Illumina MISEQ MiSeq System User Manual My

Illumina, Inc. MiSeq System My

Contents

MiSeq User Guide

FOR RESEARCH USE ONLYILLUMINA PROPRIETARY Part # 15027617 Rev. AAugust 2011MiSeq™ SystemUser Guide
MiSeq System User Guide iiThis document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and areintended solely for the contractual use of its customer in connection with the use of the product(s)described herein and for no other purpose. This document and its contents shall not be used or distributedfor any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoeverwithout the prior written consent of Illumina. Illumina does not convey any license under its patent,trademark, copyright, or common-law rights nor similar rights of any third parties by this document.The Software is licensed to you under the terms and conditions of the Illumina Sequencing SoftwareLicense Agreement in a separate document. If you do not agree to the terms and conditions therein,Illumina does not license the Software to you, and you should not use or install the SoftwareThe instructions in this document must be strictly and explicitly followed by qualified and properly trainedpersonnel in order to ensure the proper and safe use of the product(s) described herein. All of the contentsof this document must be fully read and understood prior to using such product(s).FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONSCONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS,INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY.ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THEPRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USEOF SUCH PRODUCT(S) OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES ORPERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER'S ACQUISITION OFSUCH PRODUCT(S).FOR RESEARCH USE ONLY© 2011 Illumina, Inc. All rights reserved.Illumina,illuminaDx,BeadArray,BeadXpress,cBot,CSPro,DASL,DesignStudio,Eco,GAIIx,Genetic Energy,Genome Analyzer,GenomeStudio,GoldenGate,HiScan,HiSeq,Infinium,iSelect,MiSeq,Nextera,Sentrix,Solexa,TruSeq,VeraCode, the pumpkin orange color, and the Genetic Energystreaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands andnames contained herein are the property of their respective owners.This software contains the SeqAn Library, which is licensed to Illumina and distributed under thefollowing license:Copyright © 2010, Knut Reinert, FU Berlin, All rights reserved. Redistribution and use in source andbinary forms, with or without modification, are permitted provided that the following conditions aremet:1 Redistributions of source code must retain the above copyright notice, this list of conditions andthe following disclaimer.2 Redistributions in binary form must reproduce the above copyright notice, this list of conditionsand the following disclaimer in the documentation and/or other materials provided with thedistribution.3 Neither the name of the FU Berlin or Knut Reinert nor the names of its contributors may be usedto endorse or promote products derived from this software without specific prior writtenpermission.THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS"AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THEIMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULARPURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT HOLDER ORCONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL,EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO,PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS;OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY,WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OROTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IFADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
MiSeq System User Guide iiiRevision HistoryPart # Revision Date Description of Change15027617 A August 2011 Initial release.
iv Part # 15027617 Rev. A
MiSeq System User Guide vTable of ContentsRevision History iiiTable of Contents vChapter 1 Getting Started 1Introduction 2Components 4Starting the MiSeq System 9MiSeq Workflow 11Illumina-Supplied Consumables 13User-Supplied Consumables 15Chapter 2 MiSeq Control Software 17Introduction 18MiSeq Control Software Interface 19Stopping or Pausing a Run 29Sample Sheets 30Analysis Workflow 35Run Folders 36Available Disk Space 37Chapter 3 Performing a Run 39Introduction 40Setting Up the Sample Sheet 41Preparing the Reagent Cartridge 43Preparing Your Libraries 44Clean the Flow Cell 47Loading the Flow Cell 49Load Reagents 51Starting the Run 53Chapter 4 Post-Run Procedures 55Introduction 56Performing a Post-Run Wash 57Unloading Components 59Shutting Down the Instrument 61Chapter 5 On-Instrument Analysis 63Introduction 64Primary Analysis Results 65Secondary Analysis Using MiSeq Reporter 66Using MiSeq Reporter Off-Instrument 68Chapter 6 Troubleshooting 69
vi Part # 15027617 Rev. AIntroduction 70Software Errors 71Index 73Technical Assistance 75
Chapter 1MiSeq System User Guide 1Chapter 1 Getting StartedGetting StartedIntroduction 2Components 4Starting the MiSeq System 9MiSeq Workflow 11Illumina-Supplied Consumables 13User-Supplied Consumables 15
Getting Started2Part # 15027617 Rev. AIntroductionThe Illumina MiSeq™ System combines proven sequencing by synthesis (SBS)technology with a revolutionary workflow that enables you to go from DNA to analyzeddata in as few as eight hours. The MiSeq integrates cluster amplification, sequencing,and data analysis in a single instrument.ApplicationsThe MiSeq supports a wide range of applications, including the following:}De novo sequencing}Targeted resequencing}Small genome sequencing}Highly-multiplexed PCR amplicon sequencing}Small RNA sequencing}Library QCLibrary PreparationYou can sequence libraries prepared by any of the following Illumina samplepreparation kits:}TruSeq™ DNA Sample Preparation Kit}TruSeq™ RNA Sample Preparation Kit}TruSeq™ Small RNA Sample Preparation Kit}TruSeq™ Custom Amplicon Kit}Nextera™ Sample Preparation KitFor more information, see Applications and Related Consumables on page 13.Features}Walkaway Automation—All of the reagents required for cluster generation,sequencing, and paired-end chemistry are contained in a single reagent cartridge.After run setup, no additional hands-on time is required.}Prefilled Reagent Cartridge—A specially designed single-use prefilled reagentcartridge that provides reagents for cluster generation and sequencing, includingpaired-end sequencing reagents. Radio-frequency identification (RFID) trackingenables positive consumable tracking.}Interface Controls—MiSeq Control Software (MCS) offers a simple interface toconfigure and monitor your run, and perform maintenance procedures on a touchscreen monitor.}Convenient Flow Cell Loading—A clamping mechanism auto-positions the flowcell as you load it onto the instrument.}Innovative Fluidics Architecture—Enables unmatched efficiency in chemistry cycletime during sequencing.}Real Time Analysis—Integrated analysis software performs real time on-instrumentdata analysis during the sequencing run, which includes image analysis and basecalling, and saves valuable downstream analysis time.
IntroductionMiSeq System User Guide 3Related DocumentationThis guide provides comprehensive information about the MiSeq System. However,other documentation is available for site preparation and safety guidelines, and asquick reference guides.The following list describes related documentation and availability.}MiSeq System Site Preparation Guide—Provides specifications for laboratory space,electrical requirements, and environmental considerations.This guide is available for download from the Illumina website.}MiSeq System Safety and Compliance Guide—Provides safety-related labeling andconsiderations, and information about compliance certifications and labeling.This guide is provided with the instrument and available for download from theIllumina website.}MiSeq System Quick Reference Guide—Provides an overview of the instrument, andinstructions for setting up and performing a run on the MiSeq. Use the quickreference guide after you are familiar with the contents of the MiSeq System UserGuide.This guide is provided with the instrument and available for download from theIllumina website.}MiSeq Reagent Preparation Guide—Provides a description of kit contents, andinstructions for preparing the reagent cartridge and sample libraries beforebeginning your sequencing run.This guide is provided with the MiSeq Reagent Kit and available for downloadfrom the Illumina website.}MiSeq Sample Sheet Quick Reference Guide—Provides information about samplesheets that is included in the MiSeq System User Guide, compiled into a quickreference guide.This guide is available for download from the Illumina website.Illumina WebsiteVisit the MiSeq support pages on the Illumina website(http://www.illumina.com/systems/miseq/support.ilmn) for access to documentation,related downloads, FAQs, and other support information.
Getting Started4Part # 15027617 Rev. AComponentsThe MiSeq System has the following main components.Figure 1 Main CompartmentsAFlow Cell Compartment—Contains the flow cell stage that houses the flow cellthroughout the run. The flow cell stage is controlled by motors that move the stageout of the enclosed optical module for flow cell loading, and returns it when the runbegins.BEnclosed Optics Module—Contains optical components that enable imaging of theflow cell.CStatus Bar—Uses three colors to indicate instrument status. Blue indicates theinstrument is running, orange indicates the instrument needs attention, and greenindicates that the instrument is ready to begin the next run.DTouch Screen Monitor—Enables easy run setup using the software interface and at-a-glance run monitoring.EReagent Compartment—Holds reagents used during runs, wash solutions usedduring instrument washes, and the waste bottle. The reagent compartment door issecured by a magnetic latch.The MiSeq software interface guides you through the run setup steps using the touchscreen monitor. To load run components, you need to access the reagent compartmentand the flow cell compartment.Reagent CompartmentThe reagent compartment contains the reagent chiller, and positions for theincorporation wash buffer (PR2) bottle and the waste bottle.The reagent chiller holds a single-use reagent cartridge during the run, or a washrayduring the instrument wash. The software automatically lowers sippers into thereagent wells of the cartridge at the appropriate time depending on the process beingperformed.
ComponentsMiSeq System User Guide 5Figure 2 Reagent CompartmentAReagent ChillerBSipper Handle (shown in raised position)CReagent (PR2) BottleDWaste BottleEReagent CartridgeNOTEThe reagent chiller is designed to maintain reagent temperature at 4°C. The chilleris not intended to cool down reagents; therefore, reagents must be cool beforethey are loaded onto the instrument.To the right of the reagent chiller are two form-fitted slots, one for the PR2 bottle andone for the waste bottle. After the bottles are in place, the sipper handle is lowered tolock the bottles in place and lower the appropriate sipper into each bottle.Fluidics pumps inside the instrument direct reagents through the sippers, through thefluidics lines, and then through the flow cell. Reagent waste is delivered to the wastebottle throughout the process.Reagent CartridgeThe MiSeq reagent cartridge is a single-use consumable prefilled with cluster generationand sequencing reagents.
