Illumina Knowledge is a repository of 1556 FAQs, troubleshooting articles and reference material for Illumina products and workflows, covering all Illumina Microarrays and every stage of Sequencing.

Use the navigation bar on the left of the page to find areas of interest and content types, use the search function at the top right to query keywords.

What's New

• How to Access the Linux Terminal on the MiSeq i100 Series Instruments (March 31, 2025)

• Representative library QC traces for Illumina Single Cell 3’ RNA Prep (March 31, 2025)

• Selecting cycle numbers for High and Low RNA sample types with Illumina Single Cell RNA prep kits (March 31, 2025)

• Single sample group formation to reduce GenomeStudio Methylation Module crashing during analysis (March 31, 2025)

• Viral Surveillance Panel (VSP) probes and panel design frequently asked questions (FAQs) (March 31, 2025)

Announcements

• Check out our NEW resource for End-to-end sequencing and microarray training at Illumina Solutions Centers in the Americas. (May 31, 2024)

Cloud Status

• Click the link above to check on the status of Illumina cloud software products.

Check out the MiSeq™ and NextSeq™ 500/550 Companions!

The MiSeq and NextSeq 500/550 Companions are your step-by-step guide and training resource for run preparation, run setup, instrument maintenance, instrument troubleshooting, and links to online tools and resources.

Instrumentation > General > FAQ

• Are the RFID tags for the flow cell and reagent cartridge linked?

• Differences in the percent clusters passing filter on patterned and non patterned flow cells

• End of Life Schedule for HiSeq Platforms

• How is PhiX alignment calculated by RTA (Real Time Analysis)?

• How to determine the appropriate kit configuration to use based on the data needed?

• Illumina reagent shipment packaging FAQs

• Is it possible to remove a library pool from a sequencing reagent cartridge?

• Procedures for cleaning flow cells for Illumina sequencers

• Reagent Expiration Date Definition

• Secure communication protocols on Illumina systems

• Steps if insufficient wash solution was used in a manual wash on the NextSeq 500/550 or MiniSeq

• What are Illumina's Qualification Service Offerings

• Which sequencers use patterned vs. nonpatterned flow cells?

• Check out our NEW resource for End-to-end sequencing and microarray training at Illumina Solutions Centers in the Americas. (May 31, 2024)

No. The flow cell RFID is unique to the flow cell and the reagent cartridge RFID is unique to the reagent cartridge. Each component in a reagent kit has its own RFID code that is read independently.

Note: Illumina reagents are equipped with a radio-frequency identification (RFID) tag to enable accurate consumable tracking.

The Real Time Analysis (RTA) software calculates the percentage of reads passing filter that align to the PhiX reference genome. PhiX alignment is calculated over the first 25 cycles of the insert reads. If there are >2 mismatches in the first 25 cycles, a read will not be called as PhiX.

If a library pool has already been loaded into a sequencing reagent cartridge, is it possible to remove the library and re-use the reagent kit with the same or a different library pool (if sequencing has not been performed)?

Generally, once the library is loaded into a reagent cartridge, Illumina does not recommend removing the library pool. If sequencing is significantly delayed after loading a library pool, contact Illumina Technical Support for additional information.

Instruments that use patterned flow cells have artificially lower percent clusters passing filter (%PF) metrics compared to non-patterned flow cells.

• With patterned flow cells, there is no template generation or preliminary filtration step during image analysis, which results in lower %PF values.

• Because template generation and the associated preliminary filtration steps are not applied, all nanowells, including empty wells or clusters that may be dim, low quality, or polyclonal are included in the raw cluster count, which leads to lower %PF values.

• The uniform feature sizes and optimal spacing in the patterned flow cell enable significantly increased cluster density for patterned flow cells, ultimately resulting in a greater yield capacity.

• Although the percent passing filter metric is generally lower with patterned flow cells, it does not negatively impact performance or data quality.

• Consult the specifications for the instrument and sequencing configuration to evaluate run performance, quality, and data output.

For further information, see the technical note Calculating Percent Passing Filter for Patterned and Nonpatterned Flow Cells.

In the event of an outage, please consult the Illumina Cloud Products Status page below for the latest updates and guidance.

Illumina Cloud Products Status

If you suspect that an outage is causing issues, please consult the document below for commonly recommended mitigation strategies

BaseSpace and Proactive outage resource page

iSeq 100

• No cleaning required.

• Only touch the plastic when handling the flow cell.

• Avoid touching the electrical interface, CMOS sensor, glass, and gaskets on either side of the glass.

• Reference: iSeq 100 Sequencing System Product Documentation.

MiniSeq

• Put on a new pair of powder-free gloves.

• Remove the flow cell from the container.

• Clean the glass surface of the flow cell with a lint-free alcohol wipe.

• Dry with a lint-free lens cleaning tissue. Use care around the black flow cell gasket.

• Inspect the flow cell port for obstructions. Make sure that the gasket is well-seated.

• Reference: MiniSeq System Guide.

MiSeq

• Put on a new pair of powder-free gloves.

• Remove the flow cell from the container.

• Lightly rinse the flow cell with laboratory grade water until both the glass and the plastic cartridge are thoroughly rinsed of excess salts.

• Using care around the black flow cell port gasket, thoroughly dry the flow cell glass and plastic with a lint-free lens cleaning tissue. Gently pat dry in the area of the gasket and adjacent glass.

• Clean the flow cell glass with an alcohol wipe. Make sure that the glass is free of streaks, fingerprints, and lint or tissue fibres.

• DO NOT USE THE ALCOHOL WIPE ON THE FLOW CELL PORT GASKET.

• Dry excess alcohol with a lint-free lens cleaning tissue.

• Inspect the flow cell port for obstructions. Make sure that the gasket is well-seated. If the gasket appears to be dislodged, gently press it back until it sits securely around the flow cell ports.

• Reference: MiSeq System Guide for Windows 10 and MiSeq System Guide for Windows 7.

NextSeq 500/550

• Remove the flow cell from the foil packaging.

• Open the clear plastic clamshell package and remove the flow cell.

• Clean the glass surface of the flow cell with a lint-free alcohol wipe. Dry the glass with a low-lint lab tissue.

• Reference: NextSeq 500 and NextSeq 550 Sequencing System Guide

NextSeq 1000/2000

• The flow cell MUST NOT be cleaned.

• Flow cells can look streaky; they are fine to use.

• Reference NextSeq 1000/2000 Product Documentation.

HiSeq 2500

• Remove the clustered flow cell from the container using a pair of plastistats.

• Rinse the flow cell with laboratory-grade water. Dry with a lens cleaning tissue.

• Fold an alcohol wipe to approximately the size of the flow cell.

• Hold the edges of the flow cell with 2 fingers so that the inlet and outlet ports face up.

• Wipe each side of the flow cell using a single sweeping motion. Repeat until clean, refolding the alcohol wipe with each pass.

• Dry with a dry lens cleaning tissue.

• Protect the flow cell from dust until ready to load it onto the instrument.

• Reference: HiSeq 2500 System Guide.

HiSeq 3000/4000/X

• It is not necessary to clean the flow cell.

• If desired, can clean the glass surface of the flow cell with a lint-free alcohol wipe. Dry the glass with a low-lint lab tissue.

• Reference: HiSeq 3000 System Guide, HiSeq 4000 System Guide, HiSeq X System Guide.

NovaSeq 6000

• It is not necessary to clean the flow cell.

• If desired, clean the glass surface of the flow cell with a lint-free alcohol wipe. Dry the glass with a low-lint lab tissue.

• Reference: NovaSeq 6000 Sequencing System Guide.

NovaSeq X Series

• Put on a new pair of powder-free gloves to avoid contaminating the glass surface of the flow cell.

• With the flow cell foil package over a flat surface, peel open the foil from the corner tab

• Remove the flow cell from the package. Grasp the flow cell by the sides to avoid touching the glass or the underside gaskets.

• Inspect the flow cell for any contaminants (eg, particulates, lint, or dried reagent). If contaminants are visible, clean the flow cell as follows.

  1. Wet a polynit heatseal wipe with isopropyl alcohol (70%).

  2. Gently clean the applicable surface. Wipe in a lengthwise direction only.

  3. Repeat steps a and b until surfaces are clear of all contaminants.

  4. Dry with a new polynit heatseal wipe or an unused side of the used wipe to avoid contamination.

• Reference: NovaSeq X Series.

MiSeq i100

• No cleaning required (CMOS patterned flow cell integrated into the Dry cartridge).

• Cartridge should be gripped from the side to avoid touching the flowcell circuit/CMOS sensor.

• Reference: MiSeq i100 Plus System.

What is a communication protocol, in simple terms?

