intro-to-rnaseq-with-galaxy

Gene Quantification

Counting reads for each gene

Our next step is to quantify the spliced reads that aligned to each gene in our GTF file For two non-overlapping, multiple-exon genes, our alignment may look like this:

The tool featureCounts is part of the subRead package.

• The mapped coordinates of each read are compared with the features in the GTF file
• Reads that overlap with a gene by >=1 bp are counted as belonging to that feature
• In default mode, ambiguous reads will be discarded

Source

The result is a gene count matrix:

Running featureCounts

• In the Tools panel search bar, type featureCounts
• Select featureCounts under RNA-seq
• Under Alignment file click the and select the bam collection generated by STAR
• Under Gene annotation file select in your history
• Select hg38_genes.gtf
• Click Execute
• The result will be two collections: Summary and Counts
• View the Counts file for a sample by clicking the collection and clicking the
• Run MultiQC on the Summary collection (similar to previous steps, except selecting the appropriate tool (featureCounts) and input folder (featureCounts Summary)).

Question 8: Locate the "featureCounts:Assignment" plot, which shows whether reads were assigned to genes (features) or whether they failed to be assigned. What is the main reason for reads not being "Assigned"?

Next: Differential Expression

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