Illumina NextSeq 1000 and 2000 | Run Quality and Best Practices

Name: Illumina NextSeq 1000 and 2000 | Run Quality and Best Practices
Uploaded: 2022-07-11T16:25:50.000Z
Duration: 1 h 28 min 37 s

Introduction to the Webinar

The webinar introduces the topic of run quality and best practices for the NextSeq 1000 and 2000 instruments. The learning objectives are outlined, including understanding the unique features of these instruments, providing tools for handling unexpected situations during run setup, and evaluating run performance.

System Overview

The NextSeq 1000 and 2000 systems are designed for high-throughput sequencing applications with increased sample throughput and scalability.

The pattern flow cell technology allows for higher data density and yield by utilizing nanowells with shorter distances between them.

Two-channel sequencing by synthesis chemistry with blue and green dyes improves resolution and enables higher data density.

Comparing the NextSeq 500/550 to the NextSeq 2000, there is a three times increase in data yield on the latter platform.

The main difference between the NextSeq 1000 and 2000 is the highest possible output, with the option to upgrade from NextSeq 1000 to XD2000.

Pre-run Considerations

This section covers important considerations before starting a sequencing run on the NextSeq 1000/2000 system.

Thawing Reagent Cartridge

Thawing directions for sequencing reagents are provided in the system guide.

The cartridge may rattle even if it has been thawed correctly due to integrated fluidic components.

Thawing methods include using a controlled water bath at 25 degrees Celsius or thawing via refrigerator over an extended period.

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Denature and Dilute Strategy

This section discusses the denature and dilute strategy for performing mini runs manually.

Onboard Denature and Dilute Checkbox

If you plan to perform mini runs as manual denature and dilute, you can deselect the onboard denature and dilute checkbox from the settings menu and click save.

Confirming or Changing Denature and Dilute Strategy

You have the opportunity to confirm or change the denature and dilute strategy from the run review screen.

If you are planning to perform a single run with onboard denature and dilute disabled, manually change the setting in the run setup screen.

Ensure that all settings within the run review screen are consistent with your planned run setup before beginning pre-run checks.

Validated Loading Concentrations

The system guide provides a table of validated loading concentrations for several library types.

If utilizing onboard denature and dilution strategy, first dilute the library to two nanomolars using RSV with Tween20 as a diluent.

Then, dilute this two nanomolar library to the validated loading concentration listed in the guide. For example, 7.8 microliters of two nanomolar Phi X would be combined with 16.2 microliters of RSV with Tween20 for a final loading concentration of 650 picomole.

Manual Denaturing Dilution Procedure

If your library is very low concentration and does not meet recommended requirements for onboard denaturing dilution, consider performing a manual procedure.

First, dilute the library to desired starting library concentration.

Second, denature the library with fresh 0.2 normal sodium hydroxide.

Finally, dilute the denatured library to the final loading concentration and transfer the full volume to the cartridge reservoir.

Custom Primers

This section discusses the use of custom primers and best practices for assigning them.

Assigning Custom Primers

Custom primers were enabled on the NexSeq 1000/2000 starting with control software version 1.2.

The control software allows manual selection of any combination of custom primers assigned to a given well.

Read 1 and index 1 are on the same strand, while read 2 and index 2 are also on the same strand.

It is recommended to assign read 1 and read 2 custom primers to one well, and index 1 and index 2 custom primers to another well.

Alumina Primers

When using custom primer wells, alumina primers within the cartridge will not be delivered to your sequencing run.

If including fiax in your library, you can spike in Illumina NextSeq 1000/2000 read and index primers into your custom primer wells if necessary.

Obtaining Remote Support

This section explains how to obtain remote support for technical issues.

Technical Support Availability

Technical support is available by phone and email 24 hours a day, five days a week.

For screen sharing troubleshooting sessions, a TeamViewer session is required.

If TeamViewer is uninstalled or not present, a technical support representative can assist with reinstalling it.

Initiating TeamViewer Session

To initiate a TeamViewer session, launch the application and provide the ID and password to your tech support scientist.

Illumina Proactive Support

This section discusses the Illumina Proactive Support feature.

Sensors for Performance Degradation

The NexSeq 1000/2000 system has over 20 sensors on board to detect performance degradation.

