September 2019

Differential Gene Expression Analysis Workflow


Counting

  • Use featureCounts (Liao, Smyth, and Shi 2014) programme from the subRead package

  • Need to provide featureCounts with an annotation file.

GTF File Format

GTF/GFF files define genomic regions covered by different types of genomic features, e.g. genes, transcripts, exons, or UTRs.

GTF File Format

GTF File Format

GTF File Format

GTF File Format

GTF File Format

GTF File Format

GTF File Format

GTF File Format

GTF File Format

GTF File Format

GTF File Format

Using FeatureCounts

When using a GTF/GFF file we need to tell featureCounts

  • what feature type to use to count reads

Using FeatureCounts

When using a GTF/GFF file we need to tell featureCounts

  • what feature type to use to count reads
  • what attribute type to summarise the results

For RNAseq we most commonly wish to count reads aligning to exons, and then to summarise at the gene level.

Using FeatureCounts

When using a GTF/GFF file we need to tell featureCounts

  • what feature type to use to count reads
  • what attribute type to summarise the results

For RNAseq we most commonly wish to count reads aligning to exons, and then to summarise at the gene level.

And now onto the Exercise…

Liao, Yang, Gordon K Smyth, and Wei Shi. 2014. “featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.” Bioinformatics (Oxford, England) 30 (7): 923–30. https://doi.org/10.1093/bioinformatics/btt656.