illumina MiSeq User Manual
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illumina MiSeq User Manual

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MiSeq
Sequencing System
™
User Guide
FOR RESEARCH USE ONLY
ILLUMINA PROPRIETARY
Part # 15027617 Rev. A DRAFT
July 2011

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  • Page 1 MiSeq Sequencing System ™ User Guide FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Part # 15027617 Rev. A DRAFT July 2011...
  • Page 2 License 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 Software The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein.

  • Page 3: Revision History

    Revision History Part # Revision Date Description of Change Pre-Release Early Access Draft. 15027617 DRAFT July 2011 MiSeq User Guide...
  • Page 4 Part # 15027617 Rev. A DRAFT...

  • Page 5: Table Of Contents

    Table of Contents List of Tables Chapter 1 Getting Started Introduction Components Starting the MiSeq MiSeq Workflow Consumables Chapter 2 MiSeq Control Software Introduction MiSeq Control Software Interface Stopping or Pausing a Run Sample Sheets Analysis Workflow Run Folders Available Disk Space...
  • Page 6 Software Errors Index Technical Assistance Part # 15027617 Rev. A DRAFT...

  • Page 7: List Of Tables

    List of Tables Table 1 User-Supplied Consumables Table 2 Maintenance Wash Duration Table 3 Contents of Analysis Folder Table 4 Illumina General Contact Information Table 5 Illumina Customer Support Telephone Numbers MiSeq User Guide...
  • Page 8 viii Part # 15027617 Rev. A DRAFT...

  • Page 9: Chapter 1 Getting Started

     Chapter 1  Getting Started Getting Started Introduction Components Starting the MiSeq MiSeq Workflow Consumables MiSeq User Guide...

  • Page 10: Introduction

    DNA to analyzed data in as few as eight hours. The MiSeq integrates cluster amplification, sequencing, and data analysis in a single instrument with a foot print of approximately two feet square.
  • Page 11 Audience and Purpose This guide contains information about the MiSeq. It provides an overview of instrument components and software features, and descriptions of real time analysis (RTA). This guide is for laboratory personnel and other individuals responsible for: Operating the instrument.

  • Page 12: Components

    The reagent compartment contains the reagent chiller, and positions for the incorporation wash buffer (PR2) bottle and the waste bottle. The reagent chiller holds a single-use MiSeq reagent cartridge during the run, or a wash cartridge during instrument washes. Depending on the process being performed, the software automatically lowers sippers into the reagent wells of the cartridge at the appropriate time in the process.
  • Page 13 Reagent waste is delivered to the waste bottle throughout the process. Reagent Cartridge The MiSeq reagent cartridge is a single-use consumable prefilled with cluster generation and sequencing reagents. Figure 3 MiSeq Reagent Cartridge The cartridge includes a clearly marked reservoir to load your samples.
  • Page 14 Flow Cell The MiSeq flow cell is a single-lane glass-based substrate in which clusters are generated and the sequencing reaction is performed. The single lane is divided into twelve image areas, called tiles, that are captured by the instrument camera.
  • Page 15 Samples are loaded onto the reagent cartridge and then transferred to the flow cell at the beginning of the cluster generation process. Reagents enter the flow cell through the inlet port and exit the flow cell through the outlet port. Waste exiting the flow cell is returned to the waste bottle. MiSeq User Guide...

  • Page 16: Starting The Miseq

    • User name: sbsuser • Password: sbs123 When the system is ready, the MiSeq Control Software (MCS) launches and begins initializing. Figure 7 MCS Initialization After initialization is complete, the Welcome screen appears. Part # 15027617 Rev. A DRAFT...
  • Page 17 For more information, see Shutting Down the Instrument on page If you need to turn off the instrument, wait a minimum of 60 seconds before turning the power switch back to the ON position. MiSeq User Guide...

  • Page 18: Miseq Workflow

    MiSeq. All reagents for cluster generation, sequencing, and paired-end chemistry, are loaded onto the instrument prior to the start of the run. Setting Up Your Run The following diagram illustrates the workflow for setting up a run on the MiSeq System. [DIAGRAM TBD]...

