Company: BLLN
Filing Date: 2025-08-11
Form Type: DRS/A
Source: 0000950123-25-007483
Chunk: 19

Company: BillionToOne, Inc.
Filing Date: 2025-08-11
Form: DRS/A
Chunk 19
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ends the precision-versus-scale tradeoff that has limited legacy diagnostic methods. Conventional genetic analysis techniques, such as NGS and digital droplet PCR (ddPCR), typically sacrifice either sensitivity or multiplexing, 9i.e., ability to interrogate many genomic loci at the same time, forcing compromises in clinical utility. We believe our platform eliminates this constraint, delivering both high sensitivity and broad genomic coverage simultaneously. This unique capability allows us to provide physicians and patients with more actionable information from cfDNA that was previously possible only with more invasive diagnostics.

| 8 |     | Stasik S, Mende M, Schuster C, et al. Sensitive Quantification of Cell-Free Tumor DNA for Early Detection of Recurrence in Colorectal Cancer. Front Genet. 2021;12:811291. |

| 9 |     | Tsao, D. S., Silas, S., Landry, B. P., Itzep, N. P., Nguyen, A. B., Greenberg, S., Kanne, C. K., Sheehan, V. A., & Lo, Y. H. (2019). A novel high-throughput molecular counting method with single basepair resolution 
 enables accurate single-gene NIPT. Scientific Reports, 9, 14382.                                                                                                                                                       |

5

Our smNGS platform is powered by several patented technologies, most notably the use of synthetic DNA fragments, called QCTs. QCTs encode the molecular information in sequencing data which are subsequently decoded using proprietary machine learning and bioinformatic algorithms, enabling us to precisely quantify cfDNA. The precision and multiplexability of QCT-poweredassays make them ideal for addressing clinically significant challenges where sensitivity of rare-variant detection or quantification necessary for longitudinal measurements is essential. The power of QCTs is best exemplified when the clinical problem itself is quantitative, as in the case of fetal risk assessment of recessive conditions during pregnancy and monitoring of response to therapy in a cancer patient. QCTs and our other smNGS technologies also serve as the backbone of our technical operations. We leverage this quantitative foundation to track samples throughout our workflows, to drive continuous operational improvement, and to support robust quality controls. For instance, QCTs detect cross-contamination down to the level of <0.001% and thereby enable the creation of carefully constructed and automated end-to-endlaboratory workflows, 10including special laboratory infrastructure, that prevent such cross-contamination that