expression module

The expression module is responsible for determining gene expression levels from STAR bam and count files. For the highest accuracy the strandedness of the library preparation can be specified. By default, the module assumes the data is unstranded.

The expression of a configurable set of housekeeping genes is used to normalize the expression of the genes of interest.

Tools

This module relies on the STAR count files in combination with a set of housekeeping genes to normalize gene expression levels.

Input

The minimal input for this module is one BAM file and one STAR count table specified in a PEP configuration file, as is shown below.

Minimal input for the expression module
sample_name	bam	count
SRR8615409	test/data/expression/SRR8615409.bam	test/data/expression/SRR8615409.ReadsPerGene.out.tab

For more accurate results, it is possible to specify the strandedness of your RNA library prep (unstranded, forward and reverse). If strandedness is not specified, all samples will be treated as unstranded.

Sample configuration with strandedness
sample_name	bam	count	strandedness
SRR8615409-forward	test/data/expression/SRR8615409.bam	test/data/expression/SRR8615409.ReadsPerGene.out.tab	forward
SRR8615409-reverse	test/data/expression/SRR8615409.bam	test/data/expression/SRR8615409.ReadsPerGene.out.tab	reverse
SRR8615409-unstranded	test/data/expression/SRR8615409.bam	test/data/expression/SRR8615409.ReadsPerGene.out.tab	unstranded

Output

The genes specified under the report section of the configuration will be included in the HAMLET pdf report.
All genes from the bed file and the genes_of_interest will be included in the MultiQC report. Stranded (forward and reverse) and unstranded samples will be listed separately, since their values cannot be compared directly.

Configuration

The following options are available for the expression module

Configuration options

Configuration options
Option	Description	Required
gtf	A GTF file, to look up the ENSG for the housekeeping genes	yes
housekeeping	A list of genes to use for normalizing the expression	yes
bed	A BED file with genomic regions (genes) to quantify	no
genes_of_interest	A list of gene names to quantify (must be present in the gtf)	no
report	Genes to include in the PDF report, can include names from the bed file	no

Example

{
  "gtf": "test/data/reference/hamlet-ref.gtf",
  "housekeeping": [
    "MT-CO2"
  ],
  "bed": "path/to/bed/file.bed",
  "genes_of_interest": [
    "MT-ND3",
    "MT-ND2"
  ],
  "report": [
    "MT-ND3"
  ],
}