When data is transferred from Illumina sequencers and servers to a user network storage location, there are different levels of Server Message Block (SMB) protocols that can be employed.

What are the latest secure communication protocols supported on Illumina instruments?

All Windows 10 systems support SMB 3.1.1

The NextSeq 1000/2000 (CentOS 7) supports SMB 3.0

The NovaSeq X/X Plus (Oracle Linux 8) supports SMB 3.1.1

The HiSeq platforms are all currently discontinued; however, Illumina will continue to provide reagents and full support on these instruments until the following dates. See each link for the support page of each instrument.

Articles published to Illumina Knowledge in the past 2 months:

Why are there different amounts of dry ice in different shipments? Depending on the temperatures that the shipment encounters in transit, there may be differences in the amounts of dry ice left in containers upon delivery. If there is any dry ice left in the container, then the product is good to use. Dry ice quantities have been optimized for ideal product quality in transit. Illumina continues to work on reducing environmental impact and improving customer experience.

Illumina frozen product boxes are tearing when separating them. Is this a risk to the product? Boxes may freeze together because of condensation and extreme cold due to dry ice. This damage occurs when the boxes are separated immediately after removing from contact with dry ice, is cosmetic only, and does not affect product performance. Letting the boxes sit out at room temperature for approximately 5 minutes after removal from insulated containers will allow them to separate without cosmetic damage.

Why are some shipping containers wet? Condensation on the shipping containers is due to extreme temperature differences between the dry ice and product inside the insulated container during hot and/or humid weather conditions. Shipping containers have passed rigorous thermal and simulated distribution testing, and condensation does not affect the products inside.

My product came shipped at a different temperature than the storage temperature. Is the product okay? To minimize Illumina's environmental footprint and provide an efficient unpacking experience, some products are tested to be able to ship at different temperatures than their recommended storage conditions. Always check the product label for the appropriate storage condition of your products upon receipt. These products have passed rigorous testing and evaluation for product quality throughout Illumina’s global supply chain.

What are the foil/silver bags in dry ice shipments? To minimize condensation, Illumina has implemented metallized bags inside of some longer-duration shipments.

How should shipping containers and insulation be disposed? Refer to the shipping box graphics to determine how to best dispose of (or return) specific shipping containers and insulation. If no instructions are given, boxes can be disposed of through the waste stream.

• Reduced quantity of dry ice is present with frost still visible.
• External minor damage to insulated container with no impact to product or any other contents.
• Creasing on box, surface tears or minor punctures that do not affect readability of label or integrity of box.
• Box edges slightly bent with creases or tears but integrity of box not impaired.

Patterned flow cells consist of a nanowell substrate with billions of ordered wells. Compared to nonpatterned flow cells, the uniform cluster sizes enable optimal spacing and increased cluster density. See Knowledge Article Calculating Percent Passing Filter for Patterned and Nonpatterned Flow Cells for more information.

Instruments that use patterned flow cells: NovaSeq 6000, NovaSeq X/X Plus, NextSeq 1000/2000, MiSeq i100, iSeq 100, HiSeq 3000/4000/X.

Instruments that use non-patterned flow cells: MiSeq, NextSeq 500/550, MiniSeq, HiSeq 1000/1500/2000/2500.

To determine the number of reads needed for the run (project), multiply the desired reads per sample by the number of samples in the pool. With the total number of reads, identify the kit and sequencer that fits the data needs using the sequencer specifications pages, listed below.

The control software for all instruments will allow the reagents to be used the day of expiration thus they are still within their expiration until the end of that day.

For example: If the expiration date states 25-Oct-2022, the reagents are within Illumina's consumable warranty if used on 25-Oct-2022 and can be used until 26-Oct-2022.

Instrumentation > General > Reference Material

• Adding Illumina to TeamViewer allowlist

• Amplicon Sequencing Assessing the Success of Your Run Support Webinar Video

• Antivirus recommendations for Illumina Sequencing Instruments

• Approximate sizes of sequencing run output folders

• Best practices for getting the lab back up and running after an extended shutdown

• Best practices for low diversity sequencing on the NextSeq 500/550 and MiniSeq systems

• Best practices for maintaining and cleaning Illumina sequencing system wash cartridges

• Best practices for maintaining the computer on Illumina sequencing systems

• Best practices to improve data yield when using patterned flow cells on the HiSeq 3000/4000/X

• Bluekeep and DejaBlue two vulnerabilities of the Remote Desktop Protocol within Microsoft Windows

• Calculating Percent Passing Filter for Patterned and Nonpatterned Flow Cells

• Chemistry and Imaging on NovaSeq 6000

• Cluster density considerations when migrating Illumina libraries between sequencing platforms

• Cluster density guidelines for Illumina sequencing platforms using non patterned flow cells

• Decontaminating Illumina instruments for novel coronavirus (2019 nCoV)

• Diagnosing Suboptimal Clustering in Nonpatterned Flow Cells Video

• Diagnosing Suboptimal Clustering in Patterned Flow Cells Video

• Does my sequencing run look good?

• End to end sequencing and microarray training at Illumina Solutions Centers in the Americas

• Features of Illumina Advantage (IA former TG) product

• Focus images on the MiniSeq and NextSeq 500/550

• Group Policy Object (GPO) reports

• Heat output specifications for Illumina sequencers

• HiSeq 3000/4000 Introduction Support Webinar Video

• How Do I Prepare for My Illumina Instrument Delivery? Video

• How many cycles of SBS chemistry are in my kit?

• How much PhiX spike in is recommended when sequencing low diversity libraries on Illumina platforms?

• How to Clean Illumina Sequencer Wash Cartridges Video

• How to Disable Cloud Login Feature on the MiSeq i100 and NovaSeq X Series

• How to Requeue an Analysis on BaseSpace Sequence Hub When No Changes to the Sample Sheet are Needed

• How to achieve more consistent cluster density on Illumina sequencing platforms

• How to check .NET version on Windows instruments

• How to connect benchtop instruments and Local Run Manager to a network location

• How to disable and add exceptions to the Software Restriction Policies for TeamViewer in Windows 10

• How to identify the IP and MAC address of a sequencing instrument

• How to identify the UNC path of a network server or location.

• How to import a pre existing index kits into Local Run Manager v3 and v4

• How to interpret clusters passing filter in run metrics

• How to make sure Universal Copy Service is configured to Log On as a standard (non admin) account

• How to perform System Check on the NovaSeq X Series

• How to requeue and import run data for reanalysis with Local Run Manager

• How to restart RTA on HiSeq 2500 and MiSeq systems

• How to restart a sequencer after a power outage?

• How to set up a PhiX validation run with Local Run Manager

• How to thaw and store sequencing reagents for optimal performance

• How to update Windows 10 account credentials on Benchtop Instruments

• IT Requirements for Implementing Illumina Proactive Video

• Illumina 测序平台的最大读长

• Illumina测序平台上每个测序步骤的运行时间预测

• Illumina测序平台测试Run中PhiX的上机浓度

• Illumina测序系统废液中甲酰胺的终浓度是多少？

• Illumina非Pattern Flow Cell测序平台簇密度推荐指南

• Impact of ammonium based cleaning products on sequencing run performance

• Index color balancing for the NextSeq 500/550 and MiniSeq systems

• Instrument Service Contract Plans

• Instrument maintenance and shutdown procedures for extended site closures

• Introducing TeamViewer for Remote Desktop Share with Illumina Support Teams

• Introduction to Illumina Sequencing By Synthesis Support Webinar Video

• Investigation of Log4j Vulnerability with Illumina ProActive Portal

• Investigation of Log4j Vulnerability with Illumina instruments

• Maximum read length for Illumina sequencing platforms

• Network Output Path Requirements in Local Run Manager and Universal Copy Service

• NextSeq500/550和Miniseq平台进行低多样性文库测序的最佳操作

• NextSeq和MiniSeq测序系统上index混合的指导方针

• PhiX loading concentrations for validation runs on Illumina sequencing platforms

• Plotting %Occupied by %Pass Filter to optimize loading for the NovaSeq 6000 and iSeq 100 Video

• Quality Scores for Next Generation Sequencing

• Run time estimates for each sequencing step on Illumina sequencing platforms

• Security updates to address Windows Print Spooler Remote Code Execution Vulnerability

• Sequencing Analysis Viewer (SAV) A Beginner's Guide Support Webinar Video

• Sequencing Considerations for Low Diversity Samples Support Webinar Video

• Serial Number locations on Illumina sequencers

• Sharing instrument performance data with Illumina Support

• Shelf life for Illumina reagent products

• System checks for Illumina sequencing platforms

• Technical resources for the HiSeq 3000 and 4000 systems

• Troubleshooting "Cannot initiate communication with Universal Copy Service" error

• Troubleshooting "Could not find a part of the path" errors on the NextSeq500/550 and MiniSeq

• Troubleshooting Bit Error on the NextSeq 500/550 and MiniSeq

• Troubleshooting a blank BaseSpace screen or The request timestamp is in the future during run setup

• Uninterruptible Power Supply (UPS) Models by Instrument

• Using the MyIllumina dashboard to view instrument yield, Reads PF, and Q30 data as trends over time

• What is nucleotide diversity and why is it important?

