I am performing RNA-seq analysis using the following pipe - HISAT2 - StringTie - Ballgown
I used a cell-type specific enrichment method to target reads. I performed qRT with this same sample to quantify the presence of a single transcript and saw enrichment for this transcript. For those interested in the biology, I am targeting kisspeptin neurons in brain tissue.
Now, I am attempting to analyze RNA-seq reads and would expect coverage of this transcript, as I validated its presence using an alternative method, qRT. Instead, when I run StringTie in reference only mod, by using the -e parameter;
stringtie -p 8 -G $REF_GTF -e -B -o file/transcripts.gtf -A file/gene_abundances.tsv outputfile.bam
I find 0 coverage for this transcript.
ENSMUSG00000102367 Kiss1 1 + 133309827 133329722 0.0 0.0 0.0
This transcript clearly corresponds to an Ensembl transcript ID. However, this is the strange part, when I remove the -e parameter, modifying nothing else, I suddenly have coverage.
ENSMUSG00000102367 Kiss1 1 + 133309827 133329722 0.010713 0.003730 0.004618
I confirmed this transcript is present in my reference $REF_GTF file. Thanks for an ideas or explanations for such a discrepancy.
