Using RNA-Seq to quantify gene levels and assay for differential expression including transposable elements

Background

• Transposable elements make up between 20 to 80% of the genome sequence for many eukaryotes, yet are typically excluded from the analysis that follows transcriptomic profiling with RNA-seq. This exclusion is due to the repetitive nature of transposons and the ambiguity that accompanies assigning multi-mapping reads.

Step by step analysis

• Mapping
  • Use ​STAR or another spliced mapper to map short reads to the genome of choice.
  • See our ​mapping SOP to search for details on running STAR.

• Quantification of raw counts

# Unstranded reads
TEtranscripts --format BAM --stranded no -t treat1.bam treat2.bam -c control1.bam control2.bam --GTF genes.gtf --TE transposons.gtf --mode multi --project treat_vs_control --minread 1 -i 100 --padj 0.05 --norm DESeq_default --sortByPos

# Forward stranded reads
TEtranscripts --format BAM --stranded forward -t treat1.bam treat2.bam -c control1.bam control2.bam --GTF genes.gtf --TE transposons.gtf --mode multi --project treat_vs_control --minread 1 -i 100 --padj 0.05 --norm DESeq_default --sortByPos

# Reverse stranded reads
TEtranscripts --format BAM --stranded reverse -t treat1.bam treat2.bam -c control1.bam control2.bam --GTF genes.gtf --TE transposons.gtf --mode multi --project treat_vs_control --minread 1 -i 100 --padj 0.05 --norm DESeq_default --sortByPos

• Other
  • Alternative software:
    • ​Transposable element expression in tumors is associated with immune infiltration and increased antigenicity - Yu Kong, Christopher M. Rose, Ashley A. Cass, Alexander G. Williams, Martine Darwish, Steve Lianoglou, Peter M. Haverty, Ann-Jay Tong, Craig Blanchette, Matthew L. Albert, Ira Mellman, Richard Bourgon, John Greally, Suchit Jhunjhunwala & Haiyin Chen-Harris Nature Communications 10, 5228 (2019)