Robust Detection of Variability in Multiple Sets of Data
The present teachings comprise systems and methods for calibrating the background or baseline signal in a PCR or other reaction. The background signal derived from detected emissions of sample wells can be subjected to a normalized statistical metric, and be compared to a threshold or other standard to discard outlier cycles or other extraneous data. According to various embodiments, a relative standard deviation (relativeSTD) for the background component can be generated by dividing the standard deviation by the median of differences across all wells, where the difference is defined as the difference between maximum and minimum pixel values of a well. The relativeSTD as a metric is not sensitive to machine-dependent variations in absolute signal output that can be caused by different gain settings, different LED draw currents, different optical paths, or other instrumental variations. More accurate background characterization can be achieved.
1 . A method of generating a detection signal from a reaction, comprising:
receiving reaction emission data comprising multiple sets of data;
generating a relative standard deviation (relativeSTD) representing a normalized measure of variation in the multiple sets of data, by applying the equation:
relativeSTD
=
STD
MedianDiffMinPeak
,
where STD comprises the standard deviation of the emission data, MedianDiffMinPeak comprises the median of differences across the multiple sets of data, and each of the differences comprises the difference between a respective detected maximum value and a respective detected minimum value for each respective set of data of the multiple sets of data;
determining whether to exclude one or more of the multiple sets of data based on the normalized measure;
excluding sets of data from the multiple sets of data, having relativeSTD values that exceed a predetermined value, to generate processed emission data; and
generating a detection signal based on the processed emission data.
2 . The method of claim 1 , wherein the emission data comprises emission data detected from a biological sample.
3 . The method of claim 1 , wherein the reaction comprises a polymerase chain reaction.
4 . The method of claim 1 , wherein reaction comprises a reaction with a nucleic acid.
5 . The method of claim 1 , wherein the receiving emission data comprises receiving emission data from a plurality of sample wells.
6 . The method of claim 1 , wherein the emission data comprises data detected using a plurality of filters, and the generating a relativeSTD comprises generating a relativeSTD on a per-filter basis.
7 . A system for generating a detection signal from a reaction, comprising:
an input unit configured to receive reaction emission data comprising multiple sets of data; and
a processor unit, the processor unit being configured to
generate a relative standard deviation (relativeSTD) representing a normalized measure of variation in the multiple sets of data, by applying the equation:
relativeSTD
=
STD
MedianDiffMinPeak
,
where STD comprises the standard deviation of the emission data, MedianDiffMinPeak comprises the median of differences across the multiple sets of data, and each of the differences comprises the difference between a respective detected maximum value and a respective detected minimum value for each respective set of data of the multiple sets of data,
determine whether to exclude one or more of the multiple sets of the data based on the normalized measure,
exclude sets of data from the multiple sets of data, having relativeSTD values that exceed a predetermined value, to generate processed emission data, and
generate a detection signal based on the processed emission data.
8 . The system of claim 7 , wherein the emission data comprises emission data detected from labeled nucleic acid samples.
9 . The system of claim 7 , wherein the reaction comprises a polymerase chain reaction.
10 . The system of claim 7 , wherein the input unit comprises a plurality of different filters configured to filter the emission data from the reaction.
11 . A background calibration profile, the background calibration profile being generated by a method comprising:
receiving reaction emission data comprising multiple sets of data;
generating a relative standard deviation (relativeSTD) representing a normalized measure of variation in the multiple sets of data, by applying the equation:
relativeSTD
=
STD
MedianDiffMinPeak
,
where STD comprises the standard deviation of the emission data, MedianDiffMinPeak comprises the median of differences across the multiple sets of data, and each of the differences comprises the difference between a respective detected maximum value and a respective detected minimum value for each respective set of data of the multiple sets of data;
determining whether to exclude one or more of the multiple sets of data based on the normalized measure;
excluding sets of data from the multiple sets of data, having relativeSTD values that exceed a predetermined value, to generate processed emission data; and
generating a background calibration profile based on the processed emission data.
12 . The background calibration profile of claim 11 , wherein the emission data comprises emission data detected from labeled nucleic acid samples.
13 . The background calibration profile of claim 11 , wherein the emission data comprises emission data detected from fluorescently labeled DNA samples.
14 . The background calibration profile of claim 11 , wherein the amplification reaction comprises a polymerase chain reaction.
15 . The background calibration profile of claim 11 , wherein the receiving emission data comprises receiving emission data from DNA samples loaded in a plurality of sample wells.
16 . A computer-readable medium, the computer-readable medium being readable to execute a method of generating a detection signal from a reaction, the method comprising:
receiving reaction emission data comprising multiple sets of data;
generating a relative standard deviation (relativeSTD) representing a normalized measure of variation in the multiple sets of data, by applying the equation:
relativeSTD
=
STD
MedianDiffMinPeak
,
where STD comprises the standard deviation of the emission data, MedianDiffMinPeak comprises the median of differences across the multiple sets of data, and each of the differences comprises the difference between a respective detected maximum value and a respective detected minimum value for each respective set of data of the multiple sets of data;
determining whether to exclude one or more of the multiple sets of data based on the normalized measure;
excluding sets of data from the multiple sets of data, having relativeSTD values that exceed a predetermined value, to generate processed emission data; and
generating a detection signal based on the processed emission data.
17 . The computer-readable medium of claim 16 , wherein the emission data comprises emission data detected from labeled nucleic acid samples.
18 . The computer-readable medium of claim 16 , wherein the reaction comprises a polymerase chain reaction.
19 . The computer-readable medium of claim 16 , wherein the reaction occurs within a plurality of sample wells.
20 . The computer-readable medium of claim 16 , wherein the emission data comprises data detected using a plurality of filters, and the generating a relativeSTD comprises generating a relativeSTD on a per-filter basis.