Illumina Knowledge is a repository of 1664 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 to query keywords.

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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.

• HiSeq 1500: 28-FEB-2023 (Link)

• HiSeq 2500: 28-FEB-2023 (Link)

• HiSeq 3000: 28-FEB-2023 ()

• HiSeq 4000: 31-MAR-2024 ()

• HiSeq X Five and Ten: 31-MAR-2024 ()

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.

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.

• Specifications for the iSeq 100 System

• Specifications for the MiniSeq System

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.

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.

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.

For more information, see the Support Page.

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.

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

For further information, see Cluster Optimization Overview Guide.

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.

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.

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.

Illumina next-generation sequencing technology allows for massive parallel sequencing. This support webinar video discusses Illumina library construction, cluster generation methods by platform, sequencing by synthesis, multiplexing, and primary analysis.

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Video length: 41:43 min

The Sequencing Analysis Viewer (SAV) is a software application where users can view quality metrics generated during a sequencing run by Real-Time Analysis (RTA) software on Illumina sequencing systems. RTA is compatible with iSeq 100, MiSeq, MiSeq i100, MiniSeq, NextSeq 500/550, NextSeq 1000/2000, NovaSeq 6000, NovaSeq X Series, and the available Dx versions for some of these instruments.

For viewing these metrics on SAV, the following is needed:

• InterOp folder

• RunInfo.xml

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).

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.

Instrumentation > General > FAQ

Overview

If experiencing issues related to login credentials or service authentication, checking the password history and expiration date for Windows accounts can help identify the root cause. This is especially useful when working with benchtop instruments or software like Local Run Manager (LRM).

Accurate and robust sequencing of on the NextSeq 500/550 and MiniSeq requires well-designed experiments and informatics pipelines. A common example of a low diversity library is an amplicon-based library preparation method, such as 16S metagenomics. These libraries tend to have DNA sequences that start at the same location - the probe binding site - and are mostly identical. A single represented locus causes a biased base composition that can change drastically from cycle to cycle.

The NextSeq 500/550 and MiniSeq systems use . Therefore, it is important to have all 4 DNA bases represented in every cycle, in order for the software to correctly identify DNA clusters and perform accurate base calling. To meet this requirement using a low diversity library, we recommend experimental designs that provide cycle-to-cycle diversity using the following methods:

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

该技术文档提供的信息，能够帮助您计算Illumina测序反应废液中甲酰胺的终浓度。

每个测序反应废液中甲酰胺的浓度会基于测序平台和测序长度而有所差异。您可以根据下表的说明，测量废液的总体积，并计算甲酰胺的终浓度。

*由于iSeq100试剂槽不能打开，因此它是以固体废弃物处理。

**如果剩余甲酰胺没有排入废液中，基于不同测序长度甲酰胺的浓度范围为0.35-1.5%

NovaSeq X/X Plus

The Internet Protocol (IP) and Media Access Control (MAC) addresses for an instrument are often used by IT/Networking teams to identify an instrument on the network and assign special security permissions. These will be unique for each Network Interface Card (NIC) on the instrument. Most Illumina instruments have 2-3 NICs available, generally one for external connections to a network and another for the instrument's internal connections.

To identify the IP or MAC addresses of a particular instrument, use the Command Line in Windows or the Terminal in Linux.

**In Windows Operating System (OS) Environments:**1. In the search bar in the bottom left corner, type in cmd and then press enter to open the Command Line interface. 2. In the Command Line, type in ipconfig /all and press enter. 3. The printout will display the IPv4 and Physical Address for each NIC, which are the IP and MAC addresses, respectively.

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.

The country of origin and date of manufacture of Illumina instruments are printed on the label placed on the back of the instrument.

In this video, we discuss IT requirements for implementing Illumina Proactive, an instrument performance monitoring service provided by Illumina that can increase instrument uptime, improve operational efficiency, and reduce the risk of lost resources.

Learn more about Illumina Proactive .

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Video length: 4:07 min

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.

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

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

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

The interactive catalog 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.

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

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

In the event of a power outage, an Uninterruptable Power Supply (UPS) sustains power to the instrument for a brief period of time. If the UPS fully discharges, or no UPS is connected, the sequencer will lose power and, if there is a run in progress, the run will be aborted.

