In-matrix library preparation for metagenomic sequencing of microbial cell-free DNA.

Metagenomic sequencing of microbial cell-free DNA (mcfDNA) enables comprehensive identification and quantification of diverse pathogens from blood and other biofluids. This approach enables minimally invasive diagnosis of deep-seated infectious disease, provides culture-free identification of antimicrobial resistance, and powers the discovery of novel microbial biomarkers for disease. However, widespread implementation of this approach is limited by lengthy and complex workflows, high host background cfDNA leading to high sequencing costs, and prevalent environmental DNA contamination risks. Addressing these barriers is critical for scalable deployment in both centralized and decentralized settings. To overcome these limitations, we developed Karius Helion-4 Chemistry (Helion-4), an in-matrix (DNA extraction-free) sample-to-DNA sequencing library workflow, to serve as a platform for mcfDNA sequencing applications in infectious disease, microbiome analyses, and disease biomarker discovery. We compared Helion-4 to two widely used metagenomic extraction-based sequencing workflows, as well as to the prior Karius chemistry platform (Digital Culture-3), using 36 clinical plasma specimens. Helion-4 enables end-to-end sequencing library construction for up to 96 samples in 5.25-6.1 h, including setup and final quality control evaluation, with 2.25 h of hands-on time when using automated liquid handling robots. Compared to the other methods, Helion-4 recovered 58-fold to 817-fold more endogenous mcfDNA per volume of plasma, while simultaneously demonstrating 1.8-fold to 6-fold lower exogenous background DNA contamination, likely due to the absence of DNA extraction. The fraction of mcfDNA reads among total reads was enriched by 60-fold to 164-fold for Helion-4 compared to current state-of-the-art methods, significantly lowering sequencing costs required for applications built on the Helion-4 platform vs other platforms. Collectively, these advances enable routine processing of small specimen volumes and provide a simple, efficient, and scalable approach for mcfDNA sequencing applications.IMPORTANCEMetagenomic sequencing of microbial cell-free DNA (mcfDNA) enables the identification and quantification of diverse pathogens from blood and other biofluids, providing minimally invasive and rapid diagnosis of deep-seated infectious disease. However, widespread implementation of this approach is limited by complex workflows, high sequencing costs, and prevalent contamination risks. Karius Helion-4 Chemistry, the first in-matrix (DNA extraction-free) sample-to-DNA sequencing library workflow, overcomes these limitations. Compared to the other methods, Helion-4 is faster, cleaner, and more sensitive. Helion-4 recovered up to 817-fold more endogenous mcfDNA per volume of plasma, while simultaneously demonstrating up to sixfold lower exogenous background DNA contamination. The fraction of mcfDNA reads among total reads was enriched by up to 164-fold for Helion-4, lowering sequencing costs. These advances by Helion-4 technology enable a simple, efficient, and scalable approach for mcfDNA sequencing applications and bring us closer to widespread, high-resolution, and real-time microbial profiling across diverse healthcare settings.