Company: BLLN
Filing Date: 2025-06-20
Form Type: DRS
Source: 0000950123-25-006095
Chunk: 160

Company: BillionToOne, Inc.
Filing Date: 2025-06-20
Form: DRS
Chunk 160
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DNA necessitates amplification by a factor of millions
before it can be sequenced. This 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-NGS technologies, 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

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response to therapy, as these problems require absolute quantification of a low cfDNA signal against a high background originating from other tissues.

The components of our platform

Our smNGS
platform seamlessly integrates several tools and patented technologies, as depicted in the figure below.

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

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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