  • Page 19: Consumables

    Ensure that you have all of the consumables listed in this section before you begin your run. Illumina-Supplied Consumables Consumable Catalog # Storage MiSeq Reagent Kit, 50 Cycles Catalog # XXXXXX -15° to -25°C MiSeq Reagent Kit, 300 Cycles Catalog # XXXXXX -15° to -25°C...
  • Page 20 Part # 15027617 Rev. A DRAFT...

  • Page 21: Chapter 2 Miseq Control Software

     Chapter 2  MiSeq Control Software MiSeq Control Software Introduction MiSeq Control Software Interface Stopping or Pausing a Run Sample Sheets Analysis Workflow Run Folders Available Disk Space MiSeq User Guide...

  • Page 22: Introduction

    Introduction The MiSeq Control Software (MCS) guides you through the run setup steps. After the run begins you can monitor your sequencing run as it progresses and assess quality statistics in real time. MCS has the following features: Easy to use interface that guides you through run setup options, loading of run components, and instrument washes.

  • Page 23: Miseq Control Software Interface

    Suggested Improvements—Use this option to send your suggestions to Illumina. Get Updates—This option appears on the Welcome screen only if a software update is available. Your MiSeq must be connected to a network to enable this option. For more information, see...
  • Page 24 MCS release notes. Get Updates You can update your MiSeq software from the MCS Welcome screen using the Get Updates option. Your system must be connected to a network to download updates. When software updates are available, the Get Updates button appears on the Welcome screen.
  • Page 25 Figure 11 Manage Instrument Screen Setup Options—Provides options for changing default locations of data folders. Manage Runs—Provides controls for moving and deleting files stored on the MiSeq computer. Perform Diagnostic—Provides controls for performing an instrument diagnostic. Setup Options From Setup Options, you can set the default locations for recipes, sample sheets, manifests, and the output folder.
  • Page 26 Manifests are provided when you order your custom assay (CAT). There is no need to modify the manifest. MiSeqOutput—Sets the default location for the output files. Illumina recommends that you change the default output folder location to a network location. For more...
  • Page 27 The reagent cartridge RFID is read by the instrument and the software advances to the next screen automatically. If for some reason the RFID is not read, select the online keyboard icon and manually enter the reagent cartridge ID. MiSeq User Guide...
  • Page 28 Pre-Run Check The software automatically performs a pre-run check to ensure that required run components and conditions are satisfied before allowing the run to begin. If any errors occur during the pre-run check, a message appears on the screen telling you where the error occurred and what action is needed to correct it.
  • Page 29 You can monitor your run in greater detail using the Sequencing Analysis Viewer, a downloadable software application that is installed onto a computer independent of the MiSeq. After launching the software, you can browse to the run folder for the MiSeq run you want to monitor.

  • Page 30: Stopping Or Pausing A Run

    Stopping or Pausing a Run From the Sequencing screen, you can stop a run or pause a run: You might stop a run if the run was set up incorrectly, if the data quality is bad, or if you experience a hardware error. You might pause a run if you suspect that the waste bottle is full or to check the volume in the PR2 bottle.

  • Page 31: Sample Sheets

    Sample Sheet Template A sample sheet template is available for download from the Illumina website. You can open and edit the sample sheet in Excel or in Notepad. Your sample sheet must be named with the reagent ID for your run using the following format: <ReagentID>.csv.

  • Page 32: Analysis Workflow

    Analysis Workflow The analysis workflow is a procedure performed by MiSeq Reporter, the secondary analysis software on the MiSeq. Secondary analysis begins after the Real Time Analysis (RTA) software has completed base call analysis. For more information, see Instrument Analysis on page The MiSeq supports six analysis workflows.

  • Page 33: Run Folders

    Run Folders Each run on the MiSeq generates a run folder. Real time analysis (RTA), integrated with MCS, populates the run folder with data files and log files used for secondary analysis. Run Folder Name and Path By default, the folder is named in the following format: YYMMDD_<InstrumentNumber>_<Run Number>_<Flow Cell ID>...

