Using Cufflinks for Novel Transcripts

Question

[Ron]

Hi all,

I am working on transcript level expression from RNAseq using cufflinks after STAR alignment. I am primarily interested in a gene which has a wild type transcript and a novel transcript.The wild type transcript has the coordinateds and is known so it is present in the gencode annotation file,so I have been able to calculate its expression value.

But , I also want to calculate expression of novel transcript for which I have only fasta sequence.How can I accomplish this? And also I know which exons its made up of compared to the wild type.

For e.g. if wild type has 6 exons,the novel transcript has 3 exons.

Thanks, Ron

Answer 1

[seidel]

Given that you have the fasta sequence of the novel transcript, you can map it to the genome to get the coordinates and add them to the GTF file you use when running Cufflinks. I can imagine doing this either manually - hacking your current GTF file by adding the coordinates of your novel transcript, or if your mapping program offers GTF as an output, you might be able to use cuffmerge to combine your current GTF and your newly mapped transcript GTF (might be tricky if they're not completely identical format).

On the other hand, if your novel transcript simply uses 3 of the available exons of a current transcript (i.e. mapped coordinates are identical), you can simply create a new entry in your GTF for the given gene, make up a novel transcript ID, and add the associated exons.

[Ron]

Hi Seidel,

Thanks for your response.I thought about creating a novel Transcript ID and use the necessary exons of interest.But I was not sure since this gene above has FPKM of 0.63 as well as all the exons. So if I just take a few of those exons for novel transcript ,wouldnt the expression be same again ? Since all these exons have same expression and hence resulting in the same mean expression of this gene.

chr1    Cufflinks   transcript  146386  173862  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";
chr1    Cufflinks   exon    146386  146509  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; exon_number "1"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";
chr1    Cufflinks   exon    155767  155831  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; exon_number "2"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";
chr1    Cufflinks   exon    164263  164791  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; exon_number "3"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";
chr1    Cufflinks   exon    165884  165942  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; exon_number "4"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";
chr1    Cufflinks   exon    168100  168165  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; exon_number "5"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";
chr1    Cufflinks   exon    169049  169264  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; exon_number "6"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";
chr1    Cufflinks   exon    172557  172688  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; exon_number "7"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";
chr1    Cufflinks   exon    173753  173862  1000    -   .   gene_id "ENSG00000241860.2"; transcript_id "ENST00000466557.2"; exon_number "8"; FPKM "0.6317694779"; frac "0.107515"; conf_lo "0.243566"; conf_hi "1.019973"; cov "1.602856";