Abstract:
A method comprises receiving an output signal of one of multiple detection channels. The method further includes color separating the output signal and generating a color separated signal substantially only with the peaks corresponding to the detected signals with the principle emission in the emission spectrum range of the detection channel. The method further includes estimating a time-variant amplitude of the gradually decaying tail and removing the time-variant amplitude from the color separated signal. The method further includes generating a corrected colored separated signal with substantially only the peaks corresponding to the fluorescent dyes attached to the fragments in the sample.

Description:
TECHNICAL FIELD 
       [0001]    The following generally relates to color separating multiple color signals and finds particular application to DNA analysis. However, the following is also amenable to other applications. 
       BACKGROUND 
       [0002]    DNA genotyping is the process of determining the sequence of DNA nucleotides at a position on a locus (a chromosome of a gene or other chromosome marker). For the purpose of identifying a human, certain generic loci have been selected as the standard markers to characterize the DNA. Each marker is a DNA fragment containing a repetition of a certain nucleotide sequence. Generally, there are thirteen (13) cores and several other accepted standard markers by the security authorities. These markers contain short repetitions (e.g., roughly from five (5) to forty (40)) of four nucleotides referred to as a Short Tandem Repeat (STR). 
         [0003]    The repetition numbers at these markers vary randomly from person to person. The specific form of the DNA sequence at a generic locus is called an allele, which provides sufficient differentiation among people. The STR sequence is inherited from parent&#39;s DNA. At each marker, there may be two different alleles, one from each parent, and it is called heterozygous. If the alleles from both parents have same STR numbers, it is homozygous. If the alleles of 13 core markers were heterozygous, each person will have twenty-six (26) different allele numbers. Assume each number is evenly distributed over a range of ten (10), the likelihood of having two people with the same alleles numbers from these thirteen (13) markers is extremely small. 
         [0004]    A DNA analyzer has been used to determine allele numbers in DNA samples. For this, the DNA sample is introduced into a micro-channel of a rigid sample carrier called a biochip, which generally includes multiple micro-channels in parallel to process multiple samples simultaneously. A DNA fragment containing all STR nucleotides and adjacent sections of nucleotides at each locus is copied from the DNA sample, and replicated through polymerase chain reaction (PCR). The fragments are labeled with target specific fluorescent dyes (or fluorophores) that emit radiation having different principle emission spectra in response to being excited by light. The labeled fragments are separated by size through electrophoresis. 
         [0005]    The fragment size is measured in the unit of base pairs (e.g.,  100  to  400 ), where a base pair is the size of a pair of DNA nucleotides. The separated fragments are excited by an excitation light. In response thereto, the dyes emit their characteristic radiation. A detection system includes multiple detection channels, each configured to detect radiation having emission spectra in a different emission spectrum range corresponding to a different one of the dyes. The channels detect the emission spectra and output signals with peaks indicative thereof. The peaks are used to locate fragments in the signal, the peak detection time determines the fragment size, and the fragment size identifies the locus of the fragment and is used to identify it as a DNA fragment in the locus with known STR number. 
         [0006]    Unfortunately, the emission spectra of the dyes partially overlap. As a result, a detection channel output signal will include peaks originating from dyes attached to fragments and with principle emission corresponding to the detection channel and peaks originating from dyes attached to fragments and with principle emission corresponding to other detection channels. This has been referred to as color-bleed. The output signal will also include a cluster peak of free dye peaks in which free dyes are dyes that are not attached to any fragment. The detection channel output signal will also include an offset signal including an optical and detection system offset and background signals from fluorescent emission from the sample carrier and non-fluorescent excitation light scatter. 
