Automatic threshold setting and baseline determination for real-time pcr

A method for target sequence quantitation, comprising:

• amplifying a plurality of target sequences by PCR in the presence of a detectable reporter construct;
• acquiring signal information for each target sequence of the plurality of target sequences amplified by detecting the associated detectable reporter construct;
• identifying a plurality of exponential regions, wherein each exponential region is associated with substantial increases in the signal information for each target sequence amplification and wherein each identified exponential region comprises an associated upper and lower bound;
• normalizing the signal information for each target sequence amplification using an identified baseline component for the signal information to compensate for non-specific fluorescence in the signal information;
• determining an exponential region threshold based on a comparison of the plurality of exponential regions, wherein each exponential region of the plurality of exponential regions is associated with a target sequence amplification of the plurality of target sequence amplifications;
• performing a fitting operation to at least an interval of the exponential region including the exponential region threshold;
• identifying, based on the exponential region threshold and an associated polynomial root, a threshold cycle for the at least one target sequence amplification.

A method for target sequence quantitation, comprising:

• amplifying a plurality of target sequences in the presence of a detectable reporter construct;
• acquiring signal information for the plurality of target sequences amplified by detecting the associated detectable reporter construct;
• identifying an exponential region associated with substantial increases in the signal information for each target sequence amplification, each exponential region comprising an upper bound and a lower bound;
• identifying a baseline component for the signal information associated with each target sequence amplification;
• normalizing the signal information for each target sequence amplification using the baseline component to compensate for non-specific fluorescence in the signal information;
• and
• determining an exponential region threshold for the target sequence amplifications based on a comparison of the upper bounds and lower bounds of the target sequence amplifications, wherein the determination is based on a plurality of exponential region thresholds from the plurality of target sequence amplifications.

A system for target sequence quantitation, comprising:

• a data collection module configured to collect signal information including detected reporter label intensities generated by amplifying a plurality of target sequences in the presence of a detectable reporter construct;
• and
• a data processing module configured to:
• identify a plurality of exponential regions, wherein each exponential region is associated with substantial increases in the signal information for the at least one target sequence amplification, and each exponential region comprising an upper bound and a lower bound,
• normalize the signal information for each target sequence amplification using an identified baseline component for the signal information to compensate for non-specific fluorescence in the signal information,
• determine an exponential region threshold based on a comparison of the plurality of exponential regions, wherein each exponential region of the plurality of exponential regions is associated with a target sequence amplification of the plurality of target sequence amplifications,
• perform a fitting operation to fit a polynomial to at least an interval of the exponential region including the exponential region threshold, and
• identify, based on the exponential region threshold and an associated polynomial root, a threshold cycle for the at least one target sequence amplification.

A system for target sequence quantitation, comprising:

• a data collection module that collects signal information including detected reporter label intensities generated by amplifying a plurality of target sequences in the presence of a detectable reporter construct;
• and
• a data processing module configured to identify an exponential region associated with substantial increases in the signal information for each target sequence amplification, each exponential region comprising an upper bound and a lower bound,
• identify a baseline component for the signal information associated with each target sequence amplification,
• normalize the signal information for each target sequence amplification using the baseline component to compensate for non-specific fluorescence in the signal information, and
• determine an exponential region threshold for the target sequence amplifications based on a comparison of the upper bounds and lower bounds of the target sequence amplifications, wherein the determination is based on a plurality of exponential region thresholds from the plurality of target sequence amplifications.