Development of multimodal comprehensive genomic profiling panel with on-flow cell hybrid capture

Hybrid capture is a widely used technique for comprehensive genomic profiling (CGP) due to its ability to target large number of regions and allows for the simultaneous detection of multiple types of genomic alterations. Traditional hybrid capture workflows, however, are often time-intensive and technically demanding, requiring long hybridization time, multiple wash steps, and tightly controlled thermal conditions, all of which limit throughput and turnaround time. The Element Biosciences AVITI TM sequencer, featuring its Trinity TM flow cell, introduces an integrated hybrid capture system that performs on-flow cell target capture, washing and eliminates post-capture amplification. This innovation reduces total workflow time and improves capture specificity, but also introduces new challenges for the relatively small CGP panel application due to the higher input requirements and lack of post-capture amplification. To address this limitation, a hybrid capture-based, multimodal library preparation workflow with the QIAseq xHYB CGP panel was optimized for compatibility with the AVITI system and Trinity flow cell. This involved designing AVITI-specific adapters and index primers, as well as modification of hybrid capture and library preparation protocols to suit the Trinity platform. Our study revealed that multimodal libraries prepared with AVITI-specific adapter and index are fully compatible with Trinity workflow. In addition, optimal CGP panel performance can be achieved by using a newly developed hybrid capture buffer at 71 °C for as low as 0.5 hour compared to traditional hybridization conditions of 60 °C for 16 hours. Furthermore, this workflow is compatible with both fresh DNA and FFPE DNA. Additionally, to achieve optimal Trinity output, we increased the yield of the standard multimodal workflow by implementing an enhanced amplification module. This involved increased amounts of primers and high-fidelity polymerase, along with optimized cycling conditions, resulting in a four-fold increase in library yield for both whole genome and whole transcriptome libraries. This optimized workflow enables robust CGP panel hybrid capture sequencing on the Trinity flow cell from low-input DNA, supporting small sample sets and expanding access to hybrid capture sequencing for low-throughput laboratories.