Analysis of fragmentation patterns of cell-free dna

Abstract

Factors affecting the fragmentation pattern of cell-free DNA (e.g., plasma DNA) and the applications, including those in molecular diagnostics, of the analysis of cell-free DNA fragmentation patterns are described. Various applications can use a property of a fragmentation pattern to determine a proportional contribution of a particular tissue type, to determine a genotype of a particular tissue type (e.g., fetal tissue in a maternal sample or tumor tissue in a sample from a cancer patient), and/or to identify preferred ending positions for a particular tissue type, which may then be used to determine a proportional contribution of a particular tissue type.

What is claimed is: 
     
         1 . A method of analyzing a biological sample, including a mixture of cell-free DNA molecules from a plurality of tissue types that includes a first tissue type, to determine a classification of a proportional contribution of the first tissue type in the mixture, the method comprising:
 analyzing, by a computer system, a first plurality of cell-free DNA molecules from the biological sample of a subject, wherein analyzing a cell-free DNA molecule includes:
 determining a genomic position in a reference genome corresponding to at least one end of the cell-free DNA molecule; 
   based on the analyzing of the first plurality of cell-free DNA molecules, determining, by the computer system, that a first number of the first plurality of cell-free DNA molecules end at one of a first set of genomic positions;   computing, by the computer system, a relative abundance using the first number and a second number of cell-free DNA molecules ending at a second set of genomic positions; and   determining the classification of the proportional contribution of the first tissue type by comparing the relative abundance to one or more calibration values determined from one or more calibration samples whose proportional contributions of the first tissue type are known.   
     
     
         2 . The method of  claim 1 , wherein the first plurality of cell-free DNA molecules of the first tissue type end at the first set of genomic positions at a rate above a threshold. 
     
     
         3 . The method of  claim 2 , the first set of genomic positions are identified by:
 analyzing, by a computer system, a second plurality of cell-free DNA molecules from at least one first additional sample to identify ending positions of the second plurality of cell-free DNA molecules, wherein the at least one first additional sample is known to include the first tissue type and is of a same sample type as the biological sample;   for each genomic position of a plurality of genomic positions:
 computing a corresponding number of the second plurality of cell-free DNA molecules ending on the genomic position; and 
   comparing the corresponding number to a reference value to determine whether the rate of cell-free DNA molecules ending on the genomic position is above the threshold.   
     
     
         4 . The method of  claim 3 , further comprising:
 determining a size of each of the second plurality of cell-free DNA molecules, wherein identifying the first set of genomic positions further includes:
 determining a first statistical value of a size distribution of cell-free DNA molecules of the second plurality of cell-free DNA molecules ending at a first genomic position determined to have the rate above the threshold; 
 comparing the first statistical value to a size threshold; and 
 excluding the first genomic position from the first set of genomic positions when the first statistical value does not exceed the size threshold. 
   
     
     
         5 . The method of  claim 3 , wherein the one or more calibration samples include the at least one first additional sample, the method further comprising:
 for each of the one or more calibration samples:
 measuring a corresponding proportional contribution of the first tissue type; and 
 determining a corresponding relative abundance using the corresponding numbers of the second plurality of cell-free DNA molecules ending at the first set of genomic positions, thereby obtaining one or more calibration data points, wherein each calibration data point specifies the measured proportional contribution of the first tissue type for the at least one first additional sample and the corresponding relative abundance. 
   
     
     
         6 . The method of  claim 3 , wherein each genomic position of the first set of genomic positions has at least a specified number of cell-free DNA molecules of the second plurality of cell-free DNA molecules ending on the genomic position. 
     
     
         7 . The method of  claim 3 , wherein the reference value is an expected number of cell-free DNA molecules ending at the genomic position according to a probability distribution and an average length of cell-free DNA molecules in the at least one first additional sample. 
     
     
         8 . The method of  claim 3 , wherein the genomic positions whose rate of the second plurality of cell-free DNA molecules ending on the genomic position is above the threshold comprises a first superset, and wherein the first set of genomic positions are further identified by:
 analyzing, by the computer system, a third plurality of cell-free DNA molecules from at least one second additional sample identified as having a reduced amount of the first tissue type to identify a second superset of the third plurality of cell-free DNA molecules ending on the genomic position is above the threshold; and   identifying the first set of genomic positions as including the genomic positions that are in the first superset and that are not in the second superset.   
     
     
         9 . The method of  claim 3 , wherein the reference value includes a measured number of cell-free DNA molecules ending at the genomic position, the measured number determined from a third plurality of cell-free DNA molecules of at least one second additional sample identified as not having the first tissue type. 
     
