# Re-visiting error rate in Skyline and figuring out the math in pooling samples for sequencing

Today I re-did the Skyline peak-picking error rate for the 2015 Oysterseed DIA and sent off the results to Emma for feedback. I also worked on figuring out how to make the 12 pooled samples that will be sent off for sequencing. Awaiting Steven’s feedback on that.

### 2015 Oysterseed

I made my own repo for the 2015 Oysterseed project.

Here is a link to my error rate that I did today: here

Emma responded and said that my error rate (almost 50%) looks pretty bad. I agree. So I’m going to go through again and check all my settings. Hopefully this gets sorted out quickly so that I can FINALLY continue with this process.

### Pooling samples: math thoughts

GitHub issue

Here’s what I think I’m going to do:

The pools are the yellow boxes. In the boxes, the values are the amount of ul that I should remove from that sample to get 10ng of RNA. I would then add those uls to a new tube, which will become the pooled sample (4 samples per pooled sample). Then, because the CORE wants a sample that is 50ul, I would add enough ul of 0.1% DEPC-treated H20 such that the final volume of the pooled sample is 50ul.

Here’s a screenshot of the amount of ul per sample that contains 10ng of RNA:

Waiting for Steven’s feedback on that and then the instructions for next steps.