illumina MiSeq System Parts list manual

MiSeq
System Guide
Document # 15027617 v04 Material # 20024228
July 2018
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For Research Use Only. Not for use in diagnostic procedures.

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Document # 15027617 v04 Material # 20024228
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Guide

Revision History
Document # Date Description of Change
Material # 20024228
Document # 15027617 v04
July
2018
Added new run configuration options for Local Run Manager,
Sample Sheets, and Manual sequencing modes.
Replaced references to MiSeq Reporter with Local Run
Manager.
Added Resolve Chiller Temperature Ranges to
Troubleshooting Appendix.
Material # 20024228
Document # 15027617 v03
May
2018
Removed the default user name and password required to
log on to the operating system. Illumina recommends using
site-specific credentials.
Added information about the Illumina Proactive monitoring
service in the Customize System Settings section.
Added note to Reagent Compartment regarding required
chiller temperature range.
Added back MiSeq Reporter and removed references to
Local Run Manager.
Changed the name of the guide from the MiSeq Sequencing
System Guide to the MiSeq System Guide.
Minor text edits.
Material # 20024228
Document # 15027617 v02
January
2018
Added new run configuration options for Local Run Manager,
Sample Sheets, and Manual sequencing modes.
Replaced references to MiSeq Reporter with Local Run
Manager.
Added Resolve Chiller Temperature Ranges to
Troubleshooting Appendix.
Added new instruction for collecting instrument performance
data in the Customize System Settings topic.
Changed the name of the guide from the MiSeq System
Guide to the MiSeq Sequencing System Guide.
Document # 15027617 v04 Material # 20024228
For Research Use Only. Not for use in diagnostic procedures.
iii

Document # Date Description of Change
Material # 20000262
Document # 15027617 v01
September
2015
Changed the name of the guide from the MiSeq System User
Guide to the MiSeq System Guide.
New in MCS v2.6, added BaseSpace Onsite information.
Rearranged guide information to center around 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 Guides, including flow cell types, reagent cartridge
contents, and directions for thawing the reagent cartridge.
Visit the Illumina website for all other MiSeq Reagent Kit
information at
support.illumina.com/sequencing/sequencing_kits/miseq_
reagent_kit.html.
Corrected the expected wash volume of a maintenance
wash from 17.25 ml to 51.75 ml.
Updated the order of workflow steps to first prepare the
reagent cartridge, and then if required, denature and dilute
libraries.
Moved troubleshooting information to Appendix A.
Moved output folder information and flow cell tile information
to Appendix B.
Moved information on managing files to the Maintenance
chapter.
Moved the post-run wash instructions from the Maintenance
chapter to the Sequencing chapter.
The name of the Performing a Run chapter is now
Sequencing.
Changed references from primary analysis to analysis by
RTA software.
Updated the dilution directions for the post-run wash
procedure with a template line wash from 6% NaOCl to 5%
NaOCl.
Added an NaOCl, 5% part number to the user-supplied
consumables list.
Part # 15027617 Rev. O September
2014
Updated the following information:
• New in MCS v2.5, updated post-run wash option to
include a template line wash
• Updated directions for a post-run wash with sodium
hypochlorite to the template line wash
• Added the expected wash volume for a post-run washes
Added VeriSeq PGS workflow information regarding
additional resources, run options, secondary analysis
options, instrument washes, and flow cell cap color.
Part # 15027617 Rev. N June 2014 Added information applicable to the VeriSeq PGS workflow.
Updated run metrics information for cluster generation and
density.
Removed anti-virus software information. See MiSeq System
Site Preparation Guide .
Document # 15027617 v04 Material # 20024228
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MiSeq System Guide

Document # Date Description of Change
Part # 15027617 Rev. M January
2014
Updated to change introduced in MCS v2.4:
Added bundle logs feature for sending troubleshooting files.
Part # 15027617 Rev. L October
2013
Added the reboot of system software as a pre-run step.
Added microcentrifuge tubes to the list of user-supplied
consumables.
Eliminated MiSeq Software as a separate chapter and
distributed chapter contents throughout the guide.
Removed information about folders for custom recipes.
