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how many replicates are required for discovery of novel isoform using RNA-seq and cufflinks RABT assembly

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.

rna-seq novel isoform detection
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updated by EagleEye EagleEye • written by rajeshkumar_vinod • rajeshkumar_vinod

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.

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written by Carlo Yague Carlo Yague

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?

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written by rajeshkumar_vinod • rajeshkumar_vinod

1 answer

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).

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written by EagleEye EagleEye

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)?

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written by rajeshkumar_vinod • rajeshkumar_vinod

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