These sensors help identify alerts that assist in performing maintenance and minimizing downtime.

Software Programs and Output Folder

This section explains the software programs on the system and setting the default output folder.

Control Software

The NexSeq 1000/2000 control software controls instrument operation and allows entering run parameters.

The output from the software is raw images, which are analyzed by RTA 3 to convert them into quality scored base call files and filter files.

Universal Copy Service

Universal Copy Service copies the converted files from the local run folder to the output folder, including BaseSpace if applicable.

Default Output Folder

The default output location is determined within the control software settings menu.

It can be modified for single runs from the run setup menu.

An external hard drive or network drive is recommended for your output folder, while using the instrument hard drive should be avoided.

New Section

This section discusses potential error notifications that may be encountered when mounting the output folder in the control software.

Potential Error Notifications

The selected folder is invalid, and a different folder needs to be specified. This can occur if the persistent folder is not set. In this case, power cycling the system is recommended.

Syntax errors in the f-stab file or server permission issues can also cause this error. It is advised to review the f-stab file for syntax errors or consult with the IT department for server permission issues.

Another potential cause could be that the output folder is not two folders deep from the SharePoint. To resolve this, update the output folder to be two folders deep.

New Section

This section continues discussing potential error notifications when mounting the output folder in the control software.

System Does Not Have Sufficient Permissions

Improper configuration of the group ID and fstab file can cause this error. Ensure that group ID "ilm" in users or group level has read, write, and execute permissions.

New Section

This section provides information on resolving errors related to setting up run analysis on NexSeek 1000/2000.

Run Setup Options

Four options are available for planning and analyzing sequencing runs: local mode, cloud run mode, hybrid run mode, and instrument run setup.

The choice of run setup mode depends on factors such as run setup location and analysis location.

Local mode requires run setup via a v2 sample sheet and analysis on a local dragon.

Cloud run mode requires run setup in BaseSpace instrument run setup with analysis performed in BaseSpace Dragon.

A flowchart helps determine the best run setup mode based on internet connectivity and analysis location.

New Section

This section provides further details on run setup modes for NexSeek 1000/2000.

Run Setup Modes

Internet connectivity determines whether the run is a cloud run mode or a hybrid run mode.

Cloud run mode and hybrid run mode require planning runs within instrument run setup in BaseSpace.

Instrument run setup can be accessed by logging into BaseSpace Sequence Hub, selecting "Runs," and choosing "Instrument Run Setup."

Note that DAB is only compatible with older versions of NexSeq 500, 550, and MiniSeq platforms. Runs planned in the Prep tab will not be available for sequencing on NexSeek 1000/2000.

New Section

This section explains how to select the instrument platform and analysis location in instrument run setup.

Selecting Instrument Platform and Analysis Location

From the instrument run setup screen, select the instrument platform (NexSeek 1000/2000) and then choose the analysis location.

The selection made here determines the run mode: BaseSpace for cloud run mode or local analysis location for hybrid or local mode setup.

Ensure to select the appropriate dragon version that matches the version installed on the instrument.

Enter relevant run parameters before submitting the run.

New Section

This section provides information on starting a local mode run using a v2 sample sheet.

Starting a Local Mode Run

Local mode requires a v2 sample sheet to start a run.

Two options are available for creating a v2 sample sheet:

Set up a local mode run in instrument run setup and export the sample sheet from the create run screen.

Generate a sample sheet from the v2 sample sheet template available on the support site.

Exporting from instrument run setup ensures that the sample sheet is formatted for NexSeek 1000/2000.

New Section

This section explains how to prepare and load the cartridge for sequencing.

Preparing and Loading the Cartridge

To prepare the cartridge, remove it from the bag and invert it 10 times to mix.

Remove the slow cell from its foil package and insert it into the cartridge, holding it by the gray tab. Remove the gray tab once inserted.

Load libraries into the bottom of the reagent reservoir.

Once libraries are loaded and flow cell is inserted, load the cartridge onto the sequencer with flow cell first and label facing up.

New Section

This section discusses pre-run checks performed after loading reagent cartridge.

Pre-Run Checks

Pre-run checks consist of two phases: instrument check and fluidics check.

The instrument check verifies output folder, BaseSpace connection, and performs instrument motion tests. If an error occurs during this phase, troubleshoot with reagent cartridge removal.