• What is the final formamide concentration in the waste solution of Illumina sequencing systems?

• Why sequencing 26 or more cycles in Read 1 is recommended

• 为什么Read1测序读长推荐至少需要26个循环？

• 什么是核苷酸多样性，它为什么十分重要？

• 在Illumina平台进行低多样性文库测序时，应该掺入多少PhiX？

• 场地长期关闭或仪器长期不用时illumina仪器的维护与关机程序

• 如何在HiSeq 2500和MiSeq系统上重启RTA

• 如何在Illumina测序平台上获得更稳定的簇密度

• 如何在搭载Windows 7 Professional系统的Illumina 仪器上安装相应的扩展安全更新（ESU）程序？

• 如何更新UCS和LRM在Windows10系统中的凭据 (针对装有NCS v4.0.1+, MNCS v2+, MCS v4+的平台)

• 我所购买的SBS试剂盒最多能够满足多少个循环的测序？

• 测序运行输出文件夹的近似大小

• 通过% Occupied和% Pass Filter散点图优化NovaSeq 6000和iSeq 100上样浓度

If there is not enough wash solution in the wash tray during a manual wash, this can introduce air into the system which can negatively impact subsequent sequencing run performance.

To fix this, Illumina recommends performing 2-3 maintenance washes on the instrument to flush the fluidics lines and remove any air bubbles.

This support webinar video discusses the evaluation of amplicon sequencing runs. The support video covers a review sequencing run performance for different Illumina amplicon sequencing workflows, including AmpliSeq and 16S, and analysis results. Additional topics include: optimal sequencing run set up, evaluating sequencing performance, supplementary analysis files, amplicon analysis options, and key analysis metrics.

Video link: https://youtu.be/ENAlPYK1_X0

Video length: 45:13 min

When returning to the lab after an extended shutdown or inactivity, it is important to make sure all lab resources are up and running and ready to be used. Below are best practices to check library prep, sequencing and microarray reagents and instruments to ensure a successful reopening.

Laboratory and Reagent Checks

Wipe down all lab surfaces with 10% bleach to remove any accumulated dust or contaminants.

• Pay particular attention to typical ‘hot spots’:

  • Benchtops used for processing amplified DNA

  • Door handles

  • Refrigerator and freezer door handles

  • Computer keyboard and mouse

  • Centrifuges, vortexers, and thermal cyclers

• Clean all equipment in any pre-amp areas (including pipettes)

Proper storage and handling of reagents, kits, and user-supplied materials should be followed according to documented procedures.

Check connectivity to network drives or servers used for file storage (output run folders, manifest files, cluster files, DMAPs) or data processing (IDAT repository).

Sequencing Instrumentation Considerations

For instruments that were left on:

1. Confirm the recommended maintenance/manual/standby washes were performed per the appropriate system guide.

2. Perform a full power cycle, following the steps in the system guide.

3. Leave system off for at least 5 minutes before powering back on.

Note: While the iSeq 100 and NextSeq 1000/2000 do not have onboard fluidics, a power cycle is recommended for these instruments as well.

For instruments that were turned off:

1. Power on the instrument and make sure that the control software initializes.

2. Perform two maintenance/manual washes to rehydrate the fluidics system (excluding iSeq 100 and NextSeq 1000/2000).

Note: If an additional maintenance/manual/standby wash using laboratory-grade water was not performed prior to instrument shutdown, perform three maintenance/manual washes to rehydrate the fluidics system, then run System Checks to test the fluidics delivery. System Check details are found in the appropriate system guides.

Library Preparation Considerations

Starting a New Library Prep

• When starting new library preparation, Illumina recommends reviewing the best practices for the specific library prep kit being used. This information is found on the appropriate product support page.

Proceeding to a Sequencing Run with Stored Libraries

For libraries requiring bead-based normalization, Illumina recommends storing the library at the last safe stopping point, just prior to the bead-based normalization step, as specified in the protocol guide. Libraries stored as double stranded DNA (dsDNA, before bead-based normalization) are more stable than libraries stored as single stranded DNA (ssDNA, after bead-based normalization). If a library has been stored long term after bead-based normalization, Illumina recommends repeating the bead-based normalization of the original dsDNA library.

Infinium Assay Considerations

• Check stocks of user-supplied materials (eg, cap mats, 96-well plates, gloves, etc.) that may have been consumed by other groups using the space in the interim.

Te-Flow and Water Circulator:

1. Confirm that there is no microbial growth in the water circulator and that the water is at the proper fill level.

   2. Do not use chlorine bleach to clean the water circulator.

2. Check the waste tubing lines of the Te-Flow and white reagent pan. Bleach can be squirted through the waste tubing lines leading to the waste carboy to clean them, if needed.

3. If the waste carboy has been emptied or moved, make sure it is back in its proper position, with an unimpeded, gravity-driven flow of waste from the Te-Flow Reagent pan to the waste carboy. Make sure that waste tubing is not kinked and there are no ‘uphill’ runs to the waste carboy.

Tecan/Illumina Automated Pipetting System (IAPS):

1. If the Tecan fluidics were stored dry, refill the system water carboy.

2. Empty the waste liquid container and disposable tip waste bag, if necessary.

3. Make sure that the Te-Flow is seated correctly in the pan, and that the Te-Flow is in the appropriate location on the Tecan deck.

4. Power on the Tecan.

5. 1. Preparing the Infinium Automated Pipetting System for Use (perform at least two bleach washes)

   2. Note: if the system was idle for a long time and/or dried out prior to shut down, multiple System Wash cycles are needed to purge all air from the fluidics.

   3. Testing Volume Accuracy of the IAPS Tips

iScan

1. Power on the scanner and make sure that the Archimedes drive appears on the PC before launching ICS software.

2. Clean the BeadChip carriers and iScan Nest (tray that carrier is placed on) with an Ethanol wipe to remove any dust or built up residue.

3. Turn on the scanner at least 30 minutes before use to give the lasers time to warm up.

To avoid data loss or interruptions, use the following guidelines to configure an antivirus software of your choice.

• Configure software for manual scans and do not allow automatic scanning.

• Perform manual scans only when the instrument is idle and not actively performing a sequencing run or analysis.

• If possible, perform several sequencing runs with the AV software in listening or training mode to allow it to identify normal use patterns.

• Set AV updates to download without user authorization, but to not install automatically.

  • Install updates only when the instrument is not in use and the computer can be rebooted.

  • Do not allow the control computer to reboot automatically after install.

• Exclude the application directory and data drives from any real-time file system protection. Apply this setting to the following drive locations:

  • C:\Illumina

  • C:\ProgramData\Illumina

  • D:\Illumina

  • D:\Output (NextSeq 500/550 and MiniSeq)

  • Z:\outputfolder (NovaSeq 6000 only)

  • /usr/local/illumina/images AND /usr/loca/illumina/runs (NextSeq1000/2000)

  • See the instrument Site Prep Guide for instrument specific details.

• When deploying AV software across multiple instruments of the same model, install and configure the AV software on a subset of instruments first and monitor for any issues before expanding to additional instruments.

For more details on configuring antivirus software for your system, see the Site Prep Guide for the instrument. Contact the antivirus software vendor for software-specific instructions.

• Use indexing to add multiple, indexed samples from various applications to the run.

  • Indexed samples from applications that are more diverse, such as human amplicon sequencing, enrichment, or whole-genome sequencing, can be used to balance diversity.

  • For best results, sequence multiple samples on the same flow cell using single- or dual-indexing.

• • MiniSeq reagents accommodate an optimal raw cluster density of 170-220 K/mm2 for balanced libraries

  • NextSeq 500/550 reagents accommodate an optimal raw cluster density of 170-220 K/mm2 for balanced libraries

The design recommendations in this bulletin enable the NextSeq 500/550 and MiniSeq systems to sequence low diversity libraries.

Background

The run folder sizes listed in this bulletin should be used only as an approximation. Folder size will vary depending on the raw cluster density and the number of clusters passing filter. Folder sizes are based on runs with optimal cluster densities for each platform and dual indexing. For runs on nonpatterned flow cells, the size approximations are based on runs with pass filter greater than 80%.

Additional run folder size considerations:

• iSeq 100 folder sizes include FASTQ files generated by the Local Run Manager FASTQ workflow.

