1. Upload data files (FASTQ) or select from the sample data library or history

   • If uploading data, see Uploading Data
   • If using provided sample data, see Getting Sample Data

2. Upload Reference Transcriptome (FASTA) or select from preloaded options

   • If using a preloaded reference, this is selected when starting the workflow

3. Upload Reference Transcriptome (GTF/GFF3) or select from preloaded options

   • If using a preloaded reference, this is selected when starting the workflow

4. Format data into collections if not using provided samples

   • See Formatting Collections

5. Select workflow

   • Select Workflows from the Shared Data tab in the masthead
   • Select either Single-End 2 Sample RNA-seq Differential Expression or Paired-End 2 Sample RNA-seq Differential Expression
   • Click the drop down arrow to the right of the workflow name and select Run

6. Select Input Files and add labels

   • The workflow attempts to choose the appropriate files. Confirm these are the correct selections
   • For parameter definitions, please see the full manual
   • The remaining parameters have optimized default values and do not require modification for most experiments. Detailed descriptions can be found in the full manual

Sample 1 Replicates
Select file collection for sample 1

Sample 2 Replicates
Select file collection for sample 2

Reference Transcriptome
Select the reference transcriptome file (FASTA)

Reference Transcriptome
Select the reference transcriptome file (GTF/GFF3)

Strandedness
Select how the reads relate to the reference file

Sample 1 Label
Provide a label for the first sample

Sample 2 Label
Provide a label for the second sample

Differential Expression Factor
Provide a label for the factor being compared (e.g. drug_x, time)

7. Click Run Workflow to begin analysis

8. Review output files

MultiQC Report
A compiled report of fastq and mapping QC statistics (HTML)

MultiQC Stats
Fastq and mapping QC statistics (TXT)

Sample1 Salmon Quant - Transcripts
Quantification of gene expression at transcript level (TABULAR)

Sample1 Salmon Quant - Gene
Quantification of gene expression at gene level (TABULAR)

Sample2 Salmon Quant - Transcripts
Quantification of gene expression at transcript level (TABULAR)

Sample2 Salmon Quant - Gene
Quantification of gene expression at gene level (TABULAR)

DESeq2 Results
Text report of differential gene expression analysis (TABULAR)

DESeq2 Report
PDF report of differential gene expression analysis (PDF)

Volcano Plot
Plot depicting magnitude and significance of differential expression (PDF)

Filtered Results Set of differentially expressed genes at a significance threshold of 0.05

Please contact help@galaxyworks.io for any questions or assistance.