         [0007]      FIG. 1  shows an example portion of an output signal  102  of one of the detection channels. In  FIG. 1 , a y-axis  104  represents signal amplitude and an x-axis  106  represents time. The signal  102  includes peaks  108  and  110 , which originate from dyes attached to fragments and with principle emission corresponding to detection channel, peaks  112  and  114 , which originate from other dyes attached to other fragments and with principle emission corresponding to other detection channels, a cluster peak  116 , which is an superposition of the free dye peaks, and an offset signal  118  representing optical and detection system offset and background signals. As shown, the cluster peak  116  has a tail  120  that gradually decays over time and adds to and raises the amplitudes of the peaks  108 - 114 , and the offset signal  118  raises the amplitudes of the cluster peak  116  and the peaks  108 - 114 . 
         [0008]    The raised amplitudes of the peaks  108 - 114  may introduce error in the color separation process. One approach to attempt to return the amplitudes back to their pre-raised state has been to measure the offset  118  and the cluster peak  116  and subtract them from the signal  102 .  FIG. 2  shows an example signal  202  which is the signal  102  ( FIG. 1 ) with a measured offset  118  and cluster peak  116  subtracted there from. As shown, the signal  202  includes the peaks  108 - 114  and a portion  204  of the cluster peak  116 , which was not fully removed in this example. Unfortunately, it is difficult to accurately measure the tail  120  of the cluster peak  116 , especially in the presence of the peaks  108 - 114 . Consequently, the amplitudes of the peaks  108 - 114  in the signal  202  may deviate from their true amplitudes. 
         [0009]    As the result, the color separation process may introduce pull-up (positive or negative (pull-down)) artifact into the color-separated signals, even with accurate color bleed factors.  FIG. 3  shows an example of such artifact for three color separated fragments and five dyes. In  FIG. 3 , each signal  302 ,  304 ,  306 ,  308  and  310  represents a different dye. A first fragment peak  312  corresponds to the signal  304 , is measured accurately, and does not introduce artifact into the other signals. A second fragment peak  314  corresponds to the signal  306 , includes error, and introduces pull-up artifact  316 ,  318 ,  320  and  322  into the other signals. A third fragment peak  324  corresponds to the signal  304 , includes error, and introduces pull-up artifacts  330  and  332  in signals  308  and  310  and pull-down artifacts  326  and  328  in signals  302  and  306 . 
         [0010]    Pull-up and pull-down artifacts can be detrimental to identifying fragments from the signals  302 - 310 . That is, pull-up artifact may lead to false peaks being identified as true peaks (e.g., by pull-up artifact of sufficient amplitude) in the color separated signals, and pull-down artifact may lead to true peaks being suppressed (e.g., by pull-up artifact of sufficient negative amplitude) from the color separated signals and missed. The foregoing adds uncertainty in the detection and identification of fragments in the sample and, thus, determining STR numbers in DNA samples. Furthermore, the nature of the pull-up artifact makes it difficult to effectively and reliably perform a correction for the artifact after color separation, and such a correction can substantially distort the signal, which can further add to the uncertainty. 
         [0011]    Therefore, there is an unresolved need for other approaches to removing the tail  120  of the cluster peak  116  from the peaks  108  and  110 , without introducing or reducing pull-up artifact into the color separated signals. 
       SUMMARY 
       [0012]    Aspects of the application address the above matters, and others. 