  • Page 34: Available Disk Space

    Available Disk Space The software checks available disk space during a run. If disk space drops below the safe threshold, the software pauses the run and a message alerts you. If you get this message, you need to make disk space available to continue the run. When sufficient disk space becomes available, the run automatically resumes.

  • Page 35: Chapter 3 Performing A Run

     Chapter 3  Performing a Run Performing a Run Introduction Preparing the MiSeq Reagent Cartridge Setting Up a Run Monitoring the Run MiSeq User Guide...

  • Page 36: Introduction

    Introduction To perform a sequencing run on the MiSeq, you first prepare the reagent cartridge and load your samples onto the cartridge, and then set up the run using the MCS interface. Run setup steps include entering run parameters, loading a new flow cell, and loading reagents.

  • Page 37: Preparing The Miseq Reagent Cartridge

    For more information, see the MSDS for this kit, at http://www.illumina.com/msds. Loading Samples When the reagent cartridge is thawed and ready for use on the MiSeq, you are ready to load your samples. You can load a single library or a pool of indexed libraries.
  • Page 38 If you are loading amplicon libraries, pipette 500 µl into the reservoir. Proceed directly to the run setup steps using the MCS interface. Part # 15027617 Rev. A DRAFT...

  • Page 39: Setting Up A Run

    Setting Up a Run To set up a run on the MiSeq, you first need to edit the sample sheet with parameters for your run, and then copy the sample sheet onto the instrument computer using one of the USB ports.
  • Page 40 Remove a new flow cell from the flow cell tube, and carefully dry the flow cell using lint-free tissue. Hold the flow cell by the edges of the flow cell cartridge near the Illumina label. Make sure the Illumina label is facing upward.
  • Page 41 Close Flow Cell Latch NOTE The MiSeq System reads the RFID of the flow cell. If the system is unable to register the RFID of the flow cell, you can manually enter the barcode ID printed on the flow cell using the online keyboard. Select the keyboard icon in the lower right corner of the screen, enter the barcode ID, and select Done to return to the Load Flow Cell screen.
  • Page 42 Load Reagent Cartridge NOTE The MiSeq System reads the RFID of the reagent cartridge. If the system is unable to register the RFID of the reagent cartridge, you can manually enter the barcode ID using the online keyboard. Select the keyboard icon in the lower right corner of the screen, enter the barcode ID, and select Done to return to the Load Reagents screen.
  • Page 43 When all items successfully pass the pre-run check, select Start Run. WARNING The MiSeq is sensitive to vibration. Touching the instrument after starting your run will adversely impact sequencing results. During run operation, do not touch the instrument, the touch screen monitor, or either of the compartment doors unless you plan to stop the run.

  • Page 44: Monitoring The Run

    Avoid bumping the instrument of tapping on the touch screen while the run is in progress. NOTE The MiSeq is sensitive to vibration. Vibrations occurring during a run will adversely impact sequencing results. Do not touch the instrument during normal run operations unless you plan to stop or pause your run.

  • Page 45: Chapter 4 Post-Run Procedures

     Chapter 4  Post-Run Procedures Post-Run Procedures Introduction Performing a Post-Run Wash Performing a Maintenance Wash Unloading Run Components Shutting Down the Instrument MiSeq User Guide...

  • Page 46: Introduction

    Introduction Perform an instrument wash following the completion of a sequencing run. It is important to regularly wash the fluidics lines to ensure continued performance. Two types of instrument washes are initiated from the MCS Welcome screen. Each wash has a specific purpose: Post-Run Wash—The post-run wash flushes the fluidics system with PW1 using the preloaded wash cartridge.

  • Page 47: Performing A Post-Run Wash

    Performing a Post-Run Wash Always perform a post-run wash after completing a sequencing run. If you did not perform a wash after the last run, a reminder appears on the MiSeq software interface. Illumina-Supplied Consumables MiSeq Wash Cartridge User-Supplied Consumables...

