Bulk RNAseq Quantification Walkthrough

Navigate to the Latch Data tab on the left panel.

Use the ‘Upload’ modal found on the top right, and upload your raw .fastq or .fastq.gz files to Latch Data with the upload modal.

Navigate to the Latch Registry tab on the left panel.

Create new Project (if needed) and a new Sheet under that Project for the experiment being run.

Select the ‘Import -> Bulk Import Sequencing Data’ modal on the top right of the screen.

Choose the folder containing your raw RNAseq data and click ‘Select’.

Confirm or modify name parsing method used to auto-group your samples and click ‘Next’.

Fill in ‘fastq_1’ and ‘fastq_2’ for your forward and reverse read file respectively and click ‘Import’.

This will generate a samplesheet on Latch Registry for your data.

Navigate to the Latch Workflows tab on the left panel.

Choose the ‘nf-core/rnaseq’ workflow in ‘All Workflows’ or My Workflows’ (if you have added it).

Import samplesheet with ‘Import Rows from Registry’.

Choose needed samplesheet, followed by needed samples (select all on top left if needed.

Map samplesheet columns to workflow parameters and click ‘Import’.

Click on the (x) for strandedness in most cases - only use strandedness if you have an nf-core specific strandedness column in your metadata.

Choose ‘Reference Genome’ from ‘Latch Verified Reference Genome’ tab.

Or choose your own Reference FASTA and GTF in the ‘Custom Reference Genome’ tab.

[Optional - Advanced] Choose alignment method or pseudoalignment method.

[Fill in a ‘Run Name’ and hit ‘Launch Execution’.

Do not use spaces in the Run Name.

We recommend leaving the Output Directory to its default location.

(Optional - Advanced) Choose other parameters in ‘Optional Arguments’.

There are descriptions for every parameter available when you hover above the (i) symbol on a parameter name.

[Optional] Monitor the execution under ‘All Executions’.

Double click the execution to be taken to detailed logs.

Once complete, the output will be found in the output directory under your provided run name.

Useful outputs are the run HTML report and all the computational outputs in the folder that matches the name of the aligner you used.

Navigate to the Latch Workflows tab on the left panel.

Choose the ‘DESeq2 (Differential Expression)’ workflow in ‘All Workflows’ or My Workflows’ (if you have added it).

Choose your input counts file from the outputs of ‘nf-core/rnaseq’.

All outputs from ‘nf-core/rnaseq’ have an output folder called ‘deseq2_counts’ with a TSV file that is the optimal input for DESeq2.

Choose your conditions for your samples in the ‘Manual Input’ design matrix tab.

Or choose advanced conditions for your samples in the ‘File/Registry’ tab through Latch Data or Latch Registry.

Choose ‘Report Name’ and hit ‘Launch Workflow’.

We recommend leaving the Output to its default location.

Navigate to the Latch Plots tab on the left panel.

Choose the ‘Differential Gene Expression Visualizer (0.0.6)’ template.

Once the DESeq2 run is done, choose your outputted run (or select from Latch Data if a custom output location was used) and scroll through for QC graphs and heatmaps.

Choose ‘Contrast Conditions’ and ‘Genes of Interest’ for comparison and visualize Volcano and MA Plots.

If needed, turn on ‘Dev Mode’ in the top right and add more plots or code blocks <Redirect to Plots Wiki>.

Click on the (x) for strandedness in most cases - only use strandedness if you have an nf-core specific strandedness column in your metadata.

Navigate to the Latch Workflows tab on the left panel.

Choose the ‘Pathway Enrichment Analysis’ workflow in ‘All Workflows’ or My Workflows’ (if you have added it).

Choose your needed contrast file from the DESeq2 run output.

Each run will have a ‘Data/Contrast’ folder with a file for every comparison created in DESeq2.

Choose your ‘Species’ and ‘Run Name’ and hit ‘Launch Workflow’.

Useful outputs are the final ‘Report.html’ generated.