Removed information about recommended cluster density
ranges for MiSeq reagent kits.
Removed details about MiSeq reagent kits and added an
overview of reagent kit features. For detailed information, see
the reagent prep documentation for the kit you are using.
Added content to the trademarks notice.
Part # 15027617 Rev. K August
2013
Corrected formatting errors.
Part # 15027617 Rev. J August
2013
Added run descriptions for MCS v2.3 and MiSeq Reagent Kit
v3.
Updated the following information:
• Reagent kit and version compatibility to include MiSeq
Reagent Kit v3
• Custom Recipes folder description to include a v3
subfolder
• Changed cluster density range for v2; added range for v3
• Output path for image files
Corrected flow cell bar codes for nano flow cells (D) and
micro flow cells (G).
Removed information about MiSeq Reagent Kit, including
contents and flow cell types. For more information, see
MiSeq Reagent Preparation Guide (document # 15044983) .
Part # 15027617 Rev. H March
2013
Added section titled MiSeq Concepts that introduces the
analysis workflow, manifest file, and sample sheet.
Removed information about FASTQ file generation, manifest
file formats, analysis workflow details, and sample sheet
details. For information about these topics, see the MiSeq
Reporter User Guide , part # 15028784, or the MiSeq Sample
Sheet Quick Reference Guide , part # 15028392.
Removed instructions for preparing custom primers. For more
information, see Using Custom Primers on the MiSeq , part #
15041638.
Document # 15027617 v04 Material # 20024228
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MiSeq System Guide

Document # Date Description of Change
Part # 15027617 Rev. G January
2013
Removed instructions for denaturing and diluting DNA
libraries and preparing an Illumina PhiX control. See
Preparing DNA Libraries for Sequencing on the MiSeq , part #
15039740.
Updated instrument wash instructions to add 25 ml 10%
Tween 20 to 475 ml laboratory-grade water, instead of 500
ml laboratory-grade water.
Part # 15027617 Rev. F November
2012
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, updated Perform Wash screen to add a
post-run wash option and command to raise sippers
• Updated version compatibility table to include nano and
micro kit dependencies
• Updated version compatibility information to include new
reagent kits
Part # 15027617 Rev. E October
2012
Updated the following information:
• Corrected PhiX control preparation instructions and
expected cluster density of prepared PhiX control to 1000–
1200K/mm²
• Noted that the procedure for denaturing and diluting
libraries, Preparing Your Libraries , does not apply to
Nextera XT libraries as well as TruSeq Amplicon libraries
• Changed upgrade name from MiSeq Expansion Pack to
MiSeq hardware upgrade
• Add the MiSeq Reporter User Guide to Additional
Resources list.
Document # 15027617 v04 Material # 20024228
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MiSeq System Guide

Document # Date Description of Change
Part # 15027617 Rev. D July 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 features, interface changes, and workflow
changes
• Added catalog number and description of the MiSeq
Reagent Kit v2, 500 Cycles
• Added Version Compatibility and Requirements section
• Added description of MiSeq Expansion Pack, which is
required for 14-tile dual-surface flow cell imaging
• Added description of dual-surface flow cell tile numbering
• Added the PCR Amplicon analysis workflow for Nextera XT
libraries
• Added the use of 10% Tween 20 in wash procedures and
expected wash volumes
• Added the reagent cartridge version to the RFID read
failure procedure
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 maximum recommended NaOH
concentration to 1 mM in final solution
• Noted that a maintenance wash is required to remove the
instrument from standby mode and begin the setup steps
for a subsequent run
• Removed Sample Sheet Parameters section and sample
sheet setup step in the workflow; Illumina recommends
creating the sample sheet prior to sample preparation (See
the MiSeq Sample Sheet Quick Reference Guide , part #
15028392 and the Illumina Experiment Manager User
Guide , part # 15031335).
Document # 15027617 v04 Material # 20024228
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MiSeq System Guide

Document # Date Description of Change
Part # 15027617 Rev. C April 2012 Updated software descriptions to MCS v1.2
Added the following new procedures and sections:
BaseSpace overview, Using Custom Primers, Generating
FASTQ files, Troubleshooting Flow Rate Error, Performing a
Volume Test, Performing a Maintenance Wash, and Idling
the Instrument, which includes a standby wash.
