DNA library preparation for next-generation sequencing (NGS) involves fragmentation of target DNA molecules into varying sizes, end repair or A-tailing and ligation of platform-specific adapters to the library. The libraries are then amplified and cleaned up subsequently. Depending on the nature of the DNA sample (e.g., fresh sample versus formalin-fixed paraffin embedded (FFPE) tissue), the workflow may require optimization.
Fragmentation of DNA can be achieved through mechanical or enzymatic shearing. While mechanical shearing is considered the gold standard, enzyme-based fragmentation affords speed, less hands-on time and convenience to enable automation of the workflow. Since the adapter size is constant, the library size is adjusted based on the desired insert size and the type of application. Amplification of library fragments is typically required for many applications, including target enrichment. Regions within the genome, for example those that may be GC-rich, are difficult to amplify and may introduce sequencing bias.