illumina MiSeq System User manual

FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Part # 15027617 Rev. H
March 2013
MiSeq®System
User Guide

ii Part # 15027617 Rev. H
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MiSeq System User Guide iii
Revision History
Part # Revision Date Description of Change
15027617 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.
15027617 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.
15027617 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

iv Part # 15027617 Rev. H
Part # Revision Date Description of Change
15027617 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.

Revision History
MiSeq System User Guide v
Part # Revision Date Description of Change
15027617 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)

vi Part # 15027617 Rev. H
Part # Revision Date Description of Change
15027617 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
15027617 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
15027617 A September
2011
Initial release

MiSeq System User Guide vii
Table of Contents
Revision History iii
Table of Contents vii
Chapter 1 Getting Started 1
Introduction 2
Components 5
MiSeq Reagent Kits 10
Kit Contents and Storage Requirements 12
Flow Cell Types 14
Version Compatibility and Requirements 15
User-Supplied Consumables 16
Anti-Virus Software 17
Starting the MiSeq 18
Chapter 2 MiSeq Software 19
Introduction 20
MiSeq Concepts 21
Welcome Screen 22
Perform Wash Screen 26
Manage Files Screen 28
Run Options Screen 30
Manage Instrument Screen 34
Run Setup Screens 38
Sequencing Screen 45
Run Folders 48
Run Duration 52
Required Disk Space 53
Pausing or Stopping a Run 54
Secondary Analysis Options 56
Live Help 59
Chapter 3 Performing a Run 61
Introduction 62
MiSeq Workflow 63
Preparing Reagents 65
Loading Sample Libraries onto Cartridge 67

viii Part # 15027617 Rev. H
Setting Up a Run Using MCS 68
Cleaning the Flow Cell 69
Loading the Flow Cell 71
Loading Reagents 73
Starting the Run 76
Monitoring the Run 77
Chapter 4 Maintenance Procedures 79
Introduction 80
Performing a Post-Run Wash 81
Performing a Maintenance Wash 83
Performing a Standby Wash 86
Shutting Down the Instrument 89
Chapter 5 Troubleshooting 91
Introduction 92
Resolving Run Setup Errors 93
Resolving RFID Read Failure 95
Troubleshooting Flow Rate Error 97
Performing a Volume Test 98
Measuring Expected Wash Volumes 101
Index 103
Technical Assistance 107

Chapter 1
MiSeq System User Guide 1
Chapter 1 Getting Started
Getting Started
Introduction 2
Components 5
MiSeq Reagent Kits 10
Kit Contents and Storage Requirements 12
Flow Cell Types 14
Version Compatibility and Requirements 15
User-Supplied Consumables 16
Anti-Virus Software 17
Starting the MiSeq 18

Getting Started
2Part # 15027617 Rev. H
Introduction
The Illumina MiSeq®System combines proven sequencing by synthesis (SBS) technology
with a revolutionary workflow that enables you to go from DNA to analyzed data in as
little as eight hours. The MiSeq integrates cluster generation, sequencing, and data analysis
on a single instrument.
Features
}Walkaway 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.
}Pre-filled Reagent Cartridge—A specially designed single-use pre-filled 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.
}Interface Controls—The MiSeq Control Software (MCS) interface provides controls to
configure the instrument, set up and monitor runs, and perform maintenance
procedures.
}Convenient 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.
}Innovative Fluidics Architecture—The MiSeq fluidics system enables unmatched
efficiency in chemistry cycle time during sequencing.
}Real Time Analysis (RTA)—Integrated primary 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.
}MiSeq Reporter—Integrated secondary analysis software processes data from primary
analysis to perform alignment and provide information about each sample analyzed.
Additional Resources
The following documentation is available for download from the Illumina website.