     
         10 . The method of  claim 9 , further comprising:
 determining a size of each of the second plurality of cell-free DNA molecules, wherein identifying the first set of genomic positions further includes:
 determining a first statistical value of a first size distribution of cell-free DNA molecules of the second plurality of cell-free DNA molecules ending on a first genomic position determined to have the rate above the threshold; 
 determining a second statistical value of a second size distribution of cell-free DNA molecules of the third plurality of cell-free DNA molecules ending on one or more second genomic positions determined to have the rate above the threshold; 
 comparing the first statistical value to second statistical value; and 
 excluding the first genomic position from the first set of genomic positions when the first statistical value does not exceed the second statistical value by at least a specified amount to indicate that the first size distribution is smaller than the second size distribution. 
   
     
     
         11 . The method of  claim 9 , wherein comparing the corresponding number to the reference value includes:
 computing a first ratio of the corresponding number and a third number of the second plurality of cell-free DNA molecules covering the genomic position; and   comparing the first ratio to the reference value, the reference value including a reference ratio of the measured number of reads ending within the genomic positions and a fourth number of the third plurality of cell-free DNA molecules covering the genomic position and not ending at the genomic position.   
     
     
         12 . The method of  claim 3 , wherein the sample type of the biological sample and the at least one first additional sample is selected from a group consisting of plasma, serum, cerebrospinal fluid, and urine. 
     
     
         13 . The method of  claim 2 , wherein the first tissue type has a plurality of first tissue-specific alleles in at least one additional sample, and wherein the first set of genomic positions are determined using cell-free DNA molecules of the least one additional sample that include at least one of the plurality of first tissue-specific alleles, wherein the second set of genomic positions are such that ends of cell-free DNA molecules of a second tissue type occur at a rate above the threshold in the at least one additional sample, wherein the second tissue type has a plurality of second tissue-specific alleles in the at least one additional sample, and wherein the second set of genomic positions are determined using cell-free DNA molecules of the least one additional sample that include at least one of the plurality of second tissue-specific alleles. 
     
     
         14 . The method of  claim 1 , wherein the relative abundance includes a ratio of the first number and the second number. 
     
     
         15 . The method of  claim 1 , wherein the second set of genomic positions includes all genomic positions corresponding to an end of at least one of the first plurality of cell-free DNA molecules. 
     
     
         16 . The method of  claim 1 , wherein the first tissue type is a tumor. 
     
     
         17 . The method of  claim 16 , wherein the classification is selected from a group consisting of: an amount of tumor tissue in the subject, a size of the tumor in the subject, a stage of the tumor in the subject, a tumor load in the subject, and presence of tumor metastasis in the subject. 
     
     
         18 . The method of  claim 1 , further comprising:
 obtaining template DNA molecules from the biological sample to be analyzed;   preparing a sequencing library of analyzable DNA molecules using the template DNA molecules, the preparing of the sequencing library of analyzable DNA molecules not including a step of DNA amplification of the template DNA molecules; and   sequencing the sequencing library of analyzable DNA molecules to obtain a plurality of sequence reads corresponding to the first plurality of cell-free DNA molecules,   wherein analyzing the first plurality of cell-free DNA molecules includes:
 receiving, at the computer system, the plurality of sequence reads; and 
 aligning, by the computer system, the plurality of sequence reads to the reference genome to determine genomic positions for the plurality of sequence reads. 
   
     
     
         19 . The method of  claim 1 , further comprising performing imaging of the subject based on the classification. 
     
     
         20 . A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to analyze a biological sample, including a mixture of cell-free DNA molecules from a plurality of tissues types that includes a first tissue type, to determine a classification of a proportional contribution of the first tissue type in the mixture, the instructions comprising:
 analyzing, by a computer system, a first plurality of cell-free DNA molecules from the biological sample of a subject, wherein analyzing a cell-free DNA molecule includes:
 determining a genomic position in a reference genome corresponding to at least one end of the cell-free DNA molecule; 
   based on the analyzing of the first plurality of cell-free DNA molecules, determining, by the computer system, that a first number of the first plurality of cell-free DNA molecules end at one of a first set of genomic positions;   computing, by the computer system, a relative abundance using the first number and a second number of cell-free DNA molecules ending at a second set of genomic positions; and   determining the classification of the proportional contribution of the first tissue type by comparing the relative abundance to one or more calibration values determined from one or more calibration samples whose proportional contributions of the first tissue type are known.