Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing

TLDR

The PacBio RS II enables single‑molecule, real‑time DNA sequencing and modification detection, and high‑molecular‑weight genomic DNA is essential for high‑quality sequence production. The study aims to automate PacBio SMRTbell library preparation by implementing high‑throughput methods to assess genomic DNA quality and ensure appropriate fragment size and library amounts. Automation was achieved with an Agilent NGS workstation and Bravo accessories for heating, shaking, cooling, and magnetic bead purification, while quality control from gDNA to final library was evaluated using Agilent Bioanalyzer and TapeStation systems. Automated 10‑kb PacBio library preparation yielded technical performance comparable to manual protocols, with the TapeStation system providing reliable 96‑well QC and DNA Integrity Number assessment, outperforming the Bioanalyzer for genomic DNA analysis.

Abstract

Abstract Background The PacBio RS II provides for single molecule, real-time DNA technology to sequence genomes and detect DNA modifications. The starting point for high-quality sequence production is high molecular weight genomic DNA. To automate the library preparation process, there must be high-throughput methods in place to assess the genomic DNA, to ensure the size and amounts of the sheared DNA fragments and final library. Findings The library construction automation was accomplished using the Agilent NGS workstation with Bravo accessories for heating, shaking, cooling, and magnetic bead manipulations for template purification. The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated. Conclusions Automated protocols of PacBio 10 kb library preparation produced libraries with similar technical performance to those generated manually. The TapeStation System proved to be a reliable method that could be used in a 96-well plate format to QC the DNA equivalent to the standard Bioanalyzer System results. The DNA Integrity Number that is calculated in the TapeStation System software upon analysis of genomic DNA is quite helpful to assure that the starting genomic DNA is not degraded. In this respect, the gDNA assay on the TapeStation System is preferable to the DNA 12000 assay on the Bioanalyzer System, which cannot run genomic DNA, nor can the Bioanalyzer work directly from the 96-well plates.