Create an experiment in Trovomics
The first step to analyzing your RNA-Seq data in Trovomics is to create an experiment. The Create Experiment function in Trovomics will create a new experiment that will be accessible on your Experiments Dashboard. An experiment within Trovomics will contain essential details on the RNA-Seq experiment you performed including title, description, samples, and their associated sequencing files (FASTQ). Create Experiment consists of 5 steps: Enter Experiment Details, Enter Sequencing Details, Build You Experiment, Edit Samples, and Review & Create. Once created, you will be able to manage/edit details and create an analyses within each RNA-Seq experiment.
Objective: Create a new experiment in Trovomics.
Pre-requisites:We recommend having the following RNA sequencing information and files handy before you create an experiment. Missing something? Contact your sequencing provider to request this information.
Organism your RNA-Seq samples were extracted from (i.e., Homo sapiens, Felis catus, Mus musculus, Sus scrofa, Rattus norvegicus)
Sequencing Platform (Note: Currently, only Illumina platforms are supported by Trovomics)
Platform Model
Analyzed molecule (i.e., mRNA, mRNA+lncRNA, or smallRNA)
RNA selection method (i.e., polyA selection, rRNA depletion, size fractionation)
Sequencing adapters used (i.e., Illumina TruSeq, Illumina Nextera, AmpliSeq for Illumina panels)
Sequencing read type or library layout (i.e., paired-end or single-end)
Sequencing sense (i.e. unstranded, forward, reverse)
Metadata table containing sample names, file names, strandedness, library layout, and experimental variables of your samples (see example below)
You can either read the tutorial below or watch our video! Follow along with our sales engineer, Dylan Falkowski, as he takes you through entering experiment details, uploading FASTQ files, and reviewing your experiment.
Step 1: Click Create Experiment in your Experiments Dashboard
Image 1. Experiment dashboard in Trovomics with an arrow pointing to the Create Experiment button.
Click Create Experiment in your Experiment Dashboard to begin (Image 1). You will be prompted to acknowledge that Trovomics is account for batch effects at this time (Image 2). Therefore, it is highly recommended to have all your sequencing files come from the same run.
Image 2. The batch effects warning pop-up that appears after you click Create Experiment.
TIP: At any step in the process, you can click “Save Progress and Exit” and your experiment will be saved as a draft in your Experiments Dashboard.
Step 2. Enter Experiment Details
Here you will enter basic information to identify your experiment (Image 3). We recommend being clear and descriptive to help you organize and differentiate between your experiments. The details you will need to enter are:
Name
Description
Select Organism
Sequencing type (Note: Currently, only RNA-Seq is supported.)
Image 3. The Enter Experiment Details step in Create New Experiment.
TIP: Follow your progress through Create Experiment using the flow chart on the left.
Step 3. Enter Sequencing Details
Here you will select the sequencing parameters used for your samples (Image 4). If you don’t have this information, you can request it from your sequencing provider. The parameters you will need to provide are:
Analyzed Molecule
RNA Selection Method
Sequencing Adapter
Sequencing Platform
Platform Model
Sequencing Read Type
Sequencing Sense
Image 4. The Enter Sequencing Details step in Create New Experiment.
Step 4. Build Your Experiment
Build your experiment is split into two sections: Format Your Experimental Design and Upload Your Sample Files.
Format Your Experimental Design – This is where you can upload your metadata table that contains essential information on your samples and experimental variables (Image 5). Of most importance, we use this table to assign each sequencing file to the correct sample. A template is available to help get your started with filling in your unique experimental details.
Note: Do not alter the “SampleName” or “Filename” column titles in the template. Additional columns can be added if your experiment had more experimental variables than the template contained.
Upload Your Sample Files – This is where you can bulk upload all your sequencing files at once (Image 6). It is important these file names match what is in your metadata table under the “Filename” column and they correspond to the correct sample.