  • Page 48: Performing A Maintenance Wash

    Open the reagent compartment door and remove any run components left in the reagent cartridge from a previous run. Hold the PW1 wash cartridge on the end with the Illumina label, and slide the wash cartridge into the reagent chiller until the cartridge stops.
  • Page 49 Final Water Wash Open the reagent compartment door and remove the NaOH wash cartridge. Hold a fresh PW1 wash cartridge on the end with the Illumina label, and slide the wash cartridge into the reagent chiller until the cartridge stops.

  • Page 50: Unloading Run Components

    Flow Cell Stage [INSERT IMAGE] Hold the used flow cell by the edges of the flow cell cartridge near the Illumina label, and remove it from the flow cell stage. Clean the flow cell holder using an alcohol wipe or a lint-free tissue moistened with ethanol or isopropanol.

  • Page 51: Shutting Down The Instrument

    Press down on the flow cell clamp until it locks into place. Close the flow cell compartment door. Toggle the power switch to turn off the instrument. When you are ready to restart the instrument, perform an instrument wash prior to your next sequencing run. MiSeq User Guide...
  • Page 52 Part # 15027617 Rev. A DRAFT...

  • Page 53: Chapter 5 On-Instrument Analysis

     Chapter 5  On-Instrument Analysis On-Instrument Analysis Introduction Primary Analysis Results Secondary Analysis Using MiSeq Reporter MiSeq User Guide...
  • Page 54 Introduction The MiSeq Sequencing Control Software (MCS) performs image analysis and base calling using integrated real time analysis (RTA). Analysis is performed during the sequencing run, which saves downstream analysis time. Real time analysis (RTA) runs automatically on the instrument.

  • Page 55: Table 3 Contents Of Analysis Folder

    The following table describes the folders and files generated by Real Time Analysis (RTA) during primary analysis. The majority of these files are used for secondary analysis by the MiSeq Reporter software. Other files are used for troubleshooting, if necessary.
  • Page 56 links to more detailed information in the form of graphs, such as the IVC plots and All Intensity Plots. Part # 15027617 Rev. A DRAFT...
  • Page 57 From the MiSeq Reporter interface, you can requeue runs for secondary analysis. You might need to do this if a subsequent sequencing run is started on the MiSeq before secondary analysis is complete. When this occurs, secondary analysis stops and you must requeue analysis after the subsequent sequencing run is complete.
  • Page 58 Part # 15027617 Rev. A DRAFT...

  • Page 59: Chapter 6 Troubleshooting

     Chapter 6  Troubleshooting Troubleshooting Introduction Software Errors MiSeq User Guide...

  • Page 60: Introduction

    Other errors are indicated by the status icon in the upper-right corner of the screen. For more information, see Status Icons on page For more information, visit http://www.illumina.com/support.ilmn or contact Illumina Technical Support. Part # 15027617 Rev. A DRAFT...
  • Page 61 Check the folder location for the sample sheet in Setup Options on the Welcome screen. If the sample sheet is missing, create one and copy it to the MiSeq. When you finished, select Restart Check. Pre-Run Check Improperly formatted You might have missing information or sample sheet.
  • Page 62 Part # 15027617 Rev. A DRAFT...
  • Page 63 23-24 loading reagents 33, 42 cluster generation maintenance wash 38, 40 components manifests flow cell MiSeq Control Software flow cell compartment 4, 6 MiSeq Reporter optics module monitoring the run 20, 36 reagent cartridge reagent compartment 4, 33, 42...
  • Page 64 21, 36 sequencing screen 20, 36 setup options sipper handle software disk space checking initialization instrument setup MiSeq Control run duration sample sheet 23, 31 software updates status alert icon status.xml stopping a run technical assistance trurning on the instrument...

  • Page 65: Table 4 Illumina General Contact Information

    1.800.812949 Other countries +44.1799.534000 MSDSs Material safety data sheets (MSDSs) are available on the Illumina website at http://www.illumina.com/msds. Product Documentation If you require additional product documentation, you can obtain PDFs from the Illumina website if PDFs are available. Go to http://www.illumina.com/support/documentation.ilmn.
  • Page 66 Illumina, Inc. 9885 Towne Centre Drive San Diego, CA 92121-1975 +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) techsupport@illumina.com www.illumina.com...

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