology-grade General lab spplier Denatring sample libraries and PhiX control DNA Alcohol wipes, 70% Isopropyl or Ethanol, 70% Disposable gloves, powder-free VWR, catalog # * General lab spplier General lab spplier Lab tisse, low-lint VWR, catalog # * Lens paper, 4 x 6 in. VWR, catalog # * Cleaning the flow cell holder General se Cleaning the flow cell stage and the foil seal covering the load samples reservoir Cleaning the flow cell Microcentrifge tbes General lab spplier Denatring and dilting sample libraries and PhiX control DNA MiSeq tbes NaOCl, 5% Tween 20 Tweezers, sqare-tip plastic (optional) Illmina, part # MS Sigma-Aldrich, catalog # * Sigma-Aldrich, catalog # P7949 McMaster-Carr, catalog # 7003A22* Washing the template line, for se with the VeriSeq PGS workflow (optional for other workflows) Washing the template line, for se with the VeriSeq PGS workflow (optional for other workflows) Washing the instrment Water, laboratory-grade General lab spplier Washing the instrment Removing flow cell from flow cell shipping container * or laboratory-grade eqivalent Gidelines for Laboratory-Grade Water Always se laboratory-grade water or deionized water to perform instrment procedres. Never se tap water. Use only the following grades of water or eqivalents: Deionized water Illmina PW1 18 Megohms (MΩ) water Docment # v04 Material #

24 MiSeq System Gide Milli-Q water Sper-Q water Moleclar biology grade water Docment # v04 Material #

25 Chapter 3 Seqencing Introdction 15 Rn Dration 15 MiSeq Workflow 17 Thaw Reagent Cartridge 18 Inspect the Reagent Cartridge 19 Denatre and Dilte Libraries 19 Load Sample Libraries 19 Set Up a Rn Using MCS 20 Clean the Flow Cell 21 Load the Flow Cell 23 Load Reagents 24 Start the Rn 26 Monitor the Rn 27 Perform a Post-Rn Wash 29 Introdction To perform a seqencing rn on the MiSeq, follow the setp steps described in this chapter. There are three rn setp options: Local Rn Manager Select a rn that has been prepared with Local Rn Manager. Sample Sheet Create a rn sing a sample sheets. Sample sheets are validated in Local Rn Manager. Manal Create a rn by manally entering the nmber of cycles for each read. No secondary analysis is performed when this option is selected. After the rn begins, no other ser intervention is reqired. Use one of the following to monitor the seqencing rn: On the instrment, sing the Seqencing screen Remotely, sing the Seqencing Analysis Viewer (SAV). This optional application is available for download from the Illmina website. Remotely, sing Local Rn Manager. After the seqencing rn is complete, perform an instrment wash. Rn Dration Rn dration is based on the nmber of cycles performed. Yo can perform a paired-end rn p to 2 x 301 seqencing cycles pls any Index Reads with MCS v2.3 or later. Additionally, rn dration is based on the version of MiSeq reagents yo are sing and any performance enhancing pgrades installed on yor instrment. For expected drations and other specifications, visit the MiSeq System specifications page on the Illmina website. Nmber of Cycles in a Read In a seqencing rn, the nmber of cycles performed in a read is one more cycle than the nmber of cycles analyzed. The extra cycle is reqired for phasing and prephasing calclations. Docment # v04 Material #

26 MiSeq System Gide For example, a paired-end 300-cycle rn performs two reads of 301 cycles (2 x 301) for a total of 602 cycles. At the end of the rn, 2 x 300 cycles are analyzed. Docment # v04 Material #

27 MiSeq System Gide MiSeq Workflow Prepare the prefilled reagent cartridge for se. Denatre and dilte libraries (does not apply to all library types). See Preparing Libraries for Seqencing on the MiSeq (docment # ). Load the library mix onto the reagent cartridge in the designated reservoir. From the software interface, select Seqence to start the rn setp steps. From the Seqence Mode Selection screen, select a rn setp option: Local Rn Manager, Sample Sheet, or Manal. [Optional] From Local Rn Manager selection screen select a rn. [Optional] Connect to BaseSpace or BaseSpace Onsite. [Optional] From the Sample Sheet selection screen, browse to select a sample sheet for the rn. [Optional] Connect to BaseSpace or BaseSpace Onsite. [Optional] Form the Manal mode screen, manally enter the rn parameters. No secondary analysis is available in this mode. Wash and thoroghly dry the flow cell. Load the flow cell. Load the PR2 bottle and make sre that the waste bottle is empty. Load the reagent cartridge. Review rn parameters and pre-rn check reslts. Select Start Rn. Monitor yor rn from the MCS interface or from another compter sing Local Rn Manager or Seqencing Analysis Viewer (SAV). Docment # v04 Material #

28 MiSeq System Gide Perform a post-rn wash. Clster Generation Dring clster generation, single DNA molecles are bond to the srface of the flow cell, and then bridgeamplified to form clsters. Seqencing Following clster generation, clsters are imaged sing LED and filter combinations specific to each of the for florescently labeled dideoxyncleotides. After imaging of a tile is complete, the flow cell is moved into place to expose the next tile. The process is repeated for each cycle of seqencing. Following image analysis, the software performs base calling, filtering, and qality scoring. Analysis When the rn is complete, sing Local Rn Manager or Sample Sheet mode, the Local Rn Manager analysis software lanches atomatically to perform secondary analysis, which incldes alignment and variant calling. Yo can monitor secondary analysis sing an internet connection from another compter. For more information, see the Set Local Rn Manager Option on page 20. Thaw Reagent Cartridge Thaw the reagent cartridge sing a room temperatre water bath. NOTE Alternatively, thaw reagents overnight in 2 C to 8 C storage. Reagents are stable p to one week when stored at this temperatre. 1 Remove the reagent cartridge from -25 C to -15 C storage. 2 Place the reagent cartridge in a water bath containing enogh room temperatre deionized water to sbmerge the base of the reagent cartridge. Do not allow the water to exceed the maximm water line printed on the reagent cartridge. Figre 10 Maximm Water Line 3 Allow the reagent cartridge to thaw in the room temperatre water bath ntil it is thawed completely. MiSeq v3 cartridges ~ mintes. MiSeq v2 cartridges ~ 60 mintes. 4 Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge. Dry the base of the cartridge. Docment # v04 Material #

