Controls and calibrators for tests of nucleic acid amplification performed in droplets

Abstract

System, including methods and apparatus, for performing droplet-based tests of nucleic acid amplification that are controlled and/or calibrated using signals detected from droplets.

1 . A method of performing a droplet-based assay, comprising:
 detecting a signal from each of a plurality of droplets;   comparing a width of the signal from each droplet to a permitted range;   excluding droplets for which the signal has a width that is not in the permitted range, to identify a set of included droplets; and   determining a concentration of a target provided by a sample disposed in the plurality of droplets using data collected from the included droplets and without any contribution of data collected from the excluded droplets.   
     
     
         2 . The method of  claim 1 , wherein the step of excluding droplets includes a step of comparing a width of a peak formed by the signal from each droplet to a width maximum and a step of excluding each droplet for which the corresponding peak has a width that is greater than the width maximum. 
     
     
         3 . The method of  claim 2 , wherein the step of excluding droplets includes a step of comparing a width of a peak formed by the signal from each droplet to a width minimum and a step of excluding each droplet for which the corresponding peak has a width that is less than the width minimum. 
     
     
         4 . The method of  claim 1 , wherein the width corresponds to a time interval during which the signal is detected from a droplet. 
     
     
         5 . The method of  claim 1 , wherein the step of determining a concentration is based on an intensity of the signal from included droplets. 
     
     
         6 . The method of  claim 1 , further comprising a step of thermally cycling the plurality of droplets to promote amplification of the target. 
     
     
         7 . The method of  claim 1 , wherein the step of detecting a signal includes a step of detecting a first signal and a second signal from each droplet of the plurality of droplets, and wherein the data used for determining a concentration is obtained from the first signal. 
     
     
         8 . The method of  claim 1 , wherein the step of detecting a signal includes a step of detecting a fluorescence signal. 
     
     
         9 . The method of  claim 1 , wherein the step of detecting a signal includes a step of detecting a signal from each droplet traveling through a detection region. 
     
     
         10 . The method of  claim 1 , wherein the signal has an intensity that varies according to whether or not the target is present in a droplet. 
     
     
         11 . A method of performing a droplet-based assay, comprising:
 detecting a signal from at least two types of calibration droplets, the signal for each type of calibration droplet being of different intensity;   detecting sample data from sample droplets; and   determining if amplification of a target occurred in each of the sample droplets based on the sample data and the signal of each different intensity detected from the calibration droplets.   
     
     
         12 . The method of  claim 11 , wherein the at least two types of calibration droplets include a first type and a second type configured to provide respective signal intensities corresponding at least generally to sample droplets that are negative or positive for amplification of the target. 
     
     
         13 . The method of  claim 12 , wherein each sample droplet contains a PCR mixture for amplification of the target. 
     
     
         14 . The method of  claim 11 , wherein the step of determining includes a step of determining a threshold using the signal detected from the calibration droplets and a step of comparing data for individual sample droplets to the threshold, to distinguish sample droplets that are negative from those that are positive for amplification of the target. 
     
     
         15 . The method of  claim 11 , wherein the step of detecting a signal includes a step of detecting a signal of different intensity from at least three distinct types of calibration droplets. 
     
     
         16 . The method of  claim 11 , wherein the step of detecting a signal and the step of detecting sample data are both performed at a same wavelength or wavelength range. 
     
     
         17 . The method of  claim 11 , wherein the step of detecting a signal and the step of detecting sample data are performed with a same detector. 
     
     
         18 . The method of  claim 11 , wherein the step of detecting a signal and the step of detecting sample data are performed with the calibration droplets and the sample droplets arranged in separate groups. 
     
     
         19 . The method of  claim 18 , further comprising a step of detecting a signal from the at least two types of calibration droplets with the at least two types intermixed. 
     
     
         20 . The method of  claim 11 , wherein each type of calibration droplet contains a different amount of a same dye. 
     
     
         21 . The method of  claim 11 , wherein the step of detecting a signal and the step of detecting sample data are performed on droplets flowing through a same detection region. 
     
     
         22 . The method of  claim 21 , further comprising a step of loading the calibration droplets and the sample droplets into a flow channel that intersects the detection region, wherein the calibration droplets are loaded before the sample droplets. 
     
     
         23 . The method of  claim 11 , further comprising a step of thermally cycling the sample droplets. 
     
     
         24 . The method of  claim 23 , further comprising a step of thermally cycling the calibration droplets, wherein each different intensity of the signal detected from the calibration droplets is not affected substantially by the step of thermally cycling. 
     
