Size-based analysis of fetal dna fraction in plasma

Abstract

A fractional concentration of fetal relevant DNA in a mixture of DNA from a biological sample is determined based on amounts of DNA fragments of a particular size or range of sizes. DNA fragments may be sequenced to obtain sequence reads, and the sequence reads may be aligned to a reference genome to determine sizes of the DNA fragments. Calibration data points (e.g., as a calibration function) indicate a correspondence between values of a parameter providing a statistical measure of a size profile and the fractional concentration of the fetal DNA. For a given sample, a value of the parameter can be determined from DNA fragments of a particular size or range of sizes in a sample. A comparison of the value to the calibration data points can provide the estimate of the fractional concentration of the fetal DNA.

What is claimed is: 
     
         1 . A method of analyzing a maternal plasma sample of a pregnant woman, the maternal plasma sample including cell-free DNA fragments originating from maternal cells and from fetal cells, the method comprising:
 (a) sequencing a plurality of the DNA fragments to obtain a plurality of sequence reads comprising the outermost nucleotides at each end of a plurality of DNA fragments;   (b) aligning the sequence reads to a reference genome, thereby obtaining a set of genomic coordinates including genomic coordinates of the outermost nucleotides defining a size of a DNA fragment for each of the plurality of DNA fragments;   (c) calculating a value of a parameter based on an amount of DNA fragments of a particular size or range of sizes, the parameter providing a statistical measure of a size profile of DNA fragments in the maternal plasma sample;   (d) comparing the value to a calibration value; and   (e) estimating a fractional concentration of fetal DNA in the maternal plasma sample based on the comparison.   
     
     
         2 . The method of  claim 1 , wherein the parameter represents an abundance of small DNA fragments relative to an abundance of large DNA fragments, and wherein the small DNA fragments have a smaller size than the large DNA fragments. 
     
     
         3 . The method of  claim 1 , wherein the parameter is a percentage of DNA fragments of the particular size or range of sizes relative to all DNA fragments. 
     
     
         4 . The method of  claim 1 , wherein the parameter is a percentage of DNA fragments of the particular size or range of sizes relative to DNA fragments of another size or range. 
     
     
         5 . The method of  claim 1 , wherein comparing the value to the calibration value comprises determining whether the value is higher or lower than the calibration value. 
     
     
         6 . The method of  claim 1 , wherein comparing the value to the calibration value comprises comparing the value to a calibration curve to identify a point on the calibration curve having the value of the parameter, wherein the calibration curve comprises calibration data points and each calibration data point comprises a calibration value for the parameter and a calibration fractional concentration of fetal DNA. 
     
     
         7 . The method of  claim 1 , wherein comparing the value to the calibration value comprises inputting the value into a calibration function. 
     
     
         8 . The method of  claim 7 , further comprising reading the calibration function from memory. 
     
     
         9 . The method of  claim 1 , wherein sequencing comprises obtaining a full length sequence of each of the plurality of DNA fragments. 
     
     
         10 . The method of  claim 1 , wherein sequencing comprises paired-end sequencing each of the DNA fragments. 
     
     
         11 . The method of  claim 1 , wherein the plurality of sequence reads is obtained from a circularized DNA fragment. 
     
     
         12 . The method of  claim 1 , wherein the plurality of sequence reads is obtained by massively parallel sequencing. 
     
     
         13 . The method of  claim 1 , further comprising:
 calculating one or more calibration data points by:
 for each of a plurality of calibration samples:
 measuring the fractional concentration of fetal DNA in the calibration sample; 
 measuring the amount of DNA fragments of the particular size or range of sizes; and 
 calculating a respective calibration value for the parameter based on the amounts of DNA fragments at multiple sizes, the calibration data point of the calibration sample including the calibration value and the measured fractional concentration, 
 
   wherein the calibration value is determined using at least one of the respective calibration values of at least one calibration data point.   
     
     
         14 . The method of  claim 13 , wherein the calibration value is determined from a plurality of calibration samples of one or more pregnant females, each of the plurality of calibration samples including fetal and maternal cell-free DNA fragments. 
     
     
         15 . The method of  claim 1 , further comprising including the fractional concentration of fetal DNA in a diagnostic algorithm for estimating a risk that the maternal plasma sample is obtained from an aneuploid pregnancy. 
     
     
         16 . The method of  claim 1 , further comprising determining if the value of the parameter is above or below a threshold calibration value, thereby determining if the estimated fractional concentration of the maternal plasma sample is above or below the fractional concentration corresponding to the threshold calibration value. 
     
     
         17 . The method of  claim 16 , further comprising testing the maternal plasma sample for fetal aneuploidy when the parameter is above the threshold calibration value. 
     
