Rapid advances in long-read sequencing technology have led to dramatic improvements in cost and throughput. De novo sequencing projects using long-read sequencing to generate assemblies have grown from creating single reference genomes to examining population wide diversity across progressively larger groups of individuals, paving the way for a future of clinical human whole genome sequencing. Because of its high accuracy, Pacific Biosciences HiFi sequencing is already being used to study rare genetic conditions in pediatric cancer and to generate a human pan genome reference.
However, high throughput sample and library preparation methods are needed to support the generation of such large numbers of genomes. Such workflows are commonplace in short read sequencing but do not widely exist for long-read sequencing which requires manipulation of high molecular weight (HMW) DNA.
We present a high-throughput HMW DNA extraction, library preparation, and size selection workflow capable of processing 96 samples in as little as 8 hours for HiFi sequencing on Pacific Biosciences Sequel IIe systems.
Automated HMW DNA extraction is first performed using Nanobind magnetic disk technology on Thermo Fisher KingFisher instruments. In contrast to magnetic beads, each extraction uses a single Nanobind disk that is covered with micro- and nanostructured silica wrinkles to protect the bound DNA from shearing during automated processing. The method is capable of HMW DNA extraction from 96 cell, blood, and tissue samples in 60 – 180 minutes. Fully walkaway solutions are also available for use with Hamilton NIMBUS instruments.
Automated library preparation is then performed using PacBio Express Template 2.0 Kit on Hamilton NGS Star.
Finally, high throughput size selection is performed using a 96 well plate version of Short Read Eliminator technology. Short Read Eliminator technology uses a proprietary size selective precipitation chemistry to deplete small DNA from HMW DNA samples. Alternatively, a custom magnetic bead based size selection can be used to achieve high size selection cutoffs as well.
A variety of sample types, including cultured cells, bacteria, whole blood, and tissue are demonstrated. As these methods require only common commercial instrumentation, it can be easily integrated into many existing laboratory environments.
Organization: PacBio
Year: 2022