Determining the Effects of Fragment Length on Digital PCR Dynamic Range and Optimizing Library Quantification
In order to maximize Illumina sequencing runs, the loading concentration of libraries must be carefully controlled. Both over- and under-loading will decrease the amount of usable reads by limiting the number of sequenceable clusters. One method to accurately load libraries is to use quantification with digital PCR (dPCR). dPCR provides absolute quantification, but like qPCR based methods, can be biased by libraries with extreme size distributions. Both the BioRad QX200 and the Combinati Absolute Q® were able to quantify multiple sizes of libraries, but encountered issues with an 1000 base pair sized library.