The SMRTbell Express Template Prep Kit 2.0 provides a streamlined, single-tube reaction strategy to generate SMRTbell libraries from 500 bp to >50 kb insert size targets to support large-insert genomic libraries, multiplexed microbial genomes and amplicon sequencing. With this new formulation, we have increased both the yield and efficiency of SMRTbell library preparation for SMRT Sequencing while further minimizing handling-induced DNA damage to retain the integrity of genomic DNA (gDNA). This product note highlights the key benefits, performance, and resources available for obtaining complete microbial genome assemblies with multiplexed sequencing. By using a single-tube, addition-only strategy, the streamlined workflow reduces the number of AMPure clean-up steps. This provides an opportunity to explore automation solutions for high-volume projects, while using as little as 1 ug of input DNA.