illumina iSeq 100 System Manual
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illumina iSeq 100 System Manual

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iSeq 100
Sequencing System Guide
Document # 1000000036024 v05
March 2019
For Research Use Only. Not for use in diagnostic procedures.
ILLUMINA PROPRIETARY

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  • Page 1 100 Sequencing System Guide Document # 1000000036024 v05 ILLUMINA PROPRIETARY March 2019 For Research Use Only. Not for use in diagnostic procedures.
  • Page 2 100 Sequencing 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 • Indicated that the software automatically renames sample sheets to SampleSheet.csv. Added links to the following pages: • The iSeq 100 System Sample Sheet Template for Manual Mode. • The bcl2fast Conversion Software support pages. Added volumes of 1 nM 100% PhiX and AmpliSeq Library PLUS for Illumina libraries to prepare.
  • Page 4 • Increased volume of 1 nM PhiX for a spike-in to 50 µl. Updated Illumina catalog numbers for: • iSeq 100 Spare Drip Tray Pad to 20023927. • iSeq 100 Spare Air Filter to 20023928. Updated pipette and pipette tip recommendations.
  • Page 5 100 Sequencing System Guide Document Date Description of Change Document # Added the following miscellaneous instructions: • Performing validation runs. 1000000036024 v01 2018 • Creating a sample sheet when sequencing in Manual mode. • Minimizing the control software to access other applications.

  • Page 6: Table Of Contents

    Table of Contents Chapter 1 Overview Introduction Additional Resources Instrument Components iSeq 100 i1 Reagent Chapter 2 Getting Started First Time Setup Minimize the Control Software Run Settings Instrument Customization Network Setup User-Supplied Consumables and Equipment Chapter 3 Sequencing Introduction...
  • Page 7 100 Sequencing System Guide Exchanging the iSeq 100 System Receive a Replacement System Prepare the Original System for Return Return the Original System Index Technical Assistance Document # 1000000036024 v05 For Research Use Only. Not for use in diagnostic procedures.

  • Page 8: Chapter 1 Overview

    Features Accessibility and reliability—The iSeq 100 System has a small footprint and is easy to install and use. Fluidics and imaging components are built into the consumable, simplifying instrument maintenance. Single-step consumable loading—A single-use cartridge is prefilled with all reagents required for a run.

  • Page 9: Additional Resources

    100 Sequencing System Guide Figure 1 Sample to Analysis Workflow Additional Resources iSeq 100 Sequencing System support pages on the Illumina website provide additional system resources. These resources include software, training, compatible products, and the following documentation. Always check support pages for the latest versions.

  • Page 10: Instrument Components

    100 Sequencing System Guide Instrument Components iSeq 100 Sequencing System comprises a power button, monitor, status bar, consumables compartment, and drip tray. Figure 2 External System Components A Power button—Controls instrument power and indicates whether the system is on (glows), off (dark), or off but with AC power (pulses).
  • Page 11 Do not place objects on the open door, which is not designed for use as a shelf. Reusable Test Flow Cell and Cartridge The instrument ships with the iSeq 100 Reusable Test Flow Cell and iSeq 100 Reusable Test Cartridge to use for system checks. Document # 1000000036024 v05...
  • Page 12 A Reusable test flow cell B Reusable test cartridge The reusable test components look similar to the sequencing components provided in iSeq 100 i1 Reagent, and the loading orientations are the same. However, the test cartridge does not have a library reservoir and neither test component has the chemistry needed for a run.
  • Page 13 100 Sequencing System Guide System Information The control software menu has an About section where you can view Illumina contact information and the following system information: Serial number Computer name and IP address Recipe fragment version Run count Notifications and Alerts An icon appears next to the instrument name to indicate notifications.

  • Page 14: Iseq 100 I1 Reagent

    For more information on analysis status, check the Local Run Manager software. iSeq 100 i1 Reagent Performing a run on the iSeq 100 System requires one single-use iSeq 100 i1 Reagent kit. The kit is available in one size (300-cycle) and two bundles: Single—Includes consumables for one run.
  • Page 15 100 Sequencing System Guide Flow Cell The iSeq 100 i1 Flow Cell is a patterned, single-lane flow cell built over a complementary metal oxide semiconductor (CMOS) optical sensor. A plastic cartridge encases the glass-based flow cell. Raised grip points on the plastic ensure safe handling.
  • Page 16 100 Sequencing System Guide The cartridge holds all consumables for a run: reagents, library, and flow cell. The library and flow cell are loaded into the thawed cartridge, which is then loaded onto the instrument. After the run begins, reagents and library are automatically transferred from the cartridge to the flow cell.
  • Page 17 100 Sequencing System Guide Symbol Description Indicates that caution is necessary. Indicates a health hazard. Storage temperature range in degrees Celsius. Store the consumable within the indicated range. * REF identifies the individual component, while LOT identifies the lot or batch the component belongs to.

