illumina MiSeqDx Reference Manual
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illumina MiSeqDx Reference Manual

Sequencing instrument that measures fluorescence signals of labeled nucleotides.
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MiSeqDx ® Instrument
Reference Guide
FOR IN VITRO DIAGNOSTIC USE
ILLUMINA PROPRIETARY
Document # 15038353 v04
March 2018

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Summary of Contents for illumina MiSeqDx

  • Page 1 MiSeqDx ® Instrument Reference Guide FOR IN VITRO DIAGNOSTIC USE ILLUMINA PROPRIETARY Document # 15038353 v04 March 2018...
  • Page 2 The Software is licensed to you under the terms and conditions of the Illumina Sequencing Software 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...

  • Page 3: Revision History

    MiSeqDx is intended to be used with Illumina supplied 2017 IVD assays and reagent kits." Document # 15038353 Changed the name of the guide from the MiSeqDx Reference December Guide to the MiSeqDx Instrument Reference Guide. 2016 Added a section on the Illumina Worklist Manager application.
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  • Page 5: Table Of Contents

    Chapter 1 Getting Started Intended Use Components Start the MiSeqDx Required Consumables Storage and Handling Anti-Virus Software Chapter 2 MiSeqDx Software Introduction Illumina User Management Software Illumina Worklist Manager Software MiSeq Operating Software (MOS) Real-Time Analysis (RTA) MiSeq Reporter Chapter 3 Performing a Run...
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  • Page 7: Chapter 1 Getting Started

      Chapter 1  Getting Started Getting Started Intended Use Components Start the MiSeqDx Required Consumables Storage and Handling Anti-Virus Software MiSeqDx Instrument Reference Guide...

  • Page 8: Intended Use

    Universal Kit 1.0), imaging hardware, and data analysis software. The MiSeqDx Platform is intended for targeted sequencing of human genomic DNA from peripheral whole blood samples. The MiSeqDx Platform is not intended for whole genome or de novo sequencing. Document # 15038353 v04...

  • Page 9: Components

    Reagent compartment—Holds reagents at proper temperatures, wash solutions, and the waste bottle. A magnetic latch secures the reagent compartment door. The MiSeqDx interface guides users through the run setup steps using the touch screen monitor. MiSeqDx Instrument Reference Guide...
  • Page 10 Outlet Port Imaging Area Inlet Port The MiSeqDx 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.
  • Page 11 To the right of the reagent chiller are 2 form-fitted slots, 1 for the MiSeqDx SBS Solution (PR2) bottle and 1 for the waste bottle. The sipper handle locks the bottles in place and lowers the appropriate sipper into each bottle.

  • Page 12: Start The Miseqdx

    60. Wait a minimum of 60 seconds before turning the power switch back to the ON position. If the MiSeqDx is not already on, reach around the right side of the instrument to locate the power switch on the back panel. It is in the lower corner directly above the power cord.

  • Page 13: Required Consumables

    Required Consumables Sequencing Consumables The sequencing consumables required to run the MiSeqDx are provided separately as part of an in vitro diagnostic kit. User-Supplied Consumables Make sure that the following user-supplied consumables are available before beginning a run. Consumable Purpose...

  • Page 14: Anti-Virus Software

    Anti-Virus Software Illumina strongly recommends installation of user-supplied anti-virus software to protect the computer against viruses. To avoid interfering with MiSeqDx operation or losing data, configure the anti-virus software updates as follows: Set for manual scans, not automatic scans. Perform scans only when the instrument is not in use.

  • Page 15: Chapter 2 Miseqdx Software

      Chapter 2  MiSeqDx Software MiSeqDx Software Introduction Illumina User Management Software Illumina Worklist Manager Software MiSeq Operating Software (MOS) Real-Time Analysis (RTA) MiSeq Reporter MiSeqDx Instrument Reference Guide...

  • Page 16: Introduction

    Illumina User Management Software—Is used to grant various access level permissions to users, as appropriate. This software runs off-instrument and the permissions are saved in a database file, which the MiSeqDx is configured to reference. Illumina User Management Software on page MiSeq Operating Software (MOS)—Controls the instrument operation, including...

