Hi!

I'm currently planning to perform an RNA seq experiment using the Pacbio Iso seq platform.

The goal of my experiment is to identify alternative splicing isoforms during early stages of embryonic development from pooled embryos at different time points and I have two different batches to compare male and female embryos.

I will also include Illumina RNA sequencing on the same samples as 'technical' control.

The genome of my organism is well annotated and the genome size is around 280 Mb

What is the depth that you suggest in order to be able to identify most of the alternative splicing isoforms?

For Pacbio Iso-seq is worth including biological triplicates? I was thinking of using the Iso seq results as a reference and sequencing the triplicates only with Illumina.

Thanks in advance for the help