Updated the following information:
• Updated name of Amplicon workflow to Custom Amplicon;
updated name of DenovoAssembly workflow to Assembly;
added GenerateFASTQ workflow
• Added descriptions of run folders and files; updated run
folder naming; added output file size
• Listed genome folder as required for amplicon sequencing
in Sample Sheet Parameters
• Added instructions for diluting NaOH to denature libraries
• Updated Resolving RFID Read Failure to include MiSeq
Self-Service instructions
• Listed files and folders used for troubleshooting run
performance
Part # 15027617 Rev. B December
2011
Updated software descriptions to MCS v1.1
Added information about anti-virus protection
Updated the following information:
• Instructions to resolve RFID failure
• Preparing libraries—Changed to 0.2 N NaOH
• Run folder naming convention
• Required disk space and storage capacity
• Run setup steps—Added more information to Setting Up
the Sample Sheet
• Run setup steps—Added note to dispose of remaining PR2
• Analysis duration—Added when analysis exceeds two
hours
• Analysis Input Requirements—Listed manifest files as
required for TruSeq Custom Amplicon libraries
• Corrected HT1 tube size in MiSeq Reagent Kit Contents
• Changed iCom references to MyIllumina
Part # 15027617 Rev. A September
2011
Initial release
Document # 15027617 v04 Material # 20024228
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MiSeq System Guide

Table of Contents
Chapter 1 Overview 1
Introduction 1
Additional Resources 1
Components 2
MiSeq Concepts 4
System Software 5
Secondary Analysis Options 6
Sequencing Analysis Viewer 8
Required Disk Space 8
MiSeq Reagent Kit Overview 8
Chapter 2 Getting Started 11
Start the MiSeq 11
Customize System Settings 11
Configure Notifications of BaseSpace Updates 12
Set Email Preferences 12
Set Default Folder Locations 12
User-Supplied Consumables 13
Chapter 3 Sequencing 15
Introduction 15
Run Duration 15
MiSeq Workflow 17
Thaw Reagent Cartridge 18
Inspect the Reagent Cartridge 19
Denature and Dilute Libraries 19
Load Sample Libraries 19
Set Up a Run Using MCS 20
Clean the Flow Cell 21
Load the Flow Cell 23
Load Reagents 24
Start the Run 26
Monitor the Run 27
Perform a Post-Run Wash 29
Chapter 4 Maintenance 33
Maintenance Frequency 33
Maintenance Frequency for the VeriSeq PGS Workflow 33
Perform a Maintenance Wash 34
Perform a Standby Wash 36
Manage Files 38
Software Updates 39
Shut Down the Instrument 39
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Appendix A Troubleshooting 41
Introduction 41
Bundle Logs for Troubleshooting 41
Perform a System Check 42
Pause or Stop a Run 42
Raise Reagent Cartridge Sippers Manually 44
Resolve Run Setup Errors 44
Resolve RFID Read Failure 44
Perform a Volume Test 45
Measure Expected Wash Volumes 46
Resolve Reagent Chiller Temperature Errors 46
Resolve Local Run Manager Analysis Errors 46
Configure System Settings 46
Appendix B Output Files and Folders 47
Run Folders 47
MiSeqOutput Folder Contents 47
RTA Folders and Files 49
Index 51
Technical Assistance 55
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MiSeq System Guide

Chapter 1 Overview
Introduction 1
Additional Resources 1
Components 2
MiSeq Concepts 4
System Software 5
Secondary Analysis Options 6
Sequencing Analysis Viewer 8
Required Disk Space 8
MiSeq Reagent Kit Overview 8
Introduction
The Illumina®MiSeq™system combines proven sequencing by synthesis (SBS) technology with a
revolutionary workflow that lets you go from DNA to analyzed data in as few as eight hours. The MiSeq
integrates cluster generation, sequencing, and data analysis on a single instrument.