29 MiSeq System Gide Inspect the Reagent Cartridge 1 Invert the reagent cartridge ten times to mix the thawed reagents, and then inspect that all positions are thawed. 2 Inspect the reagents in positions 1, 2, and 4 to make sre that they are flly mixed and free of precipitates. 3 Gently tap the cartridge on the bench to redce air bbbles in the reagents. NOTE The MiSeq sipper tbes go to the bottom of each reservoir to aspirate the reagents, so it is important that the reservoirs are free of air bbbles. 4 Place the reagent cartridge on ice for p to six hors, or set aside at 2 C to 8 C ntil ready to set p the rn. For best reslts, proceed directly to loading the sample and setting p the rn. WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. Denatre and Dilte Libraries If necessary for yor library type, denatre and dilte libraries, and add optional PhiX control. See MiSeq System Denatre and Dilte Libraries Gide (docment # ). If yo are performing the VeriSeq PGS workflow, see the VeriSeq PGS Library Preparation Gide (docment # ). This step does not apply to all library types. Some Illmina sample preparation methods reslt in a ready-tose normalized concentration of pooled libraries. Refer to the sample preparation gide for the kit sed to prepare sample libraries. NOTE If yo are sing cstom primers, prepare primers and set p the sample sheet as described in MiSeq Cstom Primers Gide (docment # ). Load Sample Libraries When the reagent cartridge is flly thawed and ready for se, load prepared libraries onto the cartridge. 1 Clean the foil seal covering the reservoir labeled Load Samples with a low-lint lab tisse. 2 Pierce the foil seal with a clean 1 ml pipette. 3 Pipette 600 µl of prepared libraries into the reservoir Load Samples. Avoid toching the foil seal. Docment # v04 Material #

30 MiSeq System Gide Figre 11 Load Libraries ` 4 Proceed directly to the rn setp steps sing the MiSeq Control Software (MCS) interface. Set Up a Rn Using MCS 1 From the Home screen, select Manage Instrment. 2 Select Reboot to reboot the system software. 3 [Optional] From the Rn Options screen, check the folder locations for MiSeqOtpt, recipes, sample sheets, and manifests. For more information, see Set Defalt Folder Locations on page From the Home screen, select Seqence to begin the rn setp steps. The Seqence Mode Selection screen opens. When yo select Seqence on the Home screen, a series of rn setp screens open in the following order: Seqence Mode Selection, (Local Rn Manager, Sample Sheet, Manal), BaseSpace Option, Load Flow Cell, Load Reagents, Review, and Pre-Rn Check. Set Local Rn Manager Option 1 From the Home screen, select Seqence. 2 From the Seqence Mode Selection screen, select Local Rn Manager. 3 [Optional] From the BaseSpace Options screen, select Use BaseSpace. For more information, see Set BaseSpace or BaseSpace Onsite Option on page Select Next. 5 Select a rn name from the list of available rns. 6 [Optional] Select Preview Samples to view samples that are associated with the analysis. 7 Select Next to proceed to Load the Flow Cell on page 23. NOTE Clean flow cells before loading. For more information, see Clean the Flow Cell on page 21. Set Sample Sheet Option 1 From the Home screen, select Seqence. 2 From the Seqence Mode Selection screen, select Sample Sheet. Docment # v04 Material #

31 MiSeq System Gide 3 [Optional] From the BaseSpace Options screen, select Use BaseSpace. For more information, see Set BaseSpace or BaseSpace Onsite Option on page Select Next. 5 Browse to select yor sample sheet file. The file is sent to Local Rn Manager for validation or rn creation. 6 [Optional] Select Disable Local Rn Manager Secondary Analysis to bypass Local Rn Manager secondary analysis. 7 Resolve any errors in yor sample sheet. 8 Select Next to proceed to Load the Flow Cell on page 23. NOTE Clean flow cells before loading. For more information, see Clean the Flow Cell on page 21. Set BaseSpace or BaseSpace Onsite Option Optionally rn secondary analysis sing BaseSpace or BaseSpace Onsite. If the Local Rn Manager or Sample Sheet seqencing modes are selected, yo can optionally rn secondary analysis sing BaseSpace or BaseSpace Onsite. 1 From the BaseSpace Options screen, select Use BaseSpace. Selecting this option also stores the raw data from this rn on the MiSeq. 2 Select one of the following: Send all rn data to BaseSpace Seqence Hb for remote monitoring and data analysis. Only send rn information files to BaseSpace Seqence Hb so rns can be monitored remotely. 3 Enter yor MyIllmina accont credentials. 4 Select Next. Set Manal Option 1 From the Home screen, select Seqence. 2 From the Seqence Mode Selection screen, select Manal. 3 [Optional] Select Use rn monitoring only and enter yor MyIllmina credentials. NOTE 4 Select Next. The BaseSpace Seqence Hb Analysis and Storage option is not available in manal mode. 5 Set the Read Type, Read Length and Cstom Primers for all of yor cycles. 6 Select Next to proceed to Load the Flow Cell on page 23. NOTE Clean flow cells before loading. For more information, see Clean the Flow Cell on page 21. Clean the Flow Cell 1 Pt on a new pair of powder-free gloves. Docment # v04 Material #