Getting Started6Part # 15027617 Rev. AFigure 3 Reagent CartridgeEach reservoir on the cartridge is numbered. The following table lists the reagent ineach numbered position.Position Reagent Name Description1 IMF Incorporation Mix2 SRE Scan Mix4 CMF Cleavage Mix5 AMX1 Amplification Mix6 AMX2 Read 2 Amplification Mix7 LPM Linearization Premix8 LDR Formamide9 LMX1 Linearization Mix10 LMX2 Read 2 Linearization Mix11 RMF Resynthesis Mix12 HP10 Read 1 Primer Mix13 HP12 Index Primer Mix14 HP11 Read 2 Primer Mix15 PW1 Water16 PW1 Water17 Sample Your sample libraries18 Optional Optional use for custom sequencing primers19 Optional Optional use for custom sequencing primers20 Optional Optional use for custom sequencing primers21 PW1 Water22 Empty EmptyBefore starting a run, your sample libraries must be loaded onto the reagent cartridge inthe designated reservoir. You can sequence a single library or a pool of indexedlibraries.
ComponentsMiSeq System User Guide 7Flow Cell CompartmentThe flow cell compartment houses the flow cell stage, thermal station, and fluidicsconnections to the flow cell.The flow cell is positioned on the flow cell stage, which moves in and out of the opticsmodule. The flow cell is auto-positioned on the flow cell holder and held in place by theflow cell latch. When the flow cell latch closes, two pins near the latch hinge positionthe flow cell.Figure 4 Flow Cell StageAFlow Cell StageBFlow Cell Compartment DoorCFlow Cell LatchDFlow CellThe thermal station, located beneath the flow cell stage, controls changes in flow celltemperature that are required for cluster generation and sequencing.Fluidics connections deliver reagents to the flow cell through the inlet port, and returnwaste from the flow cell through the outlet port.Flow CellThe MiSeq flow cell is a single-lane glass-based substrate in which clusters aregenerated and the sequencing reaction is performed. The single lane is divided intotwelve image areas, called tiles, that are imaged by the instrument camera.Figure 5 Flow CellAOutlet PortBImaging AreaCInlet Port
Getting Started8Part # 15027617 Rev. ASamples are loaded onto the reagent cartridge and then automatically transferred to theflow cell at the beginning of the cluster generation process.Reagents enter the flow cell through the inlet port, pass through the imaging area, andthen exit the flow cell through the outlet port. Waste exiting the flow cell is delivered tothe waste bottle.
Starting the MiSeq SystemMiSeq System User Guide 9Starting the MiSeq SystemUse the following instructions to start the MiSeq and instrument computer.1Reach around the right side of the instrument to locate the power switch on theback panel. It is in the lower corner directly above the power cord.Figure 6 Power Switch Location2Turn the power switch to the ON position. The integrated instrument computerstarts.3Log on to the operating system using the default user name. Wait until theoperating system has completely loaded.•User name: sbsuser•Password: sbs123When the system is ready, the MCS (MCS) launches and begins initialization.Figure 7 MCS InitializationAfter initialization is complete, the Welcome screen appears.
Getting Started10 Part # 15027617 Rev. AFigure 8 MCS Welcome Screen4To ensure adequate disk space, archive the data on the instrument computer fromall previous runs to a network location. For more information, see Manage Files onpage 22.Best PracticeIt is best to leave the instrument on at all times. Turn off the instrument only if it willremain idle for more than ten days. For more information, see Shutting Down theInstrument on page 61.If you need to turn off the instrument, wait a minimum of 60seconds before turning thepower switch back to the ON position.
MiSeq WorkflowMiSeq System User Guide 11MiSeq WorkflowThe MiSeq workflow consists of run setup steps, followed by automated clustergeneration, then sequencing and primary analysis, and finally on-instrument secondaryanalysis.The following steps are described in detail in Performing a Run on page 39.Setting Up Your RunCreate a sample sheet containing parametersspecific to your run.Prepare the prefilled reagent cartridge for use.Dilute and denature your libraries, and then loadyour libraries onto the reagent cartridge in thedesignated reservoir.From the MCS interface, select Sequence to start therun set up steps.Load the flow cell.Load the PR2 bottle and make sure that the wastebottle is empty.Load the reagent cartridge.Review run parameters and pre-run check results.Select Start Run.Monitor your run from the Sequencing screen or onanother computer using Sequencing AnalysisViewer (SAV).Cluster GenerationAfter the run is started, single DNA molecules are bound to the surface of the flow cell,and then bridge-amplified to form clusters.
Getting Started12 Part # 15027617 Rev. ASequencingFollowing cluster generation, clusters are imaged using LED and filter combinationsspecific to each of the four fluorescently-labeled dideoxynucleotides. After imaging ofone tile of the flow cell is complete, the flow cell is moved into place to expose the nexttile and the process is repeated. Following image analysis, Real Time Analysis (RTA)launches to perform basecalling, filtering, and quality scoring.AnalysisWhen the run is complete, the MiSeq Reporter analysis software launches automaticallyand begins secondary analysis. You can monitor secondary analysis using an internetconnection from another computer.For more information, see Secondary Analysis Using MiSeq Reporter on page 66.NOTEIf a new sequencing run is started on the MiSeq before secondary analysis iscomplete, the current analysis is stopped. From the MiSeq Reporter interface, youcan requeue secondary analysis for that run after the new sequencing run iscomplete. At that point, secondary analysis starts over from the beginning.Alternatively, MiSeq Reporter can be run off-instrument allowing secondary analysis tooccur during a subsequent sequencing run. For more information, see Using MiSeqReporter Off-Instrument on page 68.Approximate Run DurationRun duration depends on the number of cycles you perform. You can perform a paired-end sequencing run up to 2x150 cycles, or as few as 36 cycles for some applications.Generally, one cycle takes about five minutes. The following table lists the approximatedurations for cluster generation, sequencing, and on-instrument analysis.Process Read Length DurationClusterGenerationandSequencing36 Cycles (36 bp) Less than 4 hours50 Cycles (50 bp) Less than 6 hoursPaired-End 150Cycles (2x150 bp)Less than 27 hoursOn-InstrumentAnalysisand VariantCallingPaired-End 150Cycles (2x150 bp)Less than 2 hours for all analysis workflows,except 16s metagenomics.16s metagenomics completes in less than 24hours.
Illumina-Supplied ConsumablesMiSeq System User Guide 13Illumina-Supplied ConsumablesTo perform a run on the MiSeq, you need one MiSeq Reagent Kit.MiSeq ReagentsThe MiSeq Reagent Kit is available in the following sizes:}300 Cycles—Provides kitted reagents for up to 323 cycles of sequencing on theMiSeq. This includes sufficient reagents for a 151-cycle paired-end run, plus twoeight-cycle indexed reads.}50 Cycles—Provides kitted reagents for up to 73 cycles of sequencing on the MiSeq.This includes sufficient reagents for a 26-cycle paired-end run, or a 51-cycle single-read run, plus two eight-cycle indexed reads.MiSeq Reagent Kit ContentsBox1: Store at -15° to -25°CQuantity Component Storage Description1Reagent Cartridge -15° to -25°C Single-use pre-filled cartridge1HT1 -15° to -25°C 5 ml tube, Hybridization BufferBox2: Store at 2° to 8°CQuantity Component Storage Description1PR2 2° to 8°C 500 ml bottle, IncorporationBuffer1Flow Cell 2° to 8°C Single-use flow cellApplications and Related ConsumablesThe following table lists applications supported on the MiSeq, the recommended readlength for the run, and the associated Illumina-supplied consumables.Application ReadLengthMiSeqReagent Kit(# of cycles)Sample Preparation KitTruSeq CustomAmplicon2 x 151 300 Cycle Kit TruSeq Custom AmpliconLibrary PrepNextera Amplicon 1 x 36 50 Cycle Kit Nextera Sample PrepStandard Amplicon 2 x 151 300 Cycle Kit TruSeq DNA Sample PrepSmall Genome: De novo 2 x 151 300 Cycle Kit TruSeq DNA Sample PrepSmall Genome:Resequencing1 x 36 50 Cycle Kit or300 Cycle KitTruSeq DNA Sample Prep orNextera Sample Prep16S Metagenomics 2 x 151 300 Cycle Kit TruSeq DNA Sample Prep orNextera Sample Prep
Getting Started14 Part # 15027617 Rev. AApplication ReadLengthMiSeqReagent Kit(# of cycles)Sample Preparation KitLibraryQC 2 x 25 50 Cycle Kit OpenSmall RNA 1 x 36 50 Cycle Kit TruSeq Small RNA Sample PrepRNA-Seq 1 x 51 50 Cycle Kit TruSeq RNA Sample PrepTruSeq CustomEnrichment2 x 76 300 Cycle Kit TruSeq DNA Sample Prep
User-Supplied ConsumablesMiSeq System User Guide 15User-Supplied ConsumablesMake sure that you have all of the user-supplied consumables listed in this sectionbefore you begin your run.Consumable Supplier Purpose1 N NaOH General lab supplier Denaturing sample librariesDiluting PhiX controlAlcohol wipes, 70%IsopropylorEthanol, 70%VWR, catalog#15648-981General lab supplierCleaning the flow cell holderDisposable gloves, powder-freeGeneral lab supplier General useLab tissue, low-lint VWR, catalog#21905-026Cleaning the flow cell stageLens paper, 4x6in. VWR, catalog#52846-001Cleaning the flow cellTris-Cl 10mM, pH8.5 with0.1% Tween 20General lab supplier Diluting PhiX controlTweezers, square-tip plastic McMaster-Carr,catalog # 7003A22Removing flow cell from flowcell containerWater, laboratory-grade General lab supplier Washing the instrumentTable 1 User-Supplied ConsumablesGuidelines for Laboratory-Grade WaterThe following list provides acceptable examples of laboratory-grade water. Never usetap water or deionized water on the instrument.}Illumina PW1}18M Ohm water}Milli-Q water}Super-Q water}Molecular biology-grade water
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Chapter 2MiSeq System User Guide 17Chapter 2 MiSeq Control SoftwareMiSeq Control SoftwareIntroduction 18MiSeq Control Software Interface 19Stopping or Pausing a Run 29Sample Sheets 30Analysis Workflow 35Run Folders 36Available Disk Space 37
MiSeq Control Software18 Part # 15027617 Rev. AIntroductionThe MiSeq Control Software (MCS) interface guides you through the steps to load theflow cell and reagents prior to starting the run, and then provides an overview ofquality statistics that you can monitor as the run progresses.During the run, MCS operates the flow cell stage in the optical compartment, givescommands to dispense reagents, changes temperatures of the flow cell, and capturesimages of clusters on the flow cell for image analysis. MCS performs the run based onparameters specified in the sample sheet. For more information, see Sample Sheets onpage 30.Real Time Analysis (RTA)After image analysis is complete, the integrated primary analysis software, Real TimeAnalysis (RTA), performs base calling and assigns a quality score to each base for eachcycle. Quality scores can be monitored throughout the run on the MCS Sequencingscreen. For more information, see Sequencing Screen on page 27.Images are temporarily stored in the run folder for processing by RTA, and thenautomatically deleted when RTA analysis is complete.Sequencing Analysis Viewer (SAV)To monitor your run in greater detail from another computer, you can use theSequencing Analysis Viewer (SAV), an optional software application available fordownload from the Illumina website. For more information, see Sequencing AnalysisViewer on page 28.