Features
uWalk away automation—After setting up your run, which includes loading the pre-filled reagent cartridge,
buffer bottle, and flow cell, no additional hands-on time is required.
uPrefilled reagent cartridge—A specially designed single-use prefilled reagent cartridge provides reagents
for cluster generation and sequencing, including paired-end sequencing reagents and indexing reagents.
Integrated radio-frequency identification (RFID) tracking enables accurate consumable tracking.
uInterface controls—The MiSeq Control Software (MCS) interface provides controls to configure the
instrument, set up and monitor runs, and perform maintenance procedures.
uConvenient flow cell loading—A clamping mechanism auto-positions the flow cell as it is loaded onto the
instrument. Integrated radio-frequency identification (RFID) tracking enables accurate consumable
tracking.
uInnovative fluidics architecture—The MiSeq fluidics system enables unmatched efficiency in chemistry
cycle time during sequencing.
uReal-time analysis (RTA)—Integrated analysis software performs real-time on-instrument data analysis
during the sequencing run, which includes image analysis and base calling, and saves valuable
downstream analysis time.
uLocal Run Manager— Integrated secondary analysis software processes data from analysis by RTA to
perform alignment and provides information about each sample analyzed.
Additional Resources
The MiSeq system support pages on the Illumina website provide additional resources. These resources
include software, training, compatible products, and the following documentation. Always check support
pages for the latest versions.
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Resource Description
MiSeq System Site Prep
Guide (document #
15027615)
Provides specifications for laboratory space, electrical requirements, and environmental
considerations.
MiSeq System Safety and
Compliance Guide
(document # 15027616)
Provides information about instrument labeling, compliance certifications, and safety
considerations.
Illumina Experiment
Manager User Guide
(document # 15031335)
Provides instructions for creating sample plates and sample sheets for different workflows
and library types.
BlueFuse Workflow
Manager User Guide
(document #
1000000028842)
Provides instructions for creating sample plates and sample sheets for use with the
VeriSeq PGS workflow.
MiSeq Sample Sheet
Quick Reference Guide
(document # 15028392)
Provides information about adding sample sheet settings to your sample sheet.
MiSeq System Denature
and Dilute Libraries Guide
(document # 15039740)
Provides instructions for denaturing and diluting prepared sample libraries before
sequencing on the MiSeq, and preparing a PhiX control. This step applies to most library
types.
MiSeq Custom Primers
Guide (document #
15041638)
Provides instructions for preparing and loading custom primers, and editing the samples
sheet for custom primers.
Local Run Manager
Software Guide (document
# 1000000002702)
Provides an overview of the Local Run Manager software, instructions for using software
features, and instructions for installing analysis modules on the instrument computer.
BlueFuse Multi Software
Guide (document #
15053620)
Provides a comprehensive overview of analysis procedures, analysis workflows, and files
generated by BlueFuse Multi, as well as computing requirements, and troubleshooting
information. Use this guide with the VeriSeq PGS workflow.
BaseSpace User Guide
(document # 15044182)
Provides instructions for using BaseSpace and descriptions of the graphs generated for
each analysis workflow.
BaseSpace Onsite System
Guide (document #
15049148)
Provides instructions for using the BaseSpace Onsite System.
Components
The MiSeq comprises a touch screen monitor, a status bar, a power button with adjacent USBports, and
three compartments.
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MiSeq System Guide

AFlow cell compartment—Contains the flow cell stage that houses the flow cell throughout the run. Flow cell
stage motors move the stage out of the enclosed optical module for flow cell loading and returns the stage
when the run begins.
BEnclosed optics compartment—Contains optical components that enable imaging of the flow cell.
CStatus bar—Indicates flow cell status as ready to sequence (green), processing (blue), or needs attention
(orange).
DTouch screen monitor—Displays the control software interface for system configuration and run setup.
EExternal USB ports—Facilitates the transfer of files and data to the instrument computer from the touch
screen monitor.
FReagent compartment—Contains reagents at proper temperatures, wash solutions, and a bottle for used
reagents. A magnetic latch secures the reagent compartment door.
