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

Background

• Repetitive elements comprise a substantial portion of many eukaryotic genomes. In humans, for example, estimates of the repetitive fraction of the genome range from ​1/2 to more than ​2/3. Moreover, repetitive elements are known to play an important role in cellular function and disease, yet are typically excluded from the analysis of high-throughput sequencing experiments. This exclusion is due to 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)