32 MiSeq System Gide 2 Using plastic forceps, grip the flow cell by the base of the plastic cartridge and remove it from the flow cell container. Figre 12 Remove Flow Cell 3 Lightly rinse the flow cell with laboratory-grade water ntil both the glass and plastic cartridge are thoroghly rinsed of excess salts. Excess salts can affect flow cell seating on the instrment. If salts dry in the imaging area, imaging can also be affected. Figre 13 Rinse Flow Cell 4 Using care arond the black flow cell port gasket, thoroghly dry the flow cell and cartridge with a lintfree lens cleaning tisse. Gently pat dry in the area of the gasket and adjacent glass. Figre 14 Flow Cell Ports and Gasket 5 Clean the flow cell glass with an alcohol wipe. Make sre that the glass is free of streaks, fingerprints, and lint or tisse fibers. NOTE Do not se the alcohol wipe on the flow cell port gasket. Docment # v04 Material #

33 MiSeq System Gide Figre 15 Dry Flow Cell 6 Dry excess alcohol with a lint-free lens cleaning tisse. 7 Make sre that the flow cell ports are free of obstrctions and that the gasket is well-seated arond the flow cell ports. If the gasket appears to be dislodged, gently press it back into place ntil it sits secrely arond the flow cell ports. Load the Flow Cell 1 Raise the flow cell compartment door, and then press the release btton to the right of the flow cell clamp. The flow cell clamp opens. Figre 16 Open Flow Cell Clamp 2 Make sre that the flow cell stage is free of lint. If lint or other debris is present, clean the flow cell stage sing an alcohol wipe or a lint-free tisse moistened with ethanol or isopropanol. Careflly wipe the srface of the flow cell stage ntil it is clean and dry. 3 Holding the flow cell by the edges, place it on the flow cell stage. Figre 17 Place Flow Cell on Stage Docment # v04 Material #

34 MiSeq System Gide 4 Gently press down on the flow cell clamp to close it over the flow cell. As the flow cell clamp closes, alignment pins position the flow cell. An adible click indicates that the flow cell clamp is secre. Figre 18 Close Flow Cell Clamp 5 If the software does not identify the flow cell RFID, see Resolve RFID Read Failre on page Close the flow cell compartment door. 7 Select Next. Load Reagents Load PR2 and Check the Waste Bottle 1 Remove the bottle of PR2 from 2 to 8 C storage. Invert to mix, and then remove the lid. 2 Open the reagent compartment door. 3 Raise the sipper handle ntil it locks into place. 4 Remove the wash bottle and load the PR2 bottle. Figre 19 Load the PR2 Bottle 5 Empty the contents of the waste bottle into the appropriate waste container. 6 Slowly lower the sipper handle. Make sre that the sippers lower into the PR2 and waste bottles. Docment # v04 Material #

35 MiSeq System Gide Figre 20 Lower Sipper Handle 7 If the software does not identify the RFID of the PR2 bottle, see Resolve RFID Read Failre on page Select Next. Load the Reagent Cartridge 1 Open the reagent chiller door. NOTE Do not leave the reagent chiller door open for extended periods of time. 2 Hold the reagent cartridge on the end with the Illmina label, and slide the reagent cartridge into the reagent chiller ntil the cartridge stops. Always se the reagent cartridge associated with the type of flow cell that yo loaded. If the reagent cartridge is not compatible, a message appears on the screen. Select Back to load the appropriate reagent cartridge or Home to retrn to the Home screen. Figre 21 Load Reagent Cartridge 3 Close the reagent chiller door. 4 If the software does not identify the RFID of the reagent cartridge, see Resolve RFID Read Failre on page 44. Docment # v04 Material #

36 MiSeq System Gide 5 If the reagent cartridge is not compatible with the flow cell, a message appears. Select Back to load a compatible cartridge, or select Exit to retrn to the Home screen. 6 Close the reagent compartment door. 7 Select Next. Start the Rn After loading the flow cell and reagents, review the rn parameters and perform a pre-rn check before starting the rn. NOTE The rn starts atomatically if the Start rn after pre-rn check option is selected in Rn Settings. For more information, see Cstomize System Settings on page 11. Review Rn Parameters 1 Review Experiment Name, Modle Name, Read Type, Read Length, and Cstom Primers. These parameters are specified in the sample sheet. 2 [Optional] When in Local Rn Manager or Manal mode, select Edit to make changes to Read Type, Read Length, or Cstom Primers and then select Save. NOTE Changes to indexes mst be done in the sample sheet. When in Sample Sheet mode, changes mst be made to the original sample sheet and then it mst be reloaded. 3 Select Change Folder to review the folder locations. 4 Modify as needed and then select Save and Contine. 5 Select Next. Change Folders To change folder locations, select Change Folder and browse to a preferred location. Using this option from the Review screen changes folder locations for all sbseqent rns. Review Pre-Rn Check The system performs a check of all rn components, disk space, and network connections before starting the rn. If any items do not pass the pre-rn check, a message appears on the screen with instrctions on how to correct the error. For more information, see Resolve Rn Setp Errors on page 44. When all items sccessflly pass the pre-rn check, select Start Rn. Docment # v04 Material #

