A method for carrying out a process for an amplification of nucleic acids with sample nucleic acids and reference nucleic acids being amplified in separate reaction batches. Signals of the amplification are observed in real time. A number of amplification cycles and/or a duration of the amplification process are dynamically adjusted depending on the observed signals of the amplification.

A fractional concentration of tumor DNA in a plasma sample is estimated by analyzing a biological sample of an organism. One or more sequence reads obtained from a sequencing of the DNA fragment are received. The one or more sequence reads are aligned to a reference genome to obtain aligned locations for both ends of the DNA fragment. Using the aligned locations, a size of the DNA fragment is determined. For each size of a plurality of sizes, an amount of a set of the plurality of DNA fragments from the plasma sample corresponding to the size is determined. A first value of a first parameter is calculated based on the amounts of DNA fragments at multiple sizes. The first value is compared to a calibration value. A fractional concentration of tumor DNA in the plasma sample is estimated based on the comparison.

A fractional concentration of tumor DNA in a plasma sample is estimated by analyzing a biological sample of an organism. One or more sequence reads obtained from a sequencing of the DNA fragment are received. The one or more sequence reads are aligned to a reference genome to obtain aligned locations for both ends of the DNA fragment. Using the aligned locations, a size of the DNA fragment is determined. For each size of a plurality of sizes, an amount of a set of the plurality of DNA fragments from the plasma sample corresponding to the size is determined. A first value of a first parameter is calculated based on the amounts of DNA fragments at multiple sizes. The first value is compared to a calibration value. A fractional concentration of tumor DNA in the plasma sample is estimated based on the comparison.

Methods, sample vessels, and instruments are provided for quantitative and semi-quantitative amplification.

Methods, sample vessels, and instruments are provided for quantitative and semi-quantitative amplification.

A method of determining absolute quantity of a sample template is provided, comprising: i) providing a series of control templates, each having a different and known copy number; ii) amplifying each control template and the sample template by PCR and detecting a signal correlating to an amount of PCR product in each PCR cycle during an exponential phase to thereby obtain a series of control signals and a sample signal; iii) making a standard curve relating a known copy number of each control template with an intensity of each corresponding control signal for the each PCR cycle; and iv) determining a copy number of the sample template by using the standard curve to translate an intensity of the sample signal to the copy number of the sample template.

A method of determining absolute quantity of a sample template is provided, comprising: i) providing a series of control templates, each having a different and known copy number; ii) amplifying each control template and the sample template by PCR and detecting a signal correlating to an amount of PCR product in each PCR cycle during an exponential phase to thereby obtain a series of control signals and a sample signal; iii) making a standard curve relating a known copy number of each control template with an intensity of each corresponding control signal for the each PCR cycle; and iv) determining a copy number of the sample template by using the standard curve to translate an intensity of the sample signal to the copy number of the sample template.

This disclosure relates to methods, systems, computer programs and computer-readable media for the multidimensional analysis of real-time amplification data. A framework is presented that shows that the benefits of standard curves extend beyond absolute quantification when observed in a multidimensional environment. Relating to the field of Machine Learning, the disclosed method combines multiple extracted features (e.g. linear features) in order to analyse real-time amplification data using a multidimensional view. The method involves two new concepts: the multidimensional standard curve and its ‘home’, the feature space. Together they expand the capabilities of standard curves, allowing for simultaneous absolute quantification, outlier detection and providing insights into amplification kinetics. The new methodology thus enables enhanced quantification of nucleic acids, single-channel multiplexing, outlier detection, characteristic patterns in the multidimensional space related to amplification kinetics and increased robustness for sample identification and quantification.

This disclosure relates to methods, systems, computer programs and computer-readable media for the multidimensional analysis of real-time amplification data. A framework is presented that shows that the benefits of standard curves extend beyond absolute quantification when observed in a multidimensional environment. Relating to the field of Machine Learning, the disclosed method combines multiple extracted features (e.g. linear features) in order to analyse real-time amplification data using a multidimensional view. The method involves two new concepts: the multidimensional standard curve and its ‘home’, the feature space. Together they expand the capabilities of standard curves, allowing for simultaneous absolute quantification, outlier detection and providing insights into amplification kinetics. The new methodology thus enables enhanced quantification of nucleic acids, single-channel multiplexing, outlier detection, characteristic patterns in the multidimensional space related to amplification kinetics and increased robustness for sample identification and quantification.

Provided is a device including: at least one well in which an amplifiable reagent is contained in a specific copy number of less than 100; and at least one well in which the amplifiable reagent is contained in a specific copy number of 100 or greater, wherein for the at least one well in which the specific copy number of the amplifiable reagent is less than 100, a formula: CV<1/√x is established, where CV represents a coefficient of variation for the specific copy number and x represents average specific copy number of the amplifiable reagent.