Experimental Design - Replicate, Randomize, Block

The design I had set up in the last post is no good. The problem is that I am confounding technical replicates and biological replicates and have no way to tease them apart: each lane includes biological replicates, but not technical ones, so I have no idea if a difference between the lanes is due to biology or to technical differences with the sequencing.

 Here is my new solution.

I have 8 treatments: 

P. campbelli male (BBM)

P. campbelli female (BBF)

P. campbelli X P. sungorus male (BSM)

P. campbelli X P. sungorus female (BSF)

P. sungorus X P. campbelli male (SBM)

P. sungorus X P. campbelli female (SBF)

P. sungorus male (SSM)

P. sungorus female (SSF)

Each of these has 5 biological replicates (#1-5)

I will use 2 lanes and I have 24 barcodes.

So, after going over the Auer (2010) paper again, here is the new set up:

Lane 1:                      Lane 2:

BBM1+2+3              BBM3+4+5

BBF1+2+3               BBF3+4+5

BSM1+2+3              BSM3+4+5

BSF1+2+3               BSF3+4+5

SBM1+2+3              SBM3+4+5

SBF1+2+3               SBF3+4+5

SSM1+2+3              SSM3+4+5

SSF1+2+3               SSF3+4+5

This is 24 individuals per lane, meaning I have enough barcodes. And, most importantly, there is a technical replicate for each treatment --> Biological Replicate #3 appears in both lanes. This way I can test for differences between the lanes (technical replicates) and ascribe those differences to the sequencing platform while still parsing out the biological variance as well.

Auer, P. L. and R. W. Doerge. 2010. Statistical Design and Analysis of RNA Sequencing Data. Genetics 185:405–416.