37 MiSeq System Gide Important Notes Before Starting the Rn WARNING The MiSeq is sensitive to vibration. Toching the instrment after starting a rn cold adversely affect seqencing reslts. After selecting Start Rn, do not open the flow cell compartment or the reagent compartment doors, or toch the instrment monitor except to pase the rn. For more information, see Pase a Rn on page 43. WARNING Make sre to close all files on the MiSeq before starting a rn, and do not open files dring a rn. Monitor the Rn Dring a rn, monitor rn detail sing the Seqencing screen on the instrment. The Seqencing screen is view-only. Other options to monitor a rn inclde: Seqencing Analysis Viewer (SAV) installed on a compter independent of the instrment compter. A network connection is reqired. For more information, see Seqencing Analysis Viewer on page 8. Alternatively, if yo are connected to BaseSpace, the rn can be monitored sing SAV in BaseSpace. Local Rn Manager for monitoring the rn remotely if the instrment is connected to the same network. Local Rn Manager shows rn progress and seqencing information (Total Clsters, % Clsters PF, Read 1 and Read 2 %>= Q30, and Last Scored Cycle). For more information, see Local Rn Manager Software on page 7. 1 On the instrment Seqencing screen, monitor rn progress, intensities, and qality scores. Rn Progress Shows rn progress in a stats bar and lists the nmber of cycles completed. Intensity Shows the vale of clster intensities of the 90 th percentile for each tile. The graphic in the Intensity area represents the nmber of tiles and nmber of srfaces being imaged: If the flow cell is imaged on the top srface only, a single-colmn graphic appears. If the flow cell is image on the top srface and bottom srface, a 2 colmn graphic appears. A B Indicates 2 tiles, top srface only Indicates 4 tiles, top and bottom srface Q-Score All Cycles Shows the average percentage of bases greater than Q30, which is a qality score (Q-score) measrement. A Q-score is a prediction of the probability of a wrong base call. Q- scores are calclated after cycle 25. Q-Score Probability of Wrong Base Call Q40 1 in 10,000 Q30 1 in 1,000 Docment # v04 Material #

38 MiSeq System Gide Q-Score Probability of Wrong Base Call Q20 1 in 100 Q10 1 in 10 Clster Density (K/mm²) Shows the nmber of clsters per sqare millimeter for the rn. Clsters Passing Filter (%) Shows the percentage of clsters passing filter based on the Illmina chastity filter, which measres qality. This data appears only after cycle 25. NOTE The chastity of a base call is the ratio of the intensity of the greatest signal divided by the sm of the 2 greatest signals. If more than 1 base call has a chastity vale of less than 0.6 in the first 25 cycles, reads do not pass the qality filter. Estimated Yield (Mb) Shows the projected nmber of bases called for the rn, measred in megabases. This data appears only after cycle When the rn is complete, the Next btton appears. Review the reslts on the Seqencing screen before proceeding. NOTE The Seqencing screen remains viewable ntil Next is selected. After yo select Next, it is not possible to retrn to the Seqencing screen. 3 Select Next to exit the Seqencing screen and proceed to a post-rn wash. Template Generation Template generation is the process where clster positions over the entire flow cell srface are defined according to X and Y coordinate position. Real-time analysis (RTA) ses early cycles of the rn for template generation. After the template of clster positions is generated, images prodced over every sbseqent cycle of imaging are aligned against the template. Individal clster intensities in all for ncleotide color channels are extracted and base calls are prodced from the normalized clster intensities. Rn Metrics Rn metrics appear on the Seqencing screen at different points in a rn. Dring clster generation steps, no metrics appear. After seqencing begins, the following metrics appear at the indicated cycles: Metric Kit Cycle Intensity Intensity and Clster Density Intensity, Clster Density, % PF, Yield, and Q-scores MiSeq Reagent Kits, v3 MiSeq Reagent Kits, v2 MiSeq Reagent Kits, v1 MiSeq Reagent Kits, v3 MiSeq Reagent Kits, v2 MiSeq Reagent Kits, v1 MiSeq Reagent Kits, v3 MiSeq Reagent Kits, v2 MiSeq Reagent Kits, v1 Cycle 1 7 Cycle 1 4 Cycle 1 4 Cycle 8 25 Cycle 5 25 Cycle 5 25 Cycle 26 throgh rn completion Docment # v04 Material #

39 MiSeq System Gide For MiSeq rn specifications, visit the MiSeq System specifications page on the Illmina website ( RTA Analysis Reslts The RTA analysis otpt from a seqencing rn is a set of qality-scored base call files (*.bcl), which are generated from the raw image files. For a list of RTA files and folders, see RTA Folders and Files on page 49. Perform a Post-Rn Wash The post-rn wash is the standard instrment wash performed between seqencing rns. Always perform an instrment wash after completing a rn. Follow the software prompts to load the wash components and perform the wash. The post-rn wash takes approximately 20 mintes. Start the wash directly following the completion of a rn. An instrment wash is reqired before yo can set p a sbseqent rn. To perform a post-rn wash at a time other than directly following a rn, se the command on the Perform Wash screen to initiate the wash. NOTE Leave the sed flow cell on the instrment. A flow cell mst be loaded on the instrment to perform an instrment wash. Reglar instrment washes ensre contined performance in the following ways: Flshes any remaining reagents from the flidics lines and sippers Prevents salt accmlation and crystallization in the flidics lines and sippers Prevents cross-contamination from the previos rn If yo are sing MCS v2.5 or later, yo have the option to perform a post-rn wash that incldes a template line wash with sodim hypochlorite soltion (NaOCl). The wash takes approximately 30 mintes. See Procedre with Template Line Wash on page 30. NOTE If yo are sing the VeriSeq PGS workflow, perform post-rn washes that inclde a template wash. See Procedre with Template Line Wash on page 30. Consmables Tween 20 Laboratory-grade water NaOCl (se with a post-rn wash that incldes a template line wash) MiSeq tbe (part # MS ) (for post-rn washes that inclde a template line wash) Procedre 1 Prepare fresh wash soltion with Tween 20 and laboratory-grade water: a Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water. These volmes reslt in 10% Tween 20. b Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water. These volmes reslt in a 0.5% Tween 20 wash soltion. c Invert five times to mix. 2 Prepare the wash components with fresh wash soltion: Docment # v04 Material #