         [0013]    In one aspect, a method includes color separating peaks in signals output by multiple detection channels that respectively are configured to detect signals with emission spectra in different predetermined emission spectrum ranges and to generate signals indicative thereof. The method includes receiving an output signal of one of the detection channels. The output signal includes peaks corresponding to detected signals with principle emission spectra in the emission spectrum range of the detection channel and peaks corresponding to detected signals with principle emission spectra in the emission spectrum range of one or more of the other detection channels and with emission spectra that overlaps the emission spectrum range of the detection channel. The method further includes color separating the output signal and generating a color separated signal substantially only with the peaks corresponding to the detected signals with the principle emission in the emission spectrum range of the detection channel. The color separated signal includes peaks corresponding to fluorescent dyes attached to fragments in a sample and a cluster peak corresponding to fluorescent dyes that are not attached to fragments in the sample, and wherein the cluster peak includes a gradually decaying tail with a time-variant amplitude that adds to and raises amplitudes of the peaks corresponding to the fluorescent dyes attached to the fragments in the sample. The method further includes estimating the time-variant amplitude of the gradually decaying tail and removing the time-variant amplitude from the color separated signal and generating a corrected colored separated signal with substantially only the peaks corresponding to the fluorescent dyes attached to the fragments in the sample. 
         [0014]    In another aspect, a sample processing system includes a color separator that color separates a detection channel output signal, generating a color separated signal, a time-variant baseline signal determiner that determines a time-variant amplitude of a baseline signal of the color separated signal, a time-variant baseline signal remover that removes the determined time-variant amplitude from the color separated signal, generating a corrected color separated signal. 
         [0015]    In another aspect, a computer readable storage medium with computer executable instructions embedded thereon, which, when executed by a processor, cause the processor to: measure an amplitude of a time-invariant offset to an output signal of a detection channel of a plurality of detection channels processing a DNA sample and removing the amplitude from the output signal generating a time-invariant offset corrected signal, coloring separating the time-invariant offset corrected signal generating a color separated signal, and determining an amplitude of a time-variant offset to the color separated signal and removing the amplitude from the color separated signal generating a corrected color separated signal. 
         [0016]    Those skilled in the art will recognize still other aspects of the present application upon reading and understanding the attached description. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]    The application is illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like references indicate similar elements and in which: 
           [0018]      FIG. 1  illustrates an example output signal of a detection channel. 
           [0019]      FIG. 2  illustrates the output signal of  FIG. 1  with most of the cluster peak and tail and the offset and background signals removed based on a prior art approach. 
           [0020]      FIG. 3  illustrates example color separated signals with pull-up artifact for the output signal of  FIG. 2 ; 
           [0021]      FIG. 4  schematically illustrates an example sample processing apparatus; 
           [0022]      FIG. 5  illustrates an example output signal of a detection channel, similar to that in  FIG. 1 . 
           [0023]      FIG. 6  illustrates the output signal of  FIG. 5  with the offset and background signals removed. 
           [0024]      FIG. 7  illustrates an example color separated output signal for the signal of  FIG. 6 . 
           [0025]      FIG. 8  illustrates the color separated output signal of  FIG. 7  with the cluster removed. 
           [0026]      FIG. 9  illustrates an example in which the color separated signals partially overlap. 
           [0027]      FIG. 10  illustrates an example in which the partially overlapped signal of  FIG. 9  are filtered to separate the partially overlapped color separated detection signal; and 
           [0028]      FIG. 11  illustrates an example method for color separating multiple color signals while mitigating pull-up artifact. 
       