     
         25 . The method of  claim 23 , wherein the calibration droplets are not thermally cycled. 
     
     
         26 . The method of  claim 11 , wherein the step of detecting a signal includes a step of detecting a fluorescence signal from each type of calibration droplet, and wherein the step of detecting sample data includes a step of detecting sample data as fluorescence intensity. 
     
     
         27 . A method of performing a droplet-based assay, comprising:
 generating droplets from an aqueous phase including a first dye and a second dye, the second dye being an internal reference;   detecting sample data from the first dye included in the droplets, the sample data being related to a reaction performed in the droplets;   detecting reference data from the second dye included in the droplets;   transforming the sample data with the reference data to reduce variability in the sample data that is independent of the reaction; and   determining if the reaction occurred in each of the sample droplets based on sample data that has been transformed with the reference data.   
     
     
         28 . The method of  claim 27 , further comprising a step of amplifying a nucleic acid target in the droplets, wherein the step of detecting sample data includes a step of detecting amplification data from the first dye. 
     
     
         29 . The method of  claim 27 , wherein the second dye is not conjugated to a nucleic acid. 
     
     
         30 . The method of  claim 27 , wherein the step of generating droplets includes a step of generating monodisperse droplets. 
     
     
         31 . The method of  claim 27 , wherein the step of transforming the sample data includes a step of dividing a sample data value by a reference data value for each droplet. 
     
     
         32 . A method of performing a droplet-based assay, comprising:
 detecting a signal from each of a plurality of droplets flowing through a detection region;   transforming an intensity of the signal for each of the plurality of droplets according to a duration of such signal to obtain transformed signals; and   determining whether amplification of a target occurred in individual droplets based on the transformed signals.   
     
     
         33 . The method of  claim 32 , wherein the step of detecting a signal includes a step of detecting a fluorescence signal. 
     
     
         34 . The method of  claim 32 , wherein each signal forms a peak, and wherein the duration corresponds to a width of the peak. 
     
     
         35 . The method of  claim 34 , wherein the step of transforming an intensity of each signal includes a step of transforming a value corresponding to a height or an area of the peak formed by such signal. 
     
     
         36 . The method of  claim 32 , wherein the step of transforming includes a step of dividing the intensity of each signal by the duration of such signal. 
     
     
         37 . The method of  claim 32 , further comprising:
 comparing a duration of each signal to a permitted range; and   excluding each signal having a duration that is not in the permitted range.   
     
     
         38 . The method of  claim 37 , wherein the step of comparing includes a step of comparing a duration of each signal to a duration maximum and a step of excluding each signal having a duration that is greater than the duration maximum. 
     
     
         39 . The method of  claim 32 , further comprising a step of thermally cycling the plurality of droplets to promote amplification of the target. 
     
     
         40 . A method of performing a droplet-based assay, comprising:
 generating droplets from an aqueous phase including a sample and first and second dyes;   detecting sample data from the first dye in the droplets, the sample data being related to amplification of a test target from the sample;   detecting control data from the second dye in the droplets, the control data being related to amplification of a control target in individual droplets;   analyzing the sample data and the control data to determine respective concentrations of the test target and the control target; and   correlating the concentration of the test target with the concentration of the control target.   
     
     
         41 . The method of  claim 40 , wherein the test target and the control target are both endogenous to the sample. 
     
     
         42 . The method of  claim 40 , wherein the test target is endogenous to the sample and the control target is not endogenous to the sample. 
     
     
         43 . The method of  claim 40 , wherein the step of correlating includes a step of determining a validity of the test target concentration based on the control target concentration. 
     
     
         44 . A method of performing a droplet-based assay, comprising:
 obtaining a first set of droplets configured to amplify a test target from a sample disposed in the first set of droplets, and a second set of droplets configured to amplify a control target in the second set;   detecting test amplification data from the first set of droplets and control amplification data from the second set of droplets; and   analyzing the test amplification data and the control amplification data to determine a concentration of the test target and the control target; and   correlating the concentration of the test target with the concentration of the control target.   
     
     
         45 . The method of  claim 44 , wherein the step of detecting includes a step of detecting data from the first set of droplets as a group and from the second set of droplets as a separate group. 
     
     
         46 . The method of  claim 44 , wherein the first set and the second set of droplets each includes a same sample that provides the test target and the control target. 
     
     
         47 . The method of  claim 44 , wherein test target is provided by a sample that does not provide the control target. 
     
     
         48 . The method of  claim 44 , wherein the step of correlating includes a step of determining a validity of the test target concentration based on the control target concentration.