MiSeq Control Software InterfaceMiSeq System User Guide 19MiSeq Control Software InterfaceThe MiSeq Control Software (MCS) includes an intuitive interface that prompts youthrough the run setup steps, instrument setup options, and instrument washes. Theinterface includes a Sequencing screen that provides information about the run as itprogresses.Welcome ScreenThe interface opens to the Welcome screen when the software launches, and returns tothe Welcome screen when a run is complete.Figure 9 MCS Welcome ScreenThe Welcome screen has the following options:}Sequence—This option opens a series of run setup screens that guide you throughthe setup steps for your run. For more information, see Run Setup Screens on page 24.}Maintenance Wash—Use this option to perform an instrument wash before asequencing run.}Manage Instrument—Provides links to Setup Options,Manage Runs, and PerformDiagnostic. For more information, see Manage Instrument on page 20.}About—Provides information about instrument configuration and softwareversions.}Get Updates—This option appears on the Welcome screen only if a software updateis available. Your MiSeq must be connected to a network to enable this option. Formore information, see Get Updates on page 20.Activity IndicatorsIn the lower-right corner of the Welcome screen are a series of icons. Each icon is anactivity indicator that tells you which activity the instrument is performing. Theseappear throughout the interface and represent the Y-stage motor, Z-stage motor,electronics functionality, camera, and fluidics system.Figure 10 Activity Indicators
MiSeq Control Software20 Part # 15027617 Rev. AStatus IconsIn the top-right corner of the Welcome screen is a status icon that notifies you of anychange in conditions during run setup or during the run.Status Icon Status Name DescriptionStatus OK No change. System is normal.Information Information only. No action is required.Attention Information that might require attention.Warning Warnings do not stop a run, but might require actionbefore proceeding.Error Errors usually stop a run and generally require actionbefore proceeding with the run.When a change in condition occurs, the icon changes to the associated image andblinks to alert you. If this happens, select the icon to open the status window, whichcontains a general description of the condition.}Select any item listed to see a detailed description of the condition and instructionsto resolve the condition, if applicable.}Select Acknowledge to accept the message and Close to close the dialog box.You can filter the types of messages that appear in the status window by selecting theicons along the top margin of the window. Selecting an icon toggles the condition toshow or to hide.Get UpdatesYou can update the instrument software from the MCS Welcome screen using the GetUpdates feature. Your system must be connected to a network with internet access todownload updates.When software updates are available, the Get Updates button appears on the Welcomescreen. Select Get Updates. A dialog box opens asking you to confirm the command,after which a reboot of the MiSeq is required. Installation of the update beginsautomatically upon reboot.The Get Updates button appears on the Welcome screen only when updates areavailable.Manage InstrumentThe Manage Instrument screen contains controls for setting up folder locations andwash preferences, moving and deleting files, managing recipe files and genomereference files, and performing an instrument diagnostic.
MiSeq Control Software InterfaceMiSeq System User Guide 21Figure 11 Manage Instrument Screen}Setup Options—Provides options for changing default locations of data folders. Formore information, see Setup Options on page 21.}Manage Files—Provides controls for moving and deleting files on the instrumentcomputer. For more information, see Manage Files on page 22.}System Settings—Provides the option to change IP Settings, machine name, ordomain. For more information, see System Settings on page 23.}Perform Diagnostic—Provides instrument diagnostic options for use by an IlluminaTechnical Support representative during a Live Help session. Use of this feature isnot required during normal operation or for instrument maintenance.}Live Help—Connects your instrument to Illumina Technical Support, allowing arepresentative to access your instrument computer for troubleshooting. You musthave a network connection to use this feature. For more information, see Live Helpon page 23.Setup OptionsFrom Setup Options, you can set the default folder locations for recipes, sample sheets,manifests, and the output folder.Figure 12 Setup Options
MiSeq Control Software22 Part # 15027617 Rev. AFolder Locations}Recipes—Sets the default location for recipes. Recipes are XML files that thesoftware uses to perform the sequencing run. A recipe is created at the start of therun based on parameters that you provide in the sample sheet. After a run-specificrecipe is created, it is copied to the run folder. If you pause the run and resume itlater, this recipe restarts where the run was paused.}Sample Sheets—Sets the default location for sample sheets. Sample sheets are filescontaining run parameters that are copied onto the instrument before you start yourrun.For more information, see Sample Sheets on page 30.}Manifests—Sets the default location for manifests. Manifests are files used onlywith custom amplicon sequencing. They contain the amplicon sequences to whichclusters are aligned during analysis. Manifests are provided when you order yourcustom assay (CAT). There is no need to modify the manifest.}MiSeqOutput—Sets the default location for the output files. Illumina recommendsthat you change the default output folder location to a network location.For more information, see Run Folders on page 36.Manage FilesUse the features on the Manage Files screen to move, upload, or delete files on theinstrument computer. The screen is divided into five tabs: Runs, Sample Sheets,Manifests, Genomes, and Recipes.Figure 13 Manage Files Screen}Browse—Select Browse to point to the appropriate output folder or folders otherthan the default folders containing sample sheets, manifests, genomes, or recipes.}Delete—Select the checkbox next to the file or folder listed, and then select Delete.The Delete feature is available on all tabs.}Move—Available only for runs. Select the checkbox next to the folder, and thenselect Move. A window opens and prompts you to browse to an appropriatelocation.}Select All Files—Select the checkbox to the left of the Delete button, and then selectan action, Delete or Move. The action is applied to all files or folders selected.
MiSeq Control Software InterfaceMiSeq System User Guide 23}Upload Files—Available for sample sheets, manifests, genomes, and recipes. SelectUpload. A window opens that prompts you to browse to a location where the fileresides. The file is uploaded to the folder indicated in the Directory field.}Rename—Available only for sample sheets. Select the checkbox next to the file, andthen select Rename. Use the online keyboard to rename your sample sheet.Available Features By TabTab FeaturesRuns Delete or MoveSample Sheets Delete, Upload, or RenameManifests Delete or UploadGenomes Delete or UploadRecipes Delete or UploadSystem SettingsSystem Settings are normally configured when your instrument is initially installed andstarted for the first time. If you need to change your settings due to a network or facilitychange, use the System Settings feature.Figure 14 System SettingsSystem Settings configure the following components:}IP address and Domain Name Server (DNS) address}Machine name}Domain name}MiSeq Reporter Service credentialsContact your local IT representative to get information on your network settings.Live HelpThe Live Help feature is an online assistance tool that enables a member of IlluminaTechnical Support to view your screen with your permission, and share control of yourinstrument. You have overriding control and can end the screen-sharing session at anytime.To enable a connection, you must obtain a unique connection code from IlluminaTechnical Support.
MiSeq Control Software24 Part # 15027617 Rev. AFigure 15 Live Help Screen1Contact Illumina Technical Support to obtain a unique access code.2Enter your unique access code using the online keyboard.3Select Next to begin your screen-sharing session.Run Setup ScreensWhen you select Sequence on the Welcome screen, a series of four run setup screensopen in the following order: Load Flow Cell, Load Reagents, Review, and Pre-RunCheck.Set Cloud OptionThe first screen in the run setup steps prompts you to log onto the Cloud. Use the Cloudto store data analysis files. To enable access, you need a network connection and aniCom account.Figure 16 Cloud Option ScreenUsing the Cloud feature is optional. If you do not want use it, clear the checkbox on thescreen.
