An improved library preparation workflow for SMRT sequencing

The SingleMolecule Real-Time (SMRT) sequencing platforms offered by Pacific Biosciences(PacBio) provide continuous, low bias DNA reads upwards of 50kb in length. Thislong-read technology is ideally suited to provide end-to-end... [ view full abstract ]

The SingleMolecule Real-Time (SMRT) sequencing platforms offered by Pacific Biosciences(PacBio) provide continuous, low bias DNA reads upwards of 50kb in length. Thislong-read technology is ideally suited to provide end-to-end contiguity forlarge DNA assemblies, with advantages over short read length platforms inregions beset by low complexity, repetitive elements, or large structuralvariants. These capabilities ideally position the PacBio platform to provideenhanced coverage and assembly metrics for applications like de novoassembly, whole-genome sequencing, and full-length RNA isoform detection.Despite these benefits of long-read SMRT sequencing, the current limits ofsample turnaround time could be further improved to complete genome assembliesat lower cost.

Here, wedeveloped a novel library preparation workflow optimized for long-read SMRTsequencing. This method combines Swift’s enhanced repair and ligation chemistryto produce a high library conversion rate while simultaneously eliminatingformation of adapter dimers and chimeric inserts. In addition, this methodoffers a single-tube library prep workflow that can be completed in under 5hours instead of requiring multiple tube transfers and overnight enzymaticincubation. This results in a same day shearing to Blue Pippin workflow and theability to process 4 sample sets over 5 days. To validate performance, largeinsert libraries were prepared from Covaris g-TUBE and Megaruptor shears,enriched for large inserts using the BluePippin system, and run on the PacBioRS II platform. These libraries generated subreads up to 50 Kb in length withan N₅₀ subread length of up to 14 Kb. In addition,the library yields generally exceeded that of traditional SMRTBell libraries,allowing for reduced large shear DNA inputs as low as 2µg, opening up SMRTsequencing to lower input samples. Overall, the Swift library preparationprovides a convenient workflow and high quality performance optimized for theneeds of long-read SMRT sequencing.