The MiSeq interface guides you through the run setup steps using the touch screen monitor. Loading run
components requires 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 fluidics connections to the flow
cell. The flow cell stage holds the flow cell and the flow cell clamp secures and positions the flow cell. When
the flow cell clamp closes, two pins near the clamp hinge auto-position the flow cell.
The thermal station, located beneath the flow cell stage, controls changes in flow cell temperature required
for cluster generation and sequencing.
Figure 1 Flow Cell Compartment
AFlow Cell Stage
BFlow Cell Compartment Door
CFlow Cell Clamp
DFlow Cell
EFlow Cell Clamp Release Button
Reagent Compartment
The reagent compartment contains the reagent chiller, and positions for the wash buffer (PR2) bottle and the
waste bottle. To maintain a consistent temperature, open and close the reagent chiller only when instructed.
NOTE
The required temperature range of the reagent chiller is 2°C to 11°C.
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During the run, the reagent chiller holds a single-use reagent cartridge. During the instrument wash, the
reagent chiller holds the wash tray. The software automatically lowers sippers into each reservoir of the
reagent cartridge at the appropriate time during a run 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 pumped
through the sippers and fluidics lines, and then to the flow cell. Reagent waste is delivered to the waste bottle
throughout the process.
Figure 2 Reagent Compartment Components
AReagent Chiller
BSipper Handle (shown in raised position)
CPR2 Bottle
DWaste Bottle
EReagent Cartridge
MiSeq Concepts
The following concepts and terms are common to the run setup steps on the MiSeq.
Concept Description
Analysis Workflow A secondary analysis procedure is performed by Local Run Manager. The analysis
workflow for each run is specified in the sample sheet or by the selected module.
Manifest The file that specifies a reference genome and targeted reference regions to be used in
the alignment step. For workflows that require 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 Run Manager for use during secondary analysis.
Reference Genome A FASTA format file that contains the genome sequences used during analysis. For most
analysis workflows, the reference genome file is specified in the sample sheet.
Run Folder The folder structure populated by RTA software (MiSeqOutput folder) or the folder
populated by Local Run Manager (MiSeqAnalysis). For more information, see Run Folders
on page 47.
Sample Sheet A comma-separated values file (*.csv) that contains information used to set up and
analyze a sequencing run, including a list of samples and their index sequences.
The sample sheet can be provided during the run setup steps on the MiSeq. After the run
begins, the sample sheet is renamed to SampleSheet.csv and copied to the run folders:
MiSeqTemp, MiSeqOutput, and MiSeqAnalysis.
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For more information on analysis workflows and manifest file formats, see the Local Run Manager Software
Guide (document # 1000000002702) .
For more information on sample sheets, see the MiSeq Sample Sheet Quick Reference Guide (document #
15028392) .
System Software
The instrument software suite includes integrated applications that perform sequencing runs, on-instrument
analysis, and related functions.
uMiSeq Control Software (MCS)—Controls instrument operation. The MiSeq Control Software (MCS)
interface guides you through the steps to load the flow cell and reagents before beginning the run. An
overview of quality statistics appears as the run progresses.
uDuring the run, MCS operates the flow cell stage, dispenses reagents, controls flow cell temperatures,
and captures images of clusters on the flow cell. MCS performs the run according to parameters
specified in the Local Run Manager software.
uReal-time analysis (RTA) software—Performs image analysis and base calling, and assigns a quality score
to each base for each cycle. Images are temporarily stored in the run folder for processing by RTA, and
then automatically deleted when analysis by RTA is complete.
uLocal Run Manager—An on-instrument integrated solution used to create a run, monitor status, analyze
sequencing data, and view results. Local Run Manager also tracks sample information and controls user
permissions. The software runs as a Windows service on the instrument computer and is viewed through
a web browser. For more information see Local Run Manager Software on page 7.
Optional software used off-instrument includes the Sequencing Analysis Viewer (SAV). For more information,
see Sequencing Analysis Viewer on page 8.
Status Icons
A status icon on the control software interface indicates a change in conditions during run setup or during the
run. A number on the icon indicates the number of conditions for a status.
When a run status changes, the icon blinks to alert you. 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 status window by selecting the icons along the top margin of
the window. Selecting an icon toggles the condition to show or hide.
