denovo analysis of rna seq data

Question

[kartikayprasad]

Hi all,

I am a master student with biology background. I recently inherit a RNA Seq analysis project from a PhD student in my lab. We already have paired-ended RNA Seq data generated from illumina HiSeq and mapped that sequence using de novo assembly. Now I get contigs but I am now get stuck (I don't have any reference genome for aligning), now I don't know what to do next.

My aim is to find the novel genes in rna seq data. PLEASE HELP

Thanks in advance :)

[Benn]

Don't you have a supervisor?

[Medhat]

As you said you are new, so I highly advice to read this review paper it will give you in depth knowledge about what you are doing right now I think it will be so helpful;

[Biojl]

What software did you use for the de novo transcriptome assembly?

[kartikayprasad]

Right now I am using CLC.

And I am totally new in this.

Answer 1

[GouthamAtla]

Basically you have the assembled transcriptome from RNA-Seq data. May be first you need to annotate the transcriptome using some pipeline like trinotate. See few posts below:

• Annotating sequences after de-novo Trinity assembly and RSEM analysis...there must be an easier way!
• Annotating A Novel Transcriptome Assembly

Once you have annotated the transcritptome, you can think of different ways to identify novel contigs with coding potential.

P.S I have never done this sort of analysis, so its just my idea.

Answer 2

[Biogeek]

What kind of organism are you working on?

CLCBIO should have an in-software annotation mode. You now should annotate the assembly. You should also check the assembly statistics to see if it is deemed a good assembly. Additionally Have you completed QC on your reads? In order to obtain a good assembly you should remove low quality bases and short sequences.

Answer 3

[Eva_Maria]

You can use Oases and followed by cap3 to get unigene form your contigs sequence. After that you can annotate unigenes by using various tool like blast or KEGG.