Dear list,

I am not sure whether this list is the right place to ask this study design
question. But on this list, I got lots of information regarding how to
analyze RNA-Seq data, so would like to give a try.

We are going to do RNA-Sequencing using Illumina HiSeq for 200 samples.
Given that the sample size is fixed, and the budget is fixed, the following
3 options were proposed.

1.  50bp pair-end reads, sequencing each sample per lane --> we will get
~100 million reads per sample
2.  75bp pair-end reads, sequencing two samples per lane --> we will get
~50-60 million reads per sample
3.  100bp pair-end reads, sequencing four samples per lane --> we will get
~30-40 million reads per sample

Based on your experience, which option is the best or you have other
suggestions? We would like to do different kinds of analysis for these
data, i.e.,novel transcripts, lncRNA, and splicing, SNP, etc. You name it.
If we have to sort them by priority (from high to low), I would like to say
" novel transcripts, long-noncoding RNAs splicing and differential
expression".

 Currently, the majority of labs sequence 100bp pair-end, right? But I was
told that even you sequence 100bp long, after 75bp, the sequencing quality
is very bad due to the issue of sequencer itself, that is, it has nothing
with the RNA quality of samples. If this is true, why is 100bp read length
becoming more popular now?

 Many thanks,
Shirley
 <zhangxl@bu.edu>

	[[alternative HTML version deleted]]

