Dear Jen, I am not much of a Galaxy user yet. Some days ago I know something about Galaxy and found it a really wonderful tool. And I am confused by a simple question regarding how to extract intron sequences from [gtf file]; Here is a simple of a gtf file: 1 Cufflinks transcript 3 22 1000 + . gene_id "CUFF.26"; transcript_id "CUFF.26.1"; 1 Cufflinks exon 3 22 1000 + . gene_id "CUFF.26"; transcript_id "CUFF.26.1"; exon_number "1"; 1 Cufflinks transcript 10 40 1000 - . gene_id "CUFF.204"; transcript_id "CUFF.204.1"; 1 Cufflinks exon 10 15 1000 - . gene_id "CUFF.204"; transcript_id "CUFF.204.1"; exon_number "1"; 1 Cufflinks exon 30 40 1000 - . gene_id "CUFF.204"; transcript_id "CUFF.204.1"; exon_number "1"; I want to extract intron from the [gtf] file. I found 2 ways may solve the question but it is both useless; 1. I use (Filter and Sort) -> Filter to cut the [gtf] file into 2 files such as the follows: File A ( contain transcript ): 1 Cufflinks transcript 3 22 1000 + . gene_id "CUFF.26"; transcript_id "CUFF.26.1"; 1 Cufflinks transcript 10 40 1000 - . gene_id "CUFF.204"; transcript_id "CUFF.204.1"; File B ( contain exon): 1 Cufflinks exon 3 22 1000 + . gene_id "CUFF.26"; transcript_id "CUFF.26.1"; exon_number "1"; 1 Cufflinks exon 10 15 1000 - . gene_id "CUFF.204"; transcript_id "CUFF.204.1"; exon_number "1"; 1 Cufflinks exon 30 40 1000 - . gene_id "CUFF.204"; transcript_id "CUFF.204.1"; exon_number "1"; Then I use (Operate on Genomic Intervals)->Subtract to subtract File B from File A Return Non-overlapping pieces of intervals. I thought it will return a file containing intron But the result is an empty file; 2. I convert [gtf] file to [Bed] file ,and use (Extract Features)->Gene BED To Exon/Intron/Codon BED, and it return the same result, an empty file. I think it must be something wrong with my thoughts. So I really need your help. Thank you very much. sincerely yours, John