# Perfect Index Reads

# One Mismatch Index Reads

The demultiplexing stats displayed are aggregated for multi-lane flow cells (for example, P3).

The following run metrics files are included in Run_Metrics.zip and attached to the step:

• Adapter_Metrics.csv

• Demultiplex_Stats.csv

• Index_Hopping_Counts.csv

• Quality_Metrics.csv

• Top_Unknown_Barcodes.csv

Analysis Status is now visible on the step.

The integration service and integration-related properties have been moved from configuration files to database.

Supports DRAGEN BCL Convert v4.2.7 analysis, including:

• Cloud BCL Convert v4.2.7 running under the ICA Workflow Session Tracking application

• Local BCL Convert v4.2.7

Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

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. The NCS retrieves and starts the planned 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.

Automated tracking of the secondary DRAGEN analysis (up to BCL Convert only) and parsing of demultiplexing metrics from BaseSpace Sequence Hub into Clarity LIMS.

The following Cloud analysis supported applications:

• BCL Convert v4.2.7 or earlier under BaseSpace Sequence Hub DRAGEN Analysis

• BCL Convert v4.2.7 under ICA Workflow Session Tracking

Local analysis supported BCL Convert v4.2.7 and earlier.

[Optional] Preconfigured Library Prep Validation workflow used for validation purposes only. 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 v2.4 workflow.

[Optional] An integration of Clarity LIMS Product Analytics (CLPA) that make Clarity LIMS data available to the ICA and CLPA. This integration also requires installation and configuration. For more information, refer to Clarity LIMS Product Analytics Integration v1.3.1 Configuration.

Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

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. The NCS retrieves and starts the planned 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.

Automated tracking of the secondary DRAGEN analysis (up to BCL Convert only) and parsing of demultiplexing metrics from BaseSpace Sequence Hub into Clarity LIMS.

The following Cloud analysis supported applications:

• BCL Convert v4.2.7 or earlier under BaseSpace Sequence Hub DRAGEN Analysis

• BCL Convert v4.2.7 under ICA Workflow Session Tracking

Local analysis supported BCL Convert v4.2.7 and earlier.

[Optional] Preconfigured Library Prep Validation v2.3.3 workflow used for validation purposes only. 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 v2.4 workflow. For more information, refer to NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

[Optional] An integration of Clarity LIMS Product Analytics (CLPA) that make Clarity LIMS data available to the ICA and CLPA. This integration also requires installation and configuration. For more information, refer to Clarity LIMS Product Analytics Integration v1.3.0 Configuration.

AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

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 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 the integration service.

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 NextSeq 1000/2000 Product Documentation. 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 . 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 v2.4).

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 v2.4).

Select Finish Step.

On the Placement screen, perform the following actions:

1. Drag the pool into the NextSeq 1000/2000 Reagent Cartridge field in the Placed Samples area.
2. Scan or type the barcode of the reagent cartridge into the NextSeq 1000/2000 Reagent Cartridge field.
3. 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-Z]{2}[0-9]{7}-[A-Z0-9]{4}. If not, an error message displays.

⚠ The NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after a successful validation. Modifications can cause issues when Clarity LIMS tries to update the status and sample details of subsequent steps.

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 v2.4) 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 .

• 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.

• [Optional] Local Analysis Workflow Versions — Default is None. Only mandatory when Run Mode = Local/Hybrid and Analysis Workflow = GenerateFASTQ. Select according to the DRAGEN version installed on the instrument.

• [Optional] Cloud Analysis Workflow Versions — 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.

• [Optional] Adapter Sequence Read 1 — Enter the Read 1 adapter sequence of the index adapter kit.

• [Optional] Adapter Sequence Read 2 — Enter the Read 2 adapter sequence of the index adapter kit.

• [Optional] Barcode Mismatches Index 1 — Select from preset options (0, 1, 2). Leave it blank if you are unsure.

• [Optional] Barcode Mismatches Index 2 — Select from preset options (0, 1, 2). Leave it blank if you are unsure.

• [Optional] Override Cycles — String used to specify UMI cycles and mask out cycles of a read (for example, 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.

• If Run Mode is Hybrid or Cloud, create the planned run.

• 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 v2.4).

Select Finish Step.

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

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

Adapter_Metrics.csv

Demultiplex_Stats.csv

Index_Hopping_Counts.csv

Quality_Metrics.csv

Top_Unknown_Barcodes.csv

The Analysis Status step field is updated with the analysis status.

Assign QC flags to all the individual sample. There are two ways of doing this step.

1. Manually assign QC flags through the QC column in Sample Details table.
2. 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 (for example, # 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.

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.

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.

On the Placement screen, perform the following actions:

1. Drag the pool into the NextSeq 1000/2000 Reagent Cartridge field in the Placed Samples area.
2. Scan or type the barcode of the reagent cartridge into the NextSeq 1000/2000 Reagent Cartridge field.
3. 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.

⚠ The NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after a successful validation. Modifications can cause issues when Clarity LIMS tries to update the status and sample details of subsequent steps.

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 .

• 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.

• 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.

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

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

The samples in the run must not repeat.

The run must be for the single lane flow cell (P1 or P2).

Assign QC flags to all the individual sample. There are two ways of doing this step.

1. Manually assign QC flags through the QC column in Sample Details table.
2. 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.

Field Type — Text

Required Field — No

Read Only — Yes

Select Save.

Select Criteria 1 - Source Data Field.

In the Additional Options pane, remove # of > = Q30 Bases (PF) from the Dropdown Items list.

Select Criteria 2 - Source Data Field.

In the Additional Options pane, remove # of > = Q30 Bases (PF) from the Dropdown Items list.

Select Save.

In the Step File Placeholders pane, rename Demultiplex Stats to Run Metrics.

Select Save.

Add the Analysis Status field as follows.

1. In the Step Data pane, select + to open the Master Step Fields pane.

2. Select Analysis Status, and then select the check mark to add the field to the Master Step Fields list.

Update the Sample Table as follows.

1. In the Sample Table pane, navigate to the Table Columns pane and remove the following fields:

   • [Measurement] # of > = Q30 Bases (PF)

   • [Measurement] Mean Quality Score (PF)

2. Select + to open the Fields pane.

3. Select Measurement to open the field drop-down list.

4. Select Index Sequence.

5. Reorder the table columns as follows.

   • [Derived Sample] Sample Name

   • [Measurement] Index Sequence

6. Select Save.

Select Save.

[Measurement] # Reads

[Measurement] # Perfect Index Reads

[Measurement] # One Mismatch Index Reads

[Container] Container Name

[Project] Project Name

User is required to assign QC label (Pass/Fail) to the individual library in the pool and complete the step.

RunErroredOut

Set by the integration service¹

If reagent cartridge is not found, it is created and set to Active.

If reagent cartridge is not in Active status, it is set to Active.

Multiline Sequencing Log field is updated.

Multiline Sequencing Log field is updated.

The integration automatically completes the step and routes the samples in the reagent cartridge container to the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step.

Multiline Sequencing Log field is updated.

The integration does not automatically complete the step. Complete the step manually.

Multiline Sequencing Log field is updated.

The integration does not automatically complete the step. Complete the step manually.