When the power is restored, follow guidance in the instrument's system guide to power the system back on.

• If the instrument was idle at the time power was lost, no additional actions are needed, provided that the control software initializes successfully.

• If a run was in progress when power was lost, the run terminates, the data and reagents cannot be recovered, and the run cannot be resumed. After powering up the sequencer, perform a manual post-run wash before the next sequencing run.

This support webinar video is ideal for users interested in considerations for the sequencing of low diversity libraries. The video discusses the following topics: understanding and visualizing base diversity in Sequencing Analysis Viewer (SAV), strategies to optimize low diversity sequencing data, % PhiX spike-in and cluster density recommendations for different sequencing platforms, and an overview of base calling and quality score calculations.

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Video length: 35:54 min

On December 10, 2021 Illumina was made aware of a vulnerability in the Apache Log4j software suite (CVE-2021-44228, CVE-2021-45046, and CVE-2021-44832). This software component is a Java-based logging utility and part of the Apache Logging Services Foundation products.

After we became aware of the issue, we launched an investigation to identify potentially affected products and assess risk and found the Illumina ProActive Portal to be unaffected.

Illumina takes data privacy and security issues very seriously, and we hope this information helps alleviate any concerns about this vulnerability. If you have any questions, contact .

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.

Gel packs are not fully frozen. Is this an issue? Gel packs have been validated to maintain temperature for transit durations based on historical data and may not be frozen upon delivery. For any specific concerns or questions about the integrity of the product(s) ordered, contact Illumina Technical Support.

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.

Why do some shipments include both foam containers and sustainable containers? Shipping container selection depends on the site shipping the product and the availability of locally sourced materials. Illumina continues to increase the percentage of sustainable insulated containers as Illumina is committed to reducing environmental impact. See the for more information.

The packaging is damaged. Is a replacement product required? Most packaging damages are minor and do not pose a risk to the product. Outer packaging has been designed to take on impact to protect the product inside. For specific concerns or questions about the integrity of the product(s) ordered, contact . Examples of acceptable conditions include:

• 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.

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).

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

Resources

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

• As recommended in the Illumina Security Best Practices Guide, RDP should be disabled on all Illumina instruments.

• More details about the BlueKeep vulnerability can be found here.

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).

The security of your data and systems is paramount to Illumina. If you have additional questions regarding security recommendations from Illumina or about RDP on your Illumina systems, refer to the page on the Illumina website or contact [email protected].

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:

• MiniSeq and NextSeq 500/550 runs set up using the BaseSpace Sequence Hub PrepTab require different steps to edit run setup and .

• Only the owner of the run can requeue an analysis. It is possible to of a run, if necessary. A does not provide the necessary permissions needed to requeue analysis.

• There is a limit of five requeue attempts for users of BaseSpace Sequence Hub. If assistance with additional requeues is needed, email and share the run ID and sample sheet. To find the run ID, open the run in BaseSpace, and look in the web browser address bar for the numerical value after:

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.

Illumina平台和测序试剂盒支持多种 测序应用 。 为了保证最高水平的测序质量，Illumina不同测序平台以及不同的SBS试剂盒版本支持的最大读长有所不同，相关信息请参阅 我所购买的SBS试剂盒最多能够满足多少个循环的测序？ 技术文档。利用本地运行管理软件（ Local Run Manager，LRM）或仪器控制软件（Control Software）设置测序运行时，可以选择更长的读长。但是当read长度超过支持的长度时，Illumina将无法保证较高的数据质量。

表 1. 测序平台和SBS试剂盒支持的最大读长

* MO: 中通量 / HO: 高通量

Index Reads的最大读长

• 仪器控制软件：如表2所示，可在某些仪器的运行设置期间输入自定义Index读长：

• LRM v2：Index读长最多可以设置100个字符（请参阅：技术文档）。

• LRM v3：最多输入20个字符。如果需要20-100 bp读长，请选择手动上机模式。

• LRM v4：最多输入20个字符。如果需要20-1000 bp读长，请选择手动上机模式。

• 为确保Index Reads的测序质量，请勿超过Index支持的最大长度。

表 2. 测序平台的最大单个Index长度

*通过云端设置测序运行时，Index长度会受到以下列出的 BCL Convert 限制。 **Index长度为20-1000bp之间时，通过手动模式设置测序运行，因为LRM V4的上限为20bp。 ***手动设置测序运行不在测序仪进行分析的话，Index长度不受软件限制。