    
    
     DETAILED DESCRIPTION 
       [0029]      FIG. 4  illustrates a sample processing apparatus  402  configured for processing one or more samples carried by a sample carrier  404 . 
         [0030]    A suitable sample carrier  404  includes, but is not limited to, a biochip, a lab-on-a-chip, and/or other sample carrier. Such a sample carrier  404  may include one or more micro-channels for carrying and moving, in parallel and/or in series, one or more samples through a plurality of different processing regions of the sample carrier  404 . Suitable samples include, but are not limited to, a bio-sample (e.g., saliva, blood, skin cells, and/or other bio-material), a non-bio sample, etc. The sample processing apparatus  402  includes a sample carrier receptacle  406  configured to receive the sample carrier  404  for processing the sample(s) carried thereby. 
         [0031]    The sample processing apparatus  402  further includes one or more processing stations  408   1 , . . . ,  408   N  (wherein N is an integer equal to or greater than one), collectively referred to herein as processing stations  408 , configured to process samples carried by the sample carrier  404  when the sample carrier  404  is inserted in the sample carrier receptacle  406 . Such processing may includes processing DNA samples. In this instance, the processing stations  408  may be configured to extract and purify DNA fragments from the sample, replicate and label the DNA fragments with fluorescent dyes having known principle emission spectra (color), and separate the labeled fragments based on fragment size via electrophoresis. 
         [0032]    The sample processing apparatus  402  also includes an optical reader  410 . The reader  410  includes a light source that directs an excitation light beam of a predetermined wavelength range at the separated fragments. In one instance, the light source emits a 488 nm relatively narrow light beam with a diameter in the order of ten (10) to one hundred (100) microns. In another instance, the light source emits a light beam with a different wavelength, and/or with a different diameter. Examples of suitable light sources include, but are not limited to, a laser, a light emitting diode (LED), and the like. 
         [0033]    The optical reader  410  also includes an optical detection channel (e.g., a photo-multiplier tube (PMT), a charge-coupled device (CCD) camera, or the like) for each principle emission spectrum range of interest. Each detection channel detects fluorescence light, which is emitted from the dyes in the fragments in response to the dyes being illuminated by the excitation light beam and which is within its principle emission spectrum range, and generates an electrical signal in proportion to the intensity of the detected emission spectrum. Briefly turning to  FIG. 5 , an example output signal  500  of one of the detection channels for one of the dyes is shown. In  FIG. 5 , a y-axis  502  represents amplitude and an x-axis  503  represents time. 
         [0034]    The signal  500  includes a set of peaks  504  and  506  (two shown in this example) corresponding to dyes attached to fragments and having principle emission spectra in the emission spectrum range of the detection channel, and peaks  508  and  510  corresponding to other dyes attached to other fragments and having principle emission spectra in the emission spectrum range of one or more other detection channels and emission spectra of the detection channel. The signal  500  further includes a cluster peak  512 , which is a summation of free-dye peaks, which are the peaks for dyes that are not attached to any fragment. As shown, the cluster peak  512  includes a tail  514  that adds to and raises the amplitudes of the peaks  504 - 510 . The tail represents a time-variant offset or baseline of the peaks  504 - 510 . 
         [0035]    The signal  500  also includes a generally time-invariant offset signal  516 , which raises the amplitudes of the cluster peak  512  and the peaks  504 - 510 . The time-invariant offset signal  516  is a summation of an offset signal of the reader  410  and a background signal including a fluorescent emission signal from the material of the sample carrier  404  and non-fluorescent emission signal of scattered excitation light. Generally, the amplitude of offset signal  516  of the reader  40  is relatively large and can exceed the amplitude of the peaks  504 - 510 , and the amplitude of the background signal  516  is relatively small. These signals are generally constant (or vary only slightly) during data acquisition. 
         [0036]    With continuing reference to  FIGS. 4 and 5  and with reference to  FIG. 6 , a time invariant offset signal determiner  412  determines an (average) amplitude of the time invariant offset signal  516  during a predetermined time duration  518  of data acquisition before the cluster peak  512  arrives. Generally, the time invariant offset signal  516  can be readily measured with good accuracy. A time invariant offset signal remover  414  removes (e.g., subtracts) the determined time invariant offset signal amplitude from the signal  500  and generates a time invariant offset corrected signal.  FIG. 6  shows an example time invariant offset corrected signal  600 . 
