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

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NextSeq 550Dx Research Mode
Instrument Reference Guide
Document # 1000000041922 v01
March 2018
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY

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  • Page 1 NextSeq 550Dx Research Mode Instrument Reference Guide Document # 1000000041922 v01 ILLUMINA PROPRIETARY March 2018 For Research Use Only. Not for use in diagnostic procedures.
  • Page 2 NextSeq 550Dx Research Mode 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 NextSeq 550Dx Research Mode Instrument Reference Guide Revision History Document Date Description of Change Document # March Added information about the Illumina Proactive monitoring service in 1000000041922 v01 2018 the Customize System Settings section. Document # November Initial release. 1000000041922 v00...

  • Page 4: Table Of Contents

    Table of Contents Chapter 1 Overview About this Guide Introduction Additional Resources Instrument Components Sequencing Consumables Overview Chapter 2 Getting Started Starting the Instrument Customize System Settings User-Supplied Consumables and Equipment Chapter 3 Sequencing Introduction Sequencing Workflow Prepare the Reagent Cartridge Prepare the Flow Cell Prepare Libraries for Sequencing Set Up a Sequencing Run...
  • Page 5 NextSeq 550Dx Research Mode Instrument Reference Guide BeadChip and Scan Errors Custom Recipes and Recipe Folders RAID Error Message Configure System Settings Appendix B Real-Time Analysis Real-Time Analysis Overview Real-Time Analysis Workflow Appendix C Output Files and Folders Sequencing Output Files...

  • Page 6: Chapter 1 Overview

    Chapter 1 Overview About this Guide Introduction Additional Resources Instrument Components Sequencing Consumables Overview About this Guide This instrument reference guide provides instructions on the use of the NextSeq 550Dx instrument in research (RUO) mode. Introduction ® ™ The Illumina NextSeq 550Dx instrument is a single solution that provides a seamless transition between high-throughput sequencing and array scanning.

  • Page 7: Additional Resources

    Illumina website for access to documentation, software downloads, online training, and frequently asked questions. Instrument Components The NextSeq 550Dx instrument includes a touch screen monitor, a status bar, and 4 compartments. Document # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.
  • Page 8 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.
  • Page 9 Real-Time Analysis (RTA) software—For sequencing runs, RTA performs image analysis and base calling during the run. The NextSeq 550Dx instrument uses RTA v2, which includes important Real-Time architecture and feature differences from earlier versions. For more information, see...
  • Page 10 NCS is restarted. 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 11: Sequencing Consumables Overview

    Keep sequencing consumables stored in their boxes until ready for use. If you use a NextSeq 550Dx High Output Reagent Kit for a research mode run, all components must be from the same kit lot. A NextSeq 500/550 Kit cannot be used for a diagnostic mode run.
  • Page 12 NextSeq 550Dx Research Mode Instrument Reference Guide Flow Cell Overview Figure 4 Flow Cell Cartridge A Lane pair A—Lanes 1 and 3 B Lane pair B—Lanes 2 and 4 C Flow cell cartridge frame The flow cell is a glass-based substrate on which clusters are generated and the sequencing reaction is performed.
  • Page 13 NextSeq 550Dx Research Mode Instrument Reference Guide Several reservoirs are reserved for the automatic post-run wash. Wash solution is pumped from the buffer cartridge to the reserved reservoirs, through the system, and then to the spent reagents container. WARNING This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact.
  • Page 14 NextSeq 550Dx Research Mode Instrument Reference Guide Figure 7 Buffer Cartridge Document # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.
  • Page 15 NextSeq 550Dx Research Mode Instrument Reference Guide Document # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.

  • Page 16: Chapter 2 Getting Started

    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. For information on Local Run Manager passwords, see the NextSeq 550Dx Instrument Reference Guide (document # 1000000009513) . Select Login.

  • Page 17: Customize System Settings

    NextSeq 550Dx Research Mode Instrument Reference Guide Use the Reboot to RUO command in NOS to safely shut down the instrument and reboot to research mode. Select Manage Instrument. Select Reboot / Shut Down. Select Reboot to RUO. Wait until the operating system has finished loading.

  • Page 18: User-Supplied Consumables And Equipment

    NextSeq 550Dx Research Mode Instrument Reference Guide Upon instrument initialization When a run is started When certain errors occur When user interaction is required When a run has finished Select Save to save settings and exit the screen. Set Run Setup Options From the Manage Instrument screen, select System Customization.
  • Page 19 NextSeq 550Dx Research Mode 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;...