请注意，BCL Convert对Index长度是有限制的。BCL Convert 4.2以及之前版本，Index总长度不能超过27bp。 BCL Convert 4.3以及之后版本，Index长度限制提高到每个Index最长27bp。

如果使用 BCL Convert 设置override cycle，那么在Index读取中指定的用于 UMIs（U）的碱基将不会计入Index长度的计算中。

1- Is there any preparation required for the upgrade?

A: We recommend that users inform their organization's IT team regarding the upgrade to ensure rapid connection back to their network. A pre-upgrade checklist will be provided when the upgrade is scheduled.

1. Is there a way to check whether my instrument is on Windows 7 Professional or Windows 7 Embedded?

A: In the Windows 7 operating system, go to Control Panel > All Control Panel Items > System. Windows edition will show as Windows 7 Professional or Windows 7 Embedded.

1. Where are the Extended Security Update (ESU) licenses for Windows 7 Professional available?

A: See our page for more details.

1. Will there be any impact on the instrument if it stays on Windows 7?

A: While instruments will continue to function normally, we encourage the upgrade to Windows 10. Using an unsupported operating system increases the risk of exposure to malware. If users choose to remain on Windows 7, they should take appropriate steps to minimize exposure.

1. Who is responsible for upgrading the instrument?

A: When ready to upgrade an instrument, reach out to Illumina Technical Support or to your local field team to schedule a visit from an Illumina Field Service Engineer who will perform the upgrade.

1. Can I upgrade the instrument on my own?

A: No, the upgrade requires a visit from an Illumina Field Service Engineer.

1. Is there a cost to upgrade an instrument under a service contract?

A: No. Instruments under an active service contract of any tier will have their upgrade covered.

1. What is the cost to upgrade an instrument that is not under a service contract?

A: The cost will vary per instrument. Reach out to Illumina Technical Support or your local field team to request a quote.

NOTE: Microsoft has announced that standard retail versions of Windows 10 will reach End of Life (EoL) in October 2025. However, t****his does not apply to Illumina instruments, see for more information.

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.

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.

If an instrument is located in an area that is expected to be affected by a natural disaster (hurricane, tornadoes, storms, etc), see the following instructions to prepare them:

1. Perform washes as instructed in the Instrument maintenance and shutdown procedures for extended site closures

2. Power off the instrument via normal processes in the instrument system guide.

3. Unplug the instrument from the power source (remove the power cord).

Upon return to work:

1. Plug the power cord back and turn the instrument on.

2. Perform two maintenance/manual washes to rehydrate the fluidics system.

3. Perform a system check following the instrument system guide.

If system checks fail or any other errors are observed, contact .

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

For further information, see Cluster Optimization Overview Guide.

In some cases, new index kit offerings will be made available for existing library preparation kits. Index kit files (pre-existing index kit files) can be added to existing Library Prep kit options in Local Run Manager. Use the steps below to import a pre-existing index kit into Local Run Manager (LRM) v3 or v4.

1. Save the desired index kit locally on the instrument.

2. Open Local Run Manager by navigating to https://localhost/#/ via the Chromium Web Browser on the instrument.

3. Select Tools.

4. Select Index & Library Prep Kits.

5. Select the tab for Index Kit.

1. Select Add Index Kit on the right-hand side.

2. Browse to the index kit saved on the instrument and select Open.

For information on creating a custom library prep kits/index kits to import into Local Run Manager v3 and v4, see Knowledge article.

If additional assistance is needed, .

A PhiX validation run confirms proper hardware and software performance of the instrument. The Illumina PhiX control library is a well-balanced genome with relatively equal representation of A, T, G, and C nucleotides. PhiX lacks an index and is not an appropriate tool for assessing Index Read performance.