Status
Icon
Status
Name
Description
Status
OK
No change. System is normal.
Attention Important information. Action is recommended.
Warning Warnings do not stop a run. However, some warnings require action before proceeding.
Error Errors usually stop a run and generally require action before proceeding with the run.
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Activity Indicators
An activity indicator icon displays the activity the instrument is currently performing.
Figure 3 Activity Indicators
From left to right, the activity indicators represent the following activities:
uMoving the Y-stage
uMoving the Z-stage
uActivating electronics functionality
uUsing the camera
uPumping through the fluidics system
Sensor Indicators
Sensor indicators, which appear at the base of each interface screen, represent the status of instrument
components.
Figure 4 Sensor Indicators
From left to right, the sensor indicators represent the following components:
uFlow cell compartment door in the closed or open positions
uTemperature of the reagent chiller in °C
uTemperature of the flow cell in °C
uStatus of BaseSpace®connection (not connected shown)
Secondary Analysis Options
MiSeq sequencing data can be analyzed on the instrument computer using Local Run Manager, on a
networked server using BaseSpace™Onsite, or on the cloud using BaseSpace. These applications produce
information about alignment, variants, and contig assemblies for each genome requested and for each
sample of a multisample run. If performing the VeriSeq™PGS workflow, use BlueFuse™Multi software for
analysis.
BaseSpace and BaseSpace Onsite Overview
BaseSpace is the Illumina cloud computing environment. BaseSpace Onsite provides a computing
environment on a dedicated server, complete with run setup tools and analysis options.
Log in to BaseSpace or BaseSpace Onsite when you set up the sequencing run. When using BaseSpace or
BaseSpace Onsite, you have the additional option to store run data locally. For more information, see
Customize System Settings on page 11.
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When you begin your sequencing run, 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.
Figure 5 Connected to BaseSpace Icon
Figure 6 Connected to BaseSpace Onsite Icon
Using BaseSpace, data files are encrypted in transit, decrypted during analysis, and encrypted again when
stored. Using BaseSpace Onsite, data files are encrypted in transit, decrypted during analysis, and can be
optionally encrypted again when stored.
BaseSpace and BaseSpace Onsite automatically disconnect from the MiSeq at the end of the run or as soon
as all RTA analysis files have finished transfer. If the internet connection is interrupted, analysis files continue
to upload after the connection is restored from the point when the interruption occurred.
As soon as the last base call file is uploaded to BaseSpace or BaseSpace Onsite, secondary analysis of your
data begins. The same analysis workflows are supported on BaseSpace and BaseSpace Onsite as with on-
instrument analysis using Local Run Manager.
You can connect to BaseSpace at basespace.illumina.com. Log in using your MyIllumina account login. For
more information on BaseSpace, see the BaseSpace User Guide (document # 15044182) and the
BaseSpace support pages on the Illumina website.
For more information on BaseSpace Onsite, see the BaseSpace Onsite System Guide (document #
15049148) and the BaseSpace OnSite support pages on the Illumina website.
Local Run Manager Software
Local Run Manager software is an on-instrument integrated solution for creating a run, monitoring status,
analyzing data, and viewing results. The software integrates with MCS and processes base calls generated
during initial analysis. Local Run Manager performs secondary analysis automatically upon completion of a
sequencing run.
Local Run Manager is used to record sample information during library preparation and ensures positive
sample tracking throughout the process, producing information about each sample.
In addition, Local Run Manager can control user authentication if enabled, granting various access level
permissions to users. Permissions are saved in a database file, which the MiSeq references. Local Run
Manager can also monitor the sequencing run. For more information, see the Local Run Manager Software
Guide (document # 1000000002702) .
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Sequencing During Analysis
The MiSeq system computing resources are dedicated to either sequencing or analysis.
With Local Run Manager, if a new sequencing run is started on the MiSeq before secondary analysis of an
earlier run is complete, a confirmation dialog box appears. After confirming you want the new sequencing run
to start, secondary analysis of the earlier run is stopped until the new run completes sequencing.
After the new run completes sequencing, secondary analysis of the earlier run automatically starts again from
the beginning.