40 MiSeq System Gide a b Add 6 ml wash soltion to each reservoir of the wash tray. Add 350 ml wash soltion to the 500 ml wash bottle. 3 When the rn is complete, select Start Wash. The software atomatically raises the sippers in the reagent chiller. Do not select Perform optional template line wash on the Post-Rn wash screen. The template line wash reqires a different procedre. See Procedre with Template Line Wash on page Open the reagent compartment door and reagent chiller door, and slide the sed reagent cartridge from the chiller. 5 Slide the wash tray into the reagent chiller ntil it stops, and then close the reagent chiller door. 6 Raise the sipper handle in front of the PR2 bottle and waste bottle ntil it locks into place. 7 Remove the PR2 bottle and replace it with the wash bottle. NOTE Discard the PR2 bottle after each rn. Do not rese any remaining PR2. 8 Remove the waste bottle and discard the contents appropriately. Retrn the waste bottle to the reagent compartment. WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. 9 Slowly lower the sipper handle, making sre that the sippers lower into the wash bottle and waste bottle. 10 Close the reagent compartment door. 11 Select Next. When the wash is complete, leave the sed flow cell, wash tray, and wash bottle containing the remaining wash soltion on the instrment. NOTE The sippers remain in the down position, which is normal. Leave the nsed wash soltion in the wash tray and wash bottle to prevent the sippers from drying ot and air from entering the system. Procedre with Template Line Wash 1 Prepare fresh wash soltion with Tween 20 and laboratory-grade water: a Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water. These volmes reslt in 10% Tween 20. b Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water. These volmes reslt in a 0.5% Tween 20 wash soltion. c Invert five times to mix. 2 Prepare fresh NaOCl wash soltion with laboratory-grade water: a Add 36 µl of 5% NaOCl to 864 µl laboratory-grade water. These volmes reslt in a 1:25 NaOCl Docment # v04 Material #

41 MiSeq System Gide b diltion. Add 50 µl of the 1:25 NaOCl diltion to 950 µl of laboratory-grade water in a MiSeq tbe (part # MS ). NOTE Using the correct concentration of NaOCl is important. Make sre to check the percentage of NaOCl on the prodct label. If the concentration is too high, it can make clster generation fail in sbseqent rns. If 5% NaOCl is not available, make a 1 ml soltion of 0.01% NaOCl in laboratory-grade water. Do not se NaOCl with a maintenance wash or a standby wash 3 Prepare the wash components with fresh wash soltion: a b Add 6 ml wash soltion to each reservoir of the wash tray. Add 350 ml wash soltion to the 500 ml wash bottle. 4 Insert the MiSeq tbe containing 0.01% NaOCl wash soltion into position 17 of the wash tray ntil the neck of the tbe is flsh with the tray. The tbe displaces the Tween 20 and laboratory-grade water wash soltion from position 17. Figre 22 MiSeq Tbe in Position 17 of the Wash Tray NOTE Make sre to insert the MiSeq tbe with NaOCl into tray position 17 only. Inserting the tbe in another position can make clster generation fail in sbseqent rns, and can damage the flidic system of the MiSeq instrment. 5 When the rn is complete, select Start Wash. The software atomatically raises the sippers in the reagent chiller. 6 Select Perform optional template line wash on the Post-Rn Wash screen. When sing the VeriSeq PGS workflow, the option Perform optional template line wash is pre-selected for yo. The MCS tracks the type of post-rn wash performed after each rn. If Perform optional template line wash is not selected for the post-rn wash, a message on the Rn Review screen reminds yo the next time yo start a seqencing rn. 7 Open the reagent compartment door and reagent chiller door, and slide the sed reagent cartridge from the chiller. 8 Slide the wash tray into the reagent chiller ntil it stops, and then close the reagent chiller door. 9 Raise the sipper handle in front of the PR2 bottle and waste bottle ntil it locks into place. 10 Remove the PR2 bottle and replace it with the wash bottle. NOTE Discard the PR2 bottle after each rn. Do not rese any remaining PR2. Docment # v04 Material #

42 MiSeq System Gide 11 Remove the waste bottle and discard the contents appropriately. Retrn the waste bottle to the reagent compartment. WARNING This set of reagents contains potentially hazardos chemicals. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Wear protective eqipment, inclding eye protection, gloves, and laboratory coat appropriate for risk of exposre. Handle sed reagents as chemical waste and discard in accordance with applicable regional, national, and local laws and reglations. For additional environmental, health, and safety information, see the SDS at spport.illmina.com/sds.html. 12 Slowly lower the sipper handle, making sre that the sippers lower into the wash bottle and waste bottle. 13 Close the reagent compartment door. 14 Select Next. When the wash is complete, leave the sed flow cell, wash tray, and wash bottle containing the remaining wash soltion on the instrment. NOTE The sippers remain in the down position. Leave the nsed wash soltion in the wash tray and wash bottle to prevent the sippers from drying ot and air from entering the system. Docment # v04 Material #

