illumina NextSeq 500 System Manual
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illumina NextSeq 500 System Manual

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NextSeq 500
System Guide
Document # 15046563 v04
May 2018
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY

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  • Page 1 NextSeq 500 System Guide Document # 15046563 v04 ILLUMINA PROPRIETARY May 2018 For Research Use Only. Not for use in diagnostic procedures.
  • Page 2 NextSeq 500 System 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 Removed the default user name and password required to log on to the Document # 15046563 2018 operating system. Illumina recommends using site-specific credentials. Added information about the Illumina Proactive monitoring service in the Select BaseSpace Configuration section. Updated RTA v2 software references to RTA2. Material # 20006818 March Added section titled Indexing Considerations.
  • Page 4 NextSeq 500 Kit v2. For more information, see NextSeq Custom Primers Guide (document # 15057456) . Updated reagent prep instructions for using the NextSeq 500 Kit v2: Removed manual steps for loading sodium hypochlorite and dual- indexing primers to the reagent cartridge. These reagents are prefilled in the v2 reagent cartridge.
  • Page 5 April 2014 Updated software descriptions to NextSeq Control Software v1.2 and RTA v2.1: • Added recipe name NextSeq Mid for use with the NextSeq 500 Mid Output Kit. • Removed instructions to add NaOCl to the buffer wash cartridge for a manual wash.
  • Page 6 NextSeq 500 System Guide Document Date Description of Change 15046563 B February Updated software descriptions to NextSeq Control Software v1.1: • Added search feature on Run Setup screen to filter list of available 2014 runs. • Added that available recipes include NextSeq High or NextSeq Mid, depending on flow cell type.

  • Page 7: Table Of Contents

    Table of Contents Chapter 1 Overview Introduction Additional Resources Instrument Components Sequencing Consumables Overview Chapter 2 Getting Started Starting the Instrument Customize System Settings Customize Run 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 8 NextSeq 500 System Guide Flow Cell Tiles Output Folder Structure Index Technical Assistance Document # 15046563 v04 viii For Research Use Only. Not for use in diagnostic procedures.

  • Page 9: Chapter 1 Overview

    The Illumina NextSeq 500 system combines the power of high-throughput sequencing with the simplicity of a desktop sequencing instrument. Features High-throughput sequencing—The NextSeq 500 enables sequencing of exomes, whole genomes, and ® ® transcriptomes and supports TruSeq and Nextera libraries.

  • Page 10: Instrument Components

    After loading the flow cell, the imaging compartment door closes automatically and moves components into position. Reagent and Buffer Compartments Setting up a sequencing run on the NextSeq 500 requires access to the reagent compartment and buffer compartment to load run consumables and empty the spent reagents container. Document # 15046563 v04...
  • Page 11 NextSeq Control Software (NCS)—The control software guides you through the steps to set up a sequencing run. Real-Time Analysis (RTA) software—RTA performs image analysis and base calling during the run. The NextSeq 500 uses RTA v2, which includes important architecture and feature differences from earlier Real-Time Analysis on page versions. For more information, see...

  • Page 12: Sequencing Consumables Overview

    NextSeq 500 System Guide Power Button The power button on the front of the NextSeq turns on power to the instrument and instrument computer. The power button performs the following actions depending on the state of instrument power. Power State...
  • Page 13 NextSeq 500 System Guide Each flow cell and reagent cartridge is labeled High or Mid. Always check the label when you prepare consumables for a run. Kit Type Marking on Label High Output Kit Components Mid Output Kit Components Flow Cell Overview Figure 3 Flow Cell Cartridge A Lane pair A—Lanes 1 and 3...
  • Page 14 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. Reserved Reservoirs...
  • Page 15 NextSeq 500 System Guide Position Description 7, 8, and 9 Reserved for optional custom primers Load libraries For information about custom primers, see NextSeq Custom Primers Guide (document # 15057456) . Removable Reservoir in Position #6 The prefilled reagent cartridge includes a denaturation reagent in position 6 that contains formamide. To...

  • Page 16: Chapter 2 Getting Started

    Chapter 2 Getting Started Starting the Instrument Customize System Settings Customize Run Settings User-Supplied Consumables and Equipment Starting the Instrument Turn on the power toggle switch to the I (on) position. Figure 7 Power Switch Located on Back of Instrument Press the power button above the reagent compartment.