The fluidics check involves piercing the reagent cartridge, performing flow check, and locking consumable RFIDs. An audible popping noise indicates successful piercing. If an error occurs during this phase, troubleshoot with reagent cartridge removal.

New Section

This section discusses the impact of humidity on fluidic delivery and imaging, potential errors during pre-run checks, and cosmetic imperfections in flood cells.

Impact of Humidity

Humidity levels below 20% can affect fluidic delivery.

High humidity levels above 80% can impact imaging if condensation forms on the flow cell surface.

It is recommended to work with the local facilities team to maintain the lab within the recommended operating specifications.

Potential Errors During Pre-run Checks

If still is not detected during pre-run checks, remove the cartridge, power cycle the system, reseed the flow cell, and re-attempt run setup.

If the issue persists, contact Illumina technical support for further guidance.

Cosmetic Imperfections in Flood Cells

Slight cosmetic imperfections in flood cells, such as small streaks, are common but do not impact run performance or yield.

New Section

This section provides details about run performance during sequencing and real-time analysis.

Run Progress and Estimation

The control software displays a run progress screen with an estimate for run completion.

The estimate becomes more accurate over subsequent runs.

Cluster generation takes approximately four hours.

Sequencing Time

Sequencing time per cycle ranges from three to nine minutes depending on flow cell type and whether an index or reverse read is included.

Paired-end turnaround time adds an additional 45 minutes to the overall sequencing time.

At-a-Glance Overview

The progress screen provides information about which read and cycle the run is at.

After completing 25 cycles, reads past filter and initial average q30 and projected yield are displayed.

Real-Time Secondary Analysis

For certain run modes, real-time secondary analysis of read one is initiated at the start of read two.

Preliminary demultiplexing and read one alignment information are available for runs incorporating a v2.

Run Statuses

Unknown: Displayed at the start of clustering.

Not started: Displayed during cluster generation, read 1, index 1, and index reads.

In progress: Displayed during real-time analysis.

Completed: Displayed during final real-time analysis.

Error and status of erred: Indicates an analysis failure. Troubleshooting may be required.

New Section

This section discusses the auto center process on the NextSeq 1000/2000 systems.

Auto Center Process

Auto center is the process of finding the initial focus after clustering and before sequencing.

The NextSeq 1000/2000 uses patterned flow cell technology for auto centering.

Successful auto center requires some signal originating from the A or C base call at cycle one.

Certain library types may require specific adjustments to ensure successful auto centering.

New Section

This section explains potential errors that may occur during a run.

Cycle One Type Error

If a run stops unexpectedly, it may display as a failure occurred during sequencing, indicating consumables cannot be reused.

To troubleshoot this error, review library prep and run setup.

A power cycle or system check may be recommended by Illumina technical support.

New Section

This section discusses tools available for evaluating run performance.

Sequencing Analysis Viewer (SAV)

SAV is a Windows OS program that allows real-time viewing of quality metrics.

It can be installed on a separate Windows computer to review run metrics from the NextSeq 1000/2000.

Not compatible with the local Linux-based NextSeq 1000/2000.

BaseSpace Sequence Hub

Many of the same charts available in SAV are also accessible for runs sent to BaseSpace Sequence Hub as run monitoring.

Comparing Yield and Q30

When evaluating run performance, it is important to compare yield and q30 values to the specifications for the flow cell type and run read length.

The NexSeq 1000/2000 specifications page can be referenced for determining if a run has performed within specifications.

[t=0:27:16s] Overview of Phytex Validation Run

This section provides an overview of a phytex validation run, which uses the phi x viral genome as a positive control in sequencing experiments. It explains the flush chart, gradient chart, and default values within the chart.

Flush Chart and Gradient Chart

The flush chart includes a gradient chart that defaults to the highest and lowest values within the chart.

Right vs Left Side of the Chart

The right side represents the top surface, while the left side represents the bottom surface. Contrast and intensity between these surfaces are shown on the chart.

Database Cycle Chart

The database cycle chart displays run performance over time during the run. A slight bump in intensity is expected at cycle 25 to 26 due to a boost in intensity following pass filter determination.

