How do I check the expression level of a unannotated lncRNA in RNAseq dataset?

Question

[Tony]

I plan to check a lncRNA expression level in RNAseq dataset, but the lncRNA was just identified and not annotated yet.Only I know is its position in chromatin and the sequence of it. so how can I perform the RNAseq analysis to check its expression level? Thanks

[GenoMax]

is its position in chromatin

What does that mean? Do you know its location in the reference genome or you don't?

[Tony]

Yes. I know its location in the reference genome.thanks!

[Tony]

Already got it solved! thank you all.

[Taktak31]

could you explain how you solved it

Answer 1

[GenoMax]

If you know its location in reference then you can simply add an entry for the lncRNA in whatever GTF/GFF file you are planning to use for counting. Be sure to follow the format of the file as you edit this entry in.

[Tony]

which program should I use to edit GTF? thanks for your patience here

[GenoMax]

GTF files are plain text so you use any editor that you like (except that may add invisible formatting e.g. word). NotePad++ (Windows), Textedit (macOS), Nedit/Nano (Linux) are options. You should simply duplicate one relevant entry in your GTF and then edit the new info in so you can be sure that format stays intact.

[Tony]

THANK YOU!!

Answer 2

[heskett]

You need to add the gene to your reference annotation! If youre using STAR this will be in a GTF file. If you want a quick way to see how many reads are in previous alignments you can use samtools view with the region option

[ATpoint]

Alternatively, if you know the sequence you can add the transcript to the transcriptome fasta file and use selective aligners such salmon which works directly on that fasta file, without the need of a GTF file.

[Tony]

Thanks! So if I use salmon, where should I get the transcriptome fasta file, transformed from gtf file? and which program should I use to add the sequence to the fasta file? I am new in this area and thanks for your patience.

[Tony]

Thanks! so I can use STAR to add my lncRNA sequence to the gtf file, right?