This technical note outlines an alternative workflow for generating 10–15 kb HiFi reads from high-quality genomic DNA using the LongPlex Long Fragment Multiplexing Kit. In this approach, LongPlex is used to fragment and barcode samples, and PacBio’s Short Read Eliminator (SRE) is applied to size-select fragments >10 kb before SMRTbell® library preparation.

LongPlex uses plate-based transposase tagmentation for multiplexed fragmentation and barcoding, removing the need for mechanical shearing and allowing barcoded samples to be pooled before SMRTbell prep. This simplifies the workflow, increases throughput, and lowers library prep costs.

The standard LongPlex protocol generates 6–9 kb HiFi reads from high- to medium-quality DNA—ideal for microbial and other small-genome projects. However, users working with higher-quality DNA may want longer HiFi reads to maximize gigabase yield on PacBio systems.

This modified workflow is only suitable for high-quality DNA (Femto Pulse GQN30kb ≥7). Using degraded DNA will result in substantial sample loss during SRE size selection.