  • Page 18: Chapter 2 Getting Started

    Chapter 2 Getting Started First Time Setup Minimize the Control Software Run Settings Instrument Customization Network Setup User-Supplied Consumables and Equipment First Time Setup The first time the system is turned on, the control software is launched with a series of screens to guide you through first time setup.

  • Page 19: Run Settings

    Attach a keyboard and mouse to the instrument to facilitate navigation and typing outside of the control software. To maximize the control software, swipe up and select iSeq 100 System. Run Settings Configure options for run setup, run monitoring, and data analysis on the Settings tab in System Settings. This tab displays recommended express settings, which you can apply by selecting the express setup option.
  • Page 20 The service sends instrument performance data, such as temperature and run time, to Illumina. These data help Illumina detect potential failures and facilitate troubleshooting. Run data are not sent. For more information, see the Illumina Proactive Technical Note (document # 1000000052503) .

  • Page 21: Instrument Customization

    When the system is configured for Manual mode and you are analyzing data in BaseSpace Sequence Hub, each run requires a sample sheet. Create a sample sheet by editing the iSeq 100 System Sample Sheet Template for Manual Mode , then importing it into the control software during run setup. After import, the software automatically renames the sample sheet to SampleSheet.csv.

  • Page 22: Network Setup

    These settings do not need updating unless your organization has custom network requirements. If it does, consult your IT representative for help changing the default network settings. The iSeq 100 Sequencing System Site Prep Guide (document # 1000000035337) provides guidelines on network settings and control computer security.
  • Page 23 100 Sequencing System Guide Specify the Output Folder Location Universal Copy Service copies sequencing output files from the run folder to BaseSpace Sequence Hub (when applicable) and the output folder, where you can access them. An output folder is required unless the system is configured for run monitoring, analysis, collaboration, and storage using BaseSpace Sequence Hub.

  • Page 24: User-Supplied Consumables And Equipment

    100 Sequencing System Guide Connect to a Proxy Server Minimize the control software. From Windows Start, open the Run dialog box. Type cmd, and then select OK. Type the following command: C:\windows\System32\bitsadmin.exe /Util /SetIEProxy LocalSystem Manual_ proxy http://<proxyserver>:<proxy port> NULL Replace http://<proxyserver>:<proxy port>...
  • Page 25 100 Sequencing System Guide Consumable Supplier Purpose iSeq 100 i1 Spare Drip Tray Pad¹ Illumina, catalog # 20023927 Lining the drip tray to absorb any leaked fluids. iSeq 100 i1 Spare Air Filter¹ Illumina, catalog # 20023928 Replacing the air filter every six months.

  • Page 26: Chapter 3 Sequencing

    Nextera Flex for Enrichment TruSeq DNA Nano TruSeq DNA PCR-Free For other library types, Illumina recommends 50 pM as a starting loading concentration. Optimize this concentration over subsequent runs to identify a loading concentration that consistently yields data that meets specifications. Loading concentrations that are too high or too low result in suboptimal clustering and run metrics. For more information, see the Cluster Optimization Overview Guide (document # 1000000071511) .

  • Page 27: Thaw The Bagged Cartridge

    100 Sequencing System Guide The minimum and maximum cycle numbers include an extra cycle. Always add one cycle to the desired read length to correct the effects of phasing and prephasing. Read length is the number of sequencing cycles in Read 1 and Read 2, which excludes extra cycles and index cycles.

  • Page 28: Prepare The Flow Cell And Libraries

    PhiX. Libraries are denatured automatically onboard the instrument. The following dilution instructions apply to supported Illumina libraries that are double-stranded. Always perform a quality control analysis, optimize the loading concentration for your library, and use a normalization method that generates double-strand libraries.
  • Page 29 * For unlisted library types, including other Nextera libraries, start with a loading concentration of 50 pM and optimize over subsequent runs. This table provides example loading concentrations. The iSeq 100 System is compatible with all Illumina library prep kits except SureCell WTA 3′ , but the optimal loading concentration can vary.

