Hi there, We sent in a ribosomal and total extracted RNA to the company Novogene for analysis and something strange has happened with the data. A key marker gene (almost essential to the analysis) is missing reads. Every single publication concerning JA has had reads for this wound induced protein (okay, didn't check all, but I've made sure many have). It's not even a question. It's there. It's there in decently high quantities. It's a published fact. My postdoc has additionally confirmed the presence of this gene using qPCR. It's not a part of any hypothesis. It's a fact that it is induced at this point.
However, out of the whole genome, all the biosynthetic genes for JA, all the important high and low induced transcripts, this one gene is said to have 0 reads.
What gives? The bioinformatician has been very unhelpful - he/she seems nice, but hasn't been reading the emails properly and answering the wrong questions. His latest reason is that every single read (there are lots seemingly aligned to the genome (IGV viewer and bam files, TAIR10), especially when compared to other genes that have reads) fell into the 3 out of the 8 categories for being unable to have the read be quantified (HTseq - 5 out the 8 circumstances can be quantified). This gene doesn't have an overlapping genes or an intron.
Is this actually possible? That a published, verified, marker gene seriously can have no reads whatsoever? And in the same RNAseq analysis transcripts that are expected to have little to no reads can be quantified? We also have all the data showing the quality control was passed and the vast majority, almost all, reads mapped.
Sorry for the harsh tone, it's been a frustrating few weeks.