Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.0)

This step is a semiautomated step.

The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. Then review the demultiplexing result parsed into Clarity LIMS, assign QC flags, and complete the step.

For the demultiplexing information in the Sample Details table to display correctly, the following conditions must be met:

• 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).

Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.

The following automations are configured on the step:

• Assign Demultiplexing QC flags—Automatically assigns QC flags based on the criteria set on the step fields.
• Set Next Step—Automatically sets the next step to Advance and the samples complete the protocol.

There are eight fields defined on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.0) step.

The following table lists the master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.0) step.

Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.0) Step

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-Down Items

Criteria 1 - Source Data Field

Text Dropdown
• Required: No
• Read Only: No
• Custom Entries: Yes
• # Reads
• # Perfect Index Reads
• # One Mismatch Index Reads
• # of >= Q30 Bases (PF)

Criteria 2 - Source Data Field

Text Dropdown
• Required: No
• Read Only: No
• Custom Entries: Yes
• # Reads
• # Perfect Index Reads
• # One Mismatch Index Reads
• # of >= Q30 Bases (PF)

Criteria 1 - Operator

Text Dropdown
• Required: No
• Read Only: No
• Custom Entries: Yes
• >=
• <=
• =
• !=

Criteria 2 - Operator

Text Dropdown
• Required: No
• Read Only: No
• Custom Entries: Yes
• >=
• <=
• =
• !=

Criteria 1 - Threshold Value

Numeric
• Required: No
• Read Only: No
• Valid integer value

Criteria 2 - Threshold Value

Numeric
• Required: No
• Read Only: No
• Valid integer value

Log

Multiline Text
• Required: No
• Read Only: Yes

Not applicable

Reagent Cartridge ID

Text
• Required: No
• Read Only: Yes
• Hidden
• Used for parsing demultiplexing results

There are four fields defined on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.0) step. which are used to display the demultiplexing result metrics for individual library in the sample pool.

The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.0) step.

Global Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.0) Step

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-Down Items

# of >= Q30 Bases (PF)

Numeric
• Required: No
• Read Only: Yes

Not applicable

# One Mismatch Index Reads

Numeric
• Required: No
• Read Only: Yes

Not applicable

# Perfect Index Reads

Numeric
• Required: No
• Read Only: Yes

Not applicable

# Reads

Numeric
• Required: No
• Read Only: Yes

Not applicable

Refer to NextSeq 1000/2000 Integration v2.1 Validation and Troubleshooting for details.