MiSeq® System

User Guide

FOR RESEARCH USE ONLY

ILLUMINA PROPRIETARY
Part # 15027617 Rev. O
Catalog # SY-410-9001DOC
September 2014
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ii Part # 15027617 Rev. O

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MiSeq System User Guide

iii
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iv Part # 15027617 Rev. O

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MiSeq System User Guide

v
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vi Part # 15027617 Rev. O

 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)

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 Revision History
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 Updated software descriptions to MCS v1.1
2011 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 Initial release
2011

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xi
 Chapter 4 Troubleshooting 73
Introduction 74
Bundle Logs for Troubleshooting 75
Software Settings 77
Pause or Stop a Run 81
Resolve Run Setup Errors 83
Resolve RFID Read Failure 85
Troubleshoot Flow Rate Error 87
Perform a Volume Test 88
Measure Expected Wash Volumes 91

Index 93
Technical Assistance 97

xiv Part # 15027617 Rev. O

Getting Started
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 8 hours. The MiSeq integrates cluster generation, sequencing, and data analysis on
a single instrument.

Features
} Walk 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.
} 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.

2 Part # 15027617 Rev. O

 MiSeq Reagent Kit Overview
MiSeq Reagent Kit Overview
To perform a run on the MiSeq, you need one single-use MiSeq Reagent Kit, which 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 one 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 Illumina website at
support.illumina.com/sequencing/sequencing_kits/miseq_reagent_kit.ilmn.
For information about kit contents and requirements, see the reagent prep documentation
for the kit you are using.

MiSeq System User Guide

9
 Run Folders
Files
The files that are copied to the output and analysis folders include the following:
} SampleSheet.csv—Provides parameters for the run and subsequent analysis. At the
start of the run, the sample sheet is copied to the root folder and renamed
SampleSheet.csv. Copies are written to Data\Intensities and
Data\Intensities\BaseCalls.
} runParameters.xml—Contains a summary of run parameters and information about
run components, such as the RFID of the flow cell and reagents associated with the
run.
} RunInfo.xml—Contains high-level run information, such as the number of reads and
cycles in the sequencing run, and whether a read is indexed.

Folders
The folders that are copied to the output and analysis folders include the following folders
generated during the sequencing run:
} <Run folder name>\Config—Contains configuration files for the run.
} <Run folder name>\Data—Contains subfolders Intensities, BaseCalls, and Alignment.
Data generated from MiSeq Reporter are located in the Alignment subfolder.
} <Run folder name>\Data\RTA Logs—Contains log files that describe each step
performed by RTA for each Read.
} <Run folder name>\Data\Intensities\BaseCalls—Contains subfolders with base call
(*.bcl) files, matrix files, and phasing files. MiSeq Reporter writes FASTQ files to this
folder during secondary analysis. For more information, see the MiSeq Reporter User
Guide (part # 15042295).
} <Run folder name>\Recipe—Contains the recipe used for the run.
} <Run folder name>\Logs—Contains log files that describe every step performed by the
instrument for each cycle.
} <Run folder name>\InterOp—Contains binary files used by Sequencing Analysis
Viewer (SAV) to summarize various primary analysis metrics such as cluster density,
intensities, quality scores, and overall run quality.
All other files and folders created in the temporary run folder are not copied to the output
and analysis folders. They contain temporary files that are not required for analysis or
troubleshooting.

MiSeq System User Guide

29
Performing a Run
NOTE
As the flow cell latch is closed, two alignment pins near the hinge of the flow cell latch
properly align and position the flow cell. An audible click indicates that the flow cell latch
is secure.

Figure 19 Close Flow Cell Latch

5 Check the lower-left corner of the screen to confirm that the flow cell RFID was
successfully read.
NOTE
If the RFID cannot be read, the software prompts you through the steps to obtain a
temporary bypass code and proceed with setting up the run. For more information, see
Resolve RFID Read Failure on page 85.
6 Close the flow cell compartment door.
7 Select Next on the Load Flow Cell screen. The Load Reagents screen opens.