PhiX validation runs can be set up with Local Run Manager on instruments with the Local Run Manager software suite bundled for on-instrument use. These instruments are the iSeq 100; MiSeq with MiSeq Control Software v3 and v4; NextSeq 500/550 with Control Software v4; and MiniSeq. Note that MiniSeq instruments with MiniSeq Control Software v1 have Local Run Manager v1.3, while MiniSeq instruments with Control Software v2 have Local Run Manager v2. Instruments running MiSeq Control Software v3 have Local Run Manager v2 and MiSeq Control Software v4 has Local Run Manager v3.

1. Launch the Chromium browser and enter http://localhost in the address bar. If User Management is enabled in Local Run Manager v2 or v3, the login page will appear, requiring you to enter the user ID and password. If User Management is disabled, the dashboard will appear. Version 1.3 of Local Run Manager for MiniSeq always requires a login.

2. Select Create Run and choose Generate FastQ from the drop-down list.

3. Enter the following values in the highlighted fields:

• Run Name: PhiX validation run

• Library Prep Kit: Custom

• Index Reads: 0 (Note: PhiX is unindexed and selecting 1 or 2 Index reads can cause the run to terminate prematurely)

1. Keep default values in the Module-Specific Settings field.

2. Scroll to the bottom of the Create Run page. In the Import Samples table, type PhiX in the Sample ID field.

3. Select Save Run.

The run time specifications for Illumina sequencing runs include cluster generation, SBS chemistry cycles, paired-end chemistry, and wash steps. In addition, runs that use non-patterned flow cells pause for a template building step. To aid in planning sequencing workflows and in estimating overall run times, Table 1 summarizes the estimated length of each sequencing step for Illumina sequencing platforms and chemistry versions.

Table 1. Approximate times for each sequencing step per instrument and supported chemistry

* Template building time will vary based on cluster density ¤ XP workflow is approximately 150 minutes as ExAmp step is skipped

Cycles 2-5 on these runs can take up to 25 minutes each

**Purge time varies based on the number of cycles

Estimated run times will depend on cluster density and available computer resources. If the hard drive does not have sufficient space for data processing, or network location speeds are slow, run times may be prolonged. Total run times for the maximum read lengths of Illumina platforms, and other sequencing run specifications, can be found on the web page.

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 .

Illumina has recently discovered that use of ammonia-based cleaners or sanitizing products near sequencing run setup (lab benches, pipettes, etc.) can result in decreased sequencing run performance metrics. Decreased or variable cluster densities and low starting intensities have been seen by customers.

The mechanism by which this effect occurs may be linked to the effect of ammonia on DNA structure (Zinchenko et al 2004).

If you are experiencing variable cluster density (on nonpatterned flow cells), variable percent clusters passing filter (for both patterned and nonpatterned flow cells), or lower than expected starting intensities on your sequencing runs, make sure that ammonia-based cleaners are not used in the lab environment. Inspect ingredient lists of cleaners or antibacterial wipes for ammonia or quaternary ammonium compounds such as benzalkonium chloride (alkyldimethylbenzylammonium chloride) and dialkyldimethylammonium chloride.

If ammonia-based cleaners have been used, Illumina recommends:

• Extensively clean equipment and surfaces that have been in contact with the ammonia-based cleaners. To clean, use an anionic detergent such as 1% SDS or diluted dish soap, followed by water.

• Use pipettes that have not been in contact with ammonia-based cleaners when setting up clustering and/or sequencing runs.

Additional Resources:

Maximum supported read length

Illumina platforms and sequencing kits allow for a variety of sequencing applications. To ensure the highest level of quality, Illumina supports reads up to a certain length depending on the sequencing platform and SBS kit version; refer to How many cycles of SBS chemistry are in my kit?. It is possible to choose a longer read length during run setup in Local Run Manager (LRM) or the instrument control software. However, when the read length exceeds the supported length Illumina cannot guarantee maintaining high data quality.

Table 1. Maximum supported read length for sequencing platforms and SBS reagent kits.

* MO: Mid-output / HO: High-output ** Rapid Run

Maximum read length for index reads

• Instrument control software: enter custom Index Read length during run setup for certain instruments, as shown below in Table 2.

• LRM v2: enter up to 100 characters for the maximum index cycles (see for additional information on custom libraries with Local Run Manager).