  • Page 17: Customize Run Settings

    NextSeq 500 System Guide Select Start-Up Option From the Manage Instrument screen, select System Customization. Select from the following start-up options: Select Kiosk Mode to use the control software interface in full screen. Select Windows Mode to allow access to Windows on the instrument computer. Interaction with the software interface, such as button location, is likely to be altered in this mode.

  • Page 18: User-Supplied Consumables And Equipment

    Purging consumables automatically adds additional time to the workflow. Select Save to save settings and exit the screen. User-Supplied Consumables and Equipment The following consumables and equipment are used on the NextSeq 500. User-Supplied Consumables for Sequencing Runs Consumable Supplier...
  • Page 19 NextSeq 500 System Guide User-Supplied Equipment Item Source Freezer, -25°C to -15°C, frost-free General lab supplier Ice bucket General lab supplier Refrigerator, 2°C to 8°C General lab supplier Document # 15046563 v04 For Research Use Only. Not for use in diagnostic procedures.

  • Page 20: Chapter 3 Sequencing

    Automatic Post-Run Wash Introduction To perform a sequencing run on the NextSeq 500, 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.

  • Page 21: Sequencing Workflow

    NextSeq Control Software Version 2.2 is required to read NextSeq v2.5 RFID. Make sure that you complete all steps in the NextSeq 500 workflow in the specified order. For configurations using Illumina BaseSpace or BaseSpace Onsite: Set up the run on the BaseSpace Prep tab.

  • Page 22: Prepare The Flow Cell

    NextSeq 500 System Guide Thaw in a room temperature water bath until thawed (~60 minutes). Do not submerge the cartridge. Gently tap on the bench to dislodge water from the base, and then dry the base. NOTE [Alternate method] Thaw reagents overnight at 2°C to 8°C. Reagents require a minimum of 18 hours to thaw.

  • Page 23: Prepare Libraries For Sequencing

    NextSeq 500 System Guide Figure 10 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. Prepare Libraries for Sequencing The library volume and loading concentration differ depending on the version of NCS you are running.
  • Page 24 NextSeq 500 System Guide If the instrument is configured for BaseSpace, you are prompted to log in to BaseSpace. If the instrument is configured for standalone mode, the next step is loading the flow cell. Log in to BaseSpace Enter your BaseSpace user name and password.
  • Page 25 NextSeq 500 System Guide Figure 13 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. Wear protective equipment, including eye protection, gloves, and laboratory coat appropriate for risk of exposure.
  • Page 26 NextSeq 500 System Guide Load the Buffer Cartridge Remove the used buffer cartridge from the upper compartment. Slide a new buffer cartridge into the buffer compartment until it stops. An audible click indicates that the cartridge is in position, the buffer cartridge ID appears on the screen, and the sensor is checked.
  • Page 27 NextSeq 500 System Guide Figure 16 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 500 System 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 500 System Guide Select Next. Review Automated Check The software performs an automated 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 30 NextSeq 500 System Guide E Clusters Passing Filter (%)—Shows the percentage of clusters passing filter. See Clusters Passing Filter on page F Estimated Yield (Gb)—Shows the number of bases projected for the run. G Flow cell image—Shows the current process on each lane pair. One lane pair is imaging when the other lane pair is in a chemistry step.

  • Page 31: Automatic Post-Run Wash

    NextSeq 500 System Guide The Sequencing Analysis Viewer is included in the software installed on the instrument computer. You can also install Sequencing Analysis Viewer on another computer linked to the same network as the instrument to monitor run metrics remotely.

  • Page 32: Chapter 4 Maintenance

    Software Updates on page 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 33 NextSeq 500 System Guide Prepare for a Manual Post-Run Wash User-Supplied Consumables Volume and Description • NaOCl 1 ml, diluted to 0.12% Loaded onto the reagent wash cartridge (position #28) • 100% Tween 20 Used to make 125 ml 0.05% Tween 20 wash solution •...

  • Page 34: Software Updates

    NextSeq 500 System Guide Load a Used Flow Cell and the Wash Cartridges If a used flow cell is not present, load a used flow cell. Select Load, and then select Next. Remove the spent reagents container and discard the contents in accordance with applicable standards.

  • Page 35: Shut Down The Instrument

    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.
  • Page 36 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. Document # 15046563 v04 For Research Use Only. Not for use in diagnostic procedures.