• MiniSeq, NextSeq 500/550 (NextSeq Control Software v4.0 and higher) folder sizes include FASTQ files generated by the Local Run Manager FASTQ workflow.

• MiSeq folder sizes include FASTQ files generated by the MiSeq Reporter FASTQ workflow and MiSeq (MiSeq Control Software 3.1 and higher) Local Run Manager FASTQ workflow.

• NextSeq 1000/2000 folder sizes include local analysis output, which can add another 25-120+ GB depending on workflow.

• NovaSeq 6000 folders do not include FASTQ files as part of the total output folder size.

Illumina recommends storing the output folder for iSeq 100, NextSeq 500/550, and NextSeq 1000/2000 runs on a network location. NovaSeq 6000 and NovaSeq X Series Control Software requires the output folder be stored on a network location.

iSeq 100

MiniSeq

Mid Output

High Output

Rapid

MiSeq

v3 chemistry

v2 chemistry

MiSeq i100 Series

See Knowledge Article Estimating Disk Space Utilization on the MiSeq i100 Series

NextSeq 500/550

Mid Output

High Output

NextSeq 1000/2000

P1

P2

P3

NovaSeq 6000

SP

S1

S2

S4

The output folder size in the above tables is reported in Gigabytes (GB).

NovaSeq X/X Plus

Values listed below are PER FLOW cell in Terabytes (TB).

1.5B

10B

25B

DRAGEN Analysis files can ADD the following additional data in terabytes (TB) to the expected Output folder size based on Analysis options selected. Approximate file sizes are based on a single flow cell, 2x150 cycle sequencing run.

Cluster density is an important factor in optimizing data quality and yield. The following table lists the recommended raw cluster densities for balanced libraries (such as PhiX):

The iSeq 100, NextSeq 1000/NextSeq 2000, HiSeq 3000/HiSeq 4000, HiSeq X and NovaSeq 6000 systems utilize patterned flow cells, which result in a fixed cluster density, even if all nanowells are not occupied. In these systems, monitoring the Cluster PF (%) is a better measure of potential cluster occupancy.

Please see the following for further details:

• Plotting % Occupied by % Pass Filter to optimize loading concentration for NovaSeq 6000 and iSeq 100 platform

• Best practices to improve clusters passing filter and yield when using patterned flow cells

• Calculating Percent Passing filter for Patterned and Nonpatterned Flow Cells

Due to differences in chemistry and hardware, libraries exhibit different clustering efficiencies on different Illumina sequencing platforms. Migrating libraries between platforms requires instrument-specific optimization of cluster density.

Listed are guidelines for determining the loading concentration of a library that is being migrated to a different Illumina sequencing platform:

• Between HiSeq and MiSeq platforms Cluster generation on the MiSeq platform and HiSeq platform in rapid run mode typically results in approximately 15%-20% higher cluster density when compared to the same library at the same concentration clustered on a High Output flow cell.

• Between MiniSeq and NextSeq 500/550 platforms The MiniSeq and NextSeq 500/550 platforms use similar chemistries for cluster generation. As a result, the same library can be expected to cluster at a similar density.

• Between HiSeq/MiSeq and MiniSeq/NextSeq 500/550 platforms The MiniSeq and NextSeq 500/550 platforms require significantly lower cluster densities than the HiSeq and MiSeq platforms. Therefore, it is important to redo cluster density optimization when migrating a library from MiSeq or HiSeq to MiniSeq or NextSeq 500/550 platform and vice versa.

• To or from HiSeq 3000/4000 and NovaSeq 6000 The cluster generation process of the patterned flow cells on the HiSeq 3000/4000 and NovaSeq 6000 systems differs from non-patterned flow cell systems. Therefore, cluster density optimization must be performed when migrating a library from any other system to the HiSeq 3000/4000 or NovaSeq 6000 platforms, and vice versa.

When using NovaSeq Control Software version 1.2 and higher, libraries previously optimized on the HiSeq 3000/4000 platform are loaded at the same concentration on the NovaSeq 6000 platform. Optimize subsequent loading concentrations based on the pass filter percentage from primary metrics and percent duplicate metrics from the secondary analysis; refer to the Cluster Optimization Overview and the Plotting % Occupied by % Pass Filter to optimize loading concentration for NovaSeq 6000 and iSeq 100 platforms support bulletin.

• From iSeq 100 to NovaSeq 6000 Prior to performing in-depth sequencing of the library on the NovaSeq 6000 system, shallow sequencing on the iSeq 100 can provide a rapid and cost-saving quality check; refer to the Sequencing Library QC with the iSeq System application note and Step-by-step instructions for sequencing library QC with the iSeq 100 System support bulletin. When quantifying the library before sequencing on NovaSeq 6000, rebalancing the library pool based on the actual iSeq reads is as consistent as using the qPCR data.

Optimize loading concentration based on the run’s percentage occupancy and the percentage pass filter; refer to the Plotting % Occupied by % Pass Filter to optimize loading concentration for NovaSeq 6000 and iSeq 100 platforms support bulletin. The percentage of duplicate metrics from the secondary analysis also help optimize the loading concentration; refer to the Cluster Optimization Overview.

For further guidance on library loading concentrations across Illumina systems, refer to the PhiX loading concentrations for validation runs on Illumina sequencing platforms bulletin.

Use the following best practices to ensure successful clustering on patterned flow cells. Inefficient clustering on patterned flow cells can produce focusing errors, low intensities in Read 1, an unusual percent base profile, and poor pass filter rates on the HiSeq system.* Before starting the library denaturation and ExAmp reactions, complete the cBot wash, ensure that the cBot plate is fully thawed, and load the flow cell and manifold onto the cBot.

• After thawing, invert each tube of EPX1, EPX2, and EPX3 five times and then briefly centrifuge. Do not vortex EPX reagents or master mix.

• Aspirate and dispense the viscous EPX reagents slowly to ensure accurate pipetting.

• Add EPX reagents in the order listed (EPX1, then EPX2, and then EPX3). Attach the EPX master mix tube to a lab rotator and rotate for 5 minutes at room temperature to ensure proper mixing.

• Immediately add EPX mix to denatured template and then gently tap the tube strip on a hard surface to remove any air bubbles.

• Promptly start the cBot run.

For detailed information about cluster generation and sequencing using patterned flow cells, see these additional resources:

cBot System Guide HiSeq X System Guide HiSeq 3000 System Guide HiSeq 4000 System Guide

Illumina NGS (Next Generation Sequencing) platforms require a post-run wash and/or maintenance wash to be performed with regularity (refer to the appropriate instrument guide for details). To avoid contaminating the fluidics lines during these washes, Illumina recommends following these best practices.

1. After removing the wash cartridge from the instrument, properly discard any remaining wash solution (eg, do not reuse the Tween wash solution).

2. Thoroughly rinse each position of the wash cartridge with lab-grade warm water.

3. Place the wash cartridge upside down with some space to allow air flow and proper drying.

4. Inspect the wash cartridge wells regularly for contamination (refer to Fig. 1, black or pink discoloration may be noted).

Figure 1. Black ring at bottom of wash cartridge well.

1. Prepare a 1-3% lab-grade bleach solution to fill the wells of the tray.

2. Use a brush to remove the mold from the wells.

3. IMPORTANT: Thoroughly rinse the wells (at least three times) with DI or lab-grade water (residual bleach can cause clustering issues on subsequent runs).

4. Place cartridge upside down with some space to allow air flow and proper drying.

5. Repeat as necessary if mold is still present.

6. If it is not possible to remove all the mold, contact Illumina Technical Support to discuss replacing the wash cartridges.

For step-by-step guidance, refer to this.

Resources

• MiniSeq System Guide

• MiSeq System Guide

• NextSeq 550 System Guide

• NovaSeq 6000 Sequencing System Guide

IntroductionThe latest innovation in flow cell technology is the development of the patterned flow cell. Five Illumina sequencing platforms currently take advantage of this advanced technology: the iSeq 100, NextSeq 1000/NextSeq 2000, HiSeq 3000/HiSeq 4000, HiSeq X and NovaSeq 6000 System. Patterned flow cells consist of a nano well substrate with billions of ordered wells (Figure 1A). Compared to nonpatterned flow cells (Figure 1B), the uniform cluster sizes enable optima spacing and increased cluster density. Although the ordered spacing of patterned flow cells enables significantly higher cluster density, 2-4, patterned flow cells also report comparatively lower percent passing filter (%PF)\* values due to differences in the %PF calculation method. This technical note outlines these differences and describes how they lead to lower %PF metrics for patterned flow cells.

