How to generate custom fasta and gtf files for RNA spike-ins

Question

[2405592M]

Hi guys, I've been doing some RNA-seq on mice and I used 3x E. coli tRNA spike-ins. I'm assuming for analysis, I'll have to generate a fasta and GTF file for these spike-ins? If so, how do I do this? Also, I'm assuming this will have to be merged with the mouse fasta and GTF files. How do I do this as well? Once these steps are completed, can I move on to alignment? The spike-ins were custom made oligos and so I only have the 3 sequences. I don't know any gene loci/coordinates etc.

Answer 1

[michael.ante]

Assuming the tRNAs are unspliced sequences, you can add them like the ERCC92 spike in annotation: Each spike in has its own contig and each gene annotation goes from start to the end.

Something like:

>mySpikeIn1
ACGA....

and the GTF:

mySpikeIn1   spikein  exon 1  9999 0.0  +  . gene_id "mySpikeIn1"; transcript_id "mySpikeIn1.1";

Please refer to the GTF file format documentation regarding spaces/tabs and necessary fields.

Cheers,

Michael