         [0037]    With continuing reference to  FIGS. 4 and 6  and with reference to  FIG. 7 , a color separator  416  color-separates the time invariant offset corrected signal  600 , including the peaks  504 - 510  and the cluster peak  512 . The color separator  416  uses a set of color bleed factors to color-separate the time invariant offset corrected signals for all the detection channels so that each signal includes peaks corresponding to the spectrum of only one of the dyes. Color bleed factors, including an approach to calibrating and correcting them, are described in PCT application serial number PCT/US2010/53346, filed Oct. 20, 2010, and entitled “Method of Calibrating and Correcting Color-bleed Factors for Color Separation in DNA Analysis,” which is incorporated by reference herein in its entirety.  FIG. 7  shows an example color separated signal  700  for one of the detection channels. The signal  700  includes the peaks  504  and  506  and a portion  702  of the cluster peak  512  with emission spectra corresponding to the detection channel including a tail  704 . In the illustrated example, the fragment peaks  504  and  506  and the portion  702  are color separated without introducing pull-up artifacts. 
         [0038]    With continuing reference to  FIGS. 4 and 7  and with reference to  FIG. 8 , a time variant baseline signal determiner  418  determines an amplitude of the tail  704  of the portion  702  of the cluster peak  512  as a function of time. Various techniques can be used to determine the amplitude. For example, the amplitude can be determined as a baseline curve after color separation. In another instance, the time variant baseline signal determiner  418  determines an amplitude of the cluster peak  512  including the tail  704 . A time variant baseline signal remover  420  removes (e.g., subtracts) the determined amplitude of the time variant offset signal from the color separated signal as a function of time and generates time corrected color separated signals. 
         [0039]      FIG. 8  shows an example corrected color separated signal  800  for one of the detection channels. As shown, the corrected color separated signal  800  includes only the fragment peaks  504  and  506 . The corrected color separated color  800  has a flat baseline at zero amplitude. Note that any error in determining and/or removing the time variant offset signal does not introduce pull-up artifact into the signal  800 . 
         [0040]    With reference to  FIG. 9 , an optional filter  422  is used to high-pass filter corrected color separated signals. Such filtering can reduce overlap between peaks. By way of example, in  FIG. 9 , the peaks  504  and  506  overlap in a region  902 . Each peak  504  and  506  diminishes on both sides at about a same rate and can be approximated by a Gaussian function.  FIG. 10  shows the peaks  504  and  506  of  FIG. 9  after filtering. As shown, the peaks  504  and  506  no longer overlap in the region  902 . Generally, the signal shape is the result of convolving a peak shape with a narrow single-point peak. Overlapped peaks can be separated by de-convoluting the signal with the peak shape. The de-convolution is equivalent to the operation of the high-pass filtering. In an alternative embodiment, the high-pass filtering is performed prior to color separation, and, as both of the operations are linear operations, the filtering will lead to the same result. In another embodiment, the filter  422  is omitted. 
         [0041]    A STR determiner  424  identifies the peaks in the corrected color separated signals and determines STR numbers in loci of interest based on the identified peaks. It is to be appreciated that by mitigating the introduction of pull-up artifact during color separation, as described herein, allows for accurately identifying DNA allele numbers, given an accurate set of color bleed factors. 
         [0042]    Generally, the above described configuration takes into consideration that the peaks of the free dyes in the cluster peak have the same emission spectra as the fragment peaks, and, instead of removing them before color separation, they are retained to undergo color separation together with the fragment peaks. The offset and background signals, which are essentially time-invariant, are accurately measured and removed. 
         [0043]    As a result, color separation can be performed accurately with introducing pull-up artifact. After color separation, the time-variant tail of the cluster peak (or the entire cluster peak) is measured and removed from the color separated signal for subsequent processing. The foregoing allows for mitigating pull-up artifacts in the color separated signal, relative to a configuration in which the time-variant tail, which is difficult to measure accurately, is removed before color separation. 