Introduction
MiSeq System User Guide 3
Resource Description
MiSeq System Site Prep Guide Provides specifications for laboratory space, electrical
requirements, and environmental considerations.
MiSeq System Safety and
Compliance Guide
Provides information about instrument labeling, compliance
certifications, and safety considerations.
MiSeq Workflow
Quick Reference Cards
Provides a two-page graphical representation of the workflow
for the experienced user. The quick reference cards summarize
sample prep, run setup, and run monitoring, as well as
provide an overview of the analysis performed by MiSeq
Reporter.
Illumina Experiment Manager
User Guide
Provides instructions for creating sample plates and sample
sheets for different workflows and library types. Illumina
recommends that you create your sample sheet during the
sample preparation step.
MiSeq Sample Sheet
Quick Reference Guide
Provides information about sample sheet requirements. The
information in this guide is applicable whether you are
creating a sample sheet manually or using the Illumina
Experiment Manager.
MiSeq Reagent Prep Guide Provides a description of kit contents and instructions for
preparing the reagent cartridge before beginning your
sequencing run.
Preparing DNA Libraries for
Sequencing on the MiSeq
Provides instructions for denaturing and diluting prepared
sample libraries prior to sequencing on the MiSeq, and
preparing a PhiX control. This step applies to most library
types.
Using Custom Primers
on the MiSeq
Provides instructions for preparing and loading custom
primers, and editing the samples sheet for custom primers.
MiSeq ReporterUser Guide Provides a comprehensive overview of analysis procedures,
analysis workflows, and output files generated by MiSeq
Reporter, as well as computing requirements, off-instrument
installation instructions, and troubleshooting information.
MiSeq ReporterOnline Help Provides instructions for using the MiSeq Reporter interface.
BaseSpace Online Help Provides instructions for using BaseSpace and descriptions of
the graphs generated for each analysis workflow.

Getting Started
4Part # 15027617 Rev. H
Visit the MiSeq support page on the Illumina website for access to documentation, software
downloads, and frequently asked questions. To view a comprehensive list of MiSeq
training courses, go to www.illumina.com/training/miseq.

Components
MiSeq System User Guide 5
Components
The MiSeq has the following exterior components:
AFlow Cell Compartment—Contains the flow cell stage that houses the flow cell
throughout the run. The flow cell stage is controlled by motors that move the stage out
of the enclosed optical module for flow cell loading, and returns it when the run begins.
BEnclosed Optics Module—Contains optical components that enable imaging of the flow
cell.
CStatus Bar—Uses three colors to indicate instrument status. Blue indicates the
instrument is processing, orange indicates the instrument needs attention, and green
indicates that the instrument is ready to begin the next run.
DTouch Screen Monitor—Enables on-instrument configuration and run setup using the
software interface.
EExternal USB Ports—Facilitates the transfer of files and data to the instrument computer
from the touch screen monitor.
FReagent Compartment—Holds reagents at proper temperatures, wash solutions, and
the waste bottle. The reagent compartment door is secured by a magnetic latch.
The MiSeq interface guides you through the run setup steps using the touch screen
monitor. To load run components, you need to access the reagent compartment and the
flow cell compartment.

Getting Started
6Part # 15027617 Rev. H
Flow Cell Compartment
AFlow Cell Stage
BFlow Cell Compartment Door
CFlow Cell Latch
DFlow Cell
EFlow Cell Latch Release Button
The flow cell compartment houses the flow cell stage, thermal station, and fluidics
connections to the flow cell. The flow cell stage moves in and out of the optics module and
holds the flow cell, which is secured by the flow cell latch. When the flow cell latch closes,
two pins near the latch 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.

Components
MiSeq System User Guide 7
Flow Cell
AOutlet Port
BImaging Area
CInlet Port
The MiSeq flow cell is a single-use glass-based substrate on which clusters are generated
and the sequencing reaction is performed.
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.
Samples are loaded onto the reagent cartridge prior to beginning the run, and then
automatically transferred to the flow cell after the run begins.
During the sequencing run, the single lane is imaged in small imaging areas called tiles.
All MiSeq flow cells have a single lane, but the number of tiles differ depending on the type
of flow cell you are using. For more information, see Flow Cell Types on page 14.

