Normalase is an enzymatic NGS library normalization procedure that produces libraries with a specified molar concentration. This eliminates the need for library quantification and concentration adjustment prior to library pooling, resulting in optimal clustering density and index balance. Normalase is bead-free and does not use a limited capacity immobilization step which saves time and improves performance. Instead, following PCR amplification, each library is incubated with Normalase reagents to produce a set of equimolar samples for simple pooling. Normalized libraries can then be directly sequenced without additional purification. Normalase allows for more than 10-fold variation in input quantity, while generating less than 10% variation in sample clustering within a pool. Sequence analysis of normalized libraries indicates that performance metrics such as coverage, complexity, base composition and coverage uniformity are not affected. At the same time, the Normalase procedure offers a significant reduction in time, number of steps, cost and the likelihood of errors in loading when preparing samples for high throughput sequencing without affecting biological data endpoints such as allele frequency. In addition, size-independent normalization by Normalase is beneficial for libraries produced by transposon-mediated or enzymatic
fragmentation where a broad size distribution complicates molarity calculation. It can also be used for library normalization and pooling in hybridization-capture protocols.