Percent Passing Filter with Nonpatterned Flow Cells

In brief, Real-Time Analysis software proceeds through several stages including image analysis, template generation, base calling, passing filter calculations, and quality scoring. Template generation occurs during cycles 1-5 of Read 1 and defines the position of each cluster in a tile. The template is used as a reference for registration and intensity extraction during subsequent sequencing cycles. With nonpatterned flow cells, dim or low-quality clusters are removed from the raw cluster count during template generation (Figure 2). This effectively acts as a prefiltration step, removing clusters unlikely to pass filter in the first 25 sequencing cycles and yielding relatively high %PF values.

Percent Passing Filter with Patterned Flow Cells

With patterned flow cells, the calculation of %PF is different because there is no template generation step—fixed cluster locations eliminate the need for template generation. Because template generation and the associated preliminary filtration steps are not applied, empty wells or clusters that may be dim, low quality, or polyclonal are included in the raw cluster count, which leads to lower %PF values. Although the percent passing filter metric will be much lower with patterned flow cells, it will not affect performance or data quality.

Summary

Due to the differences in the %PF calculation methods, patterned flow cells have artificially lower %PF metrics compared to nonpatterned flow cells. With patterned flow cells, there is no template generation or preliminary filtration step during image analysis, which results in lower %PF values. Although the %PF values are lower, the uniform feature sizes and optimal spacing enable significantly increased cluster density for patterned flow cells

\* The %PF calculations involve the application of a chastity filter to each cluster. Chastity is defined as the ratio of the brightest base intensity divided by the sum of the brightest and second brightest base intensities. Clusters “pass filter” if no more than 1 base call has a chastity value below 0.6 in the first 25 cycles. This filtration process removes the least reliable clusters from the image analysis results.

Although over clustering is not possible on a patterned flow cell, loading a library with a suboptimal concentration negatively impacts run data. In this video, we discuss preventing and diagnosing common clustering issues on patterned flow cells.

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Video length: 4:48 min

The NovaSeq 6000 uses two-channel chemistry and Patterned Flow Cells technology.

Two-Channel SBS Chemistry, rather than using a separate dye for each base like the Four-Channel SBS Chemistry, two-channel SBS simplifies nucleotide detection by using two fluorescent dyes and two images to determine all four base calls (Figure1).

Figure1: Two-Channel SBS Chemistry by schematic representation.

Images are taken using red and green filter bands. Thymines are labeled with a green fluorophore, cytosines are labeled with a red fluorophore, and adenines are labeled with both red and green fluorophores. Guanines are permanently dark (Figure 2). Nucleotides are identified by analysis of the different emission patterns for each base across the combination of Image 1 and Image 2 that are processed by image analysis software to identify which bases are incorporated at each well position.

Figure 2: Image analysis during SBS Chemistry with Two-Channel Detection.

The system uses a patterned flow cell with nanowells (Figure 3) with 4 different flow cell options which allow the NovaSeq output to scale to different applications and throughput levels.

Clustering and sequencing occur in the nanowells with a proprietary ExAmp chemistry to ensure that each well in the flow cell generates a single clonal cluster.

Figure 3: Schematic representation of NovaSeq 6000 flow cell and the nanowells.

The NovaSeq Flow Cell specifications are further detailed in the table below.

Where can I find instrument specifications?

Follow the links below to the instrument specification pages:

• SAV v2.4.7 of SAV on all instruments except on-instrument MiSeq and NextSeq1000/2000; SAV v2.4.7 is compatible for all off-instrument (remote) use running Windows 7 or later

• SAV v1.8.37 of SAV for on-instrument MiSeq viewingOnce SAV is installed, open it and select the tab containing the desired query information.

How do I determine if my run meets spec?

Below is an example of a PhiX validation run (2 x 151 bp) on the MiSeq, using v2 reagents. The specifications for this run are as follows:

• Total data output of 4.5-5.1 gigabases (Gb)

• At least 80% of bases called with a quality score of 30 or higher (at least 80% ≥ Q30)

To determine the quality score, review the Analysis tab Q score Distribution chart and the Summary tab as shown below.

Analysis Tab:

Summary Tab:

The quality specification for a MiSeq paired-end 151-cycle run is Q30 ≥ 80%. The run meets this specification, as the percent ≥ Q30 is >94%.

To determine the yield of the run, review the information in the Summary tab as shown below.

The yield specification for a paired-end 151-cycle run is >4.4 Gb. The run meets this specification, as the total yield is 6.10 Gb.

Analysis Tab: Overview of the run metrics.

Imaging Tab: Displays thumbnails from the run if available.

Summary Tab: Provides basic data quality metrics summarized per lane and per read.

Indexing Tab: Total and Per Sample % Reads Identified if a sample sheet was used and demultiplexing was performed.

Additional Resources:

General Guidelines for Decontaminating Illumina Systems

In keeping with recommendations from the United States CDC and World Health Organization (WHO), Illumina recommends the following procedure for decontaminating instruments suspected or known to have come in contact with novel coronavirus (2019-nCoV):

Decontamination steps

1. Wear appropriate personal protective equipment before entering a contaminated space

2. Perform a maintenance wash with laboratory-grade water to flush the fluidics systems

3. Shut down the instrument and unplug the instrument from power outlets prior to applying any cleaning agent

   1. Refer to the specific instrument system guide for appropriate shutdown procedures

4. Spray all accessible components and surfaces with a 10% bleach solution and allow solution to remain on the surface for at least 10 minutes

   1. Select the appropriate spray pattern to avoid splashing of the bleach solution

   2. Use a pre saturated wipe for sensitive electronic equipment

   3. Use a pre saturated wipe to decontaminate the touchscreen, mouse, or keyboard

5. After the minimum 10 minute contact time, use a heavy-duty wipe to remove excess bleach before it dries IMPORTANT: Any exposed parts consisting of stainless steel or aluminum should then be wiped down with a 70% ethanol solution to remove excess bleach

   1. The 70% ethanol solution should be wiped off with a heavy-duty wipe before it dries

   2. Use caution when using ethanol near ignition sources such as electrical outlets

6. Make sure that all the following accessible components and surfaces are cleaned:

   1. Outer skins of the instrument

   2. Doors and parts accessible to the user

   3. Rear of instrument, accessible tubes, and cords

   4. Particular emphasis should be placed on:

   5. Areas where samples are loaded and processed

   6. Peripherals that are contacted during normal instrument use such as touch screens, mouse devices, and keyboards

Additional Resources

World Health Organization (WHO) Guidance

Illumina Support Bulletins:

Illumina Advantage (IA, formerly TG Translational Genomics) large-scale sequencing products feature lot-specific shipments and testing, extended shelf life, and advanced change notifications for greater laboratory efficiency.

This video highlights which run metrics and thumbnail images to monitor during a run to diagnose over clustering of a nonpatterned flow cell.

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Video length: 8:01 min

Companies/sites that use licensed copies of TeamViewer instead of TeamViewer QuickSupport can sometimes have security restrictions preventing Illumina Technical Support from connecting to their instruments via TeamViewer.

This article has instructions for adding connections from Illumina to the TeamViewer allow-list to facilitate these screen share connections.

Instructions for adding Illumina to TeamViewer allow-list:

1. To allow only specific TeamViewer accounts or TeamViewer IDs remote access to a device, set up an allow-list.

2. In the TeamViewer full version, select Extras > Options > Security > Block and Allow-list > Select Configure...

3. A new window will open. Activate the second option, Allow access only for the following partners, then select Add.

4. After selecting Add, either choose partners saved on the computers & contacts list, add a whole company (only visible if computer is part of company profile), or add TeamViewer IDs or contacts manually to the allow-list.

5. Select 'Manually add contact to your Allow-list' and then type ILLUMINA INC into the field.

• This allows any connections from Illumina's licensed corporate account to connect to this instrument.

Recently, several vulnerabilities around the Remote Desktop Protocol (RDP) within Microsoft Windows systems were disclosed (Bluekeep and DejaBlue). While these vulnerabilities apply to all Illumina systems, there have been no reported infections of Illumina systems.How to Secure Your Systems

Note: the BlueKeep RDP patch applies to only instruments running on Windows 7, while DejaBlue RDP patch applies to all instruments and Windows operating systems.

For DejaBlue, Illumina recommends the application of the specific patch for your instrument. Use the table below to select the appropriate patches. Due to prerequisites, the patches should be installed in numerical order (eg, Patch 1 first, Patch 2 second, Patch 3 (as applicable) last).

Proper maintenance of the instrument computer minimizes computer-related issues during sequencing runs. The following tips help maintain the instrument computer. How often these maintenance steps are recommended is dependent on the usage of the instrument.

Power cycle the computer and instrument

• Instruments should be left on at all times when not being power cycled to maintain the integrity of the instrument fluidics.

