== 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 [https://www.pnas.org/content/111/17/6131.full 1/2] to more than [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3228813/ 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 [https://github.com/alexdobin/STAR STAR] or another spliced mapper to map short reads to the genome of choice.
    * See our [http://barcwiki.wi.mit.edu/wiki/SOPs/mapping 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:
      * [[https://www.nature.com/articles/s41467-019-13035-2|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)