General Lane Mix Preparation
Libraries should ideally be pooled in an equimolar ratio for multiplexed sequencing. It is important to ensure accurate quantification of the individual libraries prior to pooling, as well as for the library pool (lane mix). To quantify your libraries:
Measure the concentration (ng/ul) of each library using either qPCR or fluorescence-based assays (e.g., QuBit, Thermo Fisher Scientific Inc.).
Determine the average library size using microcapillary electrophoresis analysis (e.g., Bioanalyzer, Agilent Technologies, Inc.). Set the range to 160 - 1,000 bp to include the whole library size distribution, and to exclude any linker-linker artifacts (150 bp when using 6nt i7 single-indexing), or overcycling bumps (>1,000 bp).
Calculate the molarity from the average library size and the concentration (ng/ul) using the following equation: Molarity = (library concentration (ng/μl) x 1,000,000 ) / (660 x average library size (bp))
A template for molarity calculation is also available for download from https://www.lexogen.com/support-tools/lane-mix-calculation/
Pool the same amounts for each library (e.g., the same number of femtomoles for each library).
After pooling the libraries, the prepared lane mix and any dilutions made for denaturing (e.g., 2 nM), should be reanalyzed to ensure the accuracy of the concentration. This can be performed according to steps 1 and 2 as above.