  • Page 17: Illumina User Management Software

    Windows platform. To start the installation, double-click the Setup.exe file. The installation wizard opens. Starting the Software—To start the software, go to Start | All Programs | Illumina | Illumina User Management Software, or click the desktop icon. The Illumina User Management Software is used off-instrument. Upon opening the application for the first time, create an Admin account and assign a password.
  • Page 18 User Information section. After making changes to user information, select Save before closing the Illumina User Management Software. To associate the database file with the MiSeqDx, go to the Run Options screen, Folder Settings tab in the MiSeq Operating Software (MOS).
  • Page 19 Select a Database section. User Access Levels User permissions are grouped into the following user levels and assigned to each user in the user authentication database file. An Admin level user type is required to change user type assignments. MiSeqDx Instrument Reference Guide...
  • Page 20 Level Permissions Admin Allows access to all software features, the Windows operating system on the instrument computer, user management activities, and data management functionality. Super User Allows mid-level access to software features, the Windows operating system on the instrument computer, and other functions. User Allows access to start, monitor, pause, and stop sequencing runs for in vitro diagnostic kits.

  • Page 21: Illumina Worklist Manager Software

    Illumina analysis software. Select sample sheet options based on the kit you use. Sample sheet creation takes place during library preparation and is described in kit reference guides. During run setup on the MiSeqDx, you select a sample sheet created with IWM software to use with the run.

  • Page 22: Miseq Operating Software (Mos)

    MiSeq Operating Software (MOS) The MiSeq Operating Software (MOS) interface guides users through the steps to load the flow cell and reagents prior to starting the run, and then provides an overview of quality statistics that can be monitored as the run progresses. During the run, MOS operates the flow cell stage, dispenses reagents, controls flow cell temperatures, and captures images of clusters on the flow cell.
  • Page 23 Flow cell compartment door in the closed or open positions Temperature of the reagent chiller in °C Temperature of the flow cell in °C ® Status of BaseSpace connection (not available for in vitro diagnostic kits) MiSeqDx Instrument Reference Guide...
  • Page 24 Perform Wash screen to initiate the wash. The MiSeqDx can be configured to perform a maintenance wash, rather than a post-run wash, between runs. For more information, see...
  • Page 25 Manage Files Screen Use the Manage Files feature to move, upload, or delete files on the instrument computer. The screen is divided into five tabs: Runs, Sample Sheets, Manifests, Genomes, and Recipes. Figure 11 Manage Files Screen MiSeqDx Instrument Reference Guide...
  • Page 26 Delete or Move. The action is applied to all files or folders. Upload Files—If the MiSeqDx is not connected to a network, use this feature to upload sample sheet or manifest files to the instrument computer from a USB drive. Select Upload and browse to the location on a USB drive where the sample sheet resides.
  • Page 27 Replicate Analysis Locally—This setting does not apply to in vitro diagnostic kits. Folder Settings Tab MiSeqDx requires access to various types of files stored in folders on a local network or on the instrument computer. The options on the Folder Settings tab tell the MiSeqDx where those folders are located.
  • Page 28 For more information, see Starting the User Management Software on page Email Notifications Tab MiSeqDx can be configured to send an email notification when primary analysis is complete, when on-instrument secondary analysis is complete, or if a critical MiSeqDx software error occurs.
  • Page 29 Minimize to Windows—Provides quick access to the instrument operating system and any folders on the instrument computer when MOS is running in kiosk mode opposed to Windows mode. Admin or SuperUser user access level is required to use this feature. MiSeqDx Instrument Reference Guide...
  • Page 30 System Settings Screen System Settings are normally configured when the instrument is initially installed and started for the first time. If any settings changes are required due to a network or facility change, use the System Settings feature. Admin user access level is required to use this feature. Figure 16 System Settings Contact the facility administrator to get information about what network settings to enter.
  • Page 31 Manual Update Screen The Manual Update feature is used to update instrument control software and analysis software. This feature is not available for the MiSeqDx. All software updates are performed by Illumina personnel to maintain proper system configuration. Run Setup Screens...
  • Page 32 If the RFID cannot be read, identifying information can be entered manually. However, the software allows only one of the three RFID-labeled components (flow cell, reagent cartridge, MiSeqDx SBS Solution (PR2)) to fail on a run. For more information, see Resolve RFID Read Failure on page Select the help icon in the upper-right corner of the screen.
  • Page 33 Load Reagents Screen The Load Reagents screen has two steps: first, load the MiSeqDx SBS Solution (PR2) bottle and empty waste bottle, and then load the reagent cartridge. When the MiSeqDx SBS Solution (PR2) bottle and reagent cartridge are loaded, the software reads and records the RFID.
  • Page 34 Change Sample Sheet Every run must have a sample sheet. By default, the software will look for a sample sheet file with a name matching the barcode number of the reagent cartridge loaded on the instrument. If a sample sheet cannot be found, a window opens for browsing to the sample sheet.
  • Page 35 For more information, see Resolve Run Setup Errors on page Figure 25 Pre-Run Check Screen When the pre-run check is successful, the Start Run button becomes active. MiSeqDx Instrument Reference Guide...
  • Page 36 Cluster Density (K/mm²)—Shows the number of clusters per square millimeter for the run. Optimally, expect a cluster density of 800K/mm². Clusters Passing Filter (%)—Shows the percentage of clusters passing filter based on the Illumina chastity filter, which measures quality. This data appears only after cycle 25. NOTE The chastity of a base call is the ratio of the intensity of the greatest signal divided by the sum of the two greatest signals.
  • Page 37 Run Folders Each run on the MiSeqDx generates 3 run folders, each with a specific purpose: D:\Illumina\MiSeqTemp—When the run begins, a temporary run folder is written to the local drive of the instrument computer and used as a working area for MOS and RTA.
  • Page 38 Restart Check. Pause or Stop a Run The MiSeqDx is designed to complete a run from beginning to end without user intervention. However, it is possible to pause a run or stop a run from the Sequencing screen.
  • Page 39 Live Help The MiSeqDx must be connected to a network with internet access to enable Live Help. The Live Help feature is an online assistance tool that enables a representative from Illumina Technical Support to view the MiSeqDx screen with your permission, and share control of the instrument.

