User Interaction, Validation and Troubleshooting
This section explains how to validate the installation of the Illumina NextSeq 1000/2000 Integration Package v2.4.0.
The validation process involves the following items:
Running samples through the Library Prep Validation v2.3.1 workflow.
The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 Sequencing v2.3 workflow.
Running the libraries through the NextSeq 1000/2000 Sequencing v2.3 workflow. This process validates the following information:
Successful sequential step advancement of samples in the following steps:
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)
Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.
Automated creation of a planned run on Illumina Connected Analytics. The NextSeq 1000/2000 Control Software retrieves the planned run to start the run via Hybrid or Cloud run mode.
Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics from BaseSpace Sequence Hub into Clarity LIMS using the SIS Integration Service.
Automated tracking of the secondary analysis using DRAGEN (up to BCL Convert only).
Parsing of demultiplexing metrics from BaseSpace Sequence Hub into Clarity LIMS using SIS integration service.
Before running the validation steps below, these steps assume that the NextSeq 1000/2000 Integration Package v2.4.0 is installed, and that you have imported the default Clarity LIMS configuration.
For information on how the integration works, refer to NextSeq 1000/2000 Integration v2.4.0 Configuration.
Activate Workflow, Create Project, Add and Assign Samples
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.1 and NextSeq 1000/2000 Sequencing v2.3 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.1 and NextSeq 1000/2000 Sequencing v2.3 workflows.
On the Projects and Samples screen, create a project and add samples to it.
Assign the samples to the Library Prep Validation v2.3.1 workflow.
Follow the steps in Library Prep Validation Protocol to run the Library Prep Validation workflow with the following:
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = NextSeq 1000/2000
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 1000/2000 Sequencing v2.3 workflow, which is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.
NextSeq 1000/2000 Sequencing v2.3 Protocol
Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)
In Lab View, locate the NextSeq 1000/2000 Sequencing v2.3 protocol. The samples are queued for the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Pooling screen, perform the following actions:
Create a pool by dragging samples into the Pool Creator.
Type a name for the pool or accept the default name (Pool #1).
Select Record Details.
On the Record Details screen, the Reagent Lot Tracking section tracks the Resuspension Buffer (RSB) lot information used in the step. If required, create new lots by referencing Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.
The Step Details area contains two required fields:
Final Loading Concentration (pM) — The value entered in this field is the recommended final loading concentration specified in the Illumina NextSeq 1000/2000 Sequencing System Guide at support.illumina.com. The value depends on the library type. The default drop-down list contains the values 650, 750, 1000, and 2000. A custom value is acceptable.
Final Loading Volume (ul) — The value in this field is the final loading volume of the pool into the reagent cartridge. The field is prepopulated with the configured default value, 24 µl, specified in the Illumina NextSeq 1000/2000 Sequencing System Guide at support.illumina.com. The value is editable when more volume is necessary.
Select Calculate Volumes.
This selection triggers the Calculate Volumes automation. This automation calculates the volume required for each library to form a pool that has the concentration and volume specified in the step details fields.
The automation also generates the Calculation File (CSV) and attaches it to the step. This file contains volume information of each of the samples and RSB buffer to add to the pool. Select the file to download it, then open it in Excel.
In the Sample Details table, select the pool icon to view details on the pool composition.
Select Next Steps.
This selection triggers the Set Next Step automation, which sets the next step for samples to ADVANCE, advancing them to the next step in the protocol. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3).
On the Assign Next Steps screen, the next step for samples is already set to the next step in the workflow. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3).
Select Finish Step.
At the end of this step, the pool of samples automatically advances to Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step.
Step 2: Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)
In Lab View, locate the NextSeq 1000/2000 Sequencing v2.3 protocol. The pool of samples is queued for the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
The Validate Single Input automation is triggered. This automation checks that there is only one container input to the step.
On the Placement screen, perform the following actions:
Drag the pool into the NextSeq 1000/2000 Reagent Cartridge field in the Placed Samples area.
Scan or type the barcode of the reagent cartridge into the NextSeq 1000/2000 Reagent Cartridge field.
Select Record Details.
On exit of the Placement screen, the Validate Reagent Cartridge Barcode automation checks that the reagent cartridge barcode conforms to the barcode mask [A-Za-z]{2}[0–9]{7}-[A-Za-z0-9]{4}. If not, an error message displays.
On entry to the Record Details screen, the Retrieve Analysis Workflow Versions automation fetches the available analysis workflow versions from ICA. It then updates the preset values of both Local Analysis Workflow Versions & Cloud Analysis Workflow Versions field.
On the Record Details screen, the Reagent Lot Tracking section tracks the NextSeq 1000/2000 Reagent Cartridge lot information used in the step. Follow the steps in Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step if you must add a lot.
In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.
The fields displayed on the Record Details screen are used to create planned run and generate the sample sheet file.
Run Name — Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
Instrument Type — Select from preset options (NextSeq 1000 or NextSeq 2000).
Run Mode — Select from preset options: Local, Hybrid, Cloud. For more information on the different run modes, refer to Run Mode Details.
Paired End — Select from preset options (True or False).
Read 1 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.
Read 2 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.
Index Reads — Select from preset options (No Index, Single Index, Dual Index).
Index Read 1 — Select from preset options (0, 6, 8) or type a custom value.
