Hi,

  I have total RNAseq data and one of my main goals is to map lncRNAs (long noncoding RNAs). I am using STAR for alignment and featureCounts for mapping (counting). For the lncRNAs, I think I need to use the annotation file gencode.v28.long_noncoding_RNAs.gtf.gz.  I will pass this annotation file on to the counter featureCounts.

I have already created a Genome Index for STAR but using the different annotation file, with --genomeFastaFiles /home/inah/RefGTF/GRCh38/sequence/Homo_sapiens.GRCh38.dna.primary_assembly.fa  --sjdbGTFfile /home/inah/RefGTF/GRCh38/annotation/Homo_sapiens.GRCh38.85.gtf .

My question is: Do I need to rerun STAR to create a Genome Index using the lncRNA annotation file gencode.v28.long_noncoding_RNAs.gtf.gz and then rerun STAR for the alignment before I run featureCounts with the lncRNA annotation file?   Or can I just run featureCounts with the lncRNA annotation file using the STAR alignments obtained with the other annotation file?  In other words, do I need to use the same annotation file for the aligner STAR and the counter featureCounts?

Thanks, Ina

Aligners like STAR, TopHat and Subread use an annotation to assist the mapping of RNA-seq reads. Since your data is total RNA sequencing data, it makes more sense to me to use a transcriptome annotation in your read mapping instead of a lncRNA annotation. You can then count mapped reads to lncRNAs using featureCounts.

Technically featureCounts works with any alignment results output from the aligner no matter which annotation was used in the mapping (or no annotation was used).