Getting Started
8Part # 15027617 Rev. H
Reagent Compartment
AReagent Chiller
BSipper Handle (shown in raised position)
CPR2 Bottle
DWaste Bottle
EReagent Cartridge
The reagent compartment contains the reagent chiller, and positions for the wash buffer
(PR2) bottle and the waste bottle.
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 two form-fitted slots, one for the PR2 bottle and one
for 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.
Reagent Cartridge
The MiSeq reagent cartridge is a single-use consumable consisting of foil-sealed reservoirs
pre-filled with clustering and sequencing reagents sufficient for sequencing one flow cell.

Components
MiSeq System User Guide 9
Each reservoir on the cartridge is numbered. Sample libraries are loaded onto the cartridge
in position 17, which is labeled Load Samples. For more information, see Reagent Cartridge
Contents on page 12.

Getting Started
10 Part # 15027617 Rev. H
MiSeq Reagent Kits
To perform a run on the MiSeq, you need one single-use MiSeq Reagent Kit, which is
available in three kit types. Each type of MiSeq reagent kit includes a kit-specific flow cell
type and reagents required for performing one run.
Kit Type Flow Cell Type Available Kit Sizes
MiSeq Reagent Kit v2 Includes a standard (14 tile) flow cell 500 cycles
300 cycles
50 cycles
MiSeq Reagent Micro Kit v2 Includes a micro (4 tile) flow cell 300 cycles
MiSeq Reagent Nano Kit v2 Includes a nano (2 tile) flow cell 500 cycles
300 cycles
The flow cell, PR2 bottle, and reagent cartridge provided in the kit use radio-frequency
identification (RFID) for accurate consumable tracking and compatibility.
You must use the reagent cartridge associated with your flow cell type. If the reagent
cartridge is not compatible, a message appears during the run setup steps. To proceed, you
must load a compatible reagent cartridge.
Reagent Kit Sizes
Reagent kit size is measured by the number of cycles that can be performed with one kit.
Possible sizes are 500 cycles, 300 cycles, and 50 cycles.
Kit Size Number of Cycles
500-Cycle Kit Provides reagents for up to 525 cycles of sequencing, which is sufficient for up
to a 251-cycle paired-end run plus two eight-cycle index reads.
300-Cycle Kit Provides reagents for up to 325 cycles of sequencing, which is sufficient for up
to a 151-cycle paired-end run plus two eight-cycle index reads.
50-Cycle Kit Provides reagents for up to 75 cycles of sequencing, which is sufficient for up
to a 26-cycle paired-end run plus two eight-cycle index reads.

MiSeq Reagent Kits
MiSeq System User Guide 11
MiSeq Reagent Kit v2
Consumable Catalog #
MiSeq Reagent Kit v2, 500 Cycles Catalog # MS-102-2003
MiSeq Reagent Kit v2, 300 Cycles Catalog # MS-102-2002
TG MiSeq Reagent Kit v2, 300 Cycles Catalog # TG-142-1003
MiSeq Reagent Kit v2, 50 Cycles Catalog # MS-102-2001
MiSeq Reagent Micro Kit v2
Consumable Catalog #
MiSeq Reagent Micro Kit v2, 300 Cycles Catalog # MS-103-1002
TGMiSeq Reagent Micro Kit v2, 300 Cycles Catalog # TG-142-1002
MiSeq Reagent Nano Kit v2
Consumable Catalog #
MiSeq Reagent Nano Kit v2, 500 Cycles Catalog # MS-103-1003
MiSeq Reagent Nano Kit v2, 300 Cycles Catalog # MS-103-1001
TG MiSeq Reagent Nano Kit v2, 300 Cycles Catalog # TG-142-1001

Getting Started
12 Part # 15027617 Rev. H
Kit Contents and Storage Requirements
Box1: Store at -15° to -25°C
Quantity Component Storage Description
1Reagent Cartridge -15° to -25°C Single-use pre-filled cartridge
1HT1 -15° to -25°C 5 ml tube, Hybridization Buffer
Box2: Store at 2° to 8°C
Quantity Component Storage Description
1PR2 Bottle 2° to 8°C 500 ml bottle, Incorporation Buffer
1MiSeq Flow Cell 2° to 8°C Single-use PE flow cell
Reagent Cartridge Contents
Figure 1 Reagent Cartridge