illumina NextSeq 550Dx Instrument Reference Manual
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illumina NextSeq 550Dx Instrument Reference Manual

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NextSeq 550Dx
Instrument Reference Guide
Document # 1000000009513 v00
November 2017
FOR IN VITRO DIAGNOSTIC USE
ILLUMINA PROPRIETARY

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  • Page 1 NextSeq 550Dx Instrument Reference Guide Document # 1000000009513 v00 ILLUMINA PROPRIETARY November 2017 FOR IN VITRO DIAGNOSTIC USE...
  • Page 2 NextSeq 550Dx Instrument Reference Guide This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose.

  • Page 3: Table Of Contents

    Chapter 1 Overview Sequencing Features Additional Resources Instrument Components Sequencing Consumables Overview User-Supplied Consumables and Equipment Chapter 2 NextSeq 550Dx Software NextSeq 550Dx Software Overview Local Run Manager Overview User Passwords Chapter 3 Getting Started Starting the Instrument Customize System Settings...
  • Page 4 NextSeq 550Dx Instrument Reference Guide Appendix B Troubleshooting Introduction System Check Troubleshooting Files Resolve Automatic Check Errors Spent Reagents Container is Full RAID Error Message Configure System Settings Appendix C Real-Time Analysis Real-Time Analysis Overview Real-Time Analysis Workflow Appendix D Output Files and Folders...

  • Page 5: Chapter 1 Overview

    Provides information about operating the NextSeq 550Dx instrument in research mode. Instrument Reference Guide (document # 1000000041922) Instrument Components The NextSeq 550Dx instrument includes a touch screen monitor, a status bar, and 4 compartments. Document # 1000000009513 v00 FOR IN VITRO DIAGNOSTIC USE...
  • Page 6 Reagent and Buffer Compartments Setting up a sequencing run on the NextSeq 550Dx instrument requires access to the reagent compartment and buffer compartment to load run consumables and empty the spent reagents container. Document # 1000000009513 v00...

  • Page 7: Sequencing Consumables Overview

    90 days. For information on replacing the filter, see Power Button The power button on the front of the NextSeq 550Dx turns on power to the instrument and instrument computer. The power button performs the following actions depending on the state of instrument power.
  • Page 8 NextSeq 550Dx Instrument Reference Guide The flow cell, reagent cartridge, and buffer cartridge use radio-frequency identification (RFID) for accurate consumable tracking and compatibility. NOTE Keep sequencing consumables stored in their boxes until ready for use. Required Storage for Sequencing Consumables...
  • Page 9 NextSeq 550Dx Instrument Reference Guide Reagent Cartridge Overview The reagent cartridge is a single-use consumable with RFID tracking and foil-sealed reservoirs that are prefilled with clustering and sequencing reagents. Figure 4 Reagent Cartridge The reagent cartridge includes a designated reservoir for loading prepared libraries. After the run begins, libraries are transferred automatically from the reservoir to the flow cell.

  • Page 10: User-Supplied Consumables And Equipment

    Figure 6 Buffer Cartridge Library Dilution Buffer Overview The library dilution buffer is in the NextSeq 550Dx Accessory Box. Use the buffer as needed to dilute libraries according to the library preparation instructions to achieve recommended cluster densities. User-Supplied Consumables and Equipment The following consumables and equipment are used for consumables preparation, sequencing, and instrument maintenance.
  • Page 11 NextSeq 550Dx Instrument Reference Guide User-Supplied Consumables for Instrument Maintenance Consumable Supplier Purpose NaOCl, 5% Sigma-Aldrich, catalog # 239305 Washing the instrument using the (sodium hypochlorite) (or laboratory-grade equivalent) manual post-run wash; diluted to 0.12% Tween 20 Sigma-Aldrich, catalog # P7949 Washing the instrument using manual wash options;...
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  • Page 13: Chapter 2 Nextseq 550Dx Software

    For more information, see NextSeq 550Dx Operating Software (NOS)—Controls instrument operation. The software is preinstalled on the NextSeq 550Dx, and runs on-instrument. The NOS performs the run according to the parameters specified in the Local Run Manager software module. Before you start the sequencing run, you select a run that you created with Local Run Manager.
  • Page 14 NextSeq 550Dx Instrument Reference Guide Status Icon Status Name Description Warning A warning has occurred. Warnings do not stop a run or require action before proceeding. Error An error has occurred. Errors require action before proceeding with the run. Service Needed A notification requiring attention has occurred.