Sequencing Analysis Viewer
You can monitor your run in greater detail without interfering with the run using the Illumina Sequencing
Analysis Viewer (SAV). Your MiSeq must be networked to view primary analysis results with SAV.
SAV allows you to review metrics during a run as metrics are generated, and later after a run has completed.
Install SAV onto a computer independent of the MiSeq with access to the same network connected to the
instrument. After launching the software, browse to the output folder for your run.
After template generation, SAV provides metrics generated by RTA and organizes the metrics into plots,
graphs, and tables.
NOTE
SAV is universal to Illumina sequencing systems, most of which use an 8-lane flow cell. Some views include
drop-down lists showing lanes 1–8. Because the MiSeq flow cell has a single lane, select All or Lane 1. For
more information, see the Sequencing Analysis Viewer User Guide (document # 15020619) .
Required Disk Space
The integrated instrument computer has approximately 550GB of storage capacity.
Before starting a run, the software checks available disk space. If there is not enough disk space for the run,
a message indicating how much disk space is required appears.
If prompted to make disk space available, move or delete older run folders as appropriate. For more
information, see Manage Files on page 38. After clearing adequate disk space, select Restart Check.
MiSeq Reagent Kit Overview
The MiSeq Reagent Kit is a single-use reagent kit required to perform a sequencing run. It is available in
different types and sizes. Each type of MiSeq Reagent Kit includes a kit-specific flow cell type and all
reagents required for performing a run.
The flow cell, PR2 bottle, and reagent cartridge provided in the kit use radio-frequency identification (RFID)
for accurate consumable tracking and compatibility.
Always use the reagent cartridge associated with your flow cell type. If the reagent cartridge is not
compatible, a message appears during run setup that prompts you to load a compatible reagent cartridge.
For a description of available reagent kits, visit the MiSeq Reagent Kits product page on the Illumina website.
Flow Cell
The MiSeq flow cell is a single-use glass-based substrate where clusters are generated and the sequencing
reaction is performed.
Document # 15027617 v04 Material # 20024228
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Guide

Reagents enter the flow cell through the inlet port, pass through the single-lane imaging area, and then exit
the flow cell through the outlet port. Waste exiting the flow cell is delivered to the waste bottle.
Libraries are loaded onto the reagent cartridge before setting up the run, and then automatically transferred
to the flow cell after the run begins.
Figure 7 MiSeq Flow Cell
AOutlet Port
BImaging Area
CInlet Port
Flow Cell Cap Color
The cap color of the flow cell container indicates the flow cell type:
Flow Cell Flow Cell Cap Color
Standard Flow Cell
PGS Flow Cell
Clear
Micro Flow Cell Green
Nano Flow Cell Yellow
Reagent Cartridge Overview
The MiSeq reagent cartridge is a single-use consumable consisting of foil-sealed reservoirs prefilled with
clustering and sequencing reagents sufficient for sequencing one flow cell.
Each reservoir on the cartridge is numbered. Sample libraries are loaded onto the cartridge in position 17,
which is labeled Load Samples.
WARNING
This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation,
ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and
laboratory coat appropriate for risk of exposure. Handle used reagents as chemical waste and discard in
accordance with applicable regional, national, and local laws and regulations. For additional environmental,
health, and safety information, see the SDS at support.illumina.com/sds.html.
Document # 15027617 v04 Material # 20024228
For Research Use Only. Not for use in diagnostic procedures.
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Reserved Reservoirs
Figure 8 Reagent Cartridge with Numbered Reservoirs
Position Reagent Name Description
8 LDR Denaturation Reagent (contains formamide)
17 Reserved Load Sample (Reserved for sample libraries)
18 Reserved Reserved for custom Read 1 primer [Optional]
19 Reserved Reserved for custom Index Read primer [Optional]
20 Reserved Reserved for custom Read 2 primer [Optional]
Table 1 Reagent Cartridge Reservoirs
NOTE
For more information about using custom primers on the MiSeq reagent cartridge, see the MiSeq Custom
Primers Guide (document # 15041638) .
Document # 15027617 v04 Material # 20024228
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Guide