MiSeq Control Software InterfaceMiSeq System User Guide 25Load Flow CellThe Load Flow Cell screen prompts you to load the flow cell. When the flow cell isloaded, the software reads the RFID of the flow cell. A confirmation that the RFID wassuccessfully read appears in the lower-right corner of the screen.Figure 17 Load Flow Cell ScreenIf for some reason the RFID is not read, select the online keyboard icon and manuallyenter the flow cell ID.After the flow cell is loaded, close the flow cell latch and flow cell compartment doorbefore proceeding. Both the latch and compartment door must be closed beforebeginning the run.Load ReagentsThe Load Reagents screen has two steps: first, you load the PR2 bottle and make surethat the waste bottle is empty, and then you load the reagent cartridge. When you loadthe PR2 bottle and reagent cartridge, the software reads the RFID of the components. Aconfirmation that the RFID was successfully read appears in the lower-right corner ofthe screen.If for some reason the RFID is not read, select the online keyboard icon and manuallyenter the barcode number located below the barcode on the component label.Figure 18 Load PR2 Bottle and Waste Bottle
MiSeq Control Software26 Part # 15027617 Rev. AFigure 19 Load Reagent CartridgeChange Sample SheetEvery run must have a unique sample sheet for the run. If the software cannot find anappropriately named sample sheet, a message appears that asks you to browse to thelocation of your sample sheet.From the Load Reagents screen, you can use Change Sample Sheet to specify adifferent sample sheet for your run. A screen opens that allows you to browse to alocation where you stored the sample sheet.ReviewThe Review screen confirms that the software has located the sample sheet for your run.The Review screen lists the sample sheet name, and the following parameters providedin the sample sheet:}Experiment name}Analysis workflow}Read lengthFigure 20 Review Screen
MiSeq Control Software InterfaceMiSeq System User Guide 27Change FoldersIn the lower-left corner of the Review screen, the current folder locations for recipes,sample sheets, manifests, and output folders are listed. If you want to change folderlocations, select Change Folders, and browse to your preferred locations. Using thisoption from the Review screen changes folder locations for the current run only.Pre-Run CheckThe software automatically performs a pre-run check to ensure that required runcomponents and conditions are satisfied before allowing the run to begin. If any errorsoccur during the pre-run check, a message appears on the screen telling you where theerror occurred and what action is needed to correct it. For more information, see SoftwareErrors on page 71.Figure 21 Pre-Run CheckWhen the pre-run check is successful, the Start Run button becomes active. You areready to start your run.Sequencing ScreenThe Sequencing screen provides a visual representation of run progress, intensities, basecall quality scores (Q-scores), and activity on the flow cell.Figure 22 Sequencing Screen
MiSeq Control Software28 Part # 15027617 Rev. A}Run Progress—Shows run progress in a status bar and lists the number of cyclescompleted.}Intensities—Shows the value of cluster intensities of 90% of data for each tile.}Q-Score All Cycle—Shows the average percentage of bases greater than Q30.A quality score (Q-score) is a prediction of the probability of an error in base calling.Q-Score Chance of Wrong Base CallQ40 1 error in 10,000Q30 1 error in 1,000Q20 1 error in 100Q10 1 error in 10}Flow Cell—Shows the current temperature of the flow cell indicated by color. Blueindicates cooler temperatures, while orange and red indicate warmer temperatures.During imaging, imaged tiles appear dark gray. During chemistry, wavy linesindicate that reagents are pumping through the flow cell.NOTEThe MiSeq is sensitive to vibration. Vibrations occurring during a run willadversely impact sequencing results. Do not touch the instrument or touch screenmonitor during normal run operations unless you plan to stop or pause your run.Sequencing Analysis ViewerYou can monitor your run in greater detail using the Sequencing Analysis Viewer, adownloadable software application that is installed onto a computer independent of theMiSeq, but with access to the same network connected to the instrument. Afterlaunching the software, you can browse to the run folder for your run.Real Time Analysis (RTA) uses the first four cycles of the sequencing run for templategeneration. Template generation is the process by which cluster positions over the entireflow cell surface are defined along the X and Y axes. After the template of clusterpositions has been generated, the images produced over every subsequent cycle ofimaging are aligned against the template so that individual cluster intensities in all fournucleotide color channels can be extracted, and basecalls produced from the normalizedcluster intensities.After cycle 4, the Sequencing Analysis Viewer provides metrics generated by RTA andorganizes the metrics into plots, graphs, and tables. Your MiSeq must be networked touse Sequencing Analysis Viewer.NOTESequencing Analysis Viewer is universal to Illumina sequencers, most of whichuse an eight-lane flow cell. Some views include drop-down lists showing lanes 1–8. The MiSeq flow cell is a single-lane flow cell, so your data appears when youselect All or Lane 1.For more information, see the Sequencing Analysis Viewer User Guide.
Stopping or Pausing a RunMiSeq System User Guide 29Stopping or Pausing a RunThe MiSeq is designed to complete a run from beginning to end without userintervention. However, it is possible to stop a run or pause a run from the Sequencingscreen.}Stop a run—You might stop a run if the run was set up incorrectly, if the dataquality is bad, or if you experience a hardware error.}Pause a run—You might pause a run if you suspect that the waste bottle is full orto check the volume in the PR2 bottle.Stopping RunYou can stop a run before it has completed using the Stop button on the Sequencingscreen.Stopping a run is final. You cannot resume a run after it was stopped using the Stopbutton. When a run is stopped, the flow cell tray moves to the forward position. You areready to unload run components and perform an instrument wash.Pausing a RunYou can temporarily interrupt a run before it has completed using the Pause button onthe Sequencing screen. When you select Pause, the current command is completed, afterwhich the run is paused and the flow cell is placed in a safe state.Best PracticeEmpty the waste bottle before each run and make sure that run consumables areproperly loaded before starting the run.NOTEOn a paused run, the flow cell is stored in safe state for a maximum of three days.After three days, the instrument flow cell temperature control will turn off.
MiSeq Control Software30 Part # 15027617 Rev. ASample SheetsThe sample sheet is a comma separated values (*.csv) file that stores much of theinformation needed to set up, perform, and analyze a sequencing run.During the run setup steps, the software automatically looks for a sample sheetassociated with reagent cartridge loaded onto the instrument in the previous run setupstep. Therefore, Illumina recommends that you name your sample sheet with thebarcode number of the reagent cartridge that you will use for your run, followed by *.csvextension. The barcode number is located on the cartridge label directly below thebarcode.Before starting your run, copy your sample sheet to the instrument computer or to thenetwork location indicated in Setup Options. When your run begins, the software copiesyour sample sheet to the root of your run folder.At the end of your run, the sample sheet is used for secondary analysis by the MiSeqReporter analysis software.Sample Sheet TemplateYou can open and edit the sample sheet template in Excel or in Notepad. A samplesheet template is available for download from the Illumina website athttp://www.illumina.com. Go to the MiSeq support page and click Downloads. AniCom account is required.NOTEDo not use commas within a field in the sample sheet. If a comma is includedwithin a field, your sample sheet will not be read correctly by the software.Illumina Experiment DesignerYou can use the Illumina Experiment Designer to create your sample sheet, a wizard-based application for creating the sample sheet. The Experiment Designer guides youthrough the steps to create your sample sheet based on the analysis workflow for yourrun.The Experiment Designer provides a feature for recording parameters for your sampleplate, such as sample ID, dual indices, and other parameters applicable to your 96-wellplate for Nextera, TruSeq DNA, TruSeq RNA, Small RNA, and Amplicon librarypreparation protocols. Using the Experiment Designer, you can import the sample plateparameters into your sample sheet. For more information, see the Experiment DesignerOnline Help integrated with the application.The Illumina Experiment Designer can be run on any Windows platform. You candownload the Experiment Designer from the Illumina website athttp://www.illumina.com. Go to the MiSeq support page and click Downloads. AniCom account is required.Sample Sheet ParametersThe sample sheet is organized by section text indicated by [ ] brackets, such as [Header],[Reads], [Manifests], and [Data]. Section text within brackets is case-sensitive. All otherfields are not case-sensitive.
Sample SheetsMiSeq System User Guide 31Figure 23 Sample Sheet Example in ExcelNot all sections of the sample sheet are applicable to every run. Applicable sectionsdepend on the analysis workflow used for your run.[Header]The Header section of the sample sheet contains information about your run. Extrafields are ignored.Parameter DescriptionInvestigator Name Your nameProject Name Project name of your preferenceExperiment Name Experiment name of your preferenceDate Date of your experimentWorkflow The analysis workflow for your run.You must type the analysis workflow name exactly as shown usingone of the following options: Resequencing, Amplicon,DenovoAssembly, SmallRNA, Metagenomics, or LibraryQC.If you are using the Experiment Designer, select your workflowfrom the drop-down menu.For more information, see Analysis Workflow on page 35.Chemistry The folder that contains recipe fragments used to build the recipe. Ifthis field is blank, the system uses the default recipe fragments.For TruSeq RNA or TruSeq DNA libraries, this field can be blankand use the default settings.For any workflows that use dual indexing, specifically Nextera andTruSeq Custom Amplicon, the chemistry field is required. Enter"amplicon" in this field.[Reads]Reads indicates the number of sequencing cycles in each read.Parameter DescriptionNumber of cycles forRead 1Read length of a target sequence.The number of cycles for the index read is indicated by the indexsequence the [Data] section.Number of cycles forRead 2Read length of a target sequence.
MiSeq Control Software32 Part # 15027617 Rev. A[Manifests]This section of the sample sheet is mandatory for the Amplicon analysis workflow. Youdo not need to populate this section if you are using other analysis workflows.Parameter DescriptionName of manifest file Name of your manifest file provided by Illumina with your customassay (CAT). The manifest file must reside in the same folderlocation as the sample sheet.When you enter your manifest name, omit the file extension (*.txt).More than one manifest can be specified: one manifest for yourcustom CAT, and one manifest for the control CAT.[SiteReports]This section of the sample sheet is used by the Resequencing and Amplicon analysisworkflows.Parameter DescriptionName of SiteReport inputfileEach line in the SiteReports section is the name of a SiteReport inputfile. This file designates positions on a given chromosome to reportthe genotype found at that position.You can use more than one input file for a run. The SiteReport inputfile must reside in the same folder location as the sample sheet.The SiteReport input file is a comma-separated value (*.csv) formatfile that must adhere to the following format:•The first line are column names. Required columns are Positionand Chromosome.•Additional user-defined columns are appended to the resultinganalysis report for that position.When you enter your SiteReport input file name, omit the fileextension (*.csv).[Data]In this section, list sample information one sample per line. There must be at least onesample in this section. Each analysis workflow requires different columns in thissection. Column order is not important.Parameter DescriptionSampleID Required. This is usually a barcode but can have any value.Sample_Name Required. This is used in the reporting web page.Index Required for multi-sample assays.Nucleotide sequence in text format. Valid characters are A, C, G, T,and N, where N matches any base.Index2 Required for multi-sample assays with dual indexing.Nucleotide sequence in text format. Valid characters are A, C, G, T,and N, where N matches any base.Additional [Data] Columns for Resequencing WorkflowParameter DescriptionGenomeFolder Required.The reference genome folder containing the FASTA files to be usedin the alignment step.
