Sequencing analysis of circulating dna to detect and monitor autoimmune diseases

Abstract

Systems, methods, and apparatuses are provided for diagnosing auto-immune diseases such as systemic lupus erythematosus (SLE) based on the sizes, methylation levels, and/or genomic characteristics of circulating DNA molecules. Patients provide blood or other tissue samples containing cell-free nucleic molecules for analysis. Massively parallel and/or methylation-aware sequencing can be used to determine the sizes and methylation levels of individual DNA molecules and identify the number of molecules originating from different genomic regions. A level of SLE can be estimated based on: the amount of molecules having sizes below a threshold value; the methylation level(s) of the entire genome or portions of the genome; correlations between the sizes and methylation levels of DNA molecules; and/or comparing the representation of DNA molecules in each of a plurality of genomic regions with a reference value for that region, and determining an amount of genomic regions having increased or decreased measured genomic representation.

What is claimed is: 
     
         1 . A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to perform a method of analyzing a biological sample of an organism, the biological sample including nucleic acid molecules, wherein at least some of the nucleic acid molecules are cell-free, the method comprising:
 analyzing sequence data of a plurality of cell-free DNA molecules from the biological sample, wherein analyzing the sequence data of a cell-free DNA molecule comprises:
 aligning the sequence data of the cell-free DNA molecule to a reference genome, 
 determining a size of the cell-free DNA molecule from the aligned portion of the sequence data of the cell-free DNA molecule, and 
 comparing the size of the cell-free DNA molecule with a threshold value; 
   determining an amount of the cell-free DNA molecules having sizes below the threshold value; and   estimating a first level of an auto-immune disease in the organism based upon the amount.   
     
     
         2 . The computer product of  claim 1 , wherein estimating the first level of the auto-immune disease in the organism comprises determining the auto-immune disease is present in the organism,
 further comprising:
 using the first level of the auto-immune disease in the organism to design a treatment regimen for the organism or determine a dose of a medication. 
   
     
     
         3 . The computer product of  claim 1 , wherein the method further comprises:
 designating a first peak size of cell-free DNA molecules, wherein the first peak size is less than the threshold value;   designating a second peak size of cell-free DNA molecules, wherein the second peak size is greater than the threshold value;   determining a first peak number, wherein the first peak number is the number of the cell-free DNA molecules having sizes within a specified range of the first peak size;   determining a second peak number, wherein the second peak number is the number of the cell-free DNA molecules having sizes within a specified range of the second peak size;   calculating a ratio of the first peak number to the second peak number; and   estimating a second level of an auto-immune disease in the organism based upon the ratio.   
     
     
         4 . The computer product of  claim 3 , wherein
 the first peak size is equal to the mean, median, or mode size of the cell-free DNA molecules having sizes less than the threshold value, and   the second peak size is equal to the mean, median, or mode size of the cell-free DNA molecules having sizes greater than the threshold value.   
     
     
         5 . The computer product of  claim 1 , wherein the biological sample is an IgG-bound fraction. 
     
     
         6 . The computer product of  claim 1 , wherein the auto-immune disease is SLE. 
     
     
         7 . The computer product of  claim 1 , wherein:
 the sequence data includes a first sequence corresponding to one end of the cell-free DNA molecule and a second sequence corresponding to the other end of the cell-free DNA molecule,   aligning the sequence data of the cell-free DNA molecule to the reference genome comprises mapping the first sequence and the second sequence to a reference genome to obtain genomic coordinates of the first sequence and the second sequence, and   determining the size of the cell-free DNA molecule comprises subtracting the genomic coordinates of the first sequence from the second sequence.   
     
     
         8 . The computer product of  claim 1 , further storing a plurality of instructions that when executed control a computer system to evaluate a treatment for an auto-immune disease in an organism by performing:
 analyzing a pre-treatment biological sample according to  claim 1 , wherein the pre-treatment biological sample is obtained from the organism prior to treatment, and estimating a pre-treatment level of the auto-immune disease in the organism;   analyzing a post-treatment biological sample according to  claim 1 , wherein the post-treatment biological sample is obtained from the organism subsequent to treatment, and estimating a post-treatment level of the auto-immune disease in the organism; and   comparing the pre-treatment level of the auto-immune disease with the post-treatment level of the auto-immune disease to determine a prognosis of the treatment.   
     