• Power cycling the instrument computer regularly can maintain consistent instrument computer performance.

• You may be asked to power cycle the instrument during troubleshooting.

• The instrument system guides contain recommendations for properly shutting down the instrument and computer for extended periods of time.

Cleaning the hard drives

iSeq 100

Temporary run folders, or “temp folders”, are not deleted by default on the iSeq 100. Without removal, old run folders will reduce hard drive space. The temp folders (D:\Illumina\iSeq Runs) can be managed by deleting the run via Process Management in the control software. Runs in the default output folder on the instrument (D:\SequencingRuns) can only be deleted via the file explorer.

MiniSeq

Temp run folders are not deleted by default on the MiniSeq. Without removal, old run folders will reduce hard drive space. Local Run Manager can be configured for automated deletion of analysis folders, and folder retention time is configured in the Local Run Manager System Settings. Otherwise, run folders require manual deletion.

MiSeq

The hard drive on the MiSeq is configured for processing runs and performing on-instrument analyses and is not meant for long-term storage. Data within the MiSeqTemp folder is automatically cleared after 7 days. It is good practice to move old run folders from the MiSeqAnalysis and MiSeqOutput folders to long-term storage regularly.

NextSeq 500/550

• With NextSeq Control Software v2The NextSeq hard drive is designed to store data for one run, which will be automatically overwritten when a new run is started.

• With NextSeq Control Software v4The NextSeq hard drive can store multiple runs, but it is recommended to use an output folder in a network location. The data in the temp folder is automatically overwritten when a new run is started. Output folders on the instrument require manual deletion.

HiSeq 1000/2000/1500/2500

HiSeq 3000/4000/X

Quick formatting is the preferred method of data deletion for the output (O:) and scratch (S:) drives. Never format the C:\ and DONOTEJECT drives. Instructions for reformatting can be found in the instrument system guides:

NovaSeq 6000

NovaSeq 6000 temp run folders are not automatically deleted. Without removal, hard drive space can become limited, which can impact data processing. Regularly deleting run folders is recommended. Delete runs from the temp folders via Process Management in the control software.

NextSeq 1000/2000

NextSeq 1000/2000 run folders are not automatically deleted. Without removal, hard drive space can become limited, which can impact data processing. Regularly deleting run folders is recommended. Delete runs from the temp folders via Disk Management in the control software. Deleting runs manually via the file navigation system can negatively impact the control software.

NovaSeq X Plus

Security best practice

It is important that installed antivirus software does not interfere with sequencing runs. For more information, refer to the site prep guide for your system.

Language and Regional settings

Instrument computer language and regional settings are set to English (United States) by default. Do not change this setting. The software is not designed to run in another language. Changing this setting can cause secondary analysis failures and communication problems between the computer and the instrument.

Daylight saving settings are enabled by default for correct syncing with BaseSpace Sequence Hub. Do not change this setting.

Illumina has Illumina Solution Centers world-wide, and offers end-to-end trainings for both sequencing and microarray workflows.

The interactive catalog here reviews the complete catalog of Illumina's End-to-End Service and Support Solutions offerings along with information on how to request trainings at Illumina Solutions Centers in the Americas. This content can also be viewed by scanning the QR code below.

This video discusses what to expect during the pre-installation, shipping, and delivery stages of instrument delivery. It also highlights important steps in the process and introduces available resources.

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Video length: 7:13 min

Note: Depending on the region, the steps shown in this video may not apply to the iSeq 100 System.

Ensuring that sufficient volumes of SBS reagents are loaded onto the sequencing instrument is crucial for a successful run. Running out of reagents during a run impacts intensity and quality metrics, and can result in total run failure.

Dual-index sequencing follows different workflows depending on the instrument, as shown in the Indexed Sequencing Overview Guide. On certain instruments, dual-index runs require an additional seven cycles of reagents for the chemistry-only cycles at the beginning of the second Index Read. The seven chemistry-only cycles are required for the workflows in which the second Index Read sequencing occurs after the forward template anneals to the grafted P5 primer on the surface of the flow cell. These seven cycles must be considered when calculating the number of cycles supported by the kit.

The following table outlines available reagent kit sizes and the maximum number of cycles each kit can perform, taking the additional seven cycles into consideration when necessary:

*The NextSeq 1000/2000 P2 v3 300 cycle kit has 338 total cycles while the P3 v3 300 cycle kit has 327 total cycles.

**All the NextSeq 1000/2000 XLEAP kits have 38 extra cycles.

For details on maximum supported cycles in each read, refer to the article Maximum read length for Illumina sequencing platforms.

The following table details the thermal output and power rating for Illumina sequencers. More information is available in the Site Prep guide for each respective instrument.

\*Excludes UPS (uninterrupted power supply) output.

At the beginning of Read 1 on the MiniSeq and NextSeq 500/550, the instrument determines the best Z position (distance between the objectives and flow cell) where the clusters are well-focused. During this process, the instrument takes and saves a series of Focus Images.

Many of the images are black with a laser dot and contain in the file name nomenclature, "Laser_AF_zpos... method_AutoFocus". These images are laser only and thus clusters are not excited and not seen.

However, some focus images show clusters and have in the file name nomenclature, "Red_IM_zpos... method_AutoFocus_BestFocus". These images show visible clusters when the Z position is correct, allowing for proper focus of the clusters on the flow cell.

Technical Support can evaluate Focus Images to diagnose clustering issues (failed to cluster/ underclustering and overclustering) or an unstable focus model.

• Location of Focus Images:

  • MiniSeq: D:\Illumina\MiniSeq Sequencing Temp[Run Folder]\Images\Focus

  • NextSeq 500/550: D:\Illumina\NextSeq Control Software Temp[Run_Folder]\Images\Focus

Example Focus Images:

This support webinar reviews the end-to-end HiSeq 3000/4000 workflow, patterned flow cell technology, ExAmp clustering tips and tricks, and tips and best practices for running and maintaining the instrument.

Video length: 40:10 min

What are Group Policy Objects (GPOs) and how do they work?

GPOs are a hierarchical infrastructure that allows a network administrator to implement specific configurations for groups of users and computers that are joined to a domain on the network.

Typically, GPOs are security policies that ensure computers on a network adhere to common safety practices. However, this can sometimes perform actions such as installing antivirus or security software, restricting programs from running, or removing permissions that can inhibit instrument operation. Running a GPO Report can provide information on what GPOs and Local Policies have been applied to the control computer on Illumina instruments.

How to generate a GPO report

Important: GPO Reports must be collected from a Command Prompt with elevated permissions. When opening the Command Prompt, select 'Run as Administrator' even if already running as a local Administrator account (this is required).

To generate a GPO report, see the following instructions:

1. In the Windows Search Bar, type 'cmd' then right-click the App and select 'Run As Administrator'

   1. Enter the sbsadmin credentials if prompted.

2. In the Command Prompt window, type gpresult /v /r > C:\Illumina\GPO-Result.txt and press Enter.

3. This will generate a GPO report and output it to a text file in the C:\Illumina folder on the instrument.

If there are any errors that present when running a GPO report, contact Illumina Technical Support.

When sequencing libraries with low base diversity, unbalanced nucleotide composition can negatively impact cluster mapping (on non-patterned flow cells) and template registration (on non-patterned flow cells) along with data quality and data output.

This table lists the percentage of PhiX Control v3 library Illumina recommends spiking in when running low diversity libraries on the indicated sequencing platforms and control software versions.

| Platform | PhiX Aligned (%)† | | iSeq 100 | Minimum 5% | | MiniSeq | 10-50%* | | MiSeq (MCS 2.2 or higher) | Minimum 5% | | NextSeq 500/550 | 10-50%* | | NextSeq 1000/2000 | 10-50%** | | HiSeq 1000/1500/2000/2500 (HCS 2.2.38 or higher) | Minimum 5% | | HiSeq 3000/4000 (HCS 3.3.76 or lower) | 10-50%* | | HiSeq 3000/4000 (HCS 3.4.0 or higher) | 5-20%* | | NovaSeq 6000 | Minimum 5% | | NovaSeq X/X Plus (control software version ≤ 1.2) | 10-20% | | NovaSeq X/X Plus (control software version 1.3) | Minimum 5% |

** For low diversity libraries using NextSeq 1000/2000 Standard P1 and P2 600 cycle kits, at least 32% of reads should align to PhiX for optimal performance. Illumina recommends spiking in 40% to account for potential variation in final % read alignment metric. Depending on the type of low diversity library, this may be reduced after optimization. With XLEAP-SBS 600 cycle kits, there is higher performance when compared to the Standard long read kits, especially at the end of the reads. The below XLEAP results are based on testing performed with P2 XLEAP-SBS 600 cycle kits.

