Project Summary Long-read sequencing has the potential to greatly simplify sequencing, helping to accelerate scientists’ ability to perform de novo sequencing, haplotype phasing and transcriptomics. This project aims to develop a method to label DNA prior to next-generation sequencing, that maintains information about the proximity of fragments in the original strand, aiding in the downstream assembly of the sequencing data. To label the DNA, transposase will be loaded with specially-designed transposons containing barcode labels, and used to fragment the DNA, prior to next-generation sequencing. The first aim of this project is to construct the transposomes used to label the DNA. The second aim is to use these transposomes to tagment and sequence a model DNA system to demonstrate read lengths of ~50 kb using next-generation sequencing on less than a picogram of DNA. If successful, the sequencing approach developed in this grant will simplify synthetic long read sequencing, making high-accuracy and inexpensive long read sequencing more accessible to the genetics community.