         [0044]      FIG. 11  illustrates a method for color separating multiple color signals without introducing pull-up artifact. 
         [0045]    It is to be appreciated that the following acts are provided for explanatory purposes and are not limiting. In other embodiments, one or more of the acts are omitted and/or one or more acts are added. In addition, the ordering of the acts may be different and/or certain acts can occur concurrently. 
         [0046]    At  1102 , a sample carrier carrying a sample is loaded in a sample processing apparatus. 
         [0047]    At  1104 , the sample is processed by one or more processing stations of the sample processing apparatus. This may include one or more of extracting and purifying a DNA fragment, replicating the fragment, labeling the fragments using nucleotide specific fluorescent dyes, or separating the labeled fragments by fragment size, as described herein. 
         [0048]    At  1106 , the separated fragments are illuminated with an excitation light source. 
         [0049]    At  1108 , emissions from the dyes in response to the illumination are detected by a plurality of detections channels, each channel being configured to detect a signal corresponding to the emission spectrum of a particular one of the dyes. 
         [0050]    As discussed herein, the output signal of each detection channel will include peaks corresponding to dyes of fragments with principle emission spectra in the spectrum of the detection channel, peaks corresponding to other dyes of the fragments with principle emission spectra in the spectrum of at least one other detection channel and emission spectra in the spectrum of the detection channel, a cluster peak with a tail that adds a baseline that raises the peaks, and offset and background signals. 
         [0051]    At  1110 , the offset and background signals of each detection signal are measured. As discussed herein, the offset and background signals are generally amplitude time-invariant signals that add to and raise the peaks and cluster peaks. 
         [0052]    At  1112 , the measured signals are removed from the corresponding detection signals. As discussed herein, this can be achieved by subtracting the measured offset and background signals from the corresponding detection signals. 
         [0053]    At  1114 , the offset corrected detection signals are color-separated. As discussed herein, color separating a detection signal removes peaks and free dye peaks having principle emission spectra not corresponding to the detection channel. 
         [0054]    At  1116 , a time-variant amplitude of the tail of the cluster of free dye peaks for each of the detection signal is estimated. Alternatively, a time variant amplitude of the cluster peak is estimated. 
         [0055]    At  1118 , the estimated amplitudes are removed from the corresponding color separated signals. 
         [0056]    At  1120 , the color separated signals is high pass filtered. As discussed herein, this can facilitate separating partially overlapping peaks. Alternatively, this filtering can be performed before the color separating. 
         [0057]    At  1122 , the filtered color separated signals are used to determine STR numbers for the DNA sample and identify DNA allele numbers. 
         [0058]    It is to be appreciated that the methods herein can be implemented via one or more processors of one or more computing systems executing one or more computer readable and/or executable instructions stored on computer storage medium such as memory local to or remote from the one or more computing systems. Additionally or alternatively, the processor can execute one or more computer readable and/or executable instructions carried in a single or carrier wave. 
         [0059]    The following describes embodiments herein in another manner. 
         [0060]    The fluorescent light intensity from a dye i is X i  and the light intensity detected through a detector channel j is Y j . The acquired signal from each detection channel contains substantial amount of offset and certain amount of background signal. The background signal is mostly the excitation light scattered by the biochip material surrounding the capillary. The amount of these offset and background signal are fairly constant throughout the data acquisition, and can be calculated and used for baseline correction. 
         [0061]    The variable Y j  is the signal amplitude after the baseline has been subtracted from the acquired signal. For an example with five (5) dyes and five (5) detection channels, the detected signal for channel j can be written as the combination of fluorescent light from five (5) dyes as shown in Equation 1: 
         [0000]        Y   j   =A   j1   *X   1   +A   j2   *X   2   +A   j3   *X   3   +A   j4   *X   4   +A   j5   *X   5 .  Equation 1:
 