Frequently Asked Questions:

• Can the PhiX Control v3 Library provide improved nucleotide diversity for low diversity index sequences?

  • No, the PhiX Control v3 Library is unindexed and does not balance signals ing index reads.

• Are there other considerations when sequencing low diversity libraries?

Background

The MiSeq i100 and NovaSeq X Series instruments require users login to the Control Software using either a Local or Cloud login. If the instrument is not connected to the internet and the Cloud login is not required, use the following steps to disable the Cloud login feature and only use the Local login.

The Cloud login should only be disabled if the instrument is completely disconnected from an internet connection. This procedure disables all Cloud upload features including both Proactive and BaseSpace features. For more information on Proactive, see the following resources:

Resolution Actions

Disabling Cloud Login

1. Ensure the instrument is idle and there are no Active sequencing Runs or DRAGEN Analyses.

2. Login to the instrument as an Administrator account.

3. Access the Settings Menu:

   1. On the MiSeq i100 Series Control Software Home screen, select the dropdown menu in the top-left corner, then select Settings.

   2. On the NovaSeq X Series Control Software Home screen, select the instrument icon, then select Settings.

4. Select Cloud Settings.

5. De-select all checkboxes under Run settings and Illumina Proactive.

6. Remove any entries in the Private domain name field.

7. Within the Hosting Location dropdown menu, select the blank entry above the BSSH USA (N. Virginia) selection (Figure 1).

   1. The Hosting location selection should be empty after selection (Figure 2).

8. Select Save.

If the Cloud Settings menu presents the message "An unexpected error has occurred. Details: Internal Server Error," see the following steps.

1. Navigate to the Cloud Settings menu.

2. Confirm that all checkboxes are de-selected and the Hosting Location is blank.

3. Select Save, then wait 2 minutes. It is expected to display a 'loading circle' that does not resolve.

4. After 2 minutes, select the X in the top-right corner of the Cloud Settings UI.

5. Navigate back to the Home screen, then select the dropdown menu in the top-left corner, and then sign out of the Admin account.

6. Click the screen to initiate Control Software login and confirm if Local Login presents as the default option.

Figure 1: Highlighting the empty Hosting location selection within the dropdown menu.

Figure 2: Highlighting the empty Hosting location selection.

Re-enabling Cloud Login

1. Ensure the instrument is idle and there are no Active sequencing Runs or DRAGEN Analyses.

2. Login to the instrument as an Administrator account.

3. Access the Settings Menu:

   1. On the MiSeq i100 Series Control Software Home screen, select the dropdown menu in the top-left corner, then select Settings.

   2. On the NovaSeq X Series Control Software Home screen, select the instrument icon, then select Settings.

4. Select Cloud Settings.

5. Select the appropriate regional Hosting Location using the dropdown menu.

6. For Enterprise users, input the Private domain name in the free text field.

7. Select the desired checkboxes under Run settings and Illumina Proactive.

   1. Illumina Proactive - Uploads hardware and run performance data to Illumina Support for troubleshooting purposes.

   2. Cloud run monitoring - Uploads run metrics to BaseSpace for user run monitoring.

   3. Cloud run storage - Uploads run metrics and data to BaseSpace, required for Cloud Analysis.

8. Select Test Configuration and confirm all required URLs are reachable.

   1. Consult the local IT team to add the affected URLs to local network security allow list if any URLs are not reachable.

9. If Test Configuration is PASS, select Save.

To check the .NET Framework version on instruments running Windows Operating Systems, follow the steps below.

1. First, open the windows file explorer and navigate to C:\Windows\Microsoft.NET\Framework. There will be a few folders with "V.x"

2. Open the folder with the highest version (V).

3. Within this folder, right click any of the .dll files and select Properties.

4. Select Details tab; the .NET framework version can be found under File version.

This video shows how to clean and maintain wash cartridges/trays for Illumina sequencers to prevent contamination.

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Video length: 4:41 min

Analyses for runs using a v1 sample sheet can be requeued through the BaseSpace Sequence Hub (BSSH) Web Interface the majority of the time. Instruments that typically use a v1 sample sheet include the iSeq 100, MiniSeq (updated to Control Software 2 and Windows 10), MiSeq, HiSeq 1000/1500/2000/2500, NextSeq 500/550 (updated to NextSeq 500/550 Control Software 4 and Windows 10), and NovaSeq 6000. Illumina recommends using the Local Run Manager software to create sample sheets, as it guides users through the process and will check for errors.

In some instances, an analysis errors out due to communications issues on BaseSpace. If an analysis needs to be requeued using the same sample sheet, follow the instructions below.

Note: Runs that use a v2 sample sheet (for example the NextSeq 1000/2000) must be requeued with the BCL Convert app on BaseSpace and cannot be requeued through the method described below.

Important Notes:

To requeue the analysis using the same sample sheet:

1. After logging in to the BaseSpace account, select the RUNS tab at the top of the page.

2. Select the blue hyperlink of the run to requeue.

3. Select the hourglass icon below the run name, then REQUEUE > SAMPLE SHEET.

1. The sample sheet may be pre-populated in the editing window. If not, select 'Load Original' to load the sample sheet.

2. Once the sample sheet has been validated, select the blue Queue Analysis button.

• If the Queue Analysis button is not active, check for and resolve any validation errors.

1. The status on the run summary page will display "Analyzing" when the analysis is ongoing and will change to "Complete" when the requeued analysis is complete.

If reformatting of the sample sheet is required prior to requeueing the analysis, search for Knowledge Base article How to edit a sample sheet and requeue an analysis in BaseSpace Sequence Hub.

Achieving optimal cluster density is critical to high-quality sequencing on MiniSeq, MiSeq, NextSeq 500/550, and HiSeq 2500 Systems. On nonpatterned flow cells, cluster density has a significant impact on run performance, specifically data quality and total data output. While underclustering can maintain high data quality, it results in lower data output. Alternatively, overclustering can lead to run failure, poor run performance, lower Q30 scores, introduction of sequencing artifacts, and lower total data output. This bulletin summarizes the resources and best practices to avoid underclustering and overclustering, and to achieve more consistent cluster densities.

• Follow the latest instrument-specific guidelines to dilute and denature libraries:
• Best practices for library denaturation:

  • The NaOH stock solution must have a pH >12.5 prior to any dilution. Check the pH of the stock solution before diluting. The recommended stock concentration is found in the appropriate instrument system guide or denature and dilution guide.

  • Always prepare freshly diluted NaOH for denaturing libraries at time of use.

  • To prevent small-volume pipetting errors from affecting the final NaOH concentration, prepare at least 1 ml of freshly diluted NaOH.

  • Follow the denature and dilute libraries guide for your instrument. - For HiSeq 2500 and the MiSeq series, it is important that the final solution of NaOH is not more than 1 mM NaOH after diluting with HT1. Greater than 1 mM NaOH inhibits template hybridization efficiency. - When denaturing libraries for NextSeq 500/550 and MiniSeq, use 200 mM Tris-HCl pH 7.0 to make sure that the NaOH is fully hydrolyzed in the final solution. As a result, template hybridization is not affected even when the final NaOH concentration is greater than 1 mM (see NextSeq 500/550 and MiniSeq denature and dilution guides, above).

• For highly structured or GC-rich libraries, cluster density consistency improves when the library is heat denatured before loading the pool onto the instrument. This method is optional for other libraries.

  • After NaOH denaturation of the sequencing library and dilution in HT1 to the final loading concentration, incubate the diluted library at 96°C for 2 minutes using a heat block.

  • After the heat incubation, invert the tube 1-2 times to mix.

  • Quickly move the library to an ice water bath for 5 minutes. The quick cooling step helps lock the library in its single stranded form.

  • Proceed immediately to cluster generation.

The Universal Naming Convention (UNC) is a standard for identifying servers, printers, and other resources on a network. A UNC network path has the following format:

\servername\share\path

Illumina recommends using the UNC path when setting a network server as the default output location and is a requirement for most Illumina platforms running Windows 10. Use the following steps to identify the UNC path for an existing mapped network location in Windows:

1. In the Windows Search bar, type in cmd and press 'Enter' to open the Command Prompt.

2. In the Command Prompt, type net use and press 'Enter' to run the command.

3. The output will list the local drive letter under the 'Local' column and UNC path under the 'Remote' column for each network resource that has been mapped to that instrument.

Figure 1: Example output of the net use command with the UNC path listed under the 'Remote' column in the output.

Introduction

Instrument control software contains several different Illumina Windows Services

• Illumina Universal Copy Service

• Illumina Local Run Manager Job Service

• Illumina Local Run Manager Analysis Service

By default, the Illumina Windows Services are configured to Log On As a predefined Windows service account called Local System. Successful data transfer from the instrument to a network output location requires that the Illumina Windows Services be reconfigured to Log On As a different local Windows account.