Data by Lane Chart

The data by lane chart summarizes how the run performed within each lane. For P1 and P2 flow cells, data is aggregated in one lane, while for P3 Plus cell, data is separated into two distinct lanes optically. The number of tiles present in the flow cell is displayed at the bottom of the chart.

Number of Tiles per Flow Cell

For P1 flow cells, there are expected to be 32 tiles, for P2 flow cells 132 tiles, and for P3 flow cells 264 total tiles.

[t=0:29:01s] Evaluating Run Performance

This section discusses additional metrics to evaluate run performance when it does not meet specifications. It covers low yield, low quality runs, and the factors to consider in these cases.

Low Yield Evaluation

For low yield, it is important to examine the cluster's passing filter and percent loading concentration. If percent loading concentration is high and percent pass filter is low, the flow cell may be overloaded, and reducing library loading concentration is recommended. Conversely, if percent loading concentration is low and percent pass filter is high, the run may be underloaded, and increasing the loading concentration is recommended.

Low Quality Run Evaluation

To evaluate a low-quality run, consider the percent clusters pass filter, percent loading concentration, phi x alignment error rate (if available), percent base profile, library quality, and consumable handling. These factors help determine the starting point for further investigation of a low-quality run.

[t=0:30:18s] Pass Filter on Pattern Flow Cells

This section explains how data output from clusters that pass filter only on pattern flow cells works and how it can be affected by underloaded or overloaded library pools.

Clusters that pass filter on pattern flow cells are those where no more than one base call in the first 25 cycles has chastity below a fixed threshold. Chastity refers to the intensity purity measurement of a base call. Underloaded or overloaded library pools can depress pass filter on pattern flow cells. Validated libraries should be loaded at recommended concentrations found in manuals or guides specific to each type of library. Custom or unvalidated libraries should start with a 650 picomole loading concentration and adjust based on percent loading concentration values obtained after completing a run.

[t=0:31:44s] Troubleshooting Incomplete Data Transfer

This section addresses troubleshooting incomplete data transfer after a successful run on the instrument.

Incomplete data transfer can be caused by a stalled control software process or a stalled UCS (Universal Copy Service) process. Rebooting the control software and restarting UCS can help resolve the issue. If these steps do not work, contacting technical support for further investigation using UCS logs and information is recommended.

[t=0:32:49s] Re-cueing Local Secondary Analysis

This section explains how to re-cue local secondary analysis on the system after completing a run.

To re-cue local secondary analysis, open process management, identify the run to be requeued, and select "requeue." A requeue analysis screen will appear, allowing you to input a new v2 sample sheet or designate a new output directory. If no new output directory is selected, the new output data will be written to a subfolder of the original output directory. The original run folder must be present on the system for re-cuing local analysis of the run.

Maintenance and Troubleshooting

This section covers maintenance and troubleshooting topics but does not provide specific details in this transcript excerpt.

New Section

This section discusses power cycling, system check, and managing the local run folder through disk management.

Power Cycling and System Check

Power cycling can help resolve transient errors in the system.

To power cycle, open the control software menu and select shutdown instrument.

Toggle the switch at the back of the instrument to the off position and wait for at least 30 seconds.

Toggle the power switch back to the on position and wait for the power button at the front to pulse.

Wait for an additional minimum of 30 seconds before pressing the power button.

A system check can confirm proper alignment and functionality of components.

It can help resolve pre-run check errors or other errors in the control software.

A system check may be requested by an Illumina technical application scientist but is not required for normal operation or instrument maintenance.

New Section

This section covers managing the local run folder through disk management.

Managing Local Run Folder

Sequencing runs require approximately 200GB to 600GB of local hard drive space.

A warning notification will display when space is low, but runs should only be deleted using disk management process.

Deleting runs manually through operating system interface can cause a disconnect between control software and ancillary programs that complete important processes on the system.

Disk management can be accessed from the control software menu. It allows viewing available space remaining on local hard drive and space used by runs.

To delete a run, select "delete run" from local drive. Confirmation prompt will appear before deleting a run. The same process applies for removing genomes from the system.

New Section

This section explains how to view available space remaining on local hard drive and how to delete runs from the local drive.

Viewing Available Space and Deleting Runs

From the disk management screen in the control software, you can view the available space remaining on the local hard drive.

To delete a run, select "delete run" from the local drive. Confirm your selection before deleting the run.