  • Page 40: Real-Time Analysis (Rta)

    Real-Time Analysis (RTA) The integrated primary analysis software, Real-Time Analysis (RTA), 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 RTA analysis is complete.

  • Page 41: Miseq Reporter

    Sequencing During Analysis The MiSeqDx instrument computing resources are dedicated to either sequencing or analysis. If a new sequencing run is started on the MiSeqDx before secondary analysis of an earlier run is complete, a confirmation dialog box appears. After confirming the sequencing run, secondary analysis stops.
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  • Page 43: Chapter 3 Performing A Run

      Chapter 3  Performing a Run Performing a Run Introduction MiSeqDx Workflow Prepare the Reagent Cartridge Log In and Select Run Type Clean the Flow Cell Load the Flow Cell Loading Reagents Starting the Run Monitoring the Run MiSeqDx Instrument Reference Guide...

  • Page 44: Introduction

    Introduction To perform a run on the MiSeqDx, follow the setup steps described in this chapter. After the run begins, no other user intervention is required. After the sequencing run is complete, perform an instrument wash. Document # 15038353 v04...

  • Page 45: Miseqdx Workflow

    Wash and thoroughly dry the flow cell. Follow the software prompts to load the flow cell. Follow the software prompts to load the MiSeqDx SBS Solution (PR2) bottle, make sure that the waste bottle is empty, load the reagent cartridge, and select a sample sheet.

  • Page 46: Prepare The Reagent Cartridge

    Gently tap the cartridge on the bench to reduce air bubbles in the reagents. NOTE The MiSeqDx sipper tubes go to the bottom of each reservoir to aspirate the reagents, so it is important that the reservoirs are free of air bubbles.
  • Page 47 Check for air bubbles in the reservoir after loading sample. If air bubbles are present, gently tap the cartridge on the bench to release the bubbles. Figure 32 Load Libraries Proceed directly to the run setup steps using the MiSeq Operating Software (MOS) interface. MiSeqDx Instrument Reference Guide...

  • Page 48: Log In And Select Run Type

    Log In and Select Run Type From the Home screen, select Sequence. If the log in screen opens, enter the appropriate user credentials, and then select Next. Select Sequence again after logging in. If the Select Run Type screen appears, select Diagnostic Run. Figure 33 Select Run Type Follow the prompts to load the flow cell and reagents and set up the run (described in...

  • Page 49: Clean The Flow Cell

    Flow Cell Ports and Gasket Clean the flow cell glass with an alcohol wipe. Make sure that the glass is free of streaks, fingerprints, and lint or tissue fibers. Avoid using the alcohol wipe on the flow cell port gasket. MiSeqDx Instrument Reference Guide...
  • Page 50 Figure 37 Dry Flow Cell Dry any excess alcohol with a lint-free lens cleaning tissue. Make sure that the flow cell ports are free of obstructions and that the gasket is well- seated around the flow cell ports. If the gasket appears to be dislodged, gently press it back into place until it sits securely around the flow cell ports.