• LRM v3: enter up to 20 characters, or use manual mode to allow for between 20 and 100 bp.

• LRM v4: enter up to 20 characters, or use manual mode to allow for between 20 and 1000 bp.

• To make sure sequencing quality of the Index Read remains as expected, do not exceed the supported read length.

Table 2. Maximum individual index length for sequencing platforms.

*When setting up runs in Cloud mode, index length is limited by the BCL Convert limitations listed below.

**For index length between 20-1000 bp, use manual mode as LRM v4 is limited to 20 bp.

***index length is not limited for runs set up in manual mode without analysis

Note: BCL Convert has its own index limitations. BCL Convert 4.2 or lower is limited to 27 index cycles total. For BCL Convert 4.3 and higher, the index length has been increased to 27 cycles for each index read.

If using the bclconvert override cycle settings, bases specified for UMIs (U) in the index reads will not count for the maximum index length.

Below is a comparison of Illumina's Qualification Service Offerings. More information can be found at .

Provides documented verification that the instrument is installed according to our specifications and safety regulations. During the IQ, a trained engineer confirms that the latest supported firmware and software versions were installed, verifies instrument setup and accessory logistics, checks that physical and environmental safety conditions are met, and provides a signed, audit-ready, digital document.

Follows a comprehensive, well-defined protocol to make sure that the system is functioning according to our preset and validated operational specifications. The OQ protocol was developed and validated in Illumina labs and is updated after each instrument hardware and software release, so you receive the most up-to-date service. Critical aspects of the OQ include motion, optics, fluidics, and thermal qualifications.

Follows a comprehensive, well-defined protocol to make sure that the instrument is functioning according to our preset and validated performance specifications. The PQ protocol was developed and validated in Illumina labs and is updated after each instrument hardware and software release, so you receive the most up-to-date service. Critical aspects of the PQ include a PhiX data run (including projected yield total), data quality, and any additional comments.

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.

For more information on the importance of base diversity on Illumina sequencing platforms, refer to the bulletin

To compensate for low base diversity in libraries, Illumina recommends spiking in the PhiX Control v3 Library (catalog number , commonly referred to as “PhiX”) for sequencing. The PhiX Control v3 Library has a diverse base composition (45% GC and 55% AT) that provides the balanced fluorescent signals that low diversity sample libraries lack during each sequencing cycle. This, in turn, assists with cluster mapping/template registration and improves overall run performance.

For more information about using PhiX Control v3 Library, refer to the bulletin

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.

Background

Illumina announced the end of life (EOL) that includes the sale and support for the MiSeq RUO system, certified pre-owned MiSeq RUO system, MiniSeq system, iSeq 100 system, and associated consumables in a Product Obsolescence Notification (PON) sent to customers on 27-MAR-2025.

This PON was distributed to North, Central, and South America, Europe, Africa, Middle East, and parts of Asia.

Key Dates

EOL Announcement: 27-MAR-2025

Instrument End of Sale (EOS): 30-SEP-2025

The Local Run Manager software is an integrated solution for recording samples for a run, specifying run parameters, monitoring run status, performing data analysis, and viewing results. Important note: The directions in this Knowledge article apply to Local Run Manager versions up to 4.X

This article describes how to requeue analysis for a run in Local Run Manager with the same module, and how to import data to analyze with a new module.

To requeue with the same module:

1. From the Local Run Manager dashboard, select Actions next to the run you wish to requeue. Select Requeue.

Background

Each Illumina platform has either a Windows or Linux OS environment that the Control Software will run in. The following lists available Illumina platforms and the associated OS version according to instrument platform or Control Software version, where applicable.

All current versions of the Windows and Linux OS on Illumina platforms use the Long Term Support (LTS) versions which have extended service lifecycles.

Illumina Sequencing Platforms

• iSeq 100 platform: Windows 10 Enterprise 2016 LTSB, Version 1607

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.

When pooling libraries for sequencing on the NextSeq 500/550 and MiniSeq systems, it is important to select compatible index combinations, otherwise, the Index Read sequencing can fail due to cluster registration failures. This bulletin provides guidelines for pooling Illumina libraries on the NextSeq 500/550 and MiniSeq systems. 2-Channel Sequencing

NextSeq 500/550 and MiniSeq systems use two images to determine all four base calls, one red channel and one green channel. Rather than using a separate fluorescent dye for each base, 2-channel sequencing uses combinations of two fluorescent dyes: red for C, green for T, green and red for A, and no dye for G.

