Enablement of long-read targeted sequencing panels using Twist hybrid capture and PacBio HiFi sequencing

¹Pacific Biosciences, Menlo Park, CA, United States; ²Twist Bioscience, South San Francisco, CA, United States; ³Leiden University Medical Center, Leiden, Netherlands

Background/Objectives:

Targeted resequencing allows for high-resolution characterization of gene panels at a scale and cost that is more accessible than whole genome sequencing. While long-read PacBio HiFi sequencing has been shown to accurately and comprehensively interrogate complex clinically actionable loci, such as pharmacogenomic targets, studies have been primarily focused on single genes using PCR amplicon- based methods. We describe a method to leverage Twist Bioscience target enrichment probes for the design of custom gene panels sequenced with HiFi reads.

Methods:

PacBio partnered with Twist Bioscience to develop a long-read hybrid capture protocol for custom gene panels of up to 2 Mb in size. The protocol follows a standard hybrid capture workflow with DNA shearing, barcoding, and sample handling optimized for long-read sequencing. The resulting enriched material is used to construct a SMRTbell library sequenced on the Sequel II system from PacBio. Up to 24 samples may be multiplexed with HiFi read length of 5-10 kb.

Results:

We demonstrate that this method efficiently and comprehensively covers gene targets using Coriell samples run with multiple gene panels of varying sizes, which include complex regions like pharmacogene CYP2D6.

Conclusion:

This protocol can be utilized with custom gene panels from Twist to efficiently capture genes of interest using long-read HiFi sequencing. The demonstrated method allows for scalable and cost- efficient hybrid capture with long read lengths, unbiased coverage, and high accuracy to fully capture all variant types, including structural variation that can be inaccessible to short-read sequencing and non-NGS technology.