Question: how many replicates are required for discovery of novel isoform using RNA-seq and cufflinks RABT assembly
0
gravatar for rajeshkumar_vinod
3.6 years ago by
india
rajeshkumar_vinod30 wrote:

Hello everyone,

can I use RNA-seq data from 3 tissues each having 3,3,2 biological replicates and NO technical replicates to identify novel transcripts using cufflinks RABT based assembly?

if not then how many replicates do i need? Thank You.

ADD COMMENTlink modified 3.6 years ago by EagleEye6.6k • written 3.6 years ago by rajeshkumar_vinod30
1

I think that sequencing depth is more critical than replicates for novel transcripts discovery so only 3 or 2 replicates might not be the biggest issue for you.

ADD REPLYlink written 3.6 years ago by Carlo Yague4.9k

Thanks for your reply.

yes it is paired end data and other things are also ok.

i was just thinking how can i say whether it is a novel transcript or not? If cufflinks reports this transcript in majority of biological replicates(eg. in 2 out of 3 or 3 out of 3 biological replicates it is present) only then?

ADD REPLYlink written 3.6 years ago by rajeshkumar_vinod30

I agree with Carlo, depth will be more important than replicates. This paper might also be of interest to you: http://biorxiv.org/content/early/2016/01/29/038224

ADD REPLYlink written 3.6 years ago by WouterDeCoster43k
0
gravatar for EagleEye
3.6 years ago by
EagleEye6.6k
Sweden
EagleEye6.6k wrote:

The isoform analysis itself is bit tricky. If you are going to discover some novel isoform, I would not recommend to just use cufflinks (though good for finding novel genes). You have to try using some specific method for isoform discovery, some useful posts

A: What is best tool to call diferentially expressed isforms ?

For sequencing I would recommend paired-end strand specific sequencing with more depth (This should compensate the need for more replicates).

ADD COMMENTlink modified 3.6 years ago • written 3.6 years ago by EagleEye6.6k

Thanks for your reply.

yes it is paired end data and other things are also ok.

i was just thinking how can i say whether it is a novel transcript or not? If cufflinks reports this transcript in majority of biological replicates(eg. in 2 out of 3 or 3 out of 3 biological replicates it is present) only then?

and what is this long RNA-seq(>200 nt long)?

ADD REPLYlink written 3.6 years ago by rajeshkumar_vinod30

It has been discussed already and this post might be useful for you.

Rna-Seq: Novel Transcripts Found. What Next?

For long RNA-seq refer,

https://www.encodeproject.org/rna-seq/long-rnas/

ADD REPLYlink written 3.6 years ago by EagleEye6.6k
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