43 Chapter 4 Maintenance Maintenance Freqency 33 Maintenance Freqency for the VeriSeq PGS Workflow 33 Perform a Maintenance Wash 34 Perform a Standby Wash 36 Manage Files 38 Software Updates 39 Sht Down the Instrment 39 Maintenance Freqency Perform the following maintenance procedres at the recommended intervals. Table 2 Activity NOTE If performing the VeriSeq PGS workflow, make sre to follow maintenance freqency gidelines for VeriSeq PGS. See Maintenance Freqency for the VeriSeq PGS Workflow on page 33. Post-Rn Wash Maintenance Wash Standby Wash Maintenance Dring Normal Operation Instrment Shtdown Table 3 Activity Standby Wash Instrment Shtdown Freqency After every rn Monthly To prepare for idle mode (if nsed for 7 days), and every 30 days the instrment remains idle As needed Maintenance Dring Idle Mode ( 7 days nsed) Freqency Monthly As needed Maintenance Freqency for the VeriSeq PGS Workflow If performing the VeriSeq PGS workflow, perform the following maintenance procedres at the recommended intervals. Table 4 Maintenance Dring Normal Operation Activity Post-Rn Wash Maintenance Wash Post-Rn Wash from the Perform Wash Screen Standby Wash Instrment Shtdown Freqency After every rn Monthly After in idle mode (nsed for > 3 days) To prepare for idle mode (if nsed for 7 days), and every 30 days the instrment remains idle As needed Docment # v04 Material #

44 MiSeq System Gide Table 5 Maintenance Dring Idle Mode ( 7 days nsed) Activity Freqency Standby Wash Monthly Instrment Shtdown As needed Perform a Maintenance Wash Perform a maintenance wash every 30 days to ensre optimal performance. The maintenance wash takes approximately 90 mintes to complete. The wash incldes a series of three wash steps that thoroghly flsh the system. Yo can configre yor instrment to perform a maintenance wash between rns. For more information, Cstomize System Settings on page 11. User-Spplied Consmables Tween 20 (Sigma-Aldrich, catalog # P7949) Laboratory-grade water Procedre 1 Make sre that a sed flow cell is loaded on the instrment. 2 From the Home screen, select Perform Wash. 3 From the Perform Wash screen, select Perform Maintenance Wash. The software atomatically raises the sippers in the reagent chiller. Perform First Wash 1 Prepare fresh wash soltion with Tween 20 and laboratory-grade water: a Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water. These volmes reslt in 10% Tween 20. b Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water. These volmes reslt in a 0.5% Tween 20 wash soltion. c Invert five times to mix. 2 Prepare the wash components with fresh wash soltion: a b Add 6 ml wash soltion to each reservoir of the wash tray. Add 350 ml wash soltion to the 500 ml wash bottle. 3 Load the wash tray and wash bottle onto the instrment: a b c Open the reagent compartment door and reagent chiller door, and slide the sed reagent cartridge or wash tray from the chiller. Slide the wash tray into the reagent chiller ntil it stops. Close the reagent chiller door. Raise the sipper handle in front of the PR2 bottle and waste bottle ntil it locks into place, and replace the PR2 bottle with the wash bottle. NOTE Discard the PR2 bottle after each rn. Do not rese any remaining PR2. Docment # v04 Material #

45 MiSeq System Gide a b c Remove the waste bottle and discard the contents appropriately. Retrn the waste bottle to the reagent compartment. Slowly lower the sipper handle, making sre that the sippers lower into the wash bottle and waste bottle. Close the reagent compartment door. 4 Select Next. The first wash begins. Perform Second Wash Always se fresh wash soltion for each wash step. Resing wash soltion from the previos wash can retrn waste to the flidics lines. 1 Prepare fresh wash soltion with Tween 20 and laboratory-grade water, as follows: a Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water. These volmes reslt in 10% Tween 20. b Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water. These volmes reslt in a 0.5% Tween 20 wash soltion. c Invert five times to mix. 2 When the first wash is complete, remove the wash tray and wash bottle, and discard the remaining wash soltion. 3 Refill the wash components with fresh wash soltion, as follows: a b Add 6 ml wash soltion to each reservoir of the wash tray. Add 350 ml wash soltion to the 500 ml wash bottle. 4 Load the wash tray and wash bottle, as follows: a b c Slide the wash tray into the reagent chiller ntil it stops. Close the reagent chiller door. Load the wash bottle and slowly lower the sipper handle, making sre that the sippers lower into the wash bottle and waste bottle. Close the reagent compartment door. 5 Select Next. The second wash begins. Perform Final Wash 1 Prepare fresh wash soltion with Tween 20 and laboratory-grade water: a Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water. These volmes reslt in 10% Tween 20. b Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water. These volmes reslt in a 0.5% Tween 20 wash soltion. c Invert five times to mix. 2 When the second wash is complete, remove the wash tray and wash bottle, and discard the remaining wash soltion. 3 Refill the wash components with fresh wash soltion: a b Add 6 ml wash soltion to each reservoir of the wash tray. Add 350 ml wash soltion to the 500 ml wash bottle. 4 Load the wash tray and wash bottle: Docment # v04 Material #