Sample SheetsMiSeq System User Guide 33Additional [Data] Columns for Amplicon WorkflowParameter DescriptionGenomeFolder Recommended.It is used to provide variant annotations, if available, and to set thechromosome sizes in the *.bam file output.Additional [Data] Columns for DenovoAssembly WorkflowParameter DescriptionGenomeFolder(or Genome)Optional.If you populate this field, list the absolute path to the genome folder.•If the folder listed does not exist, MiSeq Reporter combines theGenomePath configuration setting with the genome string.•If the path does not exist, MiSeq Reporter stops processing.Additional [Data] Columns for SmallRNA WorkflowParameter DescriptionGenomeFolder Optional.A string identifier for the sample. This is usually a barcode but canhave any value. If provided, clusters are aligned against the fullreference genome.Contaminants String; required.The path to the folder containing the FASTA files of contaminants.miRNA Required.The path to the folder containing FASTA files of mature miRNAs.RNA Required.The path to folder containing FASTA files of small RNAs.Additional [Data] Columns for Library QC WorkflowParameter DescriptionGenomeFolder Required.The reference genome folder containing the FASTA files to be usedin the alignment step.[Settings]Settings are optional in the sample sheet. This section describes possible entries in theSettings section.Parameter DescriptionFilterPCRDuplicates Setting are 0 or 1. Default is 1, no filtering.If set to 0, PCR duplicates are filtered out from subsequent analysis.Duplicates are defined as paired-end reads where both reads havethe exact same alignment positions.MinQScore Specifies the Q-score threshold for variant calling.OnlyGenerateFastq Setting are 0 or 1. Default is 0.If set to 1, MiSeq Reporter generates FASTQ files and then exits theworkflow. This option is provided as a shortcut if you prefer to usethird-party tools to process FASTQ files.
MiSeq Control Software34 Part # 15027617 Rev. AParameter DescriptionAdapter It is possible that some clusters will sequence beyond the sampleDNA and read bases from the adapter.Specify the adapter sequence here to mask the adapter sequence.Masking the adapter sequence improves accuracy and speed duringsecondary analysis.
Analysis WorkflowMiSeq System User Guide 35Analysis WorkflowThe analysis workflow is a procedure performed by MiSeq Reporter, the secondaryanalysis software on the MiSeq. Secondary analysis begins after the Real Time Analysis(RTA) software has completed base call analysis.For more information, see On-Instrument Analysis on page 63.The MiSeq supports six analysis workflows. The analysis workflow for your run isspecified in the sample sheet. When you enter the analysis workflow in the samplesheet, enter the name as shown below in the Workflow column.Workflow Applications OutputTargeted ResequencingEnter: ResequencingSequencing of an enriched portion ofthe human genome, or of a smallgenome such as E. coli.Reads are aligned against the referenceand variants are noted.Aligned reads in*.bam format.Variant calls in *.vcfformat.Amplicon SequencingEnter: AmpliconSequencing of PCR amplicons fromprobes targeting particular genomepositions (up to approx. 384 loci fromup to approx. 96 samples).A genomic reference is recommended,but not required. Including a referencemeans that the genome size in the*.bam file headers is correct, andenables annotation of variants in dbSNPor within genes.Reads are aligned against a manifest filespecified in the sample sheet. Twomanifests can be specified: one for thecontrol and one for the sample.Aligned reads in*.bam format.Variant calls in *.vcfformat.De novo AssemblyEnter: DenovoAssemblyAssembly of small (<20 Mb) genomesfrom reads without the use of agenomic reference.If a genomic reference is included, a dot-plot is generated with respect to thereference.Contigs in FASTAformat.Small RNA SequencingEnter: SmallRNASequencing of miRNA.Requirements include a genomicreference, a contaminants database, anRNA database, and a mature miRNAdatabase.Reports on therelative abundanceof each record.MetagenomicsEnter: MetagenomicsNo genomic reference is required formetagenomics workflow. Reads areclassified using a database of 16S rRNAdata, which is included with the MiSeqReporter software.Read classificationsby taxonomic group.Library QCEnter: LibraryQCFast resequencing of a referencegenome to QC the DNA library andgenerate per-sample statistics.Per-samplesummary statistics.
MiSeq Control Software36 Part # 15027617 Rev. ARun FoldersEach run on the MiSeq generates a run folder on either the local drive or a networklocation. You can specify the location of the run folder in the MiSeqOutput field on theSetup Options screen. For more information, see Setup Options on page 21.Run folders contain the data files produced during the run by Real Time Analysis(RTA), which is integrated with MCS and performs image analysis and base calling.The data files in the run folder are later used for secondary analysis. For moreinformation, see On-Instrument Analysis on page 63.Run Folder NameBy default, the folder is named in the following format:YYMMDD_<InstrumentNumber>_<Run Number>_<Flow Cell ID>Example: 110114_SN106_0016_A90095ACXXThe run number increments by one each time you perform a run on a given instrument.The flow cell ID appends to the end of the run folder name.
Available Disk SpaceMiSeq System User Guide 37Available Disk SpaceThe software checks available disk space before beginning a run. If there is not enoughdisk space for your run, the software pauses the run and a message alerts you. Requiredspace for your run depends on the number of cycles in your run and the analysisworkflow for your run. The required disk space for your run and the amount of diskspace you must clear, is listed in the message.If you get this message, you need to make disk space available to continue the run. Goto Manage Files and click the Runs tab. For more information, see Manage Files on page22. When sufficient disk space is available, the run automatically resumes.The integrated instrument computer has a storage capacity of 600GB. A 2x150-cyclerun results in approximately 4.2 GB of stored data.
38 Part # 15027617 Rev. A
Chapter 3MiSeq System User Guide 39Chapter 3 Performing a RunPerforming a RunIntroduction 40Setting Up the Sample Sheet 41Preparing the Reagent Cartridge 43Preparing Your Libraries 44Clean the Flow Cell 47Loading the Flow Cell 49Load Reagents 51Starting the Run 53
Performing a Run40 Part # 15027617 Rev. AIntroductionTo perform a run on the MiSeq, you first need to prepare for the run using the followingsteps described in this chapter:}Create a sample sheet and copy it to the instrument or an accessible location.}Prepare the reagent cartridge for use.}Denature and dilute your sample libraries.}Load your sample libraries onto the prepared reagent cartridge.}Set up the run using the MCS interface.After the run begins, the MiSeq is completely automated and no other user interventionis required. The sequencing run can be monitored from the Sequencing screen ormonitored remotely using the Sequencing Analysis Viewer, an optional softwareapplication that you can download from the Illumina website.After your run is complete, perform an instrument wash.
Setting Up the Sample SheetMiSeq System User Guide 41Setting Up the Sample SheetTo set up a run on the MiSeq, you first need to create the sample sheet for your run. Formore information, see Sample Sheets on page 30.When your sample sheet is ready, copy the sample sheet to a network locationconnected to your instrument. If your instrument is not connected to a network, you cancopy the sample sheet to the instrument using a USB flash drive.You can create the sample sheet using the sample sheet template or the IlluminaExperiment Designer, a wizard-based software application available for download fromthe Illumina website. For more information, see Illumina Experiment Designer on page 30.NOTEIllumina recommends that you name your sample sheet with the barcodenumber of the reagent cartridge that you will use for your sequencing run. Thebarcode number is located on the cartridge label.1Name your sample sheet with the reagent cartridge barcode number followed by*.csv extension.2Create your sample sheet off-instrument using the Illumina Experiment Designer orthe sample sheet template. Enter the following information:aEnter investigator name, project name, experiment name, and date.bEnter the appropriate analysis workflow (no spaces): Resequencing, Amplicon,DenovoAssembly, SmallRNA, Metagenomics, or LibraryQC.3Enter the number of cycles for each read. To add an Index Read, include an indexsequence for each sample in the [Data] section.4Complete the remaining sample sheet sections specific to your analysis workflow.AnalysisWorkflow[Manifests] [SiteReports] [Data]Resequencing NotapplicableOptional Required: SampleID, Sample_Name,GenomeFolderOptional: Index, Index 2Amplicon Required Optional Required: SampleID, Sample_NameOptional: Index, Index 2,GenomeFolderDenovoAssembly NotapplicableNotapplicableRequired: SampleID, Sample_NameOptional: Index, Index 2,GenomeFolderSmallRNA NotapplicableNotapplicableRequired: SampleID, Sample_Name,Contaminants, miRNA, RNAOptional: Index, Index 2,GenomeFolder
Performing a Run42 Part # 15027617 Rev. AAnalysisWorkflow[Manifests] [SiteReports] [Data]Metagenomics NotapplicableNotapplicableRequired: SampleID, Sample_NameOptional: Index, Index 2LibraryQC NotapplicableOptional Required: SampleID, Sample_Name,GenomeFolderOptional: Index, Index 2NOTEFor TruSeq Custom Amplicon sample sheets, see the TruSeq Custom AmpliconLibrary Preparation Guide for the appropriate index sequences.For Nextera DNA sample sheets, see the Nextera DNA Sample Preparation Guidefor the appropriate index sequences.5Copy the sample sheet to the network location specified in Setup Options, or copy itto the instrument using a USB flash drive and the Manage Files feature accessiblefrom the Welcome screen.