Figure 1: Two-channel SBS fluorescent imaging (false color image of the dyes is shown for illustration purposes). Accelerated detection of all four DNA bases is performed on the MiniSeq and NextSeq 500/550 series systems using two images to capture red and green wavelength bands. Clusters seen only in red or green images are identified as C and T bases, respectively. Clusters observed in both red and green images are identified as A bases, while unlabeled clusters are identified as G bases.

Index Considerations

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, MiSeq i100, NextSeq 1000/NextSeq 2000, NovaSeq 6000, and NovaSeq X 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.

See the following for further details:

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.

See and follow the web page for further updates.

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

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.

General antivirus (AV) and other Security information is available under the Antivirus Software and Configuration Guidelines section of the . The whole of this document also details general instrument control computer best practices that should be considered in addition to recommendations for AV software configurations.

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.

Illumina Product Support Services Plans help maximize performance and productivity with reliable, high-quality results at various cost-effective levels.

A standard 1-year base warranty is included with every new Illumina instrument purchase, along with installation* and basic applications trainings. Illumina also offers several tiered Service Plans to upgrade the base warranty to an enhanced service level or extend service coverage beyond the 1-year warranty.

1-Year Base Warranty

• Instrument repair parts, labor, and travel.

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 MiniSeq and NextSeq 500/550 platforms

在NextSeq500/550 和MiniSeq测序系统上对混合文库进行index测序时，非常重要的一点是要选择合适的index组合，否则可能会因为在读index时簇定位失败而导致index测序失败。本篇bulletin将针对NextSeq 和MiniSeq系统提供样本混合方针。

双通道测序

NextSeq 500/550和MiniSeq 测序系统只需要2张图片就可以区分4种碱基：一张图片来自红光通道，另一张图片来自绿光通道。不同于每种碱基各带一种单独的荧光标记的四通道测序，双通道测序使用两种荧光染料，C碱基标记红色，T碱基标记绿色，A碱基同时标记红色和绿色，G碱基没有荧光标记。

图1：双通道SBS荧光成像（假彩色图片仅是效果图）。为了提高4种DNA碱基的检测速度，MiniSeq和NextSeq500/550只用了2个图像分别捕捉红色和绿色波段的信号。仅在红色或绿色图像检测到的cluster分别被转译为C和T碱基。同时在红色图像和绿色图像被检测到的cluster被转译为A碱基，在两张图片中都没有检测到信号的cluster将被认为是G碱基。

簇密度是决定测序中能否获得最佳的测序质量和数据量的重要因素。下表是根据平衡文库（例如phix 文库）的结果，列出的各平台原始簇密度推荐值：

When setting a network location as the default output location on instruments that are running Local Run Manager and Universal Copy Service (UCS), there are specific requirements that must be met for UCS to recognize the path as valid.

• The path must be the fully qualified Universal Naming Convention (UNC) path.

  • Example: \server\share_folder\directory\

(BSSH) is the Illumina cloud-based platform for data management, storage, and analysis. There are three BaseSpace Sequence Hub subscription tiers - Basic, Professional, and Enterprise. The feature for the Professional and Enterprise tiers gives BaseSpace Sequence Hub users access to pooled resources. A workgroup consists of users who share data, storage space, and other resources. Only the workgroup administrator (admin) can add users to a workgroup, as described in the steps below.

1. Log into BaseSpace Sequence Hub. Switch to the workgroup by selecting the user name in the top right corner and select the workgroup. Select the user name again and select Settings. Then select manage workgroups.

Illumina guarantees that all Research Use Only (RUO) reagent products will ship with a minimum viable shelf-life of three months from the date of shipment. Actual shelf-life may be longer depending on date of manufacturing relative to shipment date. Always use reagents within the expiration date listed on the reagent packaging for optimal performance.