46 MiSeq System Gide a b c Slide the wash tray into the reagent chiller ntil it stops. Close the reagent chiller door. Load the wash bottle and slowly lower the sipper handle, making sre that the sippers lower into the wash bottle and waste bottle. Close the reagent compartment door. 5 Select Next. The final wash begins. After the Wash When the wash is complete, leave the sed flow cell, wash tray, and wash bottle containing the remaining wash soltion on the instrment. NOTE The sippers remain in the down position, which is normal. Leave the nsed wash soltion in the wash tray and wash bottle to prevent the sippers from drying ot and air from entering the system. Perform a Standby Wash When there are no plans to se the instrment within the next seven days, prepare the instrment and instrment flidics lines to sit idle by performing a standby wash. Perform a standby wash every 30 days the instrment sits idle. The standby wash takes approximately two hors to complete. The wash performs two consective washes that flsh each position of any remaining reagents or salt accmlation. Each wash takes approximately 60 mintes. When the standby wash is complete, the instrment is in standby mode and a message appears on the Home screen stating the stats of the instrment. When the instrment is in standby mode, a maintenance wash mst be performed before a seqencing rn can be initiated. User-Spplied Consmables Tween 20 (Sigma-Aldrich, catalog # P7949) Laboratory-grade water Procedre 1 Make sre that a sed flow cell is loaded on the instrment. 2 From the Home screen, select Perform Wash. 3 From the Wash Options screen, select Perform Standby Wash. The software atomatically raises the sippers in the reagent chiller. Perform First Wash 1 Prepare fresh wash soltion with Tween 20 and laboratory-grade water: a Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water. These volmes reslt in 10% Tween 20. b Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water. These volmes reslt in a 0.5% Tween 20 wash soltion. c Invert five times to mix. 2 Prepare the wash components with fresh wash soltion: Docment # v04 Material #

47 MiSeq System Gide a b Add 6 ml wash soltion to each reservoir of the wash tray. Add 350 ml wash soltion to the 500 ml wash bottle. 3 Load the wash tray and wash bottle onto the instrment: a b c Open the reagent compartment door and reagent chiller door, and slide the sed reagent cartridge or wash tray from the chiller. Slide the wash tray into the reagent chiller ntil it stops. Close the reagent chiller door. Raise the sipper handle in front of the PR2 bottle and waste bottle ntil it locks into place, and replace the PR2 bottle with the wash bottle. NOTE Discard the PR2 bottle after each rn. Do not rese any remaining PR2. a b c Remove the waste bottle and discard the contents appropriately. Retrn the waste bottle to the reagent compartment. Slowly lower the sipper handle, making sre that the sippers lower into the wash bottle and waste bottle. Close the reagent compartment door. 4 Select Next. The first wash begins. Perform Second Wash Always se fresh wash soltion for each wash step. Resing wash soltion from the previos wash can retrn waste to the flidics lines. 1 Prepare fresh wash soltion with Tween 20 and laboratory-grade water, as follows: a Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water. These volmes reslt in 10% Tween 20. b Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water. These volmes reslt in a 0.5% Tween 20 wash soltion. c Invert five times to mix. 2 When the first wash is complete, remove the wash tray and wash bottle, and discard the remaining wash soltion. 3 Refill the wash components with fresh wash soltion, as follows: a b Add 6 ml wash soltion to each reservoir of the wash tray. Add 350 ml wash soltion to the 500 ml wash bottle. 4 Load the wash tray and wash bottle, as follows: a b c Slide the wash tray into the reagent chiller ntil it stops. Close the reagent chiller door. Load the wash bottle and slowly lower the sipper handle, making sre that the sippers lower into the wash bottle and waste bottle. Close the reagent compartment door. 5 Select Next. The second wash begins. After the Wash When the wash is complete, leave the sed flow cell, wash tray, and wash bottle containing the remaining wash soltion on the instrment. Docment # v04 Material #

48 MiSeq System Gide NOTE The sippers remain in the down position, which is normal. Leave the nsed wash soltion in the wash tray and wash bottle to prevent the sippers from drying ot and air from entering the system. Manage Files Select the Manage Files on the Home screen to move, pload, delete files, or rename sample sheets on the instrment compter. Delete Files 1 From any tab on the Manage Files screen, select Browse to navigate to files accessible to the instrment. 2 Select from the following options: Select the checkbox next to individal files or folders in the list. Select the checkbox to the left of the Delete btton to select all files and folders in the list. This option is available for Rns, Sample Sheets, Manifests, Genomes, and Recipes. 3 Select Delete. NOTE The Delete command is available on all tabs except Bndle Logs. Move Rn Folders The Move command copies the rn folder to the new location and then deletes the folder from the old location. 1 From the Rns tab on the Manage Files screen, select Browse to navigate to files accessible to the instrment. 2 Select the checkbox next to individal files or folders in the list. 3 Select Move. 4 Select Browse Network and select a new location for the files or folders. 5 Select OK. Upload Files The Upload command is available for Sample Sheets, Manifests, Genomes, and Recipes. If the MiSeq is not connected to a network, se this featre to pload files to the instrment compter from a USB drive. 1 From the tab on the Manage Files screen, select Browse to navigate to files accessible to the instrment. 2 Select Upload. 3 Select Browse Network and browse to the location on a USB drive where the file resides. 4 Select OK. The file is ploaded to the folder indicated in the Directory field. Docment # v04 Material #

49 MiSeq System Gide Rename Sample Sheets 1 From the Sample Sheets tab on the Manage Files screen, select from the following options: Select the checkbox next to individal sample sheets. Select the checkbox to the left of the Delete btton to select all sample sheets in the list. 2 Select Rename. 3 Select the keyboard icon and se the on-screen keyboard to rename sample sheets. 4 Select Next. 5 Select Back. Software Updates If yor system is connected to a network with internet access, yo can atomatically pdate the instrment software from the Home screen. Yo can also configre the software to check atomatically for BaseSpace pdates. For more information, see Configre Notifications of BaseSpace Updates on page 12. If yor instrment is not connected to a network with internet access, yo can pdate the software manally. Update Software Atomatically When software pdates are available, the Update Available btton appears on the Home screen. Otherwise, this btton is not visible. Make sre yor MiSeq is connected to a network with internet access to enable this option. 1 From the Home screen, select Update Available. 2 Confirm the command to pdate in the dialog box. Reboot of the instrment is reqired. Installation of the pdate begins atomatically pon reboot. Update Software Manally Use the Manal Update featre to pdate instrment control software and analysis software from the MiSeq interface by browsing to the location of the installable software file. 1 From the Home screen, select Manage Instrment. 2 Select Software Update. 3 Select Browse to navigate to the location of the installable file for the new software version. 4 When the path to the installable software file appears on the screen, select Save and Update. 5 Confirm the command to pdate in the dialog box. Reboot of the instrment is reqired. Installation of the pdate begins atomatically pon reboot. Sht Down the Instrment It is best to leave the instrment on at all times. However, if the instrment mst be trned off, se the following procedre to sht down Windows and prepare the flidics lines. 1 Perform a maintenance wash. For more information, see Perform a Maintenance Wash on page Remove the waste bottle and discard the contents appropriately. Retrn the waste bottle to the reagent compartment. Docment # v04 Material #

