Methods for nucleic acid size detection of repeat sequences

Abstract

Disclosed herein are data processing and calculating annotation systems and devices, and corresponding methods, for nucleic acid analysis. In particular, disclosed herein are methods for sizing a repeat region of a nucleic acid sample. For example, the methods disclosed herein use a ladder of amplification products to determine nucleic acid size.

1 - 90 . (canceled) 
     
     
         91 . A method of generating an internal sizing standard, comprising:
 identifying a repeat profile in a channel of a ladder of amplification products;   iteratively generating a set of repeat peak locations, an iteration comprising:
 determining a predicted peak location in the repeat profile using a previous peak location and an interval value, 
 identifying a peak location within a first window including the predicted peak location, and 
 adding the identified peak location to the set of repeat peak locations when a signal intensity at the identified peak location satisfies an amplitude criterion; and 
   estimating a linear relationship between repeat peak location and repeat fragment size using the set of repeat peak locations and a set of corresponding fragment sizes.   
     
     
         92 . The method of  claim 91 , further comprising repeatedly updating the amplitude criterion using signal intensities of one or more of the set of repeat peak locations while iteratively generating the set of repeat peak locations. 
     
     
         93 . The method of  claim 91 , further comprising repeatedly updating the interval value using peak locations of one or more of the set of repeat peak locations while iteratively generating the set of repeat peak locations. 
     
     
         94 . The method of  claim 91 , further comprising initializing the amplitude criterion using a statistical measure of a first portion of the repeat profile before iteratively generating the set of repeat peak locations. 
     
     
         95 . The method of  claim 91 , further comprising initializing the interval value using the periodicity of a second portion of the repeat profile and iteratively generating the set of repeat peak locations. 
     
     
         96 . The method of  claim 91 , further comprising using an interpolated peak location with the set of repeat peak locations when a signal intensity at the identified peak location does not satisfy the amplitude criterion. 
     
     
         97 . A method of generating an external sizing standard, comprising:
 identifying peaks in a channel of a ladder of amplification products, the peaks exceeding a local noise threshold;   iteratively re-estimating, using identified peaks in a first region of the channel, a linear relationship between peak location and fragment size using a first set of fragment sizes and a first set of corresponding peak locations, the iterative re-estimation comprising:
 including progressively smaller fragment sizes into the first set of fragment sizes, and 
 including peak locations corresponding to the progressively smaller fragment sizes into the first set of corresponding peak locations; 
   estimating, using identified peaks in a second region of the channel, a non-linear relationship between peak location and fragment size for a second region of the channel using a second set of fragment sizes and a second set of corresponding peak locations;   generating an external sizing standard for sizing a repeat region of a nucleic acid sample by combining the linear relationship and the nonlinear relationship.   
     
     
         98 . The method of  claim 97 , wherein an iteration of the iterative re-estimation further comprises:
 determining a predicted peak location using one of the progressively smaller fragment sizes and a re-estimated linear relationship between peak location and fragment size;   determining an actual peak location in the channel within a window including the predicted peak location; and   including the actual peak location into the first set of corresponding peak locations.   
     
     
         99 . The method of  claim 97 , further comprising:
 generating an internal sizing standard by identifying a repeat profile in a channel of a ladder of amplification products, iteratively generating a set of repeat peak locations, and estimating a linear relationship between repeat peak location and repeat fragment size using the set of repeat peak locations and a set of corresponding fragment sizes; and   generating a mobility corrected sizing standard using the internal sizing standard and the external sizing standard.   
     
     
         100 . The method of  claim 99 , wherein generating a mobility corrected sizing standard comprises:
 generating an affine transformation using the internal sizing standard and the external sizing standard; and   applying the affine transformation to the external sizing standard to obtain the mobility corrected sizing standard.   
     
     
         101 . A method for genotype peak sizing, comprising:
 generating a dynamic threshold from a background model, wherein the generation comprises piecewise scaling the background model to obtain the dynamic threshold;   determining gene-specific product peak locations using the dynamic threshold;   associating repeat sizes with the gene-specific product peak locations using a sizing standard; and   outputting an indication of the gene-specific product sizes.   
     
     
         102 . The method of  claim 101 , further comprising:
 generating the sizing standard by identifying a repeat profile in a channel of a ladder of amplification products, iteratively generating a set of repeat peak locations, and estimating a linear relationship between repeat peak location and repeat fragment size using the set of repeat peak locations and a set of corresponding fragment sizes.   
     
     
         103 . The method of  claim 101 , wherein piecewise scaling the background model to obtain the dynamic threshold comprises:
 determining a first region of the background model corresponding to amplification products with sizes above a first fragment size and below a second fragment size;   determining a second region of the background model corresponding to amplification products with sizes above the second fragment size; and   multiplying the first region of the background model by a first scaling factor that varies from an initial scaling factor to a second scaling factor less than the initial scaling factor; and   multiplying the second region of the background model by the second scaling factor.   
     
