WORKING COPY_How do I make a lanemix for sequencing?

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 and pool your libraries:

1. Measure the concentration (ng/ul) of each library using either qPCR or fluorescence-based assays (e.g., QuBit Qubit, Thermo Fisher Scientific Inc.).

   1. Nanodrop is not recommended to measure the final concentration of each library as it is not sensitive enough for accurate readings. Bioanalyzer, Agilent Technologies, Inc. and other capillary electrophoresis instrument concentrations should also be confirmed with either qPCR of fluorescence-based assays.

2. 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 ~150 - 200bp depending on indexing strategy and library preparation kit used), or overcycling bumps (>1,000 bp).

3. 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))

   1. e.g., average library size: 320bp, Qubit concentration: 1.4ng/ul

   2. (1.4 ng/ul x 1,000,000) / (660 x 320) = 6.63nM

   3. A template for molarity calculation is also available for download from https://www.lexogen.com/support-tools/lane-mix-calculation/

4. Pool the same amounts for each library (e.g., the same number of femtomoles for each library).

   1. Alternatively, individual libraries can be diluted to a common molarity (e.g., 2nM) and then an equal volume per library can be added together for a final lane mix.

5. 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.