Index Read 2 — Select from preset options (0, 6, 8) or type a custom value.
Analysis Workflow — Read only. This field is set to GenerateFASTQ.
Local Analysis Workflow Versions — [Optional] (default is None). Only mandatory when Run Mode = Local/Hybrid and Analysis Workflow = GenerateFASTQ. Select according to the DRAGEN version installed on the instrument.
Cloud Analysis Workflow Versions — [Optional] (default is None). Only mandatory when Run Mode = Cloud and Analysis Workflow = GenerateFASTQ. Select the cloud DRAGEN version to be used.
FASTQ Compression Format:
If Run Mode = Local/Hybrid, Analysis Workflow = GenerateFASTQ, and Local Analysis Workflow Version >= 3.7.4, select gzip or DRAGEN.
If Run Mode = Cloud, Analysis Workflow = GenerateFASTQ, and Local Analysis Workflow Version >= 3.7.4, field is set to gzip automatically.
Otherwise, select None.
Adapter Sequence Read 1 — [Optional] Enter the Read 1 adapter sequence of the index adapter kit.
Adapter Sequence Read 2 — [Optional] Enter the Read 2 adapter sequence of the index adapter kit.
Barcode Mismatches Index 1 — [Optional] Select from preset options (0, 1, 2). Leave it blank if you are unsure.
Barcode Mismatches Index 2 — [Optional] Select from preset options (0, 1, 2). Leave it blank if you are unsure.
Override Cycles — [Optional] String used to specify UMI cycles and mask out cycles of a read (eg, N1Y150;I8;I7N1;Y141U10). Leave it blank if you are unsure.
Instructions — Read only.
On the Record Details screen, select Validate Run Setup and Create Planned Run.
This selection triggers the automation script, which does the following actions:
Validates the parameters entered on the Record Details screen.
Create the planned run and send it to ICA if Run Mode is Hybrid or Cloud.
Generates the sample sheet and attaches it to the placeholder in the Files area of the Record Details screen for all Run Mode types.
Select Next Steps.
On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow. The next step is AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3).
Select Finish Step.
At the end of this step, the pool of samples automatically advances to and is queued for the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step. The sequencing run is ready to be started. For details on how to start the sequencing run for different run modes, refer to NextSeq 1000/2000 Integration v2.4.0 Configuration.
Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
This step is fully automated.
The integration starts the step automatically and data from the run is parsed back to Clarity LIMS. No user interaction is required. However, you can open and review the various stages of the step in Clarity LIMS. Do not perform any action when reviewing the data.
Read summary metrics are recorded for the library pool in the Step Details section and the Sample Details table.
Values are populated in the following master step fields:
Run Name
Current Read
Flow Cell ID
Reagent Cartridge Lot Number
Instrument ID
Run Status
Current Cycle
Flow Cell Lot Number
Instrument Platform
Instrument Control Software Version
Output Folder
Secondary Analysis Workflow
Reagent Cartridge ID
Instrument Type
RTA Version
Sequencing Log
The summary metrics (per run level) populate in the following global fields.
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2
At the end of this step, the pool of samples automatically advances to (and queues for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step.
Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)
This step is semi-automated.
The DRAGEN version must be earlier than v3.9.3.
The samples in the run must not repeat.
The run must be for the single lane flow cell (P1 or P2).
The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. Review the data parsed and assign QC, depending on the criteria set, and complete the step.
In the Record Details screen, perform the following actions:
Review demultiplexing data.
Demultiplexing metrics are recorded for the library pool in the Sample Details table. Metrics include columns for # Reads, # Perfect Index Reads, # One Mismatch Index Reads, and # of >= Q30 Bases (PF).
The Demultiplex_stats.csv file generated by the DRAGEN onboard or Cloud analysis module is stored under the Files section.
Assign QC flags to all the individual sample. There are two ways of doing this step.
Manually assign QC flags through the QC column in Sample Details table.
Automatically assign QC flag by running Assign QC flags automation. This option is for scenarios where a huge number of libraries are involved.
In the Step Details section, the following step fields are visible. (N is the number of criteria. You can use one or more criteria.)
Criteria N - Source Data Field — Select from preset options eg # Reads.
Criteria N - Operator — Select from preset options, for example, >= (greater than or equal to).
Criteria N - Threshold Value — Enter the desired threshold value.
After filling up the criteria fields, select Assign QC flags. This selection triggers the automation script, which loops through each library in the pool and apply QC flag base on the criteria set previously.
This automation also generates an AssignQC Result file under Files section.
Select Next Steps.
On the Assign Next Steps screen, the Next Step field for all samples is prepopulated with Mark protocol as complete.
Select Finish Step.
At this point, the whole NextSeq 1000/2000 Integration workflow is fully validated.
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, refer to Troubleshooting Automation in the Clarity LIMS (API & Database) documentation.
If the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step starts but does not finish, complete the following steps:
Log in to Clarity LIMS using the default user account and use one of the following methods to open the step in Clarity LIMS:
Method 1: In Lab View, find the step in the Recent Activities pane.
Method 2: Search for the step in Clarity LIMS using reagent cartridge barcode as the search term.
On the Record Details screen, locate the Sequencing Log field. The multiline text field contains logging information.
If you cannot reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting steps already performed.
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