If this feature is not annotated, the programs for counting and measuring will not 'see' it. I would first visualize the expression by looking into the coordinates of this unannotated lncRNA using IGV or any other visual tool. If you have no reads mapping to this position, there is nothing you can do because it is not being expressed in your dataset.
If it is being expressed, you can create some 'fake' row at the annotation file (GTF file) using the coordinates of this lncRNA you have so HTSeq or Kallisto could see it.
Afterwards, you can use any statistics program (DESeq2, EdgeR) for telling if it is over or under expressed.
For Kallisto, you can transform your modified GTF to a transcriptome fasta file using gffread from the cufflinks package like this:
gffread -w transcriptome.fa -g Reference.genome.fa annotation.gtf
Afterwards, you can use this transcriptome.fa in Kallisto index and perform the counting with kallisto quant.
PS: Kallisto give you both normalized data and raw counts estimation. If you are going to use DESeq2 keep in mind that you must give only raw counts as input and never normalized data.
modified 3.6 years ago
3.6 years ago by
tiago211287 • 1.2k