44 Part # 15027617 Rev. O

Performing a Run
6 Slowly lower the sipper handle. Make sure that the sippers lower into the PR2 and
waste bottles.
Figure 21 Lower Sipper Handle

7 Check the lower-left corner of the screen to confirm that the RFID of the PR2 bottle was
read successfully.
NOTE
If the RFID cannot be read, the software prompts you through the steps to obtain a
temporary bypass code and proceed with setting up the run. For more information, see
Resolve RFID Read Failure on page 85.
8 Select Next on the Load Reagents screen.

Load the Reagent Cartridge

NOTE
Do not leave the reagent chiller door open for extended periods of time.
1 Open the reagent chiller door.
2 Hold the reagent cartridge on the end with the Illumina label, and slide the reagent
cartridge into the reagent chiller until the cartridge stops.

46 Part # 15027617 Rev. O

 Loading Reagents
Figure 22 Load Reagent Cartridge

3 Close the reagent chiller door.

4 Check the lower-left corner of the screen to confirm that the RFID of the reagent
cartridge was read successfully.
NOTE
If the RFID cannot be read, the software prompts you through the steps to obtain a
temporary bypass code and proceed with setting up the run. For more information, see
Resolve RFID Read Failure on page 85.
If the reagent cartridge is not compatible with the flow cell, a message appears. Select
Back to load a compatible cartridge, or select Exit to return to the Welcome screen.
5 Close the reagent compartment door.
6 Select Next on the Load Reagents screen. The Review screen opens.

Change Sample Sheet

Every run must have a sample sheet. By default, the software looks for a sample sheet file
with a name matching the barcode number of the reagent cartridge loaded on the
instrument. If a sample sheet is not found, a message appears that prompts you to browse
to the location of the correct sample sheet for your run.
To prevent the software from searching unsuccessfully, use the Change Sample Sheet
command on the Load Reagents screen to direct the software to the appropriate sample
sheet.

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47
 Resolve RFID Read Failure
Resolve RFID Read Failure
If the system cannot read the RFID of a consumable, you can obtain a temporary bypass
code from the Illumina website. A temporary bypass code expires in seven days.
1 Always select Retry before proceeding. If the RFID failed a second time, select Get
Code.
2 From a computer with internet access, go to my.illumina.com and log in to your
MyIllumina account.
3 From the MyIllumina page, click Account. In the Resources column, click MiSeq Self-
Service.
4 On the MiSeq Self-Service page, enter the MiSeq serial number.
5 From the Type of Override Code drop-down list, select RFID Override.
Figure 33 MiSeq Self-Service Page

6 To generate the code, select Get Code.

7 Return to the MCS interface and select Enter Code.
8 Enter the temporary bypass code using the on-screen keyboard, and then select Next.
9 Enter the barcode number of the flow cell, PR2 bottle, or reagent cartridge.

MiSeq System User Guide

85
 idling the instrument 68 reagents
Index

initialization 84 kitted 9
instrument setup 23 loading 45
InterOp folder 29 real-time analysis 2
IP address 77 results 53
run folder 28
L template generation 51
laboratory-grade water guidelines 10 rebooting software 23
Live Help 80 recipes, managing 18
loading reagents reference genome
cartridge 46 file format 13
PR2 45 RFID
PR2 45
M reagent cartridge 46
tracking 2
maintenance wash 58, 65 troubleshooting 85
manifest file RTAcomplete.txt 53
copying to instrument 18 run duration 24
definition 13 run folder
MiSeq Reporter 2 definition 13
credentials 78 run folders
overview 26 contents 28
MiSeq Self-Service 85 managing 18
monitoring the run 50 naming 28
moving files and folders 18 primary analysis files 53
temp, output, analysis 28
N run options 20
network connection 84 run setup screens 17, 39
network settings 77 RunInfo.xml 29, 53
runParameters.xml 29
O S
optics module 5
sample sheet
P changing 47
copying to instrument 18
password 12 definition 13
password, changing 78 in run folder 53
pausing a run 81 not found 84
PGS 40 secondary analysis 26
post-run wash 60 sensor indicators 16
power switch 12 sequencing 35
PR2, loading 45 Sequencing Analysis Viewer 24, 50
sequencing screen 50
R shutting down the instrument 23, 72
read length 24 sipper handle 8
reagent cartridge 8 software
reagent chiller, temperature 16 disk space checking 31
reagent compartment 5, 8 initialization 12

94 Part # 15027617 Rev. O