Preparing the Reagent CartridgeMiSeq System User Guide 43Preparing the Reagent Cartridge1Remove thereagent cartridge from -15° to -25°C storage.2Place the reagent cartridge in a water bath containing only enough roomtemperature deionized water to submerge the base of the reagent cartridge withoutreaching the clear cartridge cover.Figure 24 Maximum Water LineNOTETo minimize the risk of cross-contamination, do not allow the water to reach theclear cartridge cover.3Allow the reagent tray to thaw in the room temperature water bath forapproximately60minutes or until completely thawed.4Remove the cartridge from the water bath and gently tap it on the bench to dislodgewater from the base of the cartridge.5Invert the reagent cartridge ten times to mix the thawed reagents.6Visually inspect the reagent marked IMF to make sure that it is fully mixed and freeof precipitates.7Gently tap the cartridge on the bench to dislodge water from the base of thecartridge and reduce air bubbles in the reagents.8Place the reagent cartridge on ice until you are ready to load your sample and setup your run.WARNINGThis set of reagents contains formamide, an aliphatic amide that is a probablereproductive toxin. Personal injury can occur through inhalation, ingestion, skincontact, and eye contact.Dispose of containers and any unused contents in accordance with thegovernmental safety standards for your region.For more information, see the MSDS for this kit, at http://www.illumina.com/msds.
Performing a Run44 Part # 15027617 Rev. APreparing Your LibrariesNOTEDoes Not Apply to TruSeq Custom Amplicon Sequencing—If you prepared yourlibraries with the TruSeq Custom Amplicon protocol, do not perform this step.The TruSeq Custom Amplicon protocol results in a ready-to-use normalizedconcentration of pooled libraries for the MiSeq reagent cartridge.For all types of runs except TruSeq Custom Amplicon sequencing, you must denatureand dilute your sample DNA for cluster generation and sequencing.Illumina-Supplied Consumables}HT1 (Hybridization Buffer), pre-chilledUser-Supplied Consumables}0.1N NaOH}Tris-Cl 10mM, pH8.5 with 0.1% Tween 20Denature DNANOTEIf your application requires higher than a 20 pM final concentration of yourlibrary, please ensure your concentration of NaOH is not higher than 0.05 N inthe denaturation solution and not more than 0.001 N (1 mM) in the final solutionafter diluting with HT1.Higher concentrations of NaOH in the loaded library will inhibit libraryhybridization and decrease cluster density.Use the following instructions to denature the DNA with 0.1N NaOH to a DNAconcentration of 20pM.1Combine the following volumes of sample DNA and0.1N NaOH in amicrocentrifuge tube:•2nM sample DNA (10µl)•0.1N NaOH (10µl)2Vortex briefly to mix the sample solution.3Centrifuge the sample solution to 280xg for one minute.4Incubate for fiveminutes at room temperature to denature the DNA into singlestrands.5Add the following volume of pre-chilled HT1 to the tube containing denaturedDNA to result in a 20 pM denatured library:•Denatured DNA (20 µl)•Pre-chilled HT1 (980µl)6Place the denatured DNA on ice until you are ready to proceed to final dilution.Dilute Denatured DNAUse the following instructions to dilute the denatured DNA with pre-chilled HT1 to atotal volume of 1,000µl.
Preparing Your LibrariesMiSeq System User Guide 451Dilute the denatured DNA to the desired concentration using the followingexample:Final Concentration 6pM 8pM 10pM 12pM 15pM20 pM denatured DNA 300µl 400µl 500µl 600µl 750µlPre-chilled HT1 700µl 600µl 500µl 400µl 250µl2Invert several times to mix the DNA solution.3Pulse centrifuge the DNA solution.4Place the denatured and diluted DNA on ice until you are ready to load yoursamples onto the MiSeq reagent cartridge.Denature and Dilute PhiX ControlUse the following instructions to denature and dilute the 10nM PhiX library.1Combine the following volumes to dilute the PhiX library to 2nM:•10nM PhiX library (2µl)•10 mM Tris-Cl, pH 8.5 with 0.1% Tween 20 (8 µl)2Combine the following volumes of 2nM PhiX library and0.1N NaOH in amicrocentrifuge tube to result in a 1nM PhiX library:•2nM PhiX library (10µl)•0.1N NaOH (10µl)3Vortex briefly to mix the 1nM PhiX library solution.4Centrifuge the template solution to 280xg for one minute.5Incubate for fiveminutes at room temperature to denature the PhiX library intosingle strands.6Add the following volume of pre-chilled HT1 to the tube containing denatured PhiXlibrary to result in a 20pM PhiX library.•Denatured PhiX library (20µl)•Pre-chilled HT1 (980µl)NOTEThe denatured 20pM PhiX library can be stored up to three weeks at -15° to -25°C. After three weeks, cluster numbers tend to decrease.7Dilute the denatured 20pM PhiX library to 8 pM as follows:•20 pM denatured PhiX library (400µl)•Pre-chilled HT1 (600µl)Mix Sample Library and PhiX Control1Combine the following volumes of denatured PhiX control library and yourdenatured sample library to result in a 1% volume ratio:•8 pM PhiX control library (10 µl)•Denatured sample library (990 µl)
Performing a Run46 Part # 15027617 Rev. A2Set the combined sample library and PhiX control aside on ice until you are readyto load it onto the MiSeq reagent cartridge.Load Sample Libraries onto CartridgeWhen the reagent cartridge is prepared for use on the MiSeq, you are ready to load yoursamples. You can load a single library or a pool of indexed libraries.1Pierce the foil over the reservoir on the reagent cartridge labeled Load Samples.2Pipette 600 µl of your sample libraries into the Load Samples reservoir.Figure 25 Load Libraries3Proceed directly to the run setup steps using the MiSeq Control Software (MCS)interface.
Clean the Flow CellMiSeq System User Guide 47Clean the Flow CellThe flow cell is provided immersed in storage buffer in a flow cell container. Beforeloading the flow cell on the MiSeq, you need to rinse the flow cell and thoroughly dry it,and then clean the flow cell with an alcohol wipe.1Using plastic forceps, grip the flow cell by the base of the plastic cartridge andremove it from the flow cell container.Figure 26 Remove Flow Cell2Lightly rinse the flow cell assembly with approximately 2 ml of laboratory-gradewater, making sure that both the glass and plastic cartridge are thoroughly rinsed ofexcess salts. Excess salts can affect flow cell seating in the instrument.Figure 27 Rinse Flow Cell3Using care around the flow cell port gaskets, thoroughly dry the flow cell andcartridge using a lint-free lens cleaning tissue. Gently pat dry in the area of thegaskets and adjacent glass.4Using an alcohol wipe, clean the flow cell glass, making sure that the glass is free ofstreaks and fingerprints. Avoid using the alcohol wipe on the flow cell port gaskets.
Performing a Run48 Part # 15027617 Rev. AFigure 28 Dry Flow Cell5Dry any excess alcohol with a lint-free lens cleaning tissue, and visually inspect tomake sure that no tissue particles are obstructing the flow cell ports and the gasketswere not at all dislodged from the flow cell ports.If the gaskets appear to be dislodged at all, gently press them back into place untilthey sit squarely around the flow cell ports.
Loading the Flow CellMiSeq System User Guide 49Loading the Flow CellThe MCS interface will guide you through the run setup steps, starting from theWelcome screen.1From the Welcome screen, select Sequence. The Cloud Option screen opens.2Set your preference for using the Cloud Option and select Next. The Load Flow Cellscreen opens.3Put on a new pair of powder-free latex gloves.4Raise the flow cell compartment door.5Press the silver release button to the right of the flow cell latch. The flow cell latchraises to open.Figure 29 Open Flow Cell Latch6Visually inspect the flow cell stage to make sure it is free of lint.If lint or other debris is present, clean the flow cell stage using an alcohol wipe or alint-free tissue moistened with ethanol or isopropanol. Carefully wipe the surface ofthe flow cell stage until it is clean and dry.7Hold the flow cell by the edges of the flow cell cartridge near the Illumina label.Make sure the label is facing upward.8Place the flow cell on the flow cell stage.Figure 30 Place Flow Cell on Stage9Gently press down on the flow cell latch to close it over the flow cell. You will heara click when the flow cell latch is secure.NOTEAs you close the flow cell latch, two alignment pins near the hinge of the flow cell
Performing a Run50 Part # 15027617 Rev. Alatch align and position the flow cell into place.Figure 31 Close Flow Cell LatchNOTEThe MiSeq System reads the RFID of the flow cell. If the system is unable toregister the RFID of the flow cell, you can manually enter the barcode ID printedon the flow cell using the online keyboard. Select the keyboard icon in the lowerright corner of the screen, enter the barcode ID, and select Done to return to theLoad Flow Cell screen.10 Close the flow cell compartment door.11 Select Next on the Load Flow Cell screen. The Load Reagents screen opens.
Load ReagentsMiSeq System User Guide 51Load ReagentsFirst, load the PR2 bottle and make sure that the waste bottle is empty, and then loadthe reagent cartridge. Check for a message on the lower-right corner of the screen thatconfirms the RFID was successfully read for the PR2 bottle and the reagent cartridge.NOTEThe MiSeq System reads the RFID of the PR2 bottle and reagent cartridge. If thesystem is unable to register the RFID, you can manually enter the barcodenumber from the label using the online keyboard. Select the keyboard icon in thelower-right corner of the screen, enter the barcode number, and select Done toreturn to the Load Reagents screen.Load PR2 and Check the Waste Bottle1Remove the bottle of PR2 from 2° to 8°C storage and remove the lid.2Open the reagent compartment door.3Raise the sipper handle until it locks into place.4Place the PR2 bottle in the indentation to the right of the reagent chiller.Figure 32 Load the PR2 Bottle5Confirm that the PR2 RFID was successfully read.6If necessary, empty the waste bottle into the appropriate waste container.WARNINGThis set of reagents contains formamide, an aliphatic amide that is a probablereproductive toxin. Personal injury can occur through inhalation, ingestion, skincontact, and eye contact.Dispose of containers and any unused contents in accordance with thegovernmental safety standards for your region.For more information, see the MSDS for this kit, at http://www.illumina.com/msds.7Slowly lower the sipper handle. Make sure that the sippers lower into the PR2 andwaste bottles.
