RNA-seq Experiment Set Up

Learn to set up an RNA-seq experiment from FASTQ or Processed Counts files. Choose to follow a tutorial video or step-by-step walkthrough.

Tutorial Video:

This 10-minute video demonstrates how to set up and launch an RNA-seq experiment from FASTQ or Processed Counts files. 

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Step-by-Step Walkthrough:

Step 1: Create a new Project folder by clicking Enter a Title and Description (both can be edited at a later time).


Click the  button to proceed and automatically create a new Experiment.

Alternatively, click on an existing Project folder to open it. Then create a new Experiment by clicking . Click the  button to proceed.


Step 2: Select "Design a New Experiment" to access all available assay workflows, then click Next.


Step 3: Enter an "Experiment Title" and "Description" (both can be edited at a later time), then click Next.


Step 4a (FASTQ only): If creating an Experiment from FASTQ files, use the "Method (Technology)" drop down menu to select "Sequence." From the "Experiment Type" drop down menu, select "RNA-seq (FASTQ)." Then click Next and skip to Step 5.

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Step 4b (Processed Counts only): If creating an Experiment from a processed counts file (raw or normalized), use the "Method (Technology)" drop down menu to select "Sequence." From the "Experiment Type" drop down menu, select "RNA-seq (Processed Counts)." Then click Next.


Use the toggle to indicate if the processed counts are raw or normalized. Then click the Processed Counts File upload box ("BROWSE OR DROP FILE") to navigate to and select the input file, or drag and drop the file into the box to begin upload. Optionally, a completed Attribute File can be uploaded at this stage. If skipped, upload and creation of an Attribute File will occur later in the workflow. Click Next.


Step 5: Select the Species and Genome Build from the dropdown menus. ROSALIND offers genome builds from NCBI and Ensembl. Unless comparing the experiment to historic data, it is recommended to select the most recent build from your preferred source (NCBI or Ensembl). Click Next. If following the Processed Counts workflow, skip to Step 8.

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Step 6 (FASTQ only): Select the "Kit Vendor" and "Kit Model" from the dropdown menus. If you are unsure which kit was used for the experiment, contact your service provider to obtain that information. If the name of the kit is not listed, reach out to ROSALIND Support at support@rosalind.bio for guidance, or select "Other" to run a generic, unstranded analysis.

Click to confirm the selection, and ensure both a "Sample Kit" and "Library Kit" are registered to continue. An "Auxiliary Kit" is optional.

Click Next.


Step 7 (FASTQ only): From the drop down menu, indicate if the Experiment contains samples that are biological replicates ("Yes" or "No"). Enter the "Total number of samples" in the Experiment. Click Next.


Step 8: Specify sample attributes by typing the attribute categories into the "Enter Attributes" line.

After entering attribute categories, download an attribute file (.CSV format) by clicking . Within the file, attribute values can be manually assigned to each sample. The file can then be uploaded into ROSALIND by clicking .  Further instructions for attribute file creation can be found here or in the video at the top of this Help Center article. 

Click Next.


Step 9: ROSALIND displays a sample sheet to review for accuracy. Optionally, values for "Laboratory Identifier" and/or "Tube/Plate Barcode" can be added for internal tracking. Once reviewed, click Next.


Step 10 (FASTQ only): Indicate which samples are part of the same replicate group. This is optional though recommended to visually enhance attribute tracking. Select the drop down menu adjacent to each sample to assign replicate group membership. As replicates are assigned, replicate groups will change to coordinating colors for visual tracking. Once all replicates are assigned, click Next.


Step 11: Replicate group membership can also be used for attribute changes and tracking. If an error is noted and an edit is made, this edit will be applied to all samples in the same replicate group, reducing time for edits and risk of error. 

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Click "Next" to review attribute information for each sample, one-by-one, or click the last red dot on the right side to skip to the last sample. Click Next to proceed to Final Sample Review.


Step 12: Final sample review can be displayed in a standard table or in a table with replicate group coloring applied. Use the "Colored By" drop down menu and select "Replicates" to turn on group coloring, if desired.

After reviewing the sample information, click to confirm that the information is correct.


Step 13: Comparisons can be configured now, prior to launching the analysis, or after the experiment has been processed and QC has been reviewed. To create a new comparison now, click . Comparisons can be created at the replicate group level (using the attributes) or individual sample level (using the sample names) by dragging and dropping different attributes or samples into the condition and control boxes on the right side of the page. Combinations of two or more different attributes can be used to create comparisons with specific subsets of samples. In the example image below, a comparison is set up across two treatment groups (Radiation vs Control) at a specific time point (1 hour). Alternatively, the comparison could be set up specifically at only the 48 hour time point, or including both time points simultaneously.


Additionally, covariate correction can be added to the statistical model using the "SELECT COVARIATE" button. Click here to learn more about covariate correction for RNA-seq analysis.


Click the button to save the created comparison. Once all comparisons have been saved, click Next.

*Note: If the experiment was created using Processed Counts, then experiment will launch after comparison creation is complete.


Step 14 (FASTQ only): Lastly, upload the FASTQ files (in FASTQ, GZ, or GZIP format). Toggle between "Single-End" (one file per sample) or "Paired-End" reads (left and right read per sample) according to the sequencing strategy of your experiment. Then click the file upload box adjacent to each sample name to navigate to and select the corresponding files. Alternatively, drag and drop the files into the corresponding boxes to initiate file upload. Do not close or navigate away from the page until upload of all files is complete. After successful upload of all files, the experiment will launch. ROSALIND will notify you via email once the processing is complete and your experiment is ready for exploration.

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