MiSeq Sequencing v2.0 Protocol

1. In Lab View, locate the MiSeq Sequencing v2.0 protocol. Samples are queued for the Library Pooling (MiSeq v2.0) 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.
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 v2.0).

7. Select Finish Step.
1. In Lab View, locate the MiSeq Sequencing v2.0 protocol. The pooled samples are queued for the Denature and Dilute (MiSeq v2.0) 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. Populate the fields in the Step Details table, as needed. 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 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 v2.0) step.

7. Select Finish Step.
1. In Lab View, locate the MiSeq Sequencing v2.0 protocol. The pool of samples is queued for the MiSeq Run (MiSeq v2.0) 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 v7.0 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.