     
         104 . The method of  claim 101 , wherein determining gene-specific product peak locations in the repeat profile using the dynamic threshold comprises:
 identifying a first peak in the repeat profile at a first location; and   determining a first value of the repeat profile at the first location exceeds a first value of the dynamic threshold at the first location.   
     
     
         105 . The method of  claim 101 , wherein determining gene-specific product peak locations in the repeat profile using the dynamic threshold further comprises:
 identifying a second peak in the repeat profile at a second location, the second peak adjacent to the first peak,   determining a second value of the repeat profile at the second location satisfies an amplitude criterion, the amplitude criterion based on the first value, and   determining the second value of the repeat profile at the second location exceeds a second value of the dynamic threshold at the second location.   
     
     
         106 . A method of genotype peak sizing comprising:
 i. amplifying a repeat region of a nucleic acid sample;   ii. performing high resolution fragment analysis;   iii. obtaining a ladder of amplification products;   iv. identifying at least one gene-specific peak; and   v. sizing at least one of (a) the repeat region and (b) the at least one gene specific peak using the ladder of amplification products.   
     
     
         107 . The method of  claim 106 , wherein sizing at least one of (a) the repeat region and (b) the at least one gene-specific product peak using the ladder of amplification products comprises sizing using an internal sizing standard. 
     
     
         108 . The method of  claim 107 , wherein sizing at least one of (a) the repeat region and (b) the at least one gene-specific peak using an internal sizing standard comprises:
 generating the internal sizing standard by estimating a linear relationship between repeat peak location and repeat fragment size in a repeat profile of a first channel;   generating an external sizing standard by estimating a linear relationship between peak location and fragment size for a first region of a second channel, estimating a non-linear relationship between peak location and fragment size for a second region of the second channel, and generating the external sizing standard by combining the linear relationship and the nonlinear relationship;   generating a mobility corrected sizing standard by combining the internal sizing standard and the external sizing standard; and   sizing the at least one of the repeat region and the at least one gene-specific peak using the mobility corrected sizing standard.   
     
     
         109 . The method of  claim 106 , further comprising at least a first and a second primer for amplification of the repeat region. 
     
     
         110 . The method of  claim 109 , wherein the first primer comprises a portion of the repeat region. 
     
     
         111 . The method of  claim 109 , wherein the second primer anneals to a position outside of the repeat region. 
     
     
         112 . The method of  claim 109 , further comprising a third primer and an optional fourth primer. 
     
     
         113 . The method of  claim 106 , further comprising correcting a channel for signal artifacts. 
     
     
         114 . The method of  claim 106 , further comprising:
 identifying a window in a channel including a potential air bubble location;   determining a correlation between signal intensities for channels within the window; and   replacing, based on the determined correlation, the signal intensities for the channels within the window with simulated noise.   
     
     
         115 . The method of  claim 106 , further comprising extending the dynamic range of a first Channel, the first channel configured to detect a first electromagnetically detectable moiety, wherein extending the dynamic range comprises:
 identifying a window of the first channel including a saturated region;   determining combined signal intensities using the signal intensities for the first channel within the window and the signal intensities for a second channel configured to detect a another electromagnetically detectable moiety within the window; and   replacing the signal intensities for the first channel.   
     
     
         116 . The method of  claim 115 , wherein determining the combined signal intensities comprises extrapolating the shape of a calculated peak in the first channel. 
     
     
         117 . The method of  claim 106 , further comprising:
 identifying a bleed-over location based on signal intensities for a channel;   determining a window including the bleed-over location based on signal intensities for another channel; and   replacing the signal intensities for the other channel within the window with simulated noise.   
     
     
         118 . The method of  claim 106 , further comprising determining satisfaction of quality control criteria, the quality control criteria comprising at least one of sizing model criteria, a repeat profile signal-to-noise criterion, a repeat profile contamination criterion, and a minor allele sensitivity criterion. 
     
     
         119 . The method of  claim 118 , wherein the sizing model criteria comprise at least one of an internal sizing model goodness-of-fit criterion, an external sizing model goodness-of-fit criterion, and a consistency criterion that compares the internal sizing model to the external sizing model. 
     
     
         120 . The method of  claim 118 , wherein the repeat profile contamination criterion is failed when a repeat size associated with a location of a gene-specific peak is less than zero. 
     
     
         121 . The method of  claim 118 , wherein the minor allele sensitivity criterion is satisfied when a ratio of a noise value of a repeat profile to a maximum of the values of the repeat profile at a location of a gene-specific peak exceeds a threshold value. 
     
     
         122 . The method of  claim 106 , further comprising dynamically determining a threshold for calling peaks in a repeat profile. 
     
     
         123 . The method of  claim 106 , further comprising using a sliding window to call peaks in a repeat profile. 
     
     
         124 . The method of  claim 106 , further comprising interpolating peaks below an amplitude threshold in a repeat profile. 
     
     
         125 . The method of  claim 106 , further comprising generating a calibration curve that maps from sampling units to base pair units using estimated peak locations in a repeat profile.