     
         9 . The computer product of  claim 8 , wherein the treatment is determined to be effective if the post-treatment level of the auto-immune disease is lower than the pre-treatment level of the auto-immune disease. 
     
     
         10 . The computer product of  claim 8 , further comprising:
 determining a change between the pre-treatment level and the post-treatment level; and   determining a degree of effectiveness based on the change.   
     
     
         11 . The computer product of  claim 10 , wherein the change is determined by calculating a difference of or a ratio between the pre-treatment level and the post-treatment level. 
     
     
         12 . A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to perform a method of analyzing a biological sample of an organism, the biological sample including nucleic acid molecules, wherein at least some of the nucleic acid molecules are cell-free, the method comprising:
 analyzing a plurality of cell-free DNA molecules from the biological sample, the biological sample obtained from plasma or serum of a blood sample, wherein analyzing a cell-free DNA molecule includes:
 determining whether the cell-free DNA molecule is methylated at one or more sites on the cell-free DNA molecule; 
   calculating a first methylation level based on the methylation determined at sites of the cell-free DNA molecules;   comparing the first methylation level to a first reference value; and   estimating a first level of an auto-immune disease in the organism based upon the comparison.   
     
     
         13 . The computer product of  claim 12 , wherein the cell-free DNA molecules used to calculate the first methylation level have a specified size. 
     
     
         14 . The computer product of  claim 12 , wherein calculating the first methylation level includes:
 for each of a plurality of first sites:
 determining a respective number of cell-free DNA molecules that are methylated at the first site; 
   calculating the first methylation level based on the respective numbers of cell-free DNA molecules methylated at the plurality of first sites.   
     
     
         15 . The computer product of  claim 12 , wherein calculating the first methylation level includes:
 for each cell-free DNA molecule:
 calculating an amount of the one or more sites that are methylated; 
   summing the amounts for the cell-free DNA molecules to obtain a total amount; and   normalizing the total amount to obtain the first methylation level.   
     
     
         16 . The computer product of  claim 12 , wherein the first methylation level is calculated for a plurality of first sites of a genome of the organism, further comprising:
 for each of a plurality of second sites:
 determining a respective number of cell-free DNA molecules that are methylated at the second site; 
   calculating a second methylation level based on the respective numbers of cell-free DNA molecules methylated at the plurality of second sites;   comparing the second methylation level to a second reference value; and   estimating a second level of the auto-immune disease in the organism based upon the comparison of the second methylation level to the second reference value.   
     
     
         17 . The computer product of  claim 16 , wherein the method further comprises comparing the first level of the auto-immune disease with the second level of the auto-immune disease to determine a classification of whether the organism has the auto-immune disease. 
     
     
         18 . The computer product of  claim 17 , wherein comparing the first level with the second level includes determining a parameter between the first level and the second level, and comparing the parameter to a cutoff value. 
     
     
         19 . The computer product of  claim 18 , wherein the parameter includes a difference or a ratio between the first level and the second level. 
     
     
         20 . The computer product of  claim 16 , wherein the plurality of first sites occur in repeat regions of the genome of the organism, and the plurality of second sites occur in non-repeat regions of the genome of the organism. 
     
     
         21 . A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to perform a method of analyzing a biological sample of an organism, the biological sample including nucleic acid molecules, the biological sample obtained from plasma or serum of a blood sample, wherein at least some of the nucleic acid molecules are cell-free, the method comprising:
 analyzing a plurality of cell-free DNA molecules from the biological sample, wherein analyzing a cell-free DNA molecule includes:
 determining a location of the cell-free DNA molecule in a genome of the organism; 
 determining whether the cell-free DNA molecule is methylated at one or more sites on the cell-free DNA molecule; 
   for each of a first plurality of genomic regions:
 determining a methylation density at a plurality of sites in the genomic region based on the analysis of cell-free DNA molecules in the genomic region; 
 comparing the methylation density to a first threshold to determine whether the genomic region is hypomethylated; 
   calculating a first amount of genomic regions that are hypomethylated; and   estimating a level of an auto-immune disease in the organism based upon the first amount.   
     
     
         22 . The computer product of  claim 21 , wherein a genomic region is hypomethylated if the methylation density is less than the first threshold. 
     
     
         23 . The computer product of  claim 21 , wherein the method further comprises:
 for each of a second plurality of genomic regions:
 comparing the methylation density to a second threshold to determine whether the genomic region is hypermethylated, and 
   calculating a second amount of genomic regions that are hypermethylated,   wherein estimating a level of an auto-immune disease in the organism is further based on the second amount.   
     