  • Page 20: Chapter 3 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 21: Sequencing Workflow

    NextSeq 550Dx Research Mode Instrument Reference Guide Sequencing Workflow For configurations using Illumina BaseSpace or BaseSpace Onsite: Set up the run on the BaseSpace Prep tab. See the BaseSpace help (help.basespace.illumina.com) . Prepare a new reagent cartridge: thaw and inspect.

  • Page 22: Prepare The Flow Cell

    NextSeq 550Dx Research Mode Instrument Reference Guide NOTE If more than one cartridge is thawing in the same water bath, allow additional thawing time. Invert the cartridge 5 times to mix reagents. Inspect the bottom of the cartridge to make sure that reagents are thawed and free of precipitates.

  • Page 23: Set Up A Sequencing Run

    NextSeq 550Dx Research Mode Instrument Reference Guide Figure 10 Load Libraries Set Up a Sequencing Run From the Home screen, select Experiment, and then select Sequence. The Sequence command opens the imaging compartment door, releases consumables from a previous run, and opens the series of run setup screens. A short delay is normal.
  • Page 24 NextSeq 550Dx Research Mode Instrument Reference Guide Figure 12 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. NOTE Make sure the glass surface of the flow cell is clean. If necessary, repeat the cleaning step.
  • Page 25 NextSeq 550Dx Research Mode Instrument Reference Guide Figure 14 Remove the Spent Reagents Container NOTE As you remove the container, place your other hand underneath for support. WARNING This set of reagents contains potentially hazardous chemicals. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact.
  • Page 26 NextSeq 550Dx Research Mode Instrument Reference Guide 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 27 NextSeq 550Dx Research Mode Instrument Reference Guide Figure 17 Load Reagent Cartridge Select Load. The software moves the cartridge into position automatically (~30 seconds), the reagent cartridge ID appears on the screen, and the sensors are checked. Select Next. Remove Used Reservoir from Position #6 After you have removed the used reagent cartridge from the instrument, remove the protective rubber cover over the slot next to position #6.
  • Page 28 NextSeq 550Dx Research Mode Instrument Reference Guide Select Next. Confirm run parameters. Run Name—Name of the run as assigned in BaseSpace. Library ID—Name of the pooled libraries as assigned in BaseSpace. Recipe—Name of the recipe, either NextSeq High or NextSeq Mid depending on the reagent cartridge used for the run.

  • Page 29: Monitor Run Progress

    NextSeq 550Dx Research Mode Instrument Reference Guide Purge consumables for this run—Change the setting to purge consumables automatically after the current run. Use run monitoring for this run—Change the setting to use run monitoring in BaseSpace. Select Next. Review Pre-Run Check The software performs an automated pre-run check of the system.
  • Page 30 Run Copy Service The NextSeq 550Dx System Software Suite includes a Run 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. If an exception occurs, the file is requeued for copy based on the number of files in the copy queue.

  • Page 31: Automatic Post-Run Wash

    NextSeq 550Dx Research Mode Instrument Reference Guide Sequencing Analysis Viewer The Sequencing Analysis Viewer software shows sequencing metrics generated during the run. Metrics appear in the form of plots, graphs, and tables based on data generated by RTA and written to InterOp files.

  • Page 32: Chapter 4 Scanning

    Set Up a Scan Monitor Scan Progress Introduction To perform a scan on the NextSeq 550Dx instrument, you need the following run components: A hybridized and stained BeadChip The reusable BeadChip adapter Decode Map (DMAP) files for the BeadChip you are using...

  • Page 33: Scanning Workflow

    NextSeq 550Dx Research Mode Instrument Reference Guide When the NextSeq 550Dx instrument instrument is installed, the Illumina representative downloads these files and specifies the path in the control software. There is no need to change these files except in the case of loss or if a new version is available. For more information, see...

  • Page 34: Load The Beadchip Onto The Adapter

    NextSeq 550Dx Research Mode Instrument Reference Guide Save the DMAP folder to the specified DMAP folder location. Access DMAP Folder by BeadChip Identify BeadChips using 2 of the following options: BeadChip barcode BeadChips box ID Purchase order number Sales order number Locate the DMAP folder that you want to download.

  • Page 35: Set Up A Scan

    NextSeq 550Dx Research Mode Instrument Reference Guide Figure 22 Inspect BeadChip Position A Correct position—BeadChip is flat on adapter when clip is released. B Incorrect position—BeadChip is not flat when clip is released. Set Up a Scan From the Home screen, select Experiment, and then select Scan.
  • Page 36 NextSeq 550Dx Research Mode Instrument Reference Guide Figure 23 Load the BeadChip Adapter Select Load. The door closes automatically, the BeadChip ID appears on the screen, and the sensors are checked. A short delay is normal. If the BeadChip barcode cannot be read, a dialog box appears that allows you to enter the barcode manually.

  • Page 37: Monitor Scan Progress

    NextSeq 550Dx Research Mode Instrument Reference Guide Start the Scan When the automated check is complete, select Start. The scan begins. To configure the system to start the scan automatically after a successful check, see Set Run Setup Options on page Monitor Scan Progress Monitor scan progress using the BeadChip image.
  • Page 38 NextSeq 550Dx Research Mode Instrument Reference Guide The time required to transfer data depends on your network connection. Before beginning a subsequent scan, make sure that data have been written to the output folder. To check, make sure that GTC files are present in the barcode folder.
  • Page 39 NextSeq 550Dx Research Mode Instrument Reference Guide Document # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.

  • Page 40: 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 41 NextSeq 550Dx Research Mode Instrument Reference Guide Figure 25 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).
  • Page 42 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 43: Replace Air Filter

    Replace Air Filter The air filter ensures air flow through the instrument. The NextSeq 550Dx Operating Software in the diagnostic mode of the instrument 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 44: Software Updates

    NextSeq 550Dx Research Mode Instrument Reference Guide Figure 27 Air filter insertion Software Updates Software updates are packaged in a software bundle called the System Suite, which includes the following software: NextSeq Control Software (NCS) NextSeq recipes RTA2 NextSeq Service Software (NSS)

  • Page 45: Reboot And Shut Down Options

    If a firmware update is included, an automatic restart of the system is required after the firmware is updated. Manual Software Update Download the System Suite installer from the Illumina website and save it to a network location. Alternatively, copy the software installation file to a portable USB drive. Select Manage Instrument.
  • Page 46 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 47 NextSeq 550Dx Research Mode Instrument Reference Guide Document # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.

  • Page 48: Appendix A Troubleshooting

    Configure System Settings Introduction For technical questions, visit the NextSeq 550Dx instrument support pages on the Illumina website. The support pages provide access to documentation, downloads, and frequently asked questions. Log in to your MyIllumina account for access to support bulletins.

  • Page 49: Resolve Automatic Check Errors

    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. Troubleshooting Files for Array Scans...

  • Page 50: Spent Reagents Container Is Full

    NextSeq 550Dx Research Mode Instrument Reference Guide Imaging System Recommended Action Imaging Limits Contact Illumina Technical Support. Z Steps-and-Settle Contact Illumina Technical Support. Bit Error Rate Contact Illumina Technical Support. Flow Cell It is possible that the flow cell is not properly seated.

  • Page 51: Rehybridization Workflow

    NextSeq 550Dx Research Mode Instrument Reference Guide When the run pauses, a dialog box opens with options to raise the sippers and empty the full container. Empty Spent Reagents Container Select Raise Sippers. Remove the spent reagents container and discard the contents appropriately.
  • Page 52 NextSeq 550Dx Research Mode Instrument Reference Guide Required Consumables A rehybridization run requires a new NextSeq 550Dx reagent cartridge and buffer cartridge regardless of when the run was stopped. End the Current Run Select End Run. When prompted to confirm the command, select Yes.

  • Page 53: Beadchip And Scan Errors

    NextSeq 550Dx Research Mode Instrument Reference Guide Select Next to proceed to the pre-run check and start the run. BeadChip and Scan Errors Software Cannot Read the BeadChip Barcode When the barcode error dialog box appears, select from the following options: Select Rescan.

  • Page 54: Custom Recipes And Recipe Folders

    Wash—Contains the manual wash recipe. 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 55: Configure System Settings

    NextSeq 550Dx Research Mode Instrument Reference Guide Configure System Settings The system is configured during installation. However, if a change is required or if the system has to be reconfigured, use the system configuration options. Only a Windows administrator account has permission to access system configuration options.
  • Page 56 The name of the setting in the software interface might be different from the name in this guide, depending on the version of NCS in use. With this setting turned on, instrument performance data are sent to Illumina. This data helps Illumina troubleshoot more easily and detect potential failures, enabling proactive maintenance and maximizing instrument uptime.
  • Page 57 NextSeq 550Dx Research Mode Instrument Reference Guide Select BeadChip Scan Configuration. To specify a default DMAP folder location, select Browse and navigate to the preferred folder location on your facility network. NOTE Before each scan, download and copy the DMAP content to this location. DMAP content is required for each BeadChip and is unique to each BeadChip barcode.

