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COVIDSeq Wet Lab Demo
Video Transcript


SC2 AMD COVIDSeq Wet Lab Demo Video Transcript:

In this video we will review:
•    The LIMS Lite Launchpad
•    Starting a new workflow
•    The COVIDSeq workflow

The LIMS Lite Launchpad provides summary statistics and allows for easy navigation among modules using the left sidebar.

New Workflow Run
Once your samples are accessioned into LIMS Lite, select ‘New Workflow Run’ using the left sidebar.

On the Workflows screen you will see the COVIDSeq workflow displayed along with Test Name, Test Type, and Number of Samples. 

Samples can be prioritized to include them in the run, regardless of when they were accessioned into LIMS Lite. 

To prioritize samples, click the ‘PRIORITIZE’ button, select the check box of the samples to include, then click ‘Save’.

To start a new workflow run, click the ‘BEGIN’ button.

Enter the number of samples to include in the run and click ‘Start’.

The components are displayed according to workflow step. Select “View by Components” to toggle to a list of components grouped by kit.

Components can be selected for use in the run by clicking on the appropriate lot number next to the reagent name. 

Once selected, the lot number will turn green.

*Note* Some reagents are included in multiple steps.

If the lot number of your reagent is not already displayed, you may enter it into the space next to existing lot numbers. 

This new lot number will be retained for your run and for future runs.

After selecting all reagents to include, click ‘Continue’ to begin the workflow run. 

The COVIDSeq Workflow
This is the LIMS Lite workflow screen.

The first step of the COVIDSeq workflow is the Grid Samples step.

The top center of each workflow screen displays the current step, the run number and the workflow name.

The ordered steps of the workflow are listed in the left sidebar.

The top left and right display the current step input and output.

For the gridding step, the input is the list of samples, and the output is the plate with virus samples.

At the top right-hand corner of the page is the ‘SAVE’ button. Clicking this button will save your progress on your current step.

To view quality control checks for this step, click on ‘SHOW QCs’ at the bottom left of the screen.

In the QC Checks window, the details of each check can be viewed by clicking on the ‘QC Name’.

Clicking on the Comments button displays a window where you can enter and save any notes for this step.

The ‘Show Comments’ button will now appear, which you can click to view all entered comments for this step during the current workflow.

Clicking the ‘Instructions’ tab displays the laboratory protocol instructions that accompany the current step of the workflow.

Users may exit the workflow at any time by clicking Exit Workflow. A prompt will appear asking whether to progress. 

When finished with the workflow step, click on ‘Move to Next Step’ to proceed to the next page. 

Users may also fail the workflow run by click ‘Fail Run’.

Step 1: Grid Samples
To begin gridding, first select your negative control position.

Click the pencil icon next to NTC and then click the well assigned to the control on the plate. 

The well is now designated as an NTC position, and the well name appears next to the NTC control.

Repeat the steps to select the positive control position.

Assigning positions for samples can be done in two ways.

To grid your samples sequentially, click ‘Grid Sequential’ and click the well on the rack where you would like to assign your first sample. 

Samples selected for the run will be assigned in sequential order. Hover over the well to see the sample ID.

The ‘Clear Plate’ button can be used to remove all samples from the plate.

Samples can also be assigned to wells individually by clicking the pencil icon next to each sample, then clicking the well on the plate. 

Note that a minimum of 1 positive control, 1 negative control, and 1 sample must be assigned during this step. 

Once samples are all assigned to the correct position, click ‘Move to Next Step’ to the next step of the workflow.

Step 2: RNA Extraction
This page displays a diagram of the sample plate with positive control, negative control, and samples assigned to their respective wells. 

You will see these well positions displayed again in table format. 

RNA Extraction reagent information is displayed for easy reference, with volume per well shown with the respective lot number that was selected before beginning the run.

Click on ‘Plate Details’ to view control and sample positions entered in the Grid Samples step.

Step 3: Reverse Transcription Prep
For steps that involve a master mix, the mix will be presented on the step screen with reagent, volume per well, total volume and lot number.

Volumes are calculated according to sample count (including PTC and NTC) entered before the run, as well as additional amount factor if applicable.

Steps that include PCR information will display a field to enter in thermocycler ID and view the thermocycler profile. 

Step 4: Reverse Transcription
On this page you will view the reverse transcription master mix and PCR Information.

Step 5: Tiled Amplification
In Tiled Amplification, cDNA is amplified using two separate PCR reactions. 

This page displays these two separate PCR master mixes , as well as PCR information.

Step 6: Tagmentation
On this page you will view the tagmentation master mix, as well as PCR Information.

Step 7: Tagmentation Cleanup
This page displays the instructions for tagmentation cleanup, with step numbers, description and reagent information included.  

Scroll down to see PCR Information.

Step 8: Choose Adapters
On the Choose Adapters page, you will see barcoding kits that are entered into LIMS Lite and configured to your workflow displayed, along with their respective lot numbers.  

Click on the lot number of the kit you are using, and a pop-up window displays the barcode plate and sample plate side-by-side. 

Click on a position on the barcode plate to select the first barcode well that will be used in the barcoding workflow step.  

The barcodes for all sequential samples and control wells will populate on the barcode plate, and the sample plate will update to reflect the barcode selections.

Click Done.

To view the selected adapters and their corresponding sample/control wells, click “View Mapping”.

Step 9: Tagmentation Amplification
On this page, in addition to Master Mix and Thermocycler Instrument, is the Barcode Information table, which displays adapters linked to sample IDs.

Step 10: Library Pooling and Clean Up
This page displays the instructions for library pooling by equal volume, followed by library cleanup, with step numbers, description and reagent information included.  

Step 11: Pooled Libraries Check
The Pooled Libraries Check page includes Qubit reagent information for library quantification, as well as fields to input the quantification instrument and the observed library concentration.

Step 12: Initial Dilution for Loading
On this screen, the user will be guided through the steps to create a 4 nanomolar dilution of the pooled library. 

The concentration of the pool will be added, and the library size will default to 400 base pairs. 

Users may click “Use bioanalyzer results” to enter in average library size manually.

Target volume is customizable but should be at least 30 microliters.

With all of these inputs, the amount of library and diluent to use will populate at the bottom of the page.

Step 13: Final Dilution

This screen allows a user to select their instrument type (MiSeq or iSeq).

The instructions will change depending on the instrument selected.

Follow the denaturation and dilution instructions provided. 

The Final Series Dilution table can be referenced to dilute the final concentration to 12 pM.

Step 14: Setup Illumina Instrument
On this page, ensure that the Sequencer Start Date is correct. If incorrect, click on the calendar icon and select the correct date. 

Click “Download Sample Sheet”. A sample sheet will be automatically populated and downloaded. 

The file can be then moved to the file folder where the instrument can access it. 
Click “Finish Wet Lab Workflow” when complete.

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