Earlier today I quantified the libraries with the Qubit in preparation for sample pooling and sequencing. Before performing a full sequencing run, Mac wanted to select a subset of the libraries based on the experimental treatments to have an equal representation of samples. She also wanted to do a quick run on the MiSeq at NOAA to evaluate how well libraries map and to make sure libraries appear to be sequencing at relatively equal levels.
I created 4nM aliquots of all samples, using the average fragment length calculated by the Bioanalyzer region setting I created. The formula for determining molarity from concentration is:
Sample_Nameconcentration(ng/uL) * (660(g/mole/bp) * frag_len(bp))-1 * 1000000(uL/L) = molarity(nM)
After creating 4nM aliquots, I combined 1uL from each aliquot (per Mac’s typical procedure) and gave the pooled libraries to Mac to sequence at NOAA.
The following samples are those that were not used in the library pool:
CH05-26CH09-11CH09-29CH10-19
All these library calculations are in the principal spreadsheet for this project:
OA Crab Sample Collection 071119 (Google Sheet)
Specific columns:
qubit_concentration(ng/uL)qubit_molarity(nM)library_4nM_Vi(uL): Library volume needed for 4nM in 25uL aliquot.library_4nM_Vf(uL): Final volume of library aliquot (25uL).library_4nM_Cf(nM): Final concentration of library aliquot (4nM).library_4nM_H2O(uL): Water needed to bring aliquot to 25uL.
Additional details are available in this GitHub repo: