Automatic threshold setting and baseline determination for real-time PCR
AssigneeApplera Corporation
InventorsDavid Woo, Clinton Lewis, Nasser M. Abbasi
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Abstract
The invention discloses a system and methods for quantitating the presence of nucleic acid sequences by evaluation of amplification data generated using real-time PCR. In one aspect, the methods may be adapted to identify a threshold and threshold cycle for one or more reactions based upon evaluation of exponential and baseline regions for each amplification reaction. The methodology used in the analysis may be readily automated such that subjective user interpretation of the data is substantially reduced or eliminated.
Claims44
A method for quantifying nucleic acid sequences present in one or more amplification reactions to be collectively analyzed, the method comprising the steps of:
- acquiring intensity data for each reaction over a selected number of reaction intervals wherein the intensity data is indicative of a detected quantity of progeny sequences arising from each sequence;
- assessing the intensity data over the selected number of reaction intervals to generate an amplification profile indicative of the change in quantity of the progeny sequences for each reaction interval;
- evaluating each amplification profile to identify a corresponding exponential region, having upper and lower bounds;
- determining a threshold based upon an intersection between at least one exponential region upper bound with at least one exponential region lower bound;
- performing a polynomial fitting operation for each amplification profile that applies the threshold to determine a polynomial root which is thereafter associated with a threshold cycle for each reaction;
- and
- quantifying the sequence for each reaction using the threshold cycle.
A method for quantitating at least one nucleic acid target, the method comprising:
- performing PCR-based amplification of each target in the presence of a detectable reporter construct;
- acquiring detection information generated by the detectable reporter construct indicative of a change in the concentration of each target over the course of the amplification;
- assembling a data set comprising at least a portion of the detection information to model amplification reaction characteristics;
- identifying an exponential region for each target of the data set from the modeled amplification reaction characteristics;
- identifying a baseline component based, in part, on the exponential region;
- normalizing the data set using the baseline component;
- determining a threshold based upon a comparison of the exponential regions for the targets of the data set;
- identifying a polynomial equation whose root is identified using the threshold and wherein the root is assigned as a threshold cycle;
- and
- quantifying each target using the threshold cycle.
A system for analyzing quantitative amplification data, the system comprising:
- a reaction module used to perform PCR amplification of at least one sample target using a detectable reporter label;
- a data collection module that detects reporter label intensities over the course of the PCR amplification for the at least one target polynucleotide sequence;
- a data processing module configured to:
- receive the detected intensities for each target polynucleotide sequence and subsequently generate a corresponding amplification profile to model the PCR amplification for the sample target;
- identify an exponential region for each amplification profile, each exponential region further having upper and lower bounds;
- identify a characteristic equation for each amplification profile based, in part, from the lower bound of the exponential threshold, and thereafter generate a normalized amplification profile using the characteristic equation;
- and
- identify a threshold and threshold cycle using the normalized amplification profile.
A method for quantifying target nucleic acid sequences present in one or more amplification reactions to be collectively analyzed, the method comprising the steps of:
- acquiring intensity data for each reaction over a selected number of reaction intervals wherein the intensity data is indicative of a detected quantity of progeny sequences arising from each target sequence;
- assessing the intensity data over the selected number of reaction intervals to generate an amplification profile indicative of the change in quantity of the progeny sequences for each reaction interval;
- evaluating each amplification profile to identify a corresponding exponential region, having upper and lower bounds;
- wherein the upper bound is determined by derivatization;
- setting a threshold intersecting the exponential region between the upper and lower bounds;
- determining a fractional cycle number corresponding to the intersection of said threshold and said exponential region;
- and
- quantifying the target sequence for each reaction using the fractional cycle number.
Dependencies are inferred from the claim text.