  • Page 15: Local Run Manager Overview

    The NextSeq 550Dx system computing resources are dedicated to either sequencing or analysis. If a new sequencing run is needed on the NextSeq 550Dx before secondary analysis of an earlier run is complete, the user can cancel secondary analysis from within Local Run Manager and then start a new sequencing run.
  • Page 16 NextSeq 550Dx Instrument Reference Guide To restart the analysis performed by Local Run Manager, use the Requeue feature on the Local Run Manager interface after the new sequencing run is complete. At that point, secondary analysis starts from the beginning. See...
  • Page 17 NextSeq 550Dx Instrument Reference Guide Local Run Manager Icons The following icons are used in Local Run Manager to perform various tasks. Note that depending on the size of the screen, some icons are condensed under the More Options icon.
  • Page 18 NextSeq 550Dx Instrument Reference Guide Active Runs Page The dashboard opens to show the Active Runs page. Across the top of the Active Runs page, a summary of active runs provides a count of runs in each of the following run status categories. To filter active runs by status, click the number.
  • Page 19 NextSeq 550Dx Instrument Reference Guide Edit a Run From the Active Runs page, click the More Options icon next to the run name that you want to edit. Click Edit. When prompted to confirm the action, click Continue. Edit run parameters as needed.

  • Page 20: User Passwords

    NextSeq 550Dx Instrument Reference Guide Confirm to proceed and restore the run. The run is restored to the state it was in before the run folder was deleted. User Passwords To access the Local Run Manager interface, you need a valid user name and password to log in to the system.
  • Page 21 NextSeq 550Dx Instrument Reference Guide In the Old Password field, enter your old password. In the New Password field, enter a new password. In the Confirm New Password field, reenter your new password. Click Save. Document # 1000000009513 v00 FOR IN VITRO DIAGNOSTIC USE...
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  • Page 23: Chapter 3 Getting Started

    Figure 8 Power Button Located on Front of Instrument Wait until the operating system has finished loading. The NextSeq 550Dx Operating Software (NOS) launches and initializes the system automatically. After the initialization step is complete, the Home screen opens. Enter your Local Run Manager user name and password.

  • Page 24: Customize System Settings

    NextSeq 550Dx Instrument Reference Guide Instrument Mode Indicators The default mode of the NextSeq 550Dx is diagnostic mode. The following on the NOS screen indicate the mode of the instrument. Mode Home Screen Color Bar Status Icon Orientation Diagnostic Mode Welcome to NextSeqDx...

  • Page 25: Reboot And Shut Down Options

    [Optional] Select the Automatically start run after pre-run check checkbox to start sequencing automatically after a successful pre-run check. Select the Send Instrument Performance Data to Illumina checkbox to send information to Illumina Technical Support. Only information for troubleshooting purposes is sent.
  • Page 26 CAUTION Do not relocate the instrument. Moving the instrument improperly can affect the optical alignment and compromise data integrity. If you have to relocate the instrument, contact your Illumina representative. Exit to Windows The Exit to Windows command provides access to the instrument operating system and any folder on the instrument computer.

  • Page 27: Chapter 4 Sequencing

    Automatic Post-Run Wash Introduction To perform a sequencing run on the NextSeq 550Dx instrument, prepare a reagent cartridge and flow cell, and then follow the software prompts to set up and start the run. Cluster generation and sequencing are performed on-instrument. After the run, an instrument wash begins automatically using components already loaded on the instrument.

  • Page 28: Sequencing Workflow

    NextSeq 550Dx Instrument Reference Guide Sequencing Workflow Create Run in the Local Run Manager software module. See the analysis workflow guide for your particular module. Prepare a new reagent cartridge: thaw and inspect. Prepare a new flow cell: bring to room temperature, unwrap, and inspect.

  • Page 29: Prepare The Reagent Cartridge

    For directions on how to prepare sample libraries for sequencing, including library dilution and pooling, see the Instructions for Use section for the applicable library preparation. Optimization of cluster density on the NextSeq 550Dx is required. Document # 1000000009513 v00...