This process only deletes runs from the temporary local output folder and does not affect runs in the final output folder.

New Section

This section provides instructions for deleting runs from the local drive and removing genomes from the system.

Deleting Runs and Removing Genomes

To delete a run, select "delete run" from the local drive. Confirm your selection before deleting.

The same process applies for removing genomes from the system.

It is important to note that deleting runs or genomes only affects temporary local output folders, not final output folders.

For more detailed instructions, refer to the support short on how to complete this process.

New Section

This section explains software updates for automatic and manual installation.

Software Updates

The system can be configured for automatic or manual software updates.

Automatic updates are downloaded automatically from Space Sequence Hub for installation.

Manual updates require downloading updates manually from the web and saving them locally or to a portable drive.

An internet connection is required for enabling automatic updates but not for manual updates.

Installation instructions can be found in both the system guide and release notes of each applicable control software version.

Release notes are available within the control software downloads page for NexSeq 1000/2000 system.

New Section

This section discusses Dragon licenses provided with NexSeq 1000/2000 systems.

Dragon Licenses

At the launch of NexSeq 1000/2000, Dragon licenses were provided with a six-month trial period.

Recently, Illumina started including the Dragon license with the purchase of NexSeq 1000/2000 systems.

If your system shipped before May 2nd, 2022, and is online, no action is required as the license will update during the next regular sync to the Illumina database.

If your system is offline, contact customer care at illumina.com to obtain the license key and install it. Instructions can be found in the system guide.

Systems shipped after May 2nd will already have the license key included.

New Section

This section provides information about installing BCL Convert for local Dragon workflows.

Installing BCL Convert for Local Dragon Workflows

If utilizing local Dragon for additional workflows, install BCL Convert alongside those workflows.

Dragon workflows should only be installed from the NexSeq 1000/2000 software downloads page.

Illumina also provides Dragon workflow modules for the Dragon BioIP platform, but these are not compatible with NexSeq 1000/2000 and should not be installed on them.

New Section

This section explains how to replace the air filter in NexSeq 1000/2000 systems.

Replacing Air Filter

The air filter is a single-use cartridge that covers the fan on the right side of the instrument.

Replacement of the air filter is required every six months.

To replace it:

Press on the right side of the top panel to disengage and open it.

Press to enter and remove the air filter cartridge from the center panel. Discard it.

Insert a new air filter into the receptacle and press to secure.

Close the top panel and press it into place.

For a visual guide, refer to the support short on how to complete this process.

New Section

This section provides troubleshooting tips, including power cycling and system check.

Troubleshooting

Power cycling can help resolve transient errors in the system. It is recommended to implement a power cycle one to two times a month as part of normal maintenance.

To power cycle, open the control software menu and select shutdown instrument.

Toggle the switch at the back of the instrument to the off position and wait for at least 30 seconds.

Toggle the power switch back to the on position and wait for the power button at the front to pulse.

Wait for an additional minimum of 30 seconds before pressing the power button.

A system check can help confirm components are properly aligned and functional. It may be requested by an Illumina technical application scientist but is not required for normal operation or instrument maintenance.

New Section

This section discusses the different components of the sequencing system and provides resources for users.

Components of the Sequencing System

The enclosure checks the performance of the thermal system and visor lift mechanism motion.

The z stage, x y stage, and optics are checked to ensure proper travel limits and performance.

The imaging module's performance is assessed.

Resources for Users

Relevant documentation for the sequencing system can be found in the local illumina directory.

The NextSeq 1000/2000 Sequencing System Support page is a valuable resource with links to system-specific documentation such as guides and site prep information.

The control software downloads page provides access to software releases and release notes.

Product information, including specifications for minimum expected yield and quality scores, can be found on the specifications page.

Technical bulletins are available under product information and provide important updates related to sequencing on illumina platforms. Filters can be used to find bulletins specific to the NextSeq 1000/2000 system.

Useful bulletins highlighted include tips for navigating the Linux environment, sequencing primer compatibility, and setting up a fix validation run.

Additional support bulletins address common questions about nucleotide diversity, approximate sizes of sequencing run output folders, and assessing sequencing run quality.

Online training resources cover topics such as starting a run on NextSeq 1000/2000 systems and analysis with Dragon software.

Proactive resources are available for labs interested in evaluating its benefits.