Performing a Run52 Part # 15027617 Rev. AFigure 33 Lower Sipper Handle8Select Next on the Load Reagents screen.Load the Reagent Cartridge1Open the reagent chiller door.NOTEDo not leave the reagent chiller door open for extended periods of time.2Hold the reagent cartridge on the end with the Illumina label, and slide the reagentcartridge into the reagent chiller until the cartridge stops.Figure 34 Load Reagent Cartridge3Close the reagent chiller door.4Confirm that the reagent cartridge RFID was successfully read.5Close the reagent compartment door.6Select Next on the Load Reagents screen. The Review screen opens.
Starting the RunMiSeq System User Guide 53Starting the RunAfter you have loaded the flow cell and reagents, the MCS interface prompts you toreview run parameters and perform a pre-run check before beginning the run.Review Run ParametersNOTEThe sample sheet name must match the reagent barcode number. A messageappears if the sample sheet name and reagent barcode number are not identical,and prompts you to select a sample sheet.1Review Experiment Name, Analysis Workflow, and Read Length. These parametersare specified in the sample sheet.2Review the folder locations in the lower-left corner.If you need to change any folder locations, select Change Folders. When you havecompleted the changes, select Save and then Next.3Select Next. The Pre-Run Check screen opens.Review Pre-Run CheckThe system performs a check of all run components, disk space, and networkconnections before you start the run.If any items do not pass the pre-run check, a message appears on the screen withinstructions to correct the error. For more information, see Software Errors on page 71.When all items successfully pass the pre-run check, you are ready to start your run.Important Note Before Starting the RunWARNINGThe MiSeq is sensitive to vibration. Touching the instrument after startingyour run will adversely impact sequencing results.After you select Start Run, do not open the flow cell compartment or reagentcompartment doors, or touch the instrument monitor unless you plan to stopthe run.Wait until the run is complete before touching the monitor or openingcompartment doors.Monitoring the RunWhen the run begins, the RTA window temporarily pops up in front of the Sequencingscreen and then auto-minimizes. This is normal.
Performing a Run54 Part # 15027617 Rev. AFigure 35 Sequencing ScreenDuring your run, use the Sequencing screen to monitor run progress, intensities, qualityscores, fluidics, and imaging.For more information, see Sequencing Screen on page 27.To monitor your run in greater detail, use the Sequencing Analysis Viewer. For moreinformation, see Sequencing Analysis Viewer on page 28 and the Sequencing Analysis UserGuide.
Chapter 4MiSeq System User Guide 55Chapter 4 Post-Run ProceduresPost-Run ProceduresIntroduction 56Performing a Post-Run Wash 57Unloading Components 59Shutting Down the Instrument 61
Post-Run Procedures56 Part # 15027617 Rev. AIntroductionPerform an instrument wash following the completion of a sequencing run. It isimportant to regularly wash the fluidics lines to ensure continued performance.
Performing a Post-Run WashMiSeq System User Guide 57Performing a Post-Run WashAlways perform a post-run wash after completing a sequencing run. If you did notperform a wash after the last run, a reminder appears on the MiSeq software interface.The post-run wash takes approximately 20 minutes.Illumina-Supplied Accessories}Wash tray}Wash bottle, 500 mlUser-Supplied Consumables}Laboratory-grade waterProcedureNOTELeave the used flow cell on the instrument until the post-run wash is complete.1When your run is complete, select Start Wash.2Open the reagent compartment door.3Open the reagent chiller door and remove the used reagent cartridge from thechiller. The sippers were automatically raised at the end of the run. The reagentcartridge should slide easily from the chiller.4Prepare the wash tray by adding approximately 6ml of laboratory-grade water toeach reservoir.5Slide the wash tray into the reagent chiller until it stops. Close the reagent chillerdoor.6Raise the sipper handle in front of the PR2 bottle and waste bottle until it locks intoplace.7Prepare the 500ml wash bottle by adding at least 350ml of laboratory-grade water.8Remove the PR2 bottle from the reagent compartment and replace it with the washbottle containing laboratory-grade water.9Remove the waste bottle and discard the contents appropriately. Return the wastebottle to the reagent compartment.WARNINGThis set of reagents contains formamide, an aliphatic amide that is a probablereproductive toxin. Personal injury can occur through inhalation, ingestion, skincontact, and eye contact.Dispose of containers and any unused contents in accordance with thegovernmental safety standards for your region.For more information, see the MSDS for this kit, at http://www.illumina.com/msds.10 Slowly lower the sipper handle, making sure that the sippers lower into the washbottle and waste bottle.11 Close the reagent compartment door.12 From the Post-Run Wash screen, select Next. The post-run wash begins.When the post-run wash is complete, a message appears on the screen.
Post-Run Procedures58 Part # 15027617 Rev. A13 Select Done. The Welcome screen opens. You are ready to start another sequencingrun.
Unloading ComponentsMiSeq System User Guide 59Unloading ComponentsWhen the post-run wash is complete you are ready to unload components from theinstrument.Remove Wash Components1Open the reagent compartment door and raise the sipper handle to lift the sippersfrom the 500ml wash bottle and waste bottle. You should feel the sipper handlelock into place.2Remove the wash bottle. Wash the wash bottle with deionized water andthoroughly dry it so it is ready for a subsequent wash. Store it upside down toprotect from dust.3Remove the waste bottle and discard the contents appropriately and according tosite standards.WARNINGThis set of reagents contains formamide, an aliphatic amide that is a probablereproductive toxin. Personal injury can occur through inhalation, ingestion, skincontact, and eye contact.Dispose of containers and any unused contents in accordance with thegovernmental safety standards for your region.For more information, see the MSDS for this kit, at http://www.illumina.com/msds.4Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol,clean the floor of the reagent compartment of any visible spills or condensation.5Return the empty waste bottle to the reagent compartment.6Open the reagent chiller door.7Remove the wash tray. Wash the wash tray with deionized water and thoroughlydry it so it is ready for a subsequent wash. Store it upside down to protect fromdust.8Close the reagent chiller door and the reagent compartment door.Remove the Flow Cell1Open the flow cell compartment door.2Press the release button to the right of the flow cell latch. The flow cell latch raises.Figure 36 Flow Cell Stage
Post-Run Procedures60 Part # 15027617 Rev. A3Hold the used flow cell by the edges of the flow cell cartridge near the Illuminalabel, and remove it from the flow cell stage.4Clean the flow cell stage using an alcohol wipe or a lint-free tissue moistened withethanol or isopropanol. Carefully wipe the surface of the flow cell holder until it iscompletely clean.5Gently press down on the flow cell latch to close it. You will hear a click when theflow cell latch is secure.6Close the flow cell compartment door.
Shutting Down the InstrumentMiSeq System User Guide 61Shutting Down the InstrumentTurn off the instrument only if it will not be used for more than ten days. If theinstrument will be used within the next ten days, leave the instrument on.NOTEAny time that you turn off the instrument, wait a minimum of 60seconds beforeturning the power switch back to the ON position.Use the following procedure to safely prepare fluidics before shutting down the system.1Perform a maintenance wash to thoroughly wash the system.2Leave the used wash tray in the reagent chiller to collect any fluid that may fallback through the lines.3Empty the waste bottle and return it to the reagent compartment.4Place an empty wash bottle in the reagent compartment, lower the sipper handle,and close the reagent compartment door.5Remove the flow cell from the flow cell stage.6Using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol,carefully wipe the surface of the flow cell holder until it is completely clean and dry.7Press down on the flow cell latch until it locks into place.8Close the flow cell compartment door.9Toggle the power switch to turn off the instrument.10 When you are ready to restart the instrument, load a used flow cell and perform aninstrument wash prior to your next sequencing run.
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Chapter 5MiSeq System User Guide 63Chapter 5 On-Instrument AnalysisOn-Instrument AnalysisIntroduction 64Primary Analysis Results 65Secondary Analysis Using MiSeq Reporter 66Using MiSeq Reporter Off-Instrument 68
On-Instrument Analysis64 Part # 15027617 Rev. AIntroductionThe MiSeq Control Software (MCS) performs image analysis and base calling usingintegrated Real Time Analysis (RTA). Analysis is performed during the sequencing run,which saves downstream analysis time. RTA runs automatically on the instrument.The primary analysis output from a sequencing run is a set of quality-scored base callfiles (*.bcl files), which are generated from the raw image files and contain base calls percycle. By default, images are deleted from the instrument computer after image analysis.The raw image data are not needed for secondary analysis.MiSeq ReporterThe MiSeq is the first instrument to provide on-instrument secondary analysis using theMiSeq Reporter software. MiSeq Reporter is a Windows-based application thatprocesses base calls generated by RTA. The results include information aboutalignment, structural variants, and contig assemblies for each genome requested, andeach sample if your run is a multi-sample run or an indexed run.