  • Page 30: Load Libraries Onto The Reagent Cartridge

    NextSeq 550Dx Instrument Reference Guide Load Libraries onto the Reagent Cartridge Clean the foil seal covering reservoir #10 labeled Load Library Here using a low-lint tissue. Pierce the seal with a clean 1 ml pipette tip. Load 1.3 ml of prepared libraries into reservoir #10 labeled Load Library Here. Avoid touching the foil seal as you dispense the libraries.
  • Page 31 NextSeq 550Dx Instrument Reference Guide Figure 10 Remove from Foil Package Open the clear plastic clamshell package and remove the flow cell. Figure 11 Remove from Clamshell Package Clean the glass surface of the flow cell with a lint-free alcohol wipe. Dry the glass with a low-lint lab tissue.
  • Page 32 NextSeq 550Dx Instrument Reference Guide Select Next. Empty the Spent Reagents Container Open the buffer compartment door with the latch under the lower-left corner of the door. Remove the spent reagents container and discard the contents in accordance with applicable standards.
  • Page 33 NextSeq 550Dx Instrument Reference Guide Figure 14 Load the Empty Spent Reagents Container Load the Buffer Cartridge Remove the used buffer cartridge from the upper compartment. Some force is required to lift and then pull out the buffer cartridge. Slide a new buffer cartridge into the buffer compartment until it stops.
  • Page 34 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. NOTE To facilitate safe disposal of unused reagent, the reservoir in position 6 is removable. For more...

  • Page 35: Monitor Run Progress

    NextSeq 550Dx Instrument Reference Guide Review Pre-Run Check The software performs an automated pre-run check of the system. During the check, the following indicators appear on the screen: Gray checkmark —The check has not been performed yet. Progress icon—The check is in progress.
  • Page 36 NextSeq 550Dx Instrument Reference Guide completion is shown at the bottom. B Q-Score— Shows the distribution of quality scores (Q-scores). See Quality Scoring on page C Intensity— Shows the value of cluster intensities of the 90 percentile for each tile. Plot colors indicate each base: red is A, green is C, blue is G, and black is T.

  • Page 37: View Run And Sample Data

    NextSeq 550Dx Instrument Reference Guide View Run and Sample Data To view run and sample data, click the run name from the Local Run Manager dashboard. A summary of analysis results is presented on the following 3 tabs: Run Overview...

  • Page 38: Possible Run States

    NextSeq 550Dx Instrument Reference Guide Samples and Results Tab The Samples and Results tab provides a summary of analysis results for each sample listed in the samples table, the location of the analysis folder, and the option to requeue analysis. Information provided on the Samples and Results tab is specific to the analysis module used for the run.

  • Page 39: Automatic Post-Run Wash

    NextSeq 550Dx Instrument Reference Guide You can cancel Local Run Manager analysis, start a different sequencing run, and then return to requeue analysis on the original run later. Stop a Local Run Manager Analysis From the Active Runs page, click More Options next to the run name that you want to stop.
  • Page 40 NextSeq 550Dx Instrument Reference Guide After the Wash After the wash, the sippers remain in the down position to prevent air from entering the system. Leave the cartridges in place until the next run. Unload Consumables On rare occasions it may be necessary to unload all consumables from the instrument. To do so, use the Quick Wash screen to unload the consumables.

  • Page 41: Chapter 5 Maintenance

    Air filter replacement—Regular replacement of the air filter ensures proper air flow through the instrument. Preventive Maintenance Illumina recommends that you schedule a preventive maintenance service each year. If you are not under a service contract, contact your Territory Account Manager or Illumina Technical Support to arrange for a billable preventive maintenance service.
  • Page 42 NextSeq 550Dx Instrument Reference Guide Figure 19 Reagent Wash Cartridge and Buffer Wash Cartridge Prepare for a Manual Post-Run Wash Choose to either prepare for a manual post-run wash as described below, or prepare for a quick wash (next section). If you intend to do a manual post-run wash, skip the quick wash section and continue on...
  • Page 43 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. Slide the empty spent reagents container into the buffer compartment until it stops.

