enhancer RNA counting from RNASeq data

Question

[firatuyulur]

Hi all, My question is to find a method to count enhancer RNAs from RNASeq data. Although it may have been asked before, I could not find any post close to mine. I have a couple of fastq files of RNASeq data. I am using TopHat for alignment. When I put my .bam files to IGV, I can visually see some reads in locations where enhancers bind to. But when it comes to using a tool to count the reads in those locations, although those specific locations are included in my .gtf file with an Ensemble ID, the count is "0".

my code for featureCounts is ../anaconda/bin/featureCounts -a .../bcbio_ku/share/bcbio/genomes/Hsapiens/GRCh37/rnaseq/ref-transcripts.gtf -o .../work/my_generated.counts -t transcript -s 0 -p -C .../final/Unif_DMSO/Unif_DMSO-ready.bam

Is there anyone using an alternative method for enhancer RNA counting? I believe a lincRNA counter or miRNA counter would help me build the method too. The last option for me is to create a unique .gtf file according to "http://enhanceratlas.org/download.php" for each cell line through a python script.

Thanks already

Answer 1

[Chirag Nepal]

You could use bedtools to count number of reads mapping to interest of your region

intersectBed -wa -wb -s -a bedFile -b BAMFile

Check different parameters

Keep in mind that eRNA are generally not well detected in most RNA-seq samples because of the way how libraries are prepared.

1) As eRNA are generally non-polyadenylated, libraries should be prepared with ribo-depletion kit to make sure non polyadenylated RNAs are in the sample. If poly-A is used in library prep, eRNA are removed/depleted.

2) eRNA are degraded rapidly, so libraries should be sequenced at very high coverage to detect these events. Alternatively, instead of steady mRNA (like RNA-seq), sequencing the nascent RNA (like GRO-seq) enriches eRNA.

3) Not usually done, but if libraries are further size selected, lets says, 50-500 nt long, expected size range of eRNA, you will enhance the eRNA signals.

[PoGibas]

Great answer! +1

Answer 2

[Ming Lu]

"most lincRNAs were polyadenylated and were dynamically regulated during T cell differentiation" Expression and regulation of intergenic long noncoding RNAs during T cell development and differentiation (Guangqi HU, 2013)