How do I make a lane mix for sequencing?

General Lane Mix Preparation

To ensure an equal distribution of the reads during the sequencing step, libraries should be pooled in an equimolar ratio for multiplexed sequencing. It is important to accurately quantify the individual libraries prior to pooling, as well as the final library pool (lane mix). To quantify and pool your libraries:

1. Measure the concentration (ng/µl) of each library using either qPCR or fluorescence-based assays (e.g., Qubit, Thermo Fisher Scientific Inc.). ATTENTION! Nanodrop is not recommended to measure the final concentration of each library as it is not sensitive enough for accurate readings. The concentrations obtained with Bioanalyzer, Agilent Technologies, Inc., and other capillary electrophoresis instruments should also be confirmed by qPCR or fluorescence-based assays.

2. Determine the average library size using microcapillary electrophoresis analysis (e.g., Bioanalyzer, Agilent Technologies, Inc.). Adjust the range for the calculation to include the whole library distribution and exclude any linker-linker artifacts or overcycling bumps (generally >1,000 bp).

   1. For QuantSeq and CORALL libraries including UDIs, the range can usually be set to 180 - 1,000 bp. Linker-linker artifacts in many Lexogen libraries have a size range of 150 - 200 bp, depending on the indexing strategy and the library preparation kit used and the lower cut-off of the library range should be shifted accordingly.

   2. ATTENTION! Some library preps (e.g., small RNA-seq library prep) can have different size distributions and cut-offs. As recommendations may vary due to the specific kit technology, before proceeding to the pooling please, always consult the user guide of the specific product used, which can be found here: https://www.lexogen.com/docs/ .

3. Calculate the molarity from the average library size and the concentration (ng/µl) using the following equation: Molarity = (library concentration (ng/μl) x 1,000,000 ) / (660 x average library size (bp)) Example: average library size: 320bp, Qubit concentration: 1.4ng/µl
   Molarity: (1.4 ng/µl x 1,000,000) / (660 x 320) = 6.63nM
   NOTE: A template for molarity calculation is also available for download on our website: 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).
   NOTE: Alternatively, individual libraries can be diluted to a common molarity (e.g., 2nM) and then an equal volume of each library can be mixed together to produce the final lane mix.

5. After pooling the libraries, the 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.