Company: BLLN
Filing Date: 2025-08-11
Form Type: DRS/A
Source: 0000950123-25-007483
Chunk: 177

Company: BillionToOne, Inc.
Filing Date: 2025-08-11
Form: DRS/A
Chunk 177
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 hundred loci to cover even a few genes of interest. Traditional NGS methods have transformed many areas of clinical testing due to their ability to multiplex across thousands of loci, such as enabling a single clinical test to cover multiple genes. However, these NGS methods face significant limitations when applied to cfDNA analysis. The scarcity of cfDNA necessitates amplification by a factor of millions before it can be sequenced. 39This amplification occurs at different rates across different genomic loci, which introduces significant biases that challenge traditional NGS methods, even for relative quantification. Moreover, these NGS methods struggle with the easiest copy number analyses, such as the ability to distinguish one copy of a gene versus two copies, as may be needed for a standard germline or carrier testing. Consequently, the gold standard for clinical testing of copy number analysis is pre-NGStechnologies, such as microarray and multiplex ligation-dependent probe amplification. The identification of these copy number changes in cfDNA requires detecting a change that is more than 100 times smaller, since the fraction of cfDNA that is derived from the fetus or the tumor can constitute less than 1% of the total cfDNA. Only our smNGS platform can detect CNVs in cfDNA at these levels today. Similarly, smNGS is needed for determining fetal risk in single-gene recessive conditions and precisely quantifying response to therapy, as these problems require absolute quantification of a low cfDNA signal against a high background originating from other tissues.

| 37 |     | Hou Y, Yang J, Qi Y, et al. Factors affecting cell-free DNA fetal fraction: statistical analysis of 13,661 maternal plasmas for non-invasive prenatal screening. Hum Genomics. 2019;13(1):62. |

| 38 |     | Dang, D. K., & Park, B. H. (2022). Circulating tumor DNA: current challenges for clinical utility. Journal of Clinical Investigation, 132(12), e154941 |

| 39 |     | Bronkhorst, A. J., & Holdenrieder, S. (2023). The changing face of circulating tumor DNA (ctDNA) profiling: Factors that shape the landscape of methodologies, technologies, and commercialization. Medizinische 
 Genetik, 35(4), 201-235                                                                                                                                                                                          |

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Precision of measurement 1% BillionToOne's smNGS encoding ddPCR 10%