Illumina Library Pooling for The ENCORE Priming Project in Bermuda
Pooling Libraries using Illumina
Protocols used
Best practices for manually normalizing library concentrations
This post details the protocol used to pool the libraries constructed with the use of the Zymo-Seq SwitchFree 3’ mRNA Library Kit. Libraries were placed into 2 pools and normalized to 4nM. See here for all molecular metadata sheets. Sequencing needs a minumum of 4nM and 30uL
See here for the Genohub Sample Submission Sheet.
Steps
- Determine the library size
- Quanitfy the libraries
- Plan the dilution calculations
- Pool the normalized libraries
Determining the library size of the libray of each sample is a way to QC your libraries after constructing them. to do this I used the Tapestation. This protocol also shows any possible library issues such as adapter dimers. This was done as a form of QC for all samples. See here for all dsDNA_Qbit results.
Library quantification was done using the Qbit dsDNA HS protocol. Library concentration values were converted from ng/uL to nM using the equation below from illumina’s general reference material.
\[\frac{\text{conc in ng/µL}}{660\,\text{g/mol} \times \text{avg library size in bp}} \times 10^6 = \text{conc in nM}\]Plan the dilution calculations
This step includes the concentration normalization. A minimum if 4nM is required. Dilution was done with molecular grade water to dulute all libraries to 4nM using the equation below. Dilution volumes can be found in pool 1 and pool 2 metaddata.
(C1)(V1)=(C2)(V2)
Pool the normalized libraries After libraries were all diluted to 4nM the pool calculator was used to determine volume of each library to be used in the pool. For both pools 2uL of each sample was used.Quality control for each pool using the tapestation was done and the pools were quantified using the dsDNA Qbit protocol.
Tapestation Results of both pools found here
dsDNA Qbit results
| Sample | ng/uL #1 | ng/uL #2 | ng/uL avg |
|---|---|---|---|
| Standard 1 | 50.55 | ||
| Standard 2 | 24004.99 | ||
| Pool 1 | 1.33 | 1.30 | 1.32 |
| Pool 2 | 1.42 | 1.41 | 1.415 |
The equation below was then used to convert the conc to nM \(\frac{\text{conc in ng/µL}}{660\,\text{g/mol} \times \text{avg library size in bp}} \times 10^6 = \text{conc in nM}\)