  • Page 37: Appendix A Troubleshooting

    Configure System Settings Introduction For technical questions, visit the NextSeq 500 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 38: Resolve Automatic Check 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 39: Spent Reagents Container Is Full

    NextSeq 500 System Guide Spent Reagents Container is Full Always begin a run with an empty spent reagents container. If you begin a run without emptying the spent reagents container, system sensors trigger the software to pause the run when the container is full. System sensors cannot pause a run during clustering, paired-end resynthesis, or the automatic post-run wash.
  • Page 40 NextSeq 500 System Guide After cycle 5—Intensities appear after template registration, which requires the first 5 cycles of sequencing. Although it is safe to end a run after cycle 1, ending after cycle 5 is recommended. Do not end a run during cluster generation.

  • Page 41: Custom Recipes And Recipe Folders

    Mid—Original recipes used with a mid-output kit. Wash—Contains the manual wash recipe. 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 42 NextSeq 500 System Guide Inactive checkboxes on the Select screen indicate tests that require assistance from an Illumina field representative. Perform a System Check From the Manage Instrument screen, select System Check. When prompted to close the control software, select Yes.

  • Page 43: Raid Error Message

    RAID Error Message The NextSeq computer is equipped with 2 hard drives. If a hard drive begins to fail, the system generates a RAID error message and suggests that you contact Illumina Technical Support. Usually, a hard drive replacement is required.
  • Page 44 From the Manage Instrument screen, select System Configuration. Select BaseSpace Configuration. Select from the following options to specify a location where data are transferred for subsequent analysis. Select BaseSpace to send sequencing data to Illumina BaseSpace. [Optional] Select the Output Folder checkbox, select Browse, and navigate to a secondary network location to save BCL files in addition to BaseSpace.
  • Page 45 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 46: Appendix B Real-Time Analysis

    Output Folder Structure Real-Time Analysis Overview The NextSeq 500 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 47: Real-Time Analysis Workflow

    NextSeq 500 System 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). For the latest version of SAV, visit the SAV downloads page on the Illumina website.
  • Page 48 NextSeq 500 System Guide Phasing Correction During the sequencing reaction, each DNA strand in a cluster extends by one base per cycle. Phasing and prephasing occurs when a strand becomes out of phase with the current incorporation cycle. Phasing occurs when a base falls behind.
  • Page 49 NextSeq 500 System Guide Table 1 Base Calls in 2-Channel Sequencing Base Red Channel Green Channel Result 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 50: Sequencing Output Files

    NextSeq 500 System Guide After the Q-score is determined, results are recorded in the base call files. 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 51 NextSeq 500 System Guide Table 2 Flow Cell Tiles High Flow Cell Component Description Output Output Lanes A lane is a physical channel with dedicated input and output ports. Surfaces The flow cell is imaged on 2 surfaces, the top and bottom. The top surface of 1 tile is imaged, then the bottom surface of the same tile is imaged before moving to the next tile.
  • Page 52 NextSeq 500 System Guide Camera Numbering The NextSeq 500 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 53: Output Folder Structure

    NextSeq 500 System Guide Thumbnail Image Naming A thumbnail image for each color channel (red and green) for tiles 1, 6, and 12 are generated from all cameras, top and bottom surfaces, at every cycle during imaging. Thumbnail files are generated in the JPG file format.
  • Page 54 NextSeq 500 System Guide RTAConfiguration.xml RunInfo.xml RunNotes.xml RunParameters.xml Document # 15046563 v04 For Research Use Only. Not for use in diagnostic procedures.

  • Page 55: Index

    3 errors and warnings 3 customer support 50 status 3 customization 9 Illumina Proactive monitoring service 37 cycles in a read 13 imaging compartment 2 Document # 15046563 v04 For Research Use Only. Not for use in diagnostic procedures.
  • Page 56 NextSeq 500 System Guide imaging, 2-channel sequencing 40 indexing considerations 41 instrument Q-scores 41 configuration settings 35 quality tables 41 power button 4 start up 8 instrument maintenance consumables 10 instrument name, customizing 9 RAID error message 35 instrument wash 24...
  • Page 57 NextSeq 500 System Guide spent reagents run metrics 21 container full 31 sequencing 39 disposal 16, 26 sodium hypochlorite 25 standalone configuration 20 spent reagents 16 status alerts 3 standalone mode 20 status bar 2 swath numbering 43 system check 33...

  • Page 58: 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 # 15046563 v04 For Research Use Only. Not for use in diagnostic procedures.
  • Page 59 Document # 15046563 v04 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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