The following reagents are exceptions to this guideline and each have a minimum of six months of shelf-life from the date of shipment:

• NovaSeq X Series 1.5B, 10B, and 25B reagents

Sequencing Analysis Viewer (SAV) Software is an application where users can view important quality metrics generated during sequencing runs. This support webinar video provides a guided tour for beginners on how to use SAV, as well as tips and tricks for reviewing the most useful information for sequencing runs. The following topics are covered: how to load data into SAV, what metrics are available in each tab of the software, and understanding which are the most valuable metrics for run review and where to find them.

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Video length: 32:46 min

The %Occupied by %Pass Filter plot allows users to optimize the loading concentration for NovaSeq 6000 and iSeq 100. The %Occupied and %Pass Filter metrics can be plotted in Sequencing Analysis Viewer (SAV) to determine if a run was underloaded, optimally loaded, or overloaded. This tutorial covers how to create the plot and how to interpret it.

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Video length: 1:57 min

This information is also available in written form. For further information, search for Knowledge Base article Plotting %Occupied by %Pass Filter to optimize loading concentration for NovaSeq 6000 and iSeq 100 platforms.

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: .

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.

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.

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.

NextSeq 1000/2000

• The flow cell MUST NOT be cleaned.

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

• Reference .

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 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.

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: .

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.

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.

Background

Windows Credential Manager is a built-in tool within Windows 10 that is used to store network credentials used when authenticating connections to network storage. Adding network credentials to Credential Manager is a critical step for ensuring that Local Run Manger and Universal Copy Service have appropriate access to network storage.

If the network credentials change, see the following steps to update them within Credential Manager and restore network storage access.

Resolution Actions

1. Ensure that the user is logged in as the account that is used for sequencing, typically sbsuser.

2. In the Windows Search bar, input Credential Manager and then select the Credential Manager application (Figure 1).

3. Select Windows Credentials (Figure 2).

4. Under the Windows Credentials header, locate the mapped network storage connection, then click the down arrow to expand the selection (Figure 3).

5. Select Edit (Figure 3).

6. In the User name and Password text boxes, input the updated network credentials as necessary (Figure 4).

7. Select Save.

8. Close the Windows Credential Manager application.

9. Power cycle the instrument to refresh the mapped network connection.

Figure 1: Credential Manager application in the Windows Search menu.

Figure 2: Windows Credential tab with the Windows Credentials header showing the mapped network drive.

Figure 3: The down arrow used to expand the selection and the Edit button to update the credentials.

Figure 4: The Edit Windows credential user interface (UI) showing the User name and Password text boxes.

On December 10, 2021, Illumina was made aware of a vulnerability in the Apache Log4j software suite (CVE-2021-44228, CVE-2021-45046, and CVE-2021-44832). This software component is a Java-based logging utility and part of the Apache Logging Services Foundation products.

After Illumina became aware of the issue, we launched an investigation to identify potentially affected products and assess risk and have the following update:

The scope of products currently evaluated:

• iSeq 100

• MiSeq

• NextSeq 500/55

• NextSeq 1000/2000

• NovaSeq 6000

• HiSeq 1500/2500

• HiSeq 3000/4000

• HiSeq X

• iScan

Status of evaluation:

• For all models other than HiSeq series: the base shipping configuration is not affected.

• For all HiSeq series models: the base shipping configuration is mitigated.

• For all models: certain software installations and configurations may introduce affected components.

Known Affected Components:

• Illumina Local Run Manager (LRM)

  • This optional software module ships with an optional subcomponent, the Genome Analysis Tool Kit (GATK, MIT**),** which contains an affected version of log4j v.1.x.

  • This component is not accessible remotely, requires authenticated console access, and requires a measurable amount of preparation to execute a successful attack.

Illumina takes data privacy and security issues very seriously, and we hope this information helps alleviate any concerns about this vulnerability. If you have any questions, email .

