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denovo analysis of rna seq data

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 :)

rna-seq next-gen

Don't you have a supervisor?

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

Right now I am using CLC.

And I am totally new in this.

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;

3 answers

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:

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.

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.

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.

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