Primary Analysis ResultsMiSeq System User Guide 65Primary Analysis ResultsThe following table describes the folders and files generated by Real Time Analysis(RTA) during primary analysis. Many of these files are used for secondary analysis bythe MiSeq Reporter software.Key File Subfolder DescriptionRTAComplete.txt AnalysisFolder\ A marker file generated when base callanalysis is complete. The presence of thisfile triggers the start of secondary analysis.SampleSheet.csv AnalysisFolder\ This file is read and copied to the run folderbefore the run, and later used for secondaryanalysis.RunInfo.xml AnalysisFolder\ Identifies the boundaries of the reads(including index reads) and the quality tableselected for run.*.bcl files AnalysisFolder\Data\Intensities\BaseCalls\L001\CX.XEach *.bcl file contains RTA base calling andbase quality scoring results for one cycle,one tile.*.stats files AnalysisFolder\Data\Intensities\BaseCalls\L001\CX.X*.stats files contain RTA base callingstatistics for a given cycle/tile.*.filter files AnalysisFolder\Data\Intensities\BaseCalls*.filter files contain filter results per tile.*.txt AnalysisFolder\Data\RTALogs Log files from primary analysis.*.cif files AnalysisFolder\Data\Intensities\L001\CX.XEach binary *.cif file contains RTA imageanalysis results for one cycle, one tile. Thesefiles can be used in the OLB analysissoftware. For more information, see theOff-line Basecaller Software User Guide.*.locs files AnalysisFolder\Data\Intensities\BaseCalls\L001Reports the cluster coordinates. There isone *.locs file for each tile..*.jpg files AnalysisFolder\Thumbnail_Images\L001\CX.XThumbnail images generated for each cycleand base, and can be used to troubleshoot arun.Table 2 Contents of Analysis Folder
On-Instrument Analysis66 Part # 15027617 Rev. ASecondary Analysis Using MiSeq ReporterMiSeq Reporter runs on the instrument computer and uses the analysis workflowspecified in the sample sheet to perform secondary analysis. For more information, seeAnalysis Workflow on page 35.Even though MiSeq Reporter is running on the instrument, you view the analysis inprogress from any web browser on another computer on the same network as yourMiSeq.Viewing MiSeq ReporterMiSeq Reporter launches automatically after RTA completes primary analysis. To viewthe MiSeq Reporter interface during analysis, use any web browser on the same networkas your instrument and connect to the MiSeq HTTP service on Port 8042.}Connect using the instrument IP address:IP Address HTTP Service Port HTTP Address10.10.10.10, for example 8042 http://10.10.10.10:8042}Connect using the your network name:Network Name HTTP Service Port HTTP AddressYourNetwork, for example 8042 http://yournetwork:8042}If MiSeq Reporter is installed locally on an off-instrument computer, connect using"localhost":Off-Instrument HTTP Service Port HTTP Addresslocalhost 8042 http://localhost:8042When you are linked to your MiSeq, the MiSeq Reporter interface opens.Figure 37 MiSeq Reporter InterfaceSecondary Analysis Input RequirementsMiSeq Reporter requires the following primary analysis files to perform secondaryanalysis. There is no need to move or copy these files to another location before analysisbegins. The software automatically accesses these files in the run folder.
Secondary Analysis Using MiSeq ReporterMiSeq System User Guide 67File Type DescriptionRTAComplete.txt Marker file that indicates that RTA processing is complete.SampleSheet.csv Provided at beginning of sequencing run and contains analysisworkflow and important run information.RunInfo.xml Identifies high-level run information.*.bcl files Files contain specific run and base calling information.*.filter files Files contain filter results for each tile.*.locs files Files contain the locations of all clusters on a tile.Pre-Installed Databases and GenomesThe MiSeq includes several pre-installed databases and genomes.Databases}miRbase for human}dbSNP for human}refGene for humanGenomes}hg19 (human)}dh10b (E. coli)}S. aureus}yeast}A. thaliana}mouseYou can upload and use your own reference in FASTA format. Reference FASTA filesshould be stored in a single folder and have a *.fa extension.Use the Manage Files feature in MCS to upload files. For more information, see ManageFiles on page 22.Sequencing During AnalysisIf a new sequencing run is started on the MiSeq before secondary analysis is complete,the current secondary analysis is stopped. From the MiSeq Reporter interface, you canrequeue secondary analysis for that run after the new sequencing run is complete. Atthat point, secondary analysis starts over from the beginning.For information about using MiSeq Reporter, see MiSeq Reporter Online Help availablefrom the MiSeq Reporter interface.
On-Instrument Analysis68 Part # 15027617 Rev. AUsing MiSeq Reporter Off-InstrumentYou can install another copy of the MiSeq Reporter software on a separate computer.Doing so enables you to perform secondary analysis of run data while the MiSeq isperforming a subsequent sequencing run.Computing RequirementsTo run the MiSeq Reporter software, you will need the following computingrequirements:}64-bit Windows OS (Vista, Windows 7, Windows Server 2008 64-bit)}>8 GB RAM}>2.2 GHz}>1 TB disk spaceInstallation and Licensing}You can download a second copy of the MiSeq Reporter software from the Illuminawebsite.}After downloading, open the software installer (setup.exe), and accept the end-userlicensing agreement (EULA).}No license key is required as this additional copy is free of charge with your MiSeq.
Chapter 6MiSeq System User Guide 69Chapter 6 TroubleshootingTroubleshootingIntroduction 70Software Errors 71
Troubleshooting70 Part # 15027617 Rev. AIntroductionIf an error occurs during the run setup steps, an on-screen message appears withinstructions for correcting the error. Other errors are indicated by the status icon in theupper-right corner of the screen. For more information, see Status Icons on page 20.For more information, visit http://www.illumina.com/support.ilmn or contact IlluminaTechnical Support.
Software ErrorsMiSeq System User Guide 71Software ErrorsThis section provides an overview of possible errors using MCS. For most errors, theaction you need to take appears on the screen and the run is not impacted.Occurs During Error ActionUpon starting thesoftware.Initialization failed. The option to re-initialize appears on thescreen. Select Re-initialize.Pre-Run Check Sample sheet not found. Check the folder location for the samplesheet in Setup Options on the Welcomescreen.If the sample sheet is missing, create oneand copy it to the sample sheet locationsspecified in Setup Options.When you finished, select Restart Check.Pre-Run Check Improperly formattedsample sheet.You might have missing information orsyntax errors in your sample sheet. Correctthe errors listed on the screen, and thenselect Restart Check.Pre-Run Check Fluidics check failed. Select Pump. When fluids have beenpumped, select Restart Check.Pre-Run Check Disk space low. If disk space is low, a message appearsindicating how much disk space is required,along with the Manage Runs button. SelectManage Runs and clear the required spacefrom the instrument computer.Pre-Run Check Empty waste bottle. Open the reagent compartment door, raisethe sipper handle, and empty the wastebottle in an appropriate waste container.Pre-Run Check Network disconnected. Make sure the network cable is plugged intothe instrument.If the network connection is not resolved,reboot the instrument by turning off thepower switch. Wait at least 60 seconds, andthen turn the instrument on using thepower switch.At end of run Data files not copied. Two options appear on the screen: CopyData and Delete Data•Copy Data—Allows you to select anoutput folder and copy files.•Delete Data—Removes temporary filesfrom the instrument computer.
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IndexMiSeq System User Guide 73IndexAactivity indicators 19amplicon sequencing 2, 13analysisdatabases, genomes 67during sequencing 67input requirements 66analysis workflow 30, 35applications 13CCloud 24cluster generation 11componentsflow cell 7flow cell compartment 4, 7optics module 4reagent cartridge 5reagent compartment 4, 51, 59consumablesIllumina supplied 13user-supplied 15consumables, Illumina supplied 13copying files 20customer support 75cycle time 2Ddeleting files 20diagnostic 20disk spacechecking 37low 71documentation 75EExperiment Designer 41Fflow cellcleaning 47loading 49overview 7single-lane 28temperature 27flow cell clamp 7flow cell compartment 4, 7fluidics 27folder locations 21Hhelp, technical 75IiCloud 24iconserrors and warnings 20status alert 20initialization 71input requirements 66instrument setup 20instrument wash 56intensities 27LLive Help 20load flow cell 24load reagents 24loading reagents 51, 59Mmaintenance wash 56manifests 21, 41MiSeq Control Software 19MiSeq Reporter 66additional copy of 68monitoring the run 27, 53Nnetwork connection 71Ooptics module 4Ppausing a run 29post-run wash 57power switch 9PR2 51pre-run check 24preparing DNA 44QQ-score 27Rread length 12-13
Index74 Part # 15027617 Rev. Areagent cartridge 5, 51reagent compartment 4, 59reagentskitted 13loading 51, 59sequencing 51, 59real time analysis 2, 36, 64RFID 2, 25RTA 64results 65RTAcomplete.txt 65run duration 12run folder 36run parameters 41sample sheet 30run setup screens 24RunInfo.xml 65Ssample sheet 30, 41, 65, 71changing 25secondary analysis 66sequencing 11Sequencing Analysis Viewer 28, 53sequencing screen 27, 53setup options 21sipper handle 4site reports 41softwaredisk space checking 37initialization 9instrument setup 20MiSeq Control 19run duration 12sample sheet 30, 41software updates 20status alert icon 20status.xml 65stopping a run 29Ttechnical assistance 75trurning on the instrument 9turning off instrument 61Uuser-supplied consumables 15Vvibration 27Wwashes 21consumables 57maintenance wash 56post-run wash 57recommendations 56water wash 56waste bottle 4workflow 11analysis 35run duration 12
Technical AssistanceMiSeq System User Guide 75Technical AssistanceFor technical assistance, contact Illumina Customer Support.Illumina Website http://www.illumina.comEmail techsupport@illumina.comTable 3 Illumina General Contact InformationRegion Contact Number Region Contact NumberNorth America 1.800.809.4566 Italy 800.874909Austria 0800.296575 Netherlands 0800.0223859Belgium 0800.81102 Norway 800.16836Denmark 80882346 Spain 900.812168Finland 0800.918363 Sweden 020790181France 0800.911850 Switzerland 0800.563118Germany 0800.180.8994 United Kingdom 0800.917.0041Ireland 1.800.812949 Other countries +44.1799.534000Table 4 Illumina Customer Support Telephone NumbersMSDSsMaterial safety data sheets (MSDSs) are available on the Illumina website athttp://www.illumina.com/msds.Product DocumentationIf you require additional product documentation, you can obtain PDFs from theIllumina website if PDFs are available. Go tohttp://www.illumina.com/support/documentation.ilmn. When you click on a link, youwill be asked to log in to iCom. After you log in, you can view or save the PDF. Toregister for an iCom account, please visit https://icom.illumina.com/Account/Register.
Illumina, Inc.9885 Towne Centre DriveSan Diego, CA 92121-1975+1.800.809.ILMN (4566)+1.858.202.4566 (outside North America) techsupport@illumina.comwww.illumina.com

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