  • Page 30: Load Consumables Into The Cartridge

    100 Sequencing System Guide Combine PhiX and library: For a 2% spike-in, add 2 µl diluted PhiX to 100 µl diluted library. For a 5% spike-in, add 5 µl diluted PhiX to 100 µl diluted library. Actual PhiX percentage varies depending on library quality and quantity.

  • Page 31: Set Up A Sequencing Run (Local Run Manager)

    100 Sequencing System Guide Pull the flow cell out of the package. Touch only the plastic when handling the flow cell. Avoid touching the electrical interface, CMOS sensor, glass, and gaskets on either side of the glass. Hold the flow cell by the grip points with the label facing up.
  • Page 32 To disconnect the current run from BaseSpace Sequence Hub, select Skip BaseSpace Sequence Hub Sign In. Instrument performance data are still sent to Illumina. To change connectivity for the current run, select a Configuration option: Run Monitoring Only—Send only InterOp files to BaseSpace Sequence Hub to allow remote monitoring.
  • Page 33 100 Sequencing System Guide Enter your BaseSpace Sequence Hub credentials (email address and password), and then select Sign If the Available Workgroups list appears, select a workgroup to upload run data to. The list appears when you belong to multiple workgroups.

  • Page 34: Set Up A Sequencing Run (Manual Mode)

    Modify the template as needed and save it in CSV file format. Make sure that: The Index 2 (i5) adapter sequences are in the correct orientation. For orientations, see Illumina Adapter Sequences (document # 1000000002694) . Document # 1000000036024 v05...
  • Page 35 To disconnect the current run from BaseSpace Sequence Hub, select Skip BaseSpace Sequence Hub Sign In. Instrument performance data are still sent to Illumina. To change connectivity for the current run, select a Configuration option: Run Monitoring Only—Send only InterOp files to BaseSpace Sequence Hub to allow remote monitoring.
  • Page 36 100 Sequencing System Guide The run name can contain alphanumeric characters, hyphens, and underscores. For Read Type, select one of the following options: Single Read—Perform one sequencing read, which is the faster, simpler option. Paired End—Perform two sequencing reads, which generates higher-quality data and provides more accurate alignment.
  • Page 37 100 Sequencing System Guide Metric Description %Occupancy The percentage of flow cell wells containing clusters. Projected Total Yield The expected number of bases called for the run. To monitor file copying and other run processes, select the control software menu, and then select Process Management.

  • Page 38: Chapter 4 Maintenance

    Updating the software ensures that your system has the latest features and fixes. Software updates are bundled into a system suite, which includes the following software: iSeq Control Software iSeq 100 System recipes Universal Copy Service Real-Time Analysis Local Run Manager (framework only) NOTE Although Local Run Manager is included in the system suite, the analysis modules are not.

  • Page 39: Replace The Air Filter

    100 Sequencing System Guide To check for an update, select one of the following options: Check for Update—Check for a software update. Autocheck for Updates—Check for a software update and configure the system to automatically check for future updates.
  • Page 40 100 Sequencing System Guide Position the instrument so you can easily access the back. On the back of the instrument, press the right side of the top panel to disengage as shown in the following illustration. Remove the panel from the instrument.

  • Page 41: Relocate The Instrument

    Use the following instructions to safely relocate the instrument. Make sure that the new location meets the requirements described in the iSeq 100 Sequencing System Site Prep Guide (document # 1000000035337) . If you are returning an instrument, skip this section and see Advanced Exchange on page From the menu, select Shut Down System.