Image 5. Format Your Experimental Design section in the Build You Experiment step. A metadata table has been attached “YN NP PP upload table.csv”. Arrows designate where you click to upload a file (left) and delete a file (right).
Image 6. Upload Your Sample Files section in the Build You Experiment step with an arrow pointing where to click to upload files.
Once you select your files, you will be able to see a list of all files ready for upload. After clicking Save and Continue, your samples will immediately begin uploading. You can monitor the progress in the pop-up (Image 7). Avoid navigating away from the page to ensure a smooth uploading process. Any disruptions may require you to re-upload samples.
Image 7. You can monitor file upload progress in the pop-up (top and middle panels). Once files are done uploading, you will be automatically directed to the Edit Samples step (bottom panel)
Step 5. Edit Samples
Once your files have finished uploading, you will be able to proceed to the Edit Samples section. Here, you can view all samples along with their associated files, and experimental variables & values. Edit Samples is split into two sections:
The first table, titled Review Your Variables and Values shows your global experimental variables and values associated with your experiment (Image 8). This information is sourced from the metadata table you uploaded.
The second table titled Review Your Individual Samples, allows you to view a list of all your samples (Image 10). From here you can:
View what files were assigned to each sample by expanding the row
Select samples to be deleted
Add new samples (Image 11) or duplicate existing samples (Image 12) in your experiment. As with bulk upload, avoid navigating away from the page when your file(s) are uploading.
Image 9. Review Your Variables and Values section in the Edit Samples step.
Image 10. Review Your Individual Samples section in the Edit Samples step. Arrows are pointing to where you click to add an additional sample (top left), delete a sample (right), and view sequencing files associated with each sample (bottom left). Use the search bar to find samples by their Sample Name (middle).
Image 11. Adding a new sample called “NP+ANG4_NEW” in Review Your Individual Samples.
Image 12. Selecting a sample to duplicate, will duplicate the sample’s details (name, experimental variables and values). Sequencing files are not duplicated and will need to be uploaded separately, similar to adding a new sample.
TIP: Made a mistake? Trovomics makes it easy to edit sample details, by giving you multiple options and opportunities for updates. Edit samples globally or individually. Edit within Create Experiment or after your experiment is created.
Step 6. Edit Experimental Variables or Values (optional)
Global Edits – If you find you need to edit the labels for experimental variables or values for all samples, you can do so in the table within Review Your Experiment Variables and Values (Image 13). Any changes made here will automatically update samples accordingly.
Update a variable or value and then click the black check box that appears.
A pop-up will appear allowing you to review which samples were updated.
You can view your updates in Review Your Individual Samples.
Individual Edits – If you only want to make changes to a select samples, you can do so in Review Your Individual Samples. Here you can:
Edit Sample Name (Image 14)
Assign a different the experimental value (Image 15)
Edit a sample’s FASTQ file metadata (Image 16)
Image 13. Steps involved in updating sample experimental variables or values. Changes to experimental variables and values are made in the Review Your Variables and Values section. Once completed, changes can be reviewed in Review Your Individual Samples.
Image 14. Directly edit sample names under the Sample Name column.
Image 15. Assigned the incorrect experimental value on your metadata table? No problem, you can select from the available options using the drop-down menus.
Image 16. Edit file metadata associated with a sample. Changing file metadata is not recommended in most instances.
Step 8. Review & Create
Almost done! At this step you will be able to review all information you entered, selected, or uploaded in your experiment (Image 17). Ensure everything is correct. If you spot any errors, you can go back and make changes by clicking “Edit” within that section. Once you click “Save and Continue”, you will be directed back to the Experiments Dashboard. From here, your new experiment can be found under ALL EXPERIMENTS.
Image 17. Review & Create step within Create New Experiment with an arrow pointing to where you can click to edit details in a section.
What’s next?
After you have successfully created your experiment, you can click on it in the Experiments Dashboard to review all its details (experiment details, sequencing details, samples, and sequencing files). From here you will also be able to start analyzing your RNA-Seq samples. Login to get started with your first experiment!