         [0062]    The coefficient A ji  can be considered as the color-bleed factor from dye i to detection channel j, if i is not the same as j. For the case of i=j, the coefficient A ii  represents the detection efficiency of a dye by its principle channel. It is the principle coefficient, which has the largest value. 
         [0063]    The color bleed effect can be described through Equation 2: 
         [0000]    
       
         
           
             
               
                 
                   
                     
                       Y 
                       j 
                     
                     = 
                     
                       
                         ∑ 
                         
                           i 
                           = 
                           1 
                         
                         5 
                       
                        
                       
                         
                           A 
                           ji 
                         
                         * 
                         
                           X 
                           i 
                         
                       
                     
                   
                    
                   
                     
 
                   
                    
                   
                     
                       
                         with 
                          
                         
                             
                         
                          
                         j 
                       
                       = 
                       1 
                     
                     , 
                     2 
                     , 
                     … 
                      
                     
                         
                     
                     , 
                     5. 
                   
                 
               
               
                 
                   Equation 
                    
                   
                       
                   
                    
                   2 
                 
               
             
           
         
       
     
         [0064]    If X is the vector of the dye emission intensities, Y is the vector of the detected signal amplitudes, and A is the matrix of the color-bleed factors, the foregoing can be written as a matrix operation as shown in Equation 3: 
         [0000]        Y=AX.   Equation 3:
 
         [0000]    Equation 3 describes the relationship between the dye emission intensity and the detected signal amplitude in a set of simultaneous equations. The unknown dye emission intensity X can be solved by using the inverse matrix of A., as shown in Equation 4: 
         [0000]        B=A   −1 .  Equation 4:
 
         [0065]    Then, the dye emission intensity X is given by Equation 5: 
         [0000]        X=BY,   Equation 5:
 
         [0000]    and in expanded terms as shown in Equation 6: 
         [0000]    
       
         
           
             
               
                 
                   
                     X 
                     i 
                   
                   = 
                   
                     
                       ∑ 
                       
                         j 
                         = 
                         1 
                       
                       5 
                     
                      
                     
                       
                         B 
                         ij 
                       
                       * 
                       
                         
                           Y 
                           j 
                         
                         . 
                       
                     
                   
                 
               
               
                 
                   Equation 
                    
                   
                       
                   
                    
                   6 
                 
               
             
           
         
       
     
         [0000]    While the color-bleed factors A ji  are all positive values, the inverse matrix coefficients B ij  can be positive and negative values. 
         [0066]    The calculation for the dye emission intensities can be considered as de-convolution of the detected signal amplitudes. If the actual color-bleed factors are not accurate or if the signal amplitude Y j  is not measured accurately, the color separation will contain noticeable errors (pull-up and negative pull-up (or pull-down)). These errors are artifacts in the signal for the subsequent processing and add uncertainty to the detection and identification of DNA fragments in the sample. 
         [0067]    Recall that the output signal of each of the detection channels includes fluorescent fragment peaks, fluorescent free-dye peaks with a time-variant tail, and time-invariant offset and background signals. 
         [0068]    In order to avoid or reduce the pull-up artifact, the color-bleed factors A ji  should be accurately prepared. These factors should remain unchanged and accurate during the detection of DNA samples. Secondly, the offset and background signals should be accurately calculated. They are used to obtain the signal Y j  from the DNA fragments, by subtracting it from the acquired signal. 
         [0069]    After the subtraction, the signal amplitude Y j  still includes the free dye cluster peak, along with its gradual diminishing tail, and the fluorescent fragment peaks. All of these components in the signal Y j  are emitted from the same fluorescent sources, and they have the same the color-bleed factors A ji . These components can be separated by the inverse matrix coefficients B ij , without introducing pull-up artifacts. 
         [0070]    After the color separation, each signal contains the fragment peaks from the principle color as well as the peaks from the free dye of the principle color and its gradual diminishing tail, which are well preserved in the input variable Y j . The cluster peak can then be removed, and the resulting signal is used for subsequent analysis, STR determination, and DNA allele number identification. 
         [0071]    From the above, the time-invariant signals are measured for each detection channel output signal and subtracted there from. The signal, which still includes the peaks, is then color separated. The resulting signal includes the peaks corresponding to the detection channel and the portion of the free-dye cluster peak corresponding to the detection the detection channel. The tail or cluster is the estimated and removed from the color separated signals. Optionally, the resulting signals can be high-pass filtered facilitate separating partially overlapping peaks. The resulting signal is used for subsequent DNA analysis. 
         [0072]    The application has been described with reference to various embodiments. Modifications and alterations will occur to others upon reading the application. It is intended that the invention be construed as including all such modifications and alterations, including insofar as they come within the scope of the appended claims and the equivalents thereof.