MiSeq Sequencing v3.1 Protocol

1. In Lab View, locate the MiSeq Sequencing v3.1 protocol. Samples are queued for the Library Pooling (MiSeq v3.1) step. Select the step to proceed to the Queue screen.
2. On the Queue screen, do as follows.
a. Add the samples to the Ice Bucket.
b. In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
c. Select View Ice Bucket.
3. On the Ice Bucket screen, select Begin Work.

The Validate Maximum Number of Samples automation script runs. The maximum number of samples allowed is 1536.

4. On the Pool Samples screen, do as follows.
a. Create a pool of samples by dragging samples into the Pool Creator.
b. Name the pool or accept the default name (Pool #1).
c. Select Place Samples.
5. On the Placement screen, do as follows.
a. Select the pool from the Samples to be Placed area and drag it to the container.
b. Select Record Details.
6. On the Record Details screen, select Next Steps.

On the Assign Next Steps screen, next step is automatically set to Denature and Dilute (MiSeq v3.1).

7. Select Finish Step.
1. In Lab View, locate the MiSeq Sequencing v3.1 protocol. The pooled samples are queued for the Denature and Dilute (MiSeq v3.1) step. Select the step to proceed to the Queue screen.
2. On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.
3. On the Ice Bucket screen, select Begin Work.
4. On the Placement screen, do as follows.
a. Scan the MiSeq reagent cartridge barcode into the MiSeq Reagent Cartridge field.
b. Place the pool of samples into the reagent cartridge.
c. Select Record Details.
5. On the Record Details screen, do as follows.
a. In Reagent Lot Tracking, select the reagent lot used in the step.

If the reagent lot is not listed, add/activate the lot on the Reagents and Controls screen.

b. Use the Preset drop-down list to help populate the fields in Step Details, as needed. For more information on presets, see Using Step Details Presets. Workflow, Experiment Name, and Read 1 Cycles are required fields.
c. In the Sample Details table, enter the Final Loading Concentration. Select from the following preset options or enter a different value.
• 225 (for PCR-free workflows)
• 400 (for Nano workflows)
d. Select Validate Run Setup and Generate MiSeq SampleSheet.

Clarity LIMS generates the sample sheet and attaches it and a log file to placeholders in the Files area of the Record Details screen.

e. Download the files and validate the format and content.
6. Select Next Steps.

On the Assign Next Steps screen, samples are automatically assigned to the MiSeq Run (MiSeq v3.1) step.

7. Select Finish Step.

Most of the UDFs/custom fields in Step Details are analysis settings used by Local Run Manager and are for advanced users only. The following fields are exceptions:

• Workflow
• Experiment Name
• Description
• Read 1 Cycles
• Read 2 Cycles
• Custom Primers

To use the default analysis settings, select the analysis module from the Preset drop-down list. The default values populate the respective fields and indicate the following actions are needed:

• Required—The field is required for the specific analysis module selected. Replace the word Required with a proper value. For example, the Genome Folder field should contain the directory to the genome folder.
• None—The field is not applicable for the specific analysis module selected. Leave the field as is.

For a list of fields that are applicable for the analysis module selected, refer to the Applicable analysis fields for the selected Workflow field in Step Details.

1. In Lab View, locate the MiSeq Sequencing v3.1 protocol. The pool of samples is queued for the MiSeq Run (MiSeq v3.1) step. Select the step to proceed to the Queue screen.
2. On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.
3. On the Ice Bucket screen, select Begin Work.
4. On the Record Details screen, the fields are read-only. When the run completes, the integration automatically performs the following actions:
• Populates the fields.
• Attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders.
• Populates the fields in the Sample Details table. See MiSeq Integration v8.1 Configuration and User Interaction for details.

The Log File is attached after the next step for samples is assigned by the Next Step - Advance automation. Make sure the Illumina Run Report has been attached before continuing.

5. Select Next Steps.

On the Assign Next Steps screen, the next step is automatically set to Mark protocol as complete.

6. Select Finish Step.