  • Page 51: Load The Flow Cell

    As the flow cell latch closes, alignment pins position the flow cell. An audible click indicates that the flow cell latch is secure. Figure 40 Close Flow Cell Latch If the software does not identify the flow cell RFID, see Resolve RFID Read Failure on page NOTE MiSeqDx Instrument Reference Guide...
  • Page 52 If the RFID cannot be read, identifying information can be entered manually. However, the software allows only 1 of the 3 RFID-labeled components (flow cell, reagent cartridge, MiSeqDx SBS Solution (PR2)) to fail on an in vitro diagnostics run. For more information, see Resolve RFID Read Failure on page Close the flow cell compartment door.

  • Page 53: Loading Reagents

    Loading Reagents Load MiSeqDx SBS Solution (PR2) and Check the Waste Bottle Remove the bottle of MiSeqDx SBS Solution (PR2) from 2° to 8°C storage. Invert to mix and then remove the lid. Open the reagent compartment door. Raise the sipper handle until it locks into place.
  • Page 54 If the RFID cannot be read, identifying information can be entered manually. However, the software allows only 1 of the 3 RFID-labeled components (flow cell, reagent cartridge, MiSeqDx SBS Solution (PR2)) to fail on an in vitro diagnostics run. For more information, see Resolve RFID Read Failure on page Close the reagent compartment door.

  • Page 55: Starting The Run

    When all items successfully pass the pre-run check, select Start Run. Important Note Before Starting the Run WARNING The MiSeqDx is sensitive to vibration. Touching the instrument after starting a run could adversely affect sequencing results. After selecting Start Run, do not open the flow cell compartment or the reagent compartment doors, or touch the instrument monitor except to pause the run.

  • Page 56: Monitoring The Run

    Monitoring the Run During the run, monitor run progress, intensities, and quality scores that appear on the Sequencing screen. The Sequencing screen is view-only. For more information, see Sequencing Screen on page Figure 44 Sequencing Screen When the run is complete, the Next button appears. Review the results on the Sequencing screen before proceeding.

  • Page 57: Chapter 4 Maintenance Procedures

      Chapter 4  Maintenance Procedures Maintenance Procedures Introduction Perform a Post-Run Wash Perform a Maintenance Wash Perform a Standby Wash Shut Down the Instrument MiSeqDx Instrument Reference Guide...

  • Page 58: Introduction

    Instrument Shutdown Preventive Maintenance Illumina recommends 1 preventive maintenance per calendar year. If you are not under a service contract, please contact your Territory Account Manager or Illumina Technical Support to arrange for a billable preventive maintenance service. Document # 15038353 v04...

  • Page 59: Perform A Post-Run Wash

    Slide the wash tray into the reagent chiller until it stops, and then close the reagent chiller door. Raise the sipper handle in front of the MiSeqDx SBS Solution (PR2) bottle and waste bottle until it locks into place. Remove the MiSeqDx SBS Solution (PR2) bottle and replace it with the wash bottle.
  • Page 60 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. Slowly lower the sipper handle, making sure that the sippers lower into the wash bottle and waste bottle.

  • Page 61: Perform A Maintenance Wash

    Slide the wash tray into the reagent chiller until it stops. Close the reagent chiller door. Raise the sipper handle in front of the MiSeqDx SBS Solution (PR2) bottle and waste bottle until it locks into place, and replace the MiSeqDx SBS Solution (PR2) bottle with the wash bottle.
  • Page 62 Select Next. The first wash begins. Perform Second Wash Prepare fresh wash solution with Tween 20 and laboratory-grade water, as follows: Add 5 ml 100% Tween 20 to 45 ml laboratory-grade water. These volumes result in 10% Tween 20. Add 25 ml 10% Tween 20 to 475 ml laboratory-grade water. These volumes result in a 0.5% Tween 20 wash solution.
  • Page 63 NOTE The sippers remain in the down position, which is normal. Leave the unused wash solution in the wash tray and wash bottle to prevent the sippers from drying out and air from entering the system. MiSeqDx Instrument Reference Guide...

  • Page 64: Perform A Standby Wash

    Slide the wash tray into the reagent chiller until it stops. Close the reagent chiller door. Raise the sipper handle in front of the MiSeqDx SBS Solution (PR2) bottle and waste bottle until it locks into place, and replace the MiSeqDx SBS Solution (PR2) bottle with the wash bottle.
  • Page 65 NOTE Discard the MiSeqDx SBS Solution (PR2) bottle after each run. Do not reuse any remaining MiSeqDx SBS Solution (PR2). Remove the waste bottle and discard the contents appropriately. Return the waste bottle to the reagent compartment. Slowly lower the sipper handle, making sure that the sippers lower into the wash bottle and waste bottle.