Services are usually reconfigured to Log On As the standard local sbsuser account, which exists on all instruments. You may also choose to run the services using a different account (on-domain or local).

Note: Illumina Universal Copy Service must be configured to log on as a standard (non-administrator) Windows account.

Prerequisites and Requirements

• The Log On As account (typically sbsuser) must have read/write access, or cached credentials with read/write access, to the network storage destination.

  • It must have at least “read only” or “list folder content” permissions to all parent folders within the UNC path.

  • In the example of \server\level1\level2, the account must have access to the “level1” folder.

• Do not use a mapped network drive path (such as Z:).

• Do use the full UNC path to the output folder (with preceding double backslashes and a trailing slash, such as \server\level1\level2\ )

• The network storage destination must have enough storage for the run. The amount of storage needed can vary by flow cell type and instrument. Reference the article Approximate sizes of sequencing run output folders for estimates.

WARNING: Illumina has updated the guidance for configuring LRM and UCS service accounts and now it should be done via the Windows Services if using Local Run Manager v3.xor via the Service Accounts tab directly on Local Run Manager if on v4.x.

If on LRM v3.x:

2. Enter administrator account credentials (by default this will be sbsadmin) in the pop-up window.

3. Locate the Illumina services.

4. Right-click on Illumina Universal Copy Service.

7. Select OK to acknowledge both windows.

8. Enter the password for the selected account (typically the account is sbsuser), select Apply and OK.

9. Restart the service by right clicking and selecting Restart.

10. Repeat steps 4 - 8 for the Local Run Manager (LRM) services below (Note: some LRM modules may need to Log On As an administrator account)

• Illumina Local Run Manager Job Service

• Illumina Local Run Manager Analysis Service

If on LRM v4.x:

1. In Local Run Manager (LRM), configure the Illumina services to use the respective Log On As accounts via the admin account by accessing the Tools menu.

STEP 1: Cache credentials for access to Network Storage

1. Log in to the instrument as the Log On As account configured above (typically sbsuser).

3. Enter the credentials of the desired user who has access to the desired network storage location into the Windows Security prompt. It may be necessary to work with the local/lab IT team for assistance with these credentials.

STEP 2: Verify Write Access to Network Output Location

2. Delete the file just created.

3. If either of the actions above fails, the account does not have write permissions on the Network Output location. Work with the local/lab IT team to configure the Network Output location with appropriate permissions.

STEP 3: Configure output folder to the network

Instrument Control Software

• Set the Output Folder location as full UNC path (with trailing backslash) in the Instrument Control Software.

Local Run Manager

FAQ

1. Changing Windows Account Passwords If the Windows account passwords change, the credential configurations will also need to be updated by repeating this process for all Illumina Windows Services using the new password. Failure to update the password can cause connectivity issues and failures during sequencing runs.

2. How to access the full UNC path of a mounted drive

• Launch the Windows Command Prompt app

• Enter the command net use

• This will display the full UNC path to all network drives mounted on the system

Instruments with Windows 10 OS have the Software Restriction Policies (SRP) Windows feature enabled by default. This security feature blocks programs that are not specifically on the SRP exceptions list. When using TeamViewer QS, an exception must be added to SRP to allow TeamViewer QS to launch properly.

How to disable SRP

1. Confirm that a sequencing run is not in progress because this process could interrupt the run.

2. Log in to the system with the sbsadmin local account (see Note 1 below).

3. Open Windows File Explorer and navigate to the C:\Illumina\Security folder.

4. Double-click Disable.reg and acknowledge the prompts.

5. To reenable SRP, double-click Enable.reg and acknowledge the prompts.

Note 1: For NovaSeq 6000 with serial numbers smaller than A01535, the sbsuser account is part of the Administrators group so no need to switch accounts.

How to add exceptions for TeamViewer to SRP

1. Confirm that a sequencing run is not in progress because this process could interrupt the run.

2. Log in to the system with sbsadmin local account (see Note 2 below).

3. Disable SRP by navigating to C:\Illumina\Security, double-clicking Disable.reg, then acknowledge the prompts.

4. In the Windows search bar, type in secpol and open the Desktop App.

5. In the Local Security Policy dialog box, expand Software Restriction Policies, then select Additional Rules.

6. To add a rule:

   1. On the Action menu, select New Path Rule….

   2. In the Path field, enter the certificate, file name, file extension, or directory that you want to allow.

   3. In the Security level list, select Unrestricted.

   4. [Optional] In the Description field, enter a reason for creating the rule.

   5. Select OK to add the rule.

7. Separately add the following rules according to the previous instructions for both of the following paths:

   1. TeamViewerQS*.exe

   2. C:\Users\\AppData\Local\Temp\TeamViewer\

8. Close the Local Security Policy dialogue box.

9. Reenable SRP by navigating to C:\Illumina\Security, double-click Enable.reg, then acknowledge the prompts.

10. Sign out of sbsadmin account and then sign back in as sbsuser for the settings to take effect.

Note 2: For NovaSeq 6000 with serial numbers smaller than A01535, the sbsuser account is part of the Administrators group so no need to switch accounts.

Figure 1. Double-click Disable.reg to disable SRP.

Figure 2. Select Yes to acknowledge the prompt.

Figure 3. Select OK to acknowledge the prompt.

Figure 4. Open Local Security Policy to add exceptions for TeamViewer to SRP.

Figure 5. Expand Software Restriction Policies and then select Additional Rules.

Figure 6. Select Action and then select New Path Rule...

Figure 7. Under Path select TeamViewerQS.exe and in the Security level select Unrestricted.

Determining the number of reads passing filter (READS PF) is critical for evaluating the overall success of a sequencing run. Here are the step-by-step instructions for determining the number of clusters passing filter for a single lane, a single read, and a run using BaseSpace Sequence Hub (BSSH) or Sequencing Analysis Viewer (SAV).

BaseSpace Sequence Hub

How to access READS PF in the METRICS tab

1. Select a run.

2. Navigate to the METRICS tab.

Figure 1. Metrics tab in BSSH.

1. Locate the Per Read Metrics table.

2. Locate the READS PF column.

• Values are the calculated number of READS PF per lane.

• For a paired-end run, multiply this number by two to calculate the total number of READS PF per lane.

1. To obtain the number of READS PF for the entire run, add the total number of READS PF for each lane.

Figure 2. READS PF values are the calculated number of READS PF per lane. For a paired-end run, multiply this number by two to calculate the total number of READS PF per lane. For example, the NovaSeq run shown here has 2,313125,376 READS PF in Read 1 in lane 1. Because this is a paired-end run, the total number of READS PF in lane 1 is 4,626,250,752.

How to access READS PF in the INDEXING QC tab

1. Navigate to the INDEXING QC tab.

2. Locate the PF READS column per lane.

• To switch between lanes, use the drop-down menu.

1. To obtain the number of READS PF for the entire run, add the total number of PF READS for each lane.

Note: The INDEXING QC metrics are calculated after demultiplexing, and therefore provide a more accurate read count than the metrics tab.

Figure 3. In the INDEXING QC tab, total number of reads passing filter are shown per lane. To switch between lanes, use the drop-down menu.

Sequencing Analysis Viewer

1. Navigate to the Summary tab.

2. The Cluster Count PF (M) column shows the number of reads passing filter in millions per lane and per read.

3. To obtain the total number of reads passing filter per read, add the values for a particular read. In this case, Read 1 contains 4,659,600,000 clusters passing filter.

• Note: Unlike BaseSpace Sequence Hub, Sequencing Analysis Viewer shows metrics by read, not by lane.

Figure 4. In the Summary tab, the Cluster Count PF (M) column shows the number of reads passing filter in millions per lane and read. For example, Read 1 of lane 1 contains 2,313,130,000 reads passing filter. To obtain the total number of reads passing filter per read, add values 2,313,130,000 and 2,346,470,000. In this case, Read 1 contains 4,659,600,000 reads passing filter. The total number of reads passing filter for this run is ~9,319,200,000.

1. To determine the total number of reads passing filter for a run, add all the values in the Cluster Count PF column, excluding non-index reads.

• The precise value is in the Indexing tab under the PF Reads column.

Figure 5. Selection pane on SAV for Indexes Passing Filter

Note: Values are represented slightly differently in BSSH and SAV. In both programs, the PF Reads tabs represent identical values, as do the Reads PF and Cluster Count PF (M) in the summary tabs.

For more information about Sequencing Analysis Viewer or BaseSpace Sequence Hub, visit their respective support pages:

• Sequencing Analysis Viewer

• BaseSpace Sequence Hub