     
         24 . The computer product of  claim 23 , wherein the first plurality of genomic regions is the same as the second plurality of genomic regions. 
     
     
         25 . The computer product of  claim 23 , wherein a region is hypermethylated if the methylation density exceeds the second threshold. 
     
     
         26 . The computer product of  claim 23 , wherein a difference between the second threshold and a reference value equals the difference between the reference value and the first threshold, and wherein the reference value is a statistical value of the methylation densities determined for a plurality of genomic regions of one or more other organisms. 
     
     
         27 . The computer product of  claim 23 , wherein the first and second thresholds for a genomic region are determined based on a statistical variation in the methylation densities determined for a plurality of genomic regions. 
     
     
         28 . The computer product of  claim 21 , wherein the genomic regions are of equal size. 
     
     
         29 . The computer product of  claim 28 , wherein the size of each genomic region is from about 100 kb to about 10 Mb. 
     
     
         30 . The computer product of  claim 21 , wherein the first threshold for a genomic region reflects statistical variation in the methylation densities determined for a plurality of genomic regions. 
     
     
         31 . The computer product of  claim 12 , wherein:
 analyzing the cell-free DNA molecule further comprises:
 determining a size of the cell-free DNA molecule by sequencing the cell-free DNA molecule to obtain sequence data and aligning the sequence data to a reference genome; 
 calculating the first methylation level is based on the determined methylation for cell-free DNA molecules having a first size; 
   the first reference value is a threshold methylation level.   
     
     
         32 . The computer product of  claim 31 , wherein the first size is a range of sizes having a minimum and maximum, and the maximum is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 bp. 
     
     
         33 . The computer product of  claim 31 , wherein the first size is a range of sizes having a minimum and maximum, and the difference between the minimum and maximum is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 bp. 
     
     
         34 . The computer product of  claim 31 , wherein the method further comprises:
 for a second size:
 calculating a second methylation level based on the determined methylation for cell-free DNA molecules having the second size; 
   wherein the second size is greater than the first size,   and wherein estimating the first level of the auto-immune disease in the organism is further based on a ratio of the first methylation level to the second methylation level.   
     
     
         35 . The computer product of  claim 31 , wherein the auto-immune disease is detected if the first methylation level is less than the threshold methylation level. 
     
     
         36 . A computer product comprising a non-transitory computer readable medium storing a plurality of instructions that when executed control a computer system to perform a method of analyzing a biological sample of an organism, the biological sample including nucleic acid molecules, the biological sample obtained from plasma or serum of a blood sample, wherein at least some of the nucleic acid molecules are cell-free, the method comprising:
 for each of a plurality of nucleic acid molecules from the biological sample, identifying a location of the nucleic acid molecule in a reference genome of the organism from sequence data of the nucleic acid molecule;   for each of a plurality of genomic regions:
 identifying a respective group of nucleic acid molecules as being from the genomic region based on the identified locations; 
 calculating a respective value of the respective group of nucleic acid molecules, wherein the respective value defines a property of the nucleic acid molecules of the respective group; and 
 comparing the respective value to a reference value to determine a classification of whether the genomic region exhibits an increased or decreased measured genomic representation; 
   determining an amount of genomic regions classified as exhibiting an increased or decreased measured genomic representation;   comparing the amount to a threshold amount; and   estimating a first level of an auto-immune disease in the organism based upon the comparison.   
     
     
         37 . The computer product of  claim 36 , wherein the respective value for each genomic region is based on the number of nucleic acid molecules in the respective group for the genomic region. 
     
     
         38 . The computer product of  claim 36 , wherein the respective value for each genomic region corresponds to a statistical value of a size distribution of the nucleic acid molecules in the respective group for the genomic region. 
     
     
         39 . The computer product of  claim 36 , wherein the reference value for each genomic region is based on the respective value calculated for the genomic region using one or more control biological samples. 
     
     
         40 . The computer product of  claim 39 , wherein the reference value is the mean of the respective values calculated for the genomic region using a plurality of control biological samples. 
     
     
         41 . The computer product of  claim 36 , wherein comparing the respective value to the reference value for each genomic region comprises calculating a difference between the respective value and the reference value, and comparing the difference to a cutoff. 
     
     
         42 . The computer product of  claim 36 , wherein the amount and threshold amount are based on numbers of genomic regions of the plurality of genomic regions.