The Twist 96-Plex Library Preparation kit prepares up to 96 DNA samples for Illumina-based sequencing at a time. The protocol requires 50 ng of high quality input DNA for optimal sequencing results. The first step of the protocol, a polymerase-mediated primer extension reaction, is performed in a 96-well plate. Following this step, reaction products are combined into one pool for all subsequent steps (Figure 1). Final prepared libraries will have barcodes and adapters compatible with Illumina sequencing chemistries (Figure 2). Experienced users can expect to complete one 96 sample prep workflow within three hours.