LRN AMD Wet Lab Video Transcript:
The LIMS Lite Launchpad provides summary statistics and allows for easy navigation among modules using the left sidebar.
To upload samples into LIMS Lite, navigate to Sample Management
On the Sample Management page, samples can be accessioned by uploading sample data in .csv format.
To upload the samples file, select ‘Anthrax Sample Collection Metadata Template’ and click ‘CHOOSE FILE’. In the file select window, select your file and click ‘Open’.
You will see your file displayed in the Sample File Types window.
Click ‘UPLOAD’ to upload the file into the LIMS Lite.
A sample template can be downloaded to provide guidance for arranging data in your sample file.
Select ‘Anthrax Sample Collection Metadata Template’. Click ‘DOWNLOAD’ to download the template.
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 Anthrax 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.
After selecting the number of samples to include, click ‘Start’.
In the Components window, select ‘View by Workflow Step’ to toggle components based upon the steps in which they will be used.
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.
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. *Note* Some reagents are included in multiple steps.
The AMD LRN Workflow
This is the LIMS Lite workflow screen.
The first step of the AMD LRN workflow is the Gridding 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 the samples positioned.
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’.
This window can be closed by clicking outside of it in the gray area.
Clicking the ‘Instructions’ tab displays the laboratory protocol instructions that accompany the current step of the workflow.
Step 1: Gridding
Gridding of samples can be done through file import or through manual entry via the user interface.
Gridding by file import is not applicable to the AMD LRN Workflow.
To begin manual 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 position 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 Rack’ 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: DNA Extraction
This page displays a diagram of the sample rack with positive control, negative control, and samples assigned to their respective wells.
Scrolling down, you will see these well positions displayed again in table format.
DNA Extraction reagent information is displayed for easy reference, with volume per well and total volume shown with the respective lot number that was selected before beginning the run.
Click on ‘Rack Details’ to view control and sample positions entered in the Gridding step.
At the bottom of the page is the ‘COMMENTS’ button.
Clicking on this button displays a window where you can enter and save any notes for this step.
A ‘SHOW COMMENTS’ button will now appear, which you can click to view all entered comments for thi step during the current workflow.
Step 3: Fluorometric Quantification
Fluorometric quantification uses a dye which interacts with DNA to accurately determine the concentration of DNA in a sample.
The Fluorometric Quantification page guides you to create a master mix and record DNA concentration determined by a fluorometer.
The master mix for this step is calculated according to the sample count (including PTC and NTC) entered before the run.
An additional amount factor of 2 samples is incorporated into the calculation, and the volumes for each well and total volume are displayed accordingly.
This page also displays a section to enter in measured concentrations of controls and samples.
As you can see, positive controls and samples are highlighted in red. LIMS Lite provides an extra check to remind you of the acceptable range for the workflow.
Only a sample concentration within this range will remove the red highlight. To reference this range, hover over the exclamation point icon.
Once all sample concentrations have been entered, move to the next step.
Step 4: Spectrophotometer QC
To assess the purity of DNA, a spectrophotometer is used to measure absorbance ratios.
The Spectrophotometer QC page collects information about the spectrophotometer used and the absorbance readings observed for each control and sample.
Acceptable ratios of absorbencies are displayed for reference.
Step 5: Dilution
The Dilution step ensures that no sample exceeds 100 ng/uL. If any samples exceed 100 ng/uL, this step will quantitate the volume of sample and water that will be used to prepare 46uL of 100ng/uL.
Step 6: Gel Electrophoresis
The Oxford Nanopore Technologies sequencing platform requires high molecular weight DNA.
Gel electrophoresis is an optional step that can be used to determine the length and quality of DNA in a sample. It is also possible to skip this step by simply clicking ‘Move to Next Step’.
Select your E-gel file by clicking “Choose File”, selecting the file, and clicking upload image.
This E-gel can be compared to the Sample Gel image and a Quality Score entered that ranges from 1 (lowest) to 3 (highest).
You may also download the E-gel image from LIMS Lite for your reference.
Step 7: Barcoding Primer Selection
On the Barcoding Primer Selection 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 wells, sequence names and sequences for the selected kit in a table format.
Click on “Show Asset” to see the barcode rack and sample rack displayed side-by-side.
Click on a position on the barcode rack to select the first barcode well that will be used in the barcoding workflow step.
The barcodes for all sequential sample and control wells will populate on the barcode rack, and the sample rack will update to reflect the barcode selections.
To view the selected barcode reagents and their corresponding sample/control wells, click “View Mapping”.
Step 8: Barcoding
Now that barcodes have been assigned to sample and control wells, you will see that information displayed on this page in table format.
Enter the ID of the thermocycler being used in the barcoding step and click on Barcoding PCR program to see the thermocycling profile displayed for reference.
Step 9: Pooling
The pooling step combines equal volumes of all samples to be included in the sequencing run.
Given the target pool DNA amount of 400 ng, the volume to pool of each sample and positive control are calculated and displayed.
The negative control is not included in the pooled samples and is highlighted in yellow.
Step 10: Library Preparation
This page displays the instructions for library preparation, with step numbers, description and reagent information included.
Step 11: Instrument Set Up
On this page, you may perform a pre-run check to test the connection between the MinION and laptop, enter the date of sequencing, and review MinION and laptop information.
Step 12: Prep Flow Cell
On this page, will record the pre- and post-loading pore counts, and review the instructions for preparing the flow cell.
The provided information under “Run Info Entry in MinKNOW” is entered into the new MinKNOW browser
Step 13: Collect Genomics Data
Enter in Pore Occupancy and click ‘CHOOSE DIRECTORY’
Select the directory containing sequencing data files for your current run and click ‘FINISH’.
Click the activated ‘Confirm .fastq Files are Ready’ button.
Refer to the Estimated Time Overview for an estimate of when results will be ready for review.
Once results are ready, click ‘View Results’.
Click ‘FINISH WORKFLOW’ to complete the workflow run.