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STEP 5 - RNA-seq Counts

RNA-seq Count tables

This page covers downstream analysis of RNA-seq data, focusing on gene expression quantification and differential expression analysis.

Gene Expression Quantification

Using STAR Gene Counts

If you used STAR with --quantMode GeneCounts, you already have count tables:

# STAR produces ReadsPerGene.out.tab files
# Column 1: Gene ID
# Column 2: Counts (unstranded)
# Column 3: Counts (first read strand)
# Column 4: Counts (second read strand)

Using featureCounts

Alternative quantification with Subread’s featureCounts:

module load subread

featureCounts -p -T 8 \
  -a genes.gtf \
  -o counts.txt \
  sample1.bam sample2.bam sample3.bam

Parameters:

  • -p - Paired-end reads
  • -T - Number of threads
  • -a - GTF annotation file
  • -o - Output file

As we are interested in Hba and Hbb genes, which are homologues and have similar sequences, we will use the -M option to count multi-mapping reads:

module load subread

featureCounts -t transcript -g gene_id \
 -a refGene.gtf \
 -p --countReadPairs \
 -s 0 -M -O --ignoreDup \
 -o featurecounts_table.txt \
 sample1.bam sample2.bam sample3.bam