  • Page 44: Replace Air Filter

    NextSeq 550Dx Instrument Reference Guide Replace Air Filter The air filter ensures air flow through the instrument. The software displays a notification to change the air filter every 90 days. When prompted, select Remind in 1 day, or follow the following procedure and select Filter Changed.
  • Page 45 NextSeq 550Dx Instrument Reference Guide Figure 21 Air filter insertion Document # 1000000009513 v00 FOR IN VITRO DIAGNOSTIC USE...
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  • Page 47: Appendix A Administrative Settings And Tasks

    Manage user accounts—Adds users, assigns permissions, and sets passwords. Edit NOS system settings—Allows edits to the NOS through the Manage Instrument icon on the Home screen of the NextSeq 550Dx instrument. Manage Local Run Manager system settings—Sets system security parameters, maintenance intervals, and service accounts.
  • Page 48 NextSeq 550Dx Instrument Reference Guide User Permissions Permission Description Admin Role User Role Edit Local Run Manager System Set security, maintenance, and service Allowed Not allowed Settings accounts settings Exit to Windows Exit NOS and access instrument computer. Allowed Not allowed...

  • Page 49: System Settings

    NextSeq 550Dx Instrument Reference Guide Locate the user name you want to edit, and click the Edit icon. From the Edit User dialog box, click Unlock User. In the New Password field, enter a temporary password. In the Confirm Password field, reenter the temporary password.

  • Page 50: Module Settings

    To specify a database backup location, enter a preferred path to a backup location. To restore a database that has been backed up, contact Illumina technical support. In the Backup Period field, enter the duration in days between each backup.

  • Page 51: Audit Trails

    NextSeq 550Dx Instrument Reference Guide Navigate to the manifest file, select the manifest that you want to add, and click Open. Audit Trails Audit trails record information about specific actions, such as user access, user profile changes, and changes to system, run, or analysis parameters. Each audit trail entry includes the following information: When, represented as date YYYY-MM-DD and time in 24-hour time format.
  • Page 52 NextSeq 550Dx Instrument Reference Guide The description can be a run name, user name, analysis module name, or report name, for example. You can enter any portion of a description. Asterisks (*) are not required. Click Filter. Filter by Affected Item Category...

  • Page 53: Appendix B Troubleshooting

    For run quality or performance problems, contact Illumina Technical Support. See Technical Assistance on page System Check A system check is not required for normal operation or instrument maintenance. However, an Illumina Technical Support representative might ask you to perform a system check for troubleshooting purposes. NOTE If an instrument wash is due, perform the wash before starting a system check.
  • Page 54 NextSeq 550Dx Instrument Reference Guide a If a used flow cell is not already on the instrument, load a used flow cell. b Empty the spent reagents container and return it to the instrument. c Load the buffer wash cartridge containing 120 ml laboratory-grade water in the center reservoir.

  • Page 55: Troubleshooting Files

    RTA Errors To troubleshoot RTA errors, first check the RTA error log, which is stored in the RTALogs folder. This file is not present for successful runs. Include the error log when reporting issues to Illumina Technical Support. Resolve Automatic Check Errors If errors occur during the automatic check, use the following recommended actions to resolve the error.

  • Page 56: Spent Reagents Container Is Full

    NextSeq 550Dx Instrument Reference Guide However, the RFID is locked after the foil seals have been pierced. System Checks Recommended Action Doors Closed Make sure that the compartment doors are closed. Consumables Loaded Consumable sensors do not register. Make sure that each consumable is properly loaded.

  • Page 57: Raid Error Message

    Select Continue. The run resumes automatically. RAID Error Message The NextSeq 550Dx computer is equipped with four hard drives, two for diagnostic mode and two for research mode. If a hard drive begins to fail, the system generates a RAID error message and suggests that you contact Illumina Technical Support.
  • Page 58 NextSeq 550Dx Instrument Reference Guide The instrument computer name is assigned to the instrument computer at the time of manufacture. Any changes to the computer name can affect connectivity and require a network administrator. Connect the instrument computer to a domain or a workgroup as follows.