若希望低多样性文库在NextSeq500/550和Miniseq平台上获得精确和稳定的测序结果，需要精心设计的实验以及良好的生信分析。低多样性文库最常见的例子是基于扩增方法制备的文库，例如16S扩增子文库。这些文库扩增引物结合的位置，即起始位点的DNA序列基本是相同的。单一的位点会导致碱基组成不平衡，以至于碱基组成从一个循环到下一个循环可能会发生剧烈变化。

NextSeq500/550和Miniseq系统使用的是双通道测序技术。因此，确保每个循环中均存在4种DNA碱基是非常重要的，这样分析软件才能正确鉴定DNA簇并精确读取碱基序列。使用低多样性文库时，为了满足这种要求，我们建议在设计实验时，采用以下几种方法保证每个循环的多样性。

• 利用标签区分技术，在测序中加入多个带有标签的来自多种应用的样品。

o 那些来自多样性较高的应用的带有标签的样品，例如人类扩增子测序、富集或者全基因组测序文库，都能够用来增加文库碱基多样性。

o 为了最佳测序结果，可以利用单标签或者双标签技术，将多个样品在同一张Flow Cell上进行测序。

• 在测序中掺入

o 可以加入50% 为初始掺入比例，然后根据一级分析和二级分析的结果，逐步降低PhiX掺入比例。掺入这样的样品能提供每个循环必需的碱基多样性。

• 对于NextSeq和MiniSeq系统，建议测序时簇密度低于平衡库(如PhiX)建议的最佳簇密度的30-40%。

o Miniseq试剂建议平衡文库的原始簇密度最佳范围为170-220 K/mm2。

o NextSeq500/550试剂建议平衡文库的原始簇密度最佳范围为170-220 K/mm2。

本文上述中的这些建议能够使得NextSeq500/550和Miniseq平台进行低多样性文库测序。

Overall sequencing run performance is evaluated by determining whether the sequencing run meets the Illumina specifications for quality scores and data output. Actual run performance will vary based on sample type, quality, and clusters passing filter. Specifications are based on the Illumina control library at .

Where can I find instrument specifications?

Follow the links below to the instrument specification pages:

| | | | | | | |

The (SAV) is a free software used to assess the performance of sequencing runs, and can be downloaded from the Illumina website:

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).

How to access READS PF in the METRICS tab

1. Select a run.

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:

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

Power cycling resets the instrument computer, memory usage, and software programs. A power cycle consists of shutting down the instrument and computer, leaving the system off for two to five minutes, then restarting the instrument and computer. The instrument contain recommendations for properly power cycling.

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

For NovaSeq 6000

On the NovaSeq 6000, the Illumina Universal Copy Service runs as the currently logged in account. To perform sequencing, always login with a standard (non-administrator) account.

To verify you are sequencing using a standard account:

• Login to the instrument using the account you plan to perform sequncing with

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.

• Quality check and accurate quantification of libraries is critical. If not performed, it is the most common cause of inconsistent cluster density. Always follow methods listed in the bulletin to check Illumina libraries.

Background

The PhiX control library is a well-balanced, high diversity library that can be used as a positive control during Sequencing Runs on Illumina platforms. However, due to an average insert size of 375 bp and total library size of 500 bp, the current PhiX v3 product is not compatible with Reads longer than ~350 cycles.

The new PhiX Indexed Control (1000 Cycle) formulation addresses this by increasing the insert length, enabling users to spike-in PhiX control when performing high cycle count Runs.

Technical Details

• Insert Size

Summary A remote code execution vulnerability exists when the Windows Print Spooler service improperly performs privileged file operations. An attacker who successfully exploits this vulnerability could run arbitrary code with SYSTEM privileges. An attacker could then install programs; view, change, or delete data; or create accounts with full user rights.

Illumina recommends that customers immediately disable their printer spooler service using the instructions in the Workaround section below.

As of July 7, 2021, the security updates for Windows Server 2012, Windows Server 2016, and Windows 10, Version 1607 have been released. Refer to the Security Updates table in for the update applicable to your system.

Microsoft has released security updates to address this vulnerability. Illumina is evaluating the impact of the official Microsoft patches on the performance of Illumina Windows-based products. Until that impact testing is complete, Illumina recommends that customers immediately disable their printer spooler service (see Workaround section below).

When contacting Illumina Support for assistance, Illumina Support can efficiently access the relevant Instrument Performance Data via one or more of the following applications:

• : Select Send Instrument Performance Data to Illumina in the control software before starting a run. Provide the Run ID to Illumina Support to facilitate investigation.

• : Share the run using the Share dropdown menu, then select Get Link to generate a URL to send to Illumina Support.