Identifying novel isoforms from RNAseq data

Question

[taiber8]

Hi everyone, I'm relatively new to bioinformatics and I need some help. I have RNAseq data from a specific subset of cells from the mouse (2 ages, 3 repeats for each one) and I want to analyze it in order to find novel transcripts, and especially to find novel exons that are not annotated in the UCSC. Which tool would be suitable for that? I tried using Cufflinks and couldn't really get what I wanted. Thanks in advance, Shahar

[ATpoint]

and couldn't really get what I wanted

...because? Please be more specific.

A more recent tool would be stringtie for transcript assembly.

Answer 1

[sangram_keshari]

Have tied to look at all the options how to find novel transcripts using Cufflinks?

In that case, I think you might be missing the following things - This is from Cufflinks manual.

-g/–GTF-guide <reference_annotation.(gtf/gff)>

Tells Cufflinks to use the supplied reference annotation a GFF file to guide RABT assembly. Reference transcripts will be tiled with faux-reads to provide additional information in the assembly. The output will include all reference transcripts as well as any novel genes and isoforms that are assembled.

Then followed by looking at Cufflinks class code manual after Cuffmerge step -

Class code "J" - Potentially novel isoform (fragment): at least one splice junction is shared with a reference transcript

[Kevin Blighe]

Thanks sangram. Just adding that StringTie is an upgrade that supersedes Cufflinks. People should use StringTie in the future.

[sangram_keshari]

Yes, I completely agree. Even I encourage people to do with StrigTie (Because of its improvements in efficiency)

As a matter of doubt in the question dealing with Cufflinks, I tried to explain where it might be going wrong.