  • Page 59: Appendix C Real-Time Analysis

    Real-Time Analysis Workflow Real-Time Analysis Overview The NextSeq 550Dx instrument uses an implementation of Real-Time Analysis (RTA) software called RTA2. RTA2 runs on the instrument computer and extracts intensities from images, performs base calling, and assigns a quality score to the base call. RTA2 and the operating software communicate through a web HTTP interface and shared memory files.

  • Page 60: Real-Time Analysis Workflow

    *WarningLog*.tsv lists warnings that occurred during a run. Universal Copy Service The NextSeq 550Dx includes a Universal Copy Service. RTA v2 requests the service to copy files from a source location to a destination location and the service processes copy requests in the order received.
  • Page 61 Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. The NextSeq 550Dx instrument uses 2-channel sequencing, which requires only 2 images to encode the data for 4 DNA bases, 1 from the red channel and 1 from the green channel.
  • Page 62 NextSeq 550Dx Instrument Reference Guide Clusters Passing Filter During the run, RTA2 filters raw data to remove reads that do not meet the data quality threshold. Overlapping and low-quality clusters are removed. For 2-channel analysis, RTA2 uses a population-based system to determine the chastity of a base call.

  • Page 63: Appendix D Output Files And Folders

    Appendix D Output Files and Folders Sequencing Output Files Output Folder Structure Sequencing Output Files File Type File Description, Location, and Name Base call files Each tile analyzed is included in a base call file, aggregated in 1 file for each lane, for each cycle. The aggregated file contains the base call and encoded quality score for every cluster for that lane.
  • Page 64 Figure 26 Swath Numbering Camera Numbering The NextSeq 550Dx instrument uses 6 cameras to image the flow cell. Cameras are numbered 1–6. Cameras 1–3 image lane 1. Cameras 4–6 image lane 3. After lanes 1 and 3 are imaged, the imaging module moves on the X-axis to image lanes 2 and 4.

  • Page 65: Output Folder Structure

    NextSeq 550Dx Instrument Reference Guide Figure 27 Camera and Segment Numbering (High output flow cell shown) Tile Numbering There are 12 tiles in each swath of each camera segment. Tiles are numbered 01–12, regardless of swath number or camera segment, and represented in 2 digits.
  • Page 66 NextSeq 550Dx Instrument Reference Guide L002—Base call files for lane 2, aggregated in 1 file per cycle. L003—Base call files for lane 3, aggregated in 1 file per cycle. L004—Base call files for lane 4, aggregated in 1 file per cycle.

  • Page 67: Index

    Index reagent cartridge 5 sequencing runs 6 administrative settings and tasks 43 wash consumables 37-38 air filter 3, 40 control software 9 analysis create a run 24 output files 59 customer support 67 requeue 34 cycles in a read 23 view results 33 analysis, primary signal purity 58...
  • Page 68 NextSeq 550Dx Instrument Reference Guide maintenance, preventive 37 manage instrument shut down 21-22 icons metrics audit trails 48 base calling 57 errors and warnings 9 cluster density cycles 32 Local Run Manager 13 intensity cycles 32 minimize NOS 10 my account 16...
  • Page 69 NextSeq 550Dx Instrument Reference Guide reagent compartment 2 software reagents configuration settings 53 kitted 3 create a run 24 proper disposal 29 disk space checking 9 Real-Time Analysis software 9 image analysis, base calling 9 phasing 56 initialization 19 results 59...
  • Page 70 NextSeq 550Dx Instrument Reference Guide workflow buffer cartridge 29 flow cell 26 flow cell preparation 25 indexing considerations 58 overview 24 pre-run check 31 reagent cartridge 29 run duration 23, 25 run metrics 31 sequencing 56 sodium hypochlorite 38 spent reagents 28...

  • Page 71: Technical Assistance

    Other countries +44.1799.534000 Safety data sheets (SDSs)—Available on the Illumina website at support.illumina.com/sds.html. Product documentation—Available for download in PDF from the Illumina website. Go to support.illumina.com, select a product, then select Documentation & Literature. Document # 1000000009513 v00 FOR IN VITRO DIAGNOSTIC USE...
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  • Page 73 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 UNITED KINGDOM FOR IN VITRO DIAGNOSTIC USE © 2017 Illumina, Inc. All rights reserved.

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