  • Page 42: Software Restriction Policies

    Software Restriction Policies Windows Software Restriction Policies (SRP) use rules to allow only specified software to run. For the iSeq 100 System, SRP rules are based on certificates, file names and extensions, and directories. By default, SRP is turned on to prevent unwanted software from running on the control computer. An IT representative or system administrator can add and remove rules to customize the security level.
  • Page 43 100 Sequencing System Guide Executable Files Portmon.exe Procmon.exe Procmon64.exe Tcpview.exe File Extensions *.cfg *.lnk *.png *.tif *.txt *.xml Directories %HKEY_LOCAL_MACHINE\SOFTWARE\Microsoft\Windows\CurrentVersion\ProgramFilesDir% %HKEY_LOCAL_MACHINE\SOFTWARE\Microsoft\Windows NT\CurrentVersion\SystemRoot% C:\CrashDumps\* C:\Illumina Maintenance Logs\* C:\Illumina Manufacturing Test Results\* C:\Illumina\* C:\Program Files (x86)\Chromium\Application\* C:\Program Files (x86)\Illumina\* C:\Program Files (x86)\Internet Explorer\*...
  • Page 44 100 Sequencing System Guide When using the touch-screen interface, tapping and holding for about 2 seconds is equivalent to a right-click. Select Start, and then select Run. In the Open field, enter secpol.msc. In the Local Security Policy dialog box, expand Software Restriction Policies, and then select Additional Rules.

  • Page 45: Appendix A Sequencing Output

    100 Sequencing System uses the RTA2 implementation of Real-Time Analysis. RTA2 and the control software communicate through a web HTTP interface and shared memory files. If RTA2 is terminated, processing does not resume and run data are not saved.
  • Page 46 100 Sequencing System Guide File Type File Description, Location, and Name InterOp files Real-time metrics of run quality that are updated throughout the run. These binary files contain tile, cycle, and read-level metrics, and are needed to view metrics in Sequencing Analysis Viewer.

  • Page 47: Real-Time Analysis Workflow

    100 Sequencing System Guide Output Folder Structure Recipe—Run-specific recipe file. Logs—Log files describing instrument analytics, operational steps, and other events. Config—Configuration settings for the run. RunParameters.xml RunInfo.xml CopyComplete.txt RunCompletionStatus.txt RTAComplete.txt RTAConfiguration.xml Data Intensities BaseCalls L001 s.locs InterOp Images SampleSheet.csv—Sample sheet or the sample manifest.
  • Page 48 Base Calling Base calling determines a base (A, C, G, or T) for every cluster of a given tile at a specific cycle. The iSeq 100 System uses one-dye sequencing, which requires one dye and two images to encode data for the four bases.
  • Page 49 (preferably both images) for every cycle. The plate layout and sequences provided in IDT for Illumina TruSeq UD Indexes are designed to have the proper balance.
  • Page 50 100 Sequencing System 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 51: Appendix B Troubleshooting

    Appendix B Troubleshooting Error Message Resolution Cancel a Started Run Power Cycle the Instrument Perform a System Check Leak Mitigation Restore to Factory Settings Error Message Resolution This appendix provides detailed instructions for various troubleshooting procedures. The following flowchart shows the workflow for troubleshooting error messages that appear during initialization, run setup, pre-run checks, or sequencing that a retry does not resolve.

  • Page 52: Cancel A Started Run

    100 Sequencing System Guide Cancel a Started Run After a run starts, you can cancel it to end the run, eject the cartridge, and return to the Sequence screen. CAUTION Canceling a run is final . The software cannot resume the run and consumables cannot be reused after the instrument check portion of pre-run checks.

  • Page 53: Perform A System Check

    100 Sequencing System Guide Figure 12 Toggle Switch Location Wait 30 seconds. Press the power on (I) side of the toggle switch. When the power button pulses, press it. Figure 13 Power Button Location When the operating system is loaded, log on to Windows.
  • Page 54 100 Sequencing System Guide From the control software menu, select System Check. The System Check dialog box appears with the mechanical, thermal, optical, and sensor tests selected. Select Unload to open cartridge compartment door and eject the tray. If present, remove the used cartridge from the tray.

  • Page 55: Leak Mitigation

    After cleaning, a system check confirms normal operation. If a significant amount of fluid is visible on the flow cell, cartridge, or instrument, shutdown and disconnect as follows and contact Illumina Technical Support. From the menu, select Shut Down System.
  • Page 56 100 Sequencing System Guide When the power button pulses, press the power off (Ο) side of the toggle switch on the back of the instrument. d Wait 30 seconds. Disconnect the power cord from the wall outlet, and then from the AC power outlet on the rear panel.