  • Page 58: Appendix B 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 control software communicate through a web HTTP interface and shared memory files.

  • Page 59: Real-Time Analysis Workflow

    NextSeq 550Dx Research Mode Instrument Reference Guide RTA v2 provides real-time metrics of run quality stored as InterOp files. InterOp files are a binary output containing tile, cycle, and read-level metrics, and are required to view real-time metrics using the Sequencing Analysis Viewer (SAV).
  • Page 60 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 61 NextSeq 550Dx Research Mode Instrument Reference Guide Table 1 Base Calls in 2-Channel Sequencing Green Base Red Channel Result Channel 1 (on) 1 (on) Clusters that show intensity in both the red and green channels. 1 (on) 0 (off) Clusters that show intensity in the red channel only.
  • Page 62 NextSeq 550Dx Research Mode Instrument Reference Guide Quality scoring calculates a set of predictors for each base call, and then uses the predictor values to look up the Q-score in a quality table. Quality tables are created to provide optimally accurate quality predictions for runs generated by a specific configuration of sequencing platform and version of chemistry.
  • Page 63 NextSeq 550Dx Research Mode Instrument Reference Guide Document # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.

  • Page 64: Appendix C Output Files And Folders

    Appendix C Output Files and Folders Sequencing Output Files Sequencing Output Folder Structure Scanning Output Files Scanning 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.
  • Page 65 Each lane is imaged in 3 swaths. Swaths are numbered 1–3 for high output flow cells. Figure 32 Swath Numbering Camera Numbering The NextSeq 550Dx instrument uses 6 cameras to image the flow cell. Document # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.

  • Page 66: Sequencing Output Folder Structure

    NextSeq 550Dx Research Mode Instrument Reference Guide 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 67: Scanning Output Files

    NextSeq 550Dx Research Mode Instrument Reference Guide BaseCalls L001—Base call files for lane 1, aggregated in 1 file per cycle. 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.

  • Page 68: Scanning Output Folder Structure

    NextSeq 550Dx Research Mode Instrument Reference Guide Scanning Output Folder Structure [Date]_[Instrument Name]_[Scan#]_[Barcode] [Barcode] Config Effective.cfg—Records config settings used during the scan. Focus—Contains image files used to focus the scan. Logs—Contains log files that list each step performed during the scan.
  • Page 69 NextSeq 550Dx Research Mode Instrument Reference Guide Document # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.

  • Page 70: Index

    Index imaging compartment 3 reagent compartment 3 adapter status bar 3 BeadChip loading 30 Configuration 51 BeadChip orientation 29 configuration settings 50 overview 5 consumables 6 advanced loading option 13 buffer cartridge 8 air filter 4, 38 flow cell 7 analysis instrument maintenance 14 output files 59...
  • Page 71 NextSeq 550Dx Research Mode Instrument Reference Guide packaging 17 rehybridization 46 swath number 60 keyboard 12 tile numbering 61 tiles 59 types 1 folder location 23 formamide, position 6 22 laboratory-grade water guidelines 14 lane numbering 60 lane pairs 60...
  • Page 72 NextSeq 550Dx Research Mode Instrument Reference Guide on-instrument 4 run duration 15-16 spent reagents RAID error message 49 container full 46 read length 15-16 disposal 19, 37 reagent cartridge standalone configuration 23 overview 7 status alerts 4 reservoir #28 36...
  • Page 73 NextSeq 550Dx Research Mode Instrument Reference Guide flow cell preparation 17 indexing considerations 56 overview 16, 28 pre-run check 24, 31 reagent cartridge 21 run duration 15-16 run metrics 24 sequencing 54 sodium hypochlorite 36 spent reagents 19 standalone mode 23 Document # 1000000041922 v01 For Research Use Only.

  • Page 74: 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 # 1000000041922 v01 For Research Use Only. Not for use in diagnostic procedures.
  • Page 75 Document # 1000000041922 v01 Illumina 5200 Illumina Way San Diego, California 92122 U.S.A. +1.800.809.ILMN (4566) +1.858.202.4566 (outside North America) techsupport@illumina.com www.illumina.com For Research Use Only. Not for use in diagnostic procedures. © 2018 Illumina, Inc. All rights reserved.

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