  • Page 66: Shut Down The Instrument

    Shut Down the Instrument It is best to leave the instrument on at all times. However, if the instrument must be turned off, use the following procedure to shut down Windows and prepare the fluidics lines. Perform a maintenance wash. For more information, see Procedure on page Remove the waste bottle and discard the contents appropriately.

  • Page 67: Chapter 5 Troubleshooting

      Chapter 5  Troubleshooting Troubleshooting Introduction Resolve Run Setup Errors Resolve RFID Read Failure Prevent Reboots During a Run Troubleshoot Flow Rate Error Perform a Volume Test Resolve MiSeq Reporter Analysis Errors MiSeqDx Instrument Reference Guide...

  • Page 68: Introduction

    Technical Support. For most errors, an on-screen message appears with instructions for correcting the error. For technical questions, visit the MiSeqDx support pages on the Illumina website for access to frequently asked questions, or log in to your MyIllumina account for access to support bulletins.

  • Page 69: Resolve Run Setup Errors

    Aspirate Rate: 2500 • Dispense Rate: 2500 Select Pump. If the error persists, set the volume to pump 500 µl MiSeqDx SBS Solution (PR2) and repeat the process. When fluids have been pumped, select Restart Check. When the pre-run check is successful, the Start Run button becomes active.

  • Page 70: Resolve Rfid Read Failure

    Enter the temporary bypass code using the on-screen keyboard. Select Next. Enter the following information: Barcode number of the MiSeqDx SBS Solution (PR2) bottle, which is located on the MiSeqDx SBS Solution (PR2) bottle label directly below the barcode MiSeqDx SBS Solution (PR2) bottle part number Select Next to proceed to the Load Reagents screen.
  • Page 71 If the RFID fails a second time, select Get Code. Contact Illumina Technical Support to obtain a temporary RFID bypass code. A temporary bypass code expires in 7 days. Enter the temporary bypass code using the on-screen keyboard.

  • Page 72: Prevent Reboots During A Run

    Prevent Reboots During a Run If the MiSeqDx restarts in the middle of a run, it might mean the Windows Update software on the network is configured to automatically install software updates. (This setting should have been turned off during installation.) Contact the local IT department for help to disable automatic updates to the Windows operating system running in the background on the MiSeqDx.

  • Page 73: Troubleshoot Flow Rate Error

    Measure Flow Rate When the pump step is complete, select Restart Check. If the error persists, set the volume to pump 500 µl MiSeqDx SBS Solution (PR2) and repeat the process 1 more time. Contact Illumina Technical Support if the second attempt does not resolve the error.

  • Page 74: Perform A Volume Test

    Perform a Volume Test An obstruction in the fluidics lines can cause poor reagent delivery and affect sequencing results. If an obstruction in the fluidics lines is suspected, perform a volume test. A volume test checks the health of the fluidics system by estimating the volume between 2 bubbles as they pass by the sensors.
  • Page 75 When the volume test is complete, the results appear on the screen. Figure 50 Volume Test Results If the test did not pass, perform a maintenance wash. See Procedure on page When the maintenance wash is complete, repeat the volume test. MiSeqDx Instrument Reference Guide...

  • Page 76: Resolve Miseq Reporter Analysis Errors

    Resolve MiSeq Reporter Analysis Errors For troubleshooting information related to analysis errors, see the MiSeq Reporter Software Reference Guide (document # 15038356). It includes instructions on how to re-queue analysis if the wrong sample sheet is used. Document # 15038353 v04...

  • Page 77: Index

    6 flow rate, troubleshooting 67 password, changing 24 fluidics passwords 11 troubleshooting 67-68 pausing a run 32 washing 55, 58 post-run wash 52-53 folder locations power switch 6 default settings 20 for current run 28 MiSeqDx Instrument Reference Guide...
  • Page 78 50 sequencing screen 30, 50 shutting down the instrument 23, 52, sipper handle 5 software anti-virus 8 disk space checking 31 Illumina User Management Software 11 Illumina Worklist Manager Software 15 initialization 6 instrument setup 23 run duration 31...

  • Page 79: Technical Assistance

    Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.html. Product Documentation Product documentation in PDF is available for download from the Illumina website. Go to support.illumina.com, select a product, then click Documentation & Literature. MiSeqDx Instrument Reference Guide...
  • Page 80 Australian Sponsor Illumina Australia 1 International Court Scoresby, Victoria, 3179 Australia Illumina 5200 Illumina Way San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) Illumina Cambridge Limited +1.858.202.4566 (outside North America) Chesterford Research Park, Little Chesterford techsupport@illumina.com Saffron Walden, CB10 1XL www.illumina.com...

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