  • Page 57: Restore To Factory Settings

    Restore to Factory Settings Restore the system to factory defaults to downgrade the software, recover from an undesirable configuration, or clear user data before returning an instrument to Illumina. Restoring the system uninstalls control software and clears drive C. If the reference genomes repository for Local Run Manager resides on drive C: Document # 1000000036024 v05 For Research Use Only.
  • Page 58 The restoration can include several reboots. When complete, the system reboots with the original factory settings, less the control software. Install the control software: Download the software installer from the iSeq 100 Sequencing System support pages. Save the installer to a network location or portable USB drive. d Copy the installer to C:\Illumina.

  • Page 59: Appendix C Advanced Exchange

    Return the Original System Exchanging the iSeq 100 System Because the air filter and drip tray pad are the only serviceable parts the iSeq 100 System has, Illumina uses advanced exchange to fix issues that cannot be resolved remotely. Advanced exchange replaces a damaged or defective system with a refurbished system. To minimize downtime, you receive the replacement system before shipping back the original.
  • Page 60 The Ethernet cable from the wall outlet, and then from the Ethernet port on the rear panel. The keyboard and mouse from the USB ports on the rear panel. Decontaminate the Instrument Shipping an instrument requires the following decontamination procedure, which Illumina confirms is complete. Site-specific hazards can require additional decontamination. Decontaminate With Bleach Put on a new pair of powder-free gloves.
  • Page 61 100 Sequencing System Guide Gently pull the cartridge compartment door from the side edges to open. Clean the entire compartment door with a bleach wipe: Door interior Door exterior Door hinges Close the cartridge compartment door. Locate the drip tray door below the cartridge compartment on the front of the instrument, and then lower the door.

  • Page 62: Return The Original System

    Dispose of the pad and other consumables in accordance with applicable standards, which vary by region. For more information, see the Safety Data Sheet (SDS) at support.illumina.com/sds.html. 10 Clean the drip tray with a bleach wipe. 11 Wait 15 minutes for the bleach to take effect.
  • Page 63 Close the front flap, and then close the top of the box. Package the Reusable Test Components Put the iSeq 100 i1 Reusable Test Cartridge into the larger resealable bag and seal. Put the iSeq 100 i1 Reusable Test Flow Cell into the clamshell case.
  • Page 64 100 Sequencing System Guide Ship the System If it was removed, place the protective foam base into the bottom of the brown shipping box. Lift the white box by the handles (a two-person lift is recommended), and then lower the white box into the brown box.
  • Page 65 If your lab has daily deliveries scheduled with UPS, give the labeled shipping box to the driver. If your lab does not have daily deliveries with UPS, notify Illumina Customer Service so that they can schedule the return shipment for you.

  • Page 66: Index

    1 alcohol wipes 17 clusters alerts 31 filtering 42 amplification 19 locations 38 AmpliSeq Library PLUS for Illumina 19 optimizing 19 analysis CMOS sensors 8, 23, 39 methods 5, 19 command lines 17 off-instrument 19 commercial invoice 57...
  • Page 67 Ethernet port 49, 52 i7 index adapters 28 Ethernet, turning on 16 icons 6, 9 expiration dates 9, 32 IDT for Illumina TruSeq UD Indexes 42 express setup settings 12 Illumina Proactive Support 12-13 external drives 16 illuminator 8 extra cycles 19...
  • Page 68 InterOp files 38 manufacturer 9 IP addresses 6 maximizing control software 11 iSeq 100 i1 Reagents 7, 17 maximum cycles 19 iSeq 100 System Test Kit 17, 46 minimum cycles 19 monitor 3 mouse 3, 11 moving 3, 34 keyboards 3, 11...
  • Page 69 100 Sequencing System Guide output folder 6, 26, 28, 31, 38 accessing 11 default location 16, 39 read cycles 28 read lengths 19 read types 19, 28 reagent kits 7 packaging reagents 7-8 cartridge 20 disposal 27, 30, 45...
  • Page 70 100 Sequencing System Guide sample manifest 40 System Settings 11, 14 sample sheets 25, 27-28, 40 System Suite installer 31 naming 14 templates 14, 27 sbsadmin vs. sbsuser 11 SDS 49, 54 technical assistance 65 security 35 template, sample sheet 14, 27...
  • Page 71 100 Sequencing System Guide yield metrics 26, 29 Document # 1000000036024 v05 For Research Use Only. Not for use in diagnostic procedures.

  • Page 72: 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 # 1000000036024 v05 For Research Use Only. Not for use in diagnostic procedures.
  • Page 73 Document # 1000000036024 v05 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. © 2019 Illumina, Inc. All rights reserved.

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