High quality and high yield of enriched DNA-Seq libraries possible using automated library preparation and capture methods

Abstract: Genomes hold an abundance of information, both invaluable and inscrutable. Whole-genome sequencing with Next-Generation Sequencing (NGS) technology has driven a wide diversity of investigations and applications, especially in the field of human health. Technological advances have improved throughput capacity and decreased cost-per-base, thereby increasing the feasibility of large-scale studies and sequencing-based diagnostic tools. Even so, the financial and computational costs of sequencing remain non-trivial. NGS can be economized by limiting the sequencing libraries to specific genomic regions during preparation thereby accommodating higher throughput and more replicates for equivalent sequencing and computational resources. 

Expanding the scope and scale of sequencing-based experiments through targeted enrichment adds logistical complexity to sample preparation.
Larger sample sizes impose greater labor demands, longer processing time, and more opportunities for errors. Automating sample preparations on liquid handling
instruments resolves many of these issues. Standardized NGS liquid handling platforms, such as the Sciclone G3 NGSx and NGSx iQ workstations from Perkin Elmer,
expedite implementation of NGS workflows by offering out-of-the box solutions for many popular reagent kits, including the Roche KAPA NGS workflows.
The KAPA HyperCap v3 workflow offers high-quality, automation-friendly library preparation and target enrichment and has been implemented on both the Sciclone
G3 NGSx and NGSx iQ systems. Sequencing libraries are constructed by using either the KAPA HyperPrep kit or the KAPA HyperPlus kit, ligation-based preparations with
an optional upfront enzymatic fragmentation step in the KAPA HyperPlus kit. Libraries are then enriched using the KAPA Target Enrichment Kit, an in-solution probe
hybridization process compatible with the KAPA HyperExome human exome probe panel or other custom probe panels. 

The automated workflow was validated on the Sciclone G3 NGSx workstations by processing replicates of human gDNA samples
in a low-throughput (16 samples) and high-throughput (96 samples) format. An additional 16 replicate samples were prepared on the NGSx iQ workstation using a
similar automated workflow that also implements an integrated robot and an on-deck thermocycler, ultimately reducing hands-on time. Libraries were prepared
using the KAPA HyperPlus kit, and then combined into pools of 8 libraries each for target enrichment using the KAPA HyperExome panel. All libraries were assessed
for quantity and fragment distribution before and after target enrichment. Results demonstrated consistent yields and expected fragment distributions for both
low-throughput and high-throughput processing, as well as across platforms, with minimal variation. Sequencing metrics revealed successful enrichment of high-quality
sequencing libraries, including low duplication rates, high on-target rates, and sufficient depth with uniform coverage across the targeted regions.

For Research Use Only. Not for use in diagnostics procedures. KAPA is a trademark of Roche. Sciclone is a trademark of Perkin Elmer, Inc.