Company: BLLN
Filing Date: 2025-09-17
Form Type: DRS/A
Source: 0001193125-25-206347
Chunk: 195

Company: BillionToOne, Inc.
Filing Date: 2025-09-17
Form: DRS/A
Chunk 195
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STM)* Specialized & scalable infrastructure Our smNGS platform is protected by an expanding moat. Custom bioinformatics with machine learning Engineering biology approach 50+ issued patents** & knowhow * patented ** as of Jun 30, 2025

Quantitative Counting Templates (QCTs)

QCTs are the foundation of our smNGS
technology. They are artificial DNA fragments that we design and synthesize to mimic the properties of the human DNA loci that are being interrogated in an assay. A specific identifier is embedded into the sequence that flags the QCTs as synthetic
controls, and a randomized embedded sequence ensures that each QCT is unique. QCTs are added to the biological sample at the start of the testing process so that they are subject to the same laboratory processes, including amplification and
sequencing, as the disease-associated genetic molecules that the assay targets. Importantly, QCTs amplify at the same rate as the genetic targets. We can then precisely calculate the amplification and sequencing biases that were introduced during
the sample processing and remove their effects from the data to absolutely quantify the number of cfDNA molecules that are present in the sample. The precise number of QCT molecules added to each specimen is calculated bioinformatically through the
counting of the number of diversity regions in the sequencing data. This method is therefore “calibration-free” and does not require the exact measurement and titration of spike-in DNA
concentrations, a feature that is particularly advantageous for quantitative assessments across multiple timepoints in longitudinal monitoring contexts.

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QCTs are compatible with almost all NGS library preparation methods and can be used to measure both genetic and epigenetic changes, such as methylation. Our ability to absolutely quantify methylation across genomic loci allows us to combine signals from thousands of genomic loci to reduce the amplification and sample processing noise to negligible levels, thereby converting a 0.01% signal to the equivalent of a 10+% signal. Our patented smNGS platform has single-molecule precision. Proprietary Design** Original sample Artificial molecules (QCTSTM*) Amplification and sequencing Proprietary Machine Learning** Decode molecular counts from sequencing data Unique variable regions Both amplify at same, unknown rate * Quantitative Counting Templates ** multiple issued patents QCTs are used in our testing workflow with the following steps:

| (1) |     | Add an aliquot of traceable and specific QCTs into the patient’s blood sample, which contains