Company: BLLN
Filing Date: 2025-10-17
Form Type: S-1/A
Source: 0001193125-25-242632
Chunk: 20

Company: BillionToOne, Inc.
Filing Date: 2025-10-17
Form: S-1/A
Chunk 20
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 monitor disease, as both genetic and epigenetic properties of cfDNA directly reflect the originating tissue. Despite its potential, the detection of cfDNA is fundamentally challenging due to its limited quantity in blood and short half-life. Certain conditions that stem from large scale genetic changes or generate abundant quantities of cfDNA in the bloodstream can be identified by previous testing approaches. However, other serious conditions may have lower DNA shedding rates which can result in very few relevant DNA fragments in the blood. These conditions are nearly undetectable with previous cfDNA detection techniques. For example, even in a late-stage cancer patient, there may only be one mutated cell-free tumor DNA molecule found in one tube of blood. While traditional NGS has revolutionized genomic medicine over the past decade, it faces inherent limitations when applied to the analysis of cfDNA. Conventional NGS technologies are primarily limited to presence or absence detection. For example, within germline testing, which includes nearly unlimited input DNA material, the clinically relevant changes are detected at around 50% additional disease burden. However, in cfDNA applications, these clinically relevant changes can be at the level of 0.01% of cfDNA in blood. These applications require ultrasensitive quantification at the single molecule level that we believe is only possible with our smNGS platform. 12Further complicating the challenge, cfDNA samples undergo numerous enzymatic, amplification, and other biochemical steps prior to sequencing. Each of these steps introduces technical noise that makes it harder for traditional sequencing technologies to accurately quantify the absolute and relative abundance of cfDNA sequences in the biological specimen, resulting in lower sensitivity and specificity. Our proprietary single-molecule next-generation sequencing platform (smNGS) We have developed a transformative technology platform that redefines the possibilities of cfDNA analysis. In the past, the significant advancements from polymerase chain reaction (PCR) to Sanger Sequencing to NGS expanded molecular diagnostics from being limited to infectious disease testing and human genome mapping to now becoming standard of care for genetic screening. Today, we are experiencing another step change as we believe our smNGS platform enables the absolute quantification of cfDNA and we believe overcomes the technical noise that restrains the traditional NGS testing methods used by other diagnostic companies. This breakthrough capability enables our non-invasivediagnostic tests to achieve performance characteristics previously only possible with invasive tissue biopsies from the affected organ. The cornerstone of our platform is its ability to resolve and quantify individual DNA molecules with absolute precision. This single-molecule resolution provides extraordinary