troubleshooting the nextseq system · •perform a manual software update to restore all software...

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Troubleshooting the

NextSeq System

2

After completing this lesson, you will be able to:

Troubleshoot pre-run check errors

Identify helpful troubleshooting files

Locate troubleshooting resources

Learning Objectives

3

Agenda

ReagentsBaseSpace

ErrorsPre-Run and

System ChecksRe-hyb

WorkflowTroubleshooting

Files

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BaseSpace Errors

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• Ensure that the reagent barcode entered in Prep tab matches that of the reagent cartridge

• Or leave Reagent Barcode field blank

Reagent Barcode was entered incorrectly

Planned Run Not Available

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• Option to “Automatically adjust for Daylight Savings Time” under the computer date and time settings is unchecked. Ensure that the following are CHECKED:

• “Automatically adjust for Daylight Savings Time”

• Network Time Server

• Correct local time zone of instrument

Daylight Savings Option

• Ensure network upload speed is at least 10 mbps

Network upload speed is too slow

BaseSpace Connection Errors

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NextSeq Pre-Run Checks

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Status Icons

Status icons signal any change

in the conditions during run

setup or during the run

When a change in condition

occurs, the icon image changes

and blinks

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NextSeq Sequencing Pre-Run checks

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Pre-Check Errors: Systems Checks

• Ensure that buffer compartment and reagent compartment doors are closed

Doors Closed

• Consumable sensors do not register

• Make sure that each consumable is properly loaded and repeat run set-up

Consumables Loaded

• Critical components of the software are missing

• Perform a manual software update to restore all software components

Required Software

• There is not enough disk space

• Free up disk space on the Network or remove any previously saved runs on the instrument

Instrument or Network Disk Space

• The network connection has been interrupted

• Confirm network status and physical network connection

Network Connection

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Pre-Check Errors: Temperature

• Checks the temperature ramp time of the flow cell heater plate

• If this check fails, contact Illumina Technical Support

Temperature Ramp

• Checks the speed of system fans in pulse per minute (PPM) to confirm fans are functioning


Fans

• Checks the average temperature of each thermal sensor


Temperature Sensors

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Pre-Check Errors: Imaging System

• Sends stages to their sensor limits


Imaging Limits

• Measures ability for the 6 z-stages to move and stop moving in a set time


Z Step-and-Settle

• Each camera sends a bit to the computer to make sure the circuits to each of the camera are working properly


Bit Error Rate

• Verifies registration of the flow cell in the correct imaging position

• If fails, it is possible that the flow cell is not properly seated

• Unload and reload the flow cell to make sure that it is seated properly

Flow Cell Registration

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Pre-Check Errors: Reagent Delivery

• Tests the accuracy of valve and pump syringe rate movements


Valve Response

• Moves both pump syringes to their min and max positions


Pump

• Moves the sipper assay in the Buffer Straw Mechanism to it’s min and max sensor limits


Buffer Mechanism

• Verifies spent reagents tray is empty

• If fails, empty spent reagents tray and return to the instrument

Spent Reagents Empty

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NextSeq System Check

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System Check

• Used during Live Help session with assistance from an Illumina representative

• Not required during normal operation or for instrument maintenance

When to use

• Select System Check from Manage Instrument screen

• Load consumables when prompted:

o Used flow cell

o Empty spent reagents tray

o Buffer wash tray with lab-grade H2O

o Empty reagent wash tray unless otherwise prompted

• Select tests to run

How to use

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Live Help

Online assistance tool enabling

an Illumina Tech Support

representative to view the

NextSeq screen with permission

Obtain unique access code from

representative

Support.illumina.com/share_desktop.html

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The following tests can be performed without assistance of an Illumina field representative:

• Motion tests

• Fluidics tests

• Thermal test

System Checks Tests

Note: Selecting Systems Check from the management screen will close the NextSeq Control

Software (NCS) and will open the NextSeq Service Software (NSS)

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Rehybridization Workflow

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Rehybridization Run

• Rehybridization of the Read 1 sequencing primer onto clusters

What is it

• If metrics generated during the first few cycles show intensities below 2500• Note: some low-diversity libraries can show intensities

<1000 which is expected

When to use it

• NextSeq is using NCS v1.3 or later

• Run must be stopped before paired-end synthesis

• Requires new NextSeq 500 kit

• Not all poor quality runs can be corrected with a rehybridization run

Considerations

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End Current

Run

Save Flow Cell

Perform Manual Wash

Set up New Run on Prep

Tab

Start Run on

Instrument

Rehybridization Workflow

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End Current Run

Select End Run

Select Yes to

save the flow cell

Brief wait while flow

cell is placed in safe state

RFID of saved flow

cell is unlocked

Automatic post-run wash is

bypassed

NOTE: The End Run command is final. The run cannot be resumed, run

consumables cannot be reused, and sequencing data from the run is not saved.

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Perform a Manual Post-Run Wash

Reagent and buffer cartridges from the stopped run can be used for the

manual wash if they have not yet been removed

Perform Manual Wash

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Set up a new run using the same parameters as the original run

Click the Pools tab, select the appropriate Pool ID to retain the previous

run settings, and then assign a unique name for the new run

Set Up New Run in Prep Tab

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Prepare a new reagent cartridge

Allow saved flow cell to reach room temperature (15-

30’)

Load saved flow cell

Empty spent reagents

Load the new buffer and reagent

cartridges

Select the run from the run set

up screen*

Select Next to proceed to the

pre-run check and start the run

Start New Run on Instrument

*If using Standalone Mode: enter the

name of the run and specify the same

parameters as the original run

32 COMPANY CONFIDENTIAL – INTERNAL USE ONLY

Troubleshooting Files


An Illumina Technical Support representative

might request copies of run-specific files for

troubleshooting purposes

– These files can be found from the run

folder

Troubleshooting files

Run folder in Windows

Run folder in BaseSpace

Links to BaseSpace runs

are also very useful for

Technical Support

Troubleshooting


Run Information and Run Parameters

Location: root folder

Run Info.xml description: contains name of the run, number of cycles in the run, number of cycles in each read, whether the read is an indexed read, and the number of swatch and tiles on the flow cell

Runparameters.xml description: contains a summary of run parameters and information about run components, such as the RFID of the flow cell and reagents associated with the run

Run information (RunInfo.xml)


RTA Configuration File


Description: contains the RTA configuration settings for the run. The RTAConfiguration.xml file is created at the beginning of the run

RTA configuration file


InterOp files


Description: Binary reporting files used for Sequencing Analysis Viewer.

InterOp files are updated throughout the run.

InterOp files


RTA Logs

Location: RTA Logs

Description: Log of RTA errors. Error log files are updated whenever an error occurs.

Location: RTA Logs

Description: Log of all RTA events. Global log files are updated throughout the run.

Error log files

Global log files

Location: RTA Logs

Description: Logs RTA processing events. Lane log files are updated throughout the run.

Lane log files

RTA Logs


Some pre-run check errors are easily resolvable by the user

– See the Troubleshooting section of the NextSeq System User Guide for more detail

System Checks are typically performed during a Live Help session with

assistance from an Illumina Technical Support representative

Rehybridization of read 1 sequencing primer can be performed as potential run

rescue

Troubleshooting files found in the run folder help Illumina representatives

diagnose sequencing run issues more effectively

Session Summary

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Questions?

troubleshooting the nextseq system · •perform a manual software update to restore all software...

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