Nucleic Acid Analysis Calibration Curve Threshold

The patent applies preliminary action by introducing standard nucleic acid samples with known copy numbers before analyzing the actual analyte samples. These standard samples are processed through the same library preparation and sequencing workflow, allowing the system to establish a calibration curve that maps read numbers to actual copy numbers in advance. This pre-established reference framework enables precise threshold determination for data splitting without requiring complex real-time calculations during analyte analysis.

The patent employs parameter changes by utilizing standard nucleic acid samples with specifically controlled copy numbers (e.g., 1, 10, 100, 1000 copies) to generate a calibration curve. By varying the copy number parameter across multiple known standards, the system establishes a reference relationship between read numbers and actual concentrations. This parameter variation approach transforms the threshold determination from a complex theoretical calculation into a straightforward comparison against empirically derived reference values.

The patent uses standard nucleic acid samples as an intermediary between the sequencing process and the analyte analysis. These standards with known copy numbers serve as a mediating reference that translates raw read numbers into meaningful quantitative information. By comparing analyte read numbers against the calibration curve generated from standards, the system can objectively determine which sequences represent true biological signals versus those arising from errors or contamination, thus filtering data based on reliability rather than arbitrary thresholds.

The patent implements feedback by using the standard samples to establish a reference framework that feeds back into the analysis of analyte samples. The calibration curve generated from standards provides continuous feedback criteria for evaluating analyte data quality. This feedback mechanism allows the system to dynamically adjust its interpretation of read numbers based on the established relationship between read counts and actual copy numbers, enabling reliable distinction between usable and non-usable data without losing valuable low-abundance sequences.

The patent applies universality by designing standard nucleic acid samples that can be used across multiple analyte samples and experimental conditions. A single set of standards with known copy numbers serves as a universal reference for calibrating the entire sequencing run, eliminating the need for separate threshold determination for each sample. This multi-functional approach allows the same standard samples to provide calibration data, establish thresholds, and validate the sequencing performance across different analytes, simplifying the overall process despite the added initial step.

The patent uses copying by creating multiple copies of standard nucleic acid sequences at known concentrations (e.g., 1, 10, 100, 1000 copies). These replicated standard samples are processed alongside analyte samples through the same library preparation workflow. By having multiple copied instances of known standards, the system generates robust calibration data that establishes precise threshold criteria without requiring complex custom preparations for each experimental condition, making the process more reproducible and easier to standardize.