50 MiSeq System Gide 3 Close the reagent compartment door. 4 From the Home screen, select Manage Instrment. 5 Select Sht Down. This command shts down the software. 6 Toggle the power switch to the OFF position. NOTE Any time that yo trn off the instrment, wait a minimm of 60 seconds before trning the power switch back to the ON position. Docment # v04 Material #

51 Appendix A Trobleshooting Introdction 41 Bndle Logs for Trobleshooting 41 Perform a System Check 42 Pase or Stop a Rn 42 Raise Reagent Cartridge Sippers Manally 44 Resolve Rn Setp Errors 44 Resolve RFID Read Failre 44 Perform a Volme Test 45 Measre Expected Wash Volmes 46 Resolve Reagent Chiller Temperatre Errors 46 Resolve Local Rn Manager Analysis Errors 46 Configre System Settings 46 Introdction This section describes common trobleshooting steps to take before contacting Illmina Technical Spport. For most errors, an on-screen message appears with instrctions for correcting the error. For technical qestions, visit the MiSeq spport pages on the Illmina website for access to freqently asked qestions, or log in to yor MyIllmina accont for access to spport blletins. For problems with rn qality or performance, contact Illmina Technical Spport. For more information, see Technical Assistance on page 55. Illmina Technical Spport representatives typically reqest copies of rn-specific files for trobleshooting prposes. Yo can se the Bndle Logs tab on the Manage Files screen to combine and zip the files reqired for trobleshooting. See Bndle Logs for Trobleshooting on page 41. Bndle Logs for Trobleshooting Bndle Logs is a featre that bndles files to send to Illmina Technical Spport for trobleshooting. Use the Bndle Logs tab on the Manage Files screen to select a grop of files, called a bndle. The bndle is zipped atomatically. The Bndle Logs featre grops the files from a rn into one bndle type at time. Repeat the Bndle Logs procedre for each rn and bndle type IllminaTechnical Spport reqests. 1 On the Manage Files screen, select the Bndle Logs tab. 2 Select Browse to navigate to the location of the MiSeqOtpt folder. 3 Click in the ble box next to the rn, and in the ble circle next to the bndle type reqested by Illmina Technical Spport. 4 Select Bndle Logs. A Bndle Files screen opens with information abot the bndle, inclding a list of individal files the bndle contains. For more information on the individal folders and files of the Bndle Logs featre, see MiSeq Otpt and Analysis Folders Qick Reference Card (docment # ). 5 Select Next. 6 Navigate to a location where yo want the zipped bndle files saved. 7 Select Save. Docment # v04material #

52 MiSeq System Gide When the files finish bndling, the Bndle Logs tab reopens. 8 Send the zipped bndle to Illmina Technical Spport. Figre 23 Bndle Logs Tab Perform a System Check The System Check screen is typically sed to connect with an Illmina Technical Spport representative dring a Live Help session. Use of this featre is not reqired dring normal operation or for instrment maintenance. Some system checks can be performed before contacting Illmina Technical Spport, sch as the Volme Test. A volme test checks the health of the flidics system by estimating the flow volme as bbbles pass by the sensors. For more information, see Perform a Volme Test on page From the Home screen, select Manage Instrment. 2 Select System Check. 3 Do one of the following: Select the individal tests yo want to perform. Choose Select All to perform all tests. 4 Select Next. When complete, the test reslts appear on the screen. 5 [Optional] Select Show Details to see a smmary of the reslts on the software interface. 6 [Optional] Select Export Reslts to export the reslts in a *.csv file format to a USB drive. 7 Select Done. Pase or Stop a Rn The MiSeq is designed to complete a rn from beginning to end withot ser intervention. However, it is possible to pase a rn or stop a rn from the Seqencing screen. Docment # v04material #

53 MiSeq System Gide Pase a Rn Yo can temporarily pase a rn before it has completed. For example, a rn can be pased if yo sspect that the waste bottle is fll. Pased rns can be resmed. When yo select Pase, the crrent command is completed before pasing the rn and placing the flow cell in a safe state. To pase a rn from the Seqencing screen, select Pase. The btton changes to Resme. When yo are ready to resme the rn, select Resme. Figre 24 Seqence Screen of a Pased Rn Stop a Rn Yo can stop a rn dring seqencing before the rn has completed sing the Stop btton on the Seqencing screen. Yo might stop a rn if the rn was set p incorrectly, if the data qality is bad, or if yo experience a hardware error. When a rn is stopped, the crrent command is not completed and the flow cell stage moves to the forward position. Real-time Analysis software contines analysis for the last completed cycle. Figre 25 Stopping a Rn Stopping a rn is final. A stopped rn cannot be resmed. The only option is to proceed to an instrment wash. Docment # v04material #