     
         18 . A method of estimating a fractional concentration of clinically-relevant DNA in a plasma sample, the plasma sample including the clinically-relevant DNA and other DNA, the plasma sample being from a female pregnant with a fetus, the clinically-relevant DNA being fetal DNA, the method comprising:
 for each size of a plurality of sizes:
 measuring an amount of a plurality of DNA fragments from the plasma sample corresponding to the size; 
   calculating, with a computer system, a first value of a first parameter based on the amounts of DNA fragments at multiple sizes, the first parameter providing a statistical measure of a size profile of DNA fragments in the plasma sample;   obtaining one or more first calibration data points, wherein each first calibration data point specifies a fractional concentration of clinically-relevant DNA corresponding to a calibration value of the first parameter, and wherein the one or more first calibration data points are determined from a plurality of calibration samples, wherein each calibration sample of the plurality of calibration samples is a plasma sample including the clinically-relevant DNA and the other DNA;   comparing the first value to a calibration value of at least one first calibration data point; and   estimating the fractional concentration of the clinically-relevant DNA in the plasma sample based on the comparison.   
     
     
         19 . The method of  claim 18 , wherein the DNA fragments are chosen at random. 
     
     
         20 . The method of  claim 18 , wherein the DNA fragments are chosen from more than one region of a genome. 
     
     
         21 . The method of  claim 20 , wherein the DNA fragments are chosen from more than one predetermined region of the genome. 
     
     
         22 . The method of  claim 20 , wherein the DNA fragments are chosen from more than one chromosome. 
     
     
         23 . The method of  claim 20 , wherein measuring the amount of the plurality of DNA fragments from the plasma sample corresponding to the size includes paired-end massively parallel sequencing. 
     
     
         24 . The method of  claim 20 , wherein the first parameter represents an abundance of small DNA fragments relative to an abundance of large DNA fragments, and wherein the small DNA fragments have a smaller size than the large DNA fragments. 
     
     
         25 . The method of  claim 20 , further comprising:
 calculating one or more second values of one or more second parameters based on the amounts of DNA fragments at multiple sizes, the one or more second parameters providing different statistical measures of the size profile of DNA fragments in the plasma sample;   obtaining one or more second calibration data points corresponding to the one or more second parameters;   comparing the one or more second values to corresponding second calibration values of the second calibration data points; and   estimating the fractional concentration of the clinically-relevant DNA in the plasma sample based on the comparisons involving the first value and the one or more second values.   
     
     
         26 . The method of  claim 25 , wherein the first calibration data points and the second calibration data points are points on a multidimensional curve and the comparison includes identifying a multidimensional point having coordinates corresponding to the first value and the one or more second values. 
     
     
         27 . The method of  claim 20 , wherein each first calibration data point is determined from a histogram corresponding to a different calibration sample, wherein a histogram provides amounts of DNA fragments at a plurality of sizes, and wherein at least a portion of the different calibration samples have different fractional concentrations. 
     
     
         28 . The method of  claim 20 , wherein measuring amounts of DNA fragments corresponding to the sizes includes:
 for each of the plurality of DNA fragments from the plasma sample:
 measuring a size of the DNA fragment, 
   wherein measuring a size of a DNA fragment includes:
 performing paired-end sequencing of the DNA fragment to obtain paired sequence reads; 
 aligning the paired sequence reads to a reference genome; and 
 using the aligned locations to determine the size of the DNA fragment. 
   
     
     
         29 . The method of  claim 20 , further comprising:
 determining a function that approximates the calibration value.   
     
     
         30 . A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to estimate a fractional concentration of clinically-relevant DNA in a plasma sample, the plasma sample including the clinically-relevant DNA and other DNA, the plasma sample being from a female pregnant with a fetus, the clinically-relevant DNA being fetal DNA, the plurality of instructions comprising:
 for each size of a plurality of sizes:
 measuring an amount of a plurality of DNA fragments from the plasma sample corresponding to the size; 
   calculating, with a computer system, a first value of a first parameter based on the amounts of DNA fragments at multiple sizes, the first parameter providing a statistical measure of a size profile of DNA fragments in the plasma sample;   obtaining one or more first calibration data points, wherein each first calibration data point specifies a fractional concentration of clinically-relevant DNA corresponding to a calibration value of the first parameter, and wherein the one or more first calibration data points are determined from a plurality of calibration samples, wherein each calibration sample of the plurality of calibration samples is a plasma sample including the clinically-relevant DNA and the other DNA;   comparing the first value to a calibration value of at least one first calibration data point; and   estimating the fractional concentration of the clinically-relevant DNA in the plasma sample based on the comparison.