Cancer screening using methylation of cpg islands

Abstract

Systems, methods, and apparatuses can determine and use methylation profiles of various tissues and samples. Examples are provided. A methylation profile can be deduced for fetal/tumor tissue based on a comparison of plasma methylation (or other sample with cell-free DNA) to a methylation profile of the mother/patient. A methylation profile can be determined for fetal/tumor tissue using tissue-specific alleles to identify DNA from the fetus/tumor when the sample has a mixture of DNA. A methylation profile can be used to determine copy number variations in genome of a fetus/tumor. Methylation markers for a fetus have been identified via various techniques. The methylation profile can be determined by determining a size parameter of a size distribution of DNA fragments, where reference values for the size parameter can be used to determine methylation levels. Additionally, a methylation level can be used to determine a level of cancer.

1 . (canceled) 
     
     
         2 . A method of analyzing a biological sample of a human organism, the biological sample comprising cell-free DNA originating from normal cells and potentially from cells associated with cancer, the method comprising:
 analyzing a plurality of cell-free DNA molecules from the biological sample, wherein analyzing each of the plurality of cell-free DNA molecules includes:
 determining a location of the cell-free DNA molecule in a genome of the human organism by mapping, via sequence reads, the cell-free DNA molecule to a part of a human genome; and 
 determining whether the cell-free DNA molecule is methylated at one or more CpG sites of a plurality of CpG sites organized into a plurality of CpG islands, each of the plurality of CpG islands including more than one CpG site; 
   determining a respective number of cell-free DNA molecules at each of the plurality of CpG islands that are methylated; and   calculating a first methylation level using the respective numbers of cell-free DNA molecules that are methylated at the plurality of CpG islands.   
     
     
         3 . The method of  claim 2 , wherein determining whether the cell-free DNA molecule is methylated at the one or more CpG sites comprises performing methylation-aware sequencing. 
     
     
         4 . The method of  claim 3 , wherein performing the methylation-aware sequencing comprises performing methylation-aware massively parallel sequencing. 
     
     
         5 . The method of  claim 2 , wherein performing methylation-aware sequencing generates at least 10 million reads. 
     
     
         6 . The method of  claim 2 , wherein performing methylation-aware sequencing includes:
 treating the cell-free DNA molecules with sodium bisulfite; and   performing sequencing of the treated cell-free DNA molecules.   
     
     
         7 . The method of  claim 6 , wherein treating the cell-free DNA molecules with the sodium bisulfite is part of Tet-assisted bisulfite conversion or oxidative bisulfite sequencing for a detection of 5-hydroxymethylcytosine. 
     
     
         8 . The method of  claim 2 , wherein performing methylation-aware sequencing comprises sequencing of at least 60,000 cell-free DNA molecules. 
     
     
         9 . The method of  claim 2 , wherein the plurality of CpG sites are on a plurality of chromosomes. 
     
     
         10 . The method of  claim 2 , wherein the plurality of CpG islands are determined to be hypermethylated CpG islands compared to a reference group. 
     
     
         11 . The method of  claim 2 , wherein the first methylation level is calculated for a methylation profile. 
     
     
         12 . The method of  claim 2 , further comprising determining a first classification of a level of cancer based on the first methylation level. 
     
     
         13 . The method of  claim 12 , wherein the first classification indicates that cancer exists for the human organism, the method further comprising identifying a type of cancer associated with the human organism by comparing the first methylation level to a corresponding value determined from other human organisms, wherein at least two of the other human organisms are identified as having different types of cancer. 
     
     
         14 . The method of  claim 12 , wherein determining the first classification of the level of cancer based on the first methylation level comprises:
 comparing the first methylation level to a cutoff value; and   determining the first classification of the level of cancer based on the comparison.   
     
     
         15 . The method of  claim 14 , wherein the cutoff value is a specified distance from a reference methylation level established from another biological sample obtained from a healthy organism. 
     
     
         16 . The method of  claim 15 , wherein the specified distance is a specified number of standard deviations from the reference methylation level. 
     
     
         17 . The method of  claim 14 , wherein the cutoff value is established from a reference methylation level determined from a previous biological sample of the human organism obtained previous to the biological sample being tested. 
     
     
         18 . The method of  claim 14 , wherein comparing the first methylation level to the cutoff value includes:
 determining a difference between the first methylation level and a reference methylation level; and   comparing the difference to a threshold corresponding to the cutoff value.   
     
     
         19 . The method of  claim 14 , further comprising:
 determining a fractional concentration of tumor DNA in the biological sample; and   calculating the cutoff value based on the fractional concentration of tumor DNA in the biological sample.   
     
     
         20 . The method of  claim 14 , further comprising:
 measuring a size of the cell-free DNA molecules at the plurality of CpG sites, thereby obtaining measured sizes; and   before comparing the first methylation level to the cutoff value, normalizing the first methylation level using cell-free DNA molecules having a first size.   
     
     
         21 . The method of  claim 20 , wherein the first size is a range of lengths. 
     
     
         22 . The method of  claim 20 , wherein the cell-free DNA molecules having the first size are selected based on a physical separation that is dependent on size. 
     
     
         23 . The method of  claim 20 , further comprising selecting the cell-free DNA molecules having the first size by:
 performing paired-end massively parallel sequencing of the plurality of cell-free DNA molecules to obtain pairs of sequences for each of the cell-free DNA molecules;   determining a size of a cell-free DNA molecule by comparing the pair of sequences to a reference genome; and   selecting the cell-free DNA molecules having the first size.   
     
     
         24 . The method of  claim 20 , wherein normalizing the first methylation level using the cell-free DNA molecules having the first size includes:
 obtaining a functional relationship between size and methylation levels; and   using the functional relationship to normalize the first methylation level, wherein the functional relationship provides scaling values corresponding to respective sizes.   
     
     
         25 . The method of  claim 24 , further comprising:
 computing an average size corresponding to the cell-free DNA molecules used to calculate the first methylation level; and   multiplying the first methylation level by a corresponding scaling value.   
     
     
         26 . The method of  claim 24 , further comprising:
 for each site of the plurality of sites:
 for each of the cell-free DNA molecules located at the site:
 obtaining a respective size of the cell-free DNA molecule at the site; and 
 using a scaling value corresponding to the respective size to normalize a contribution of the cell-free DNA molecule to the respective number of cell-free DNA molecules that are hypermethylated at the site. 
 
   
     
     
         27 . The method of  claim 2 , further comprising:
 determining whether a fractional concentration of tumor DNA in the biological sample is greater than a minimum value; and   if the fractional concentration of tumor DNA is not greater than the minimum value, flagging the biological sample.   
     
     
         28 . The method of  claim 27 , wherein the minimum value is determined based on an expected difference in methylation levels for a tumor relative to a reference methylation level. 
     
     
         29 . The method of  claim 2 , wherein the plurality of CpG sites are from disjointed regions separated from each other. 
     
     
         30 . The method of  claim 2 , wherein the biological sample is a plasma sample or a serum sample. 
     
     
         31 . The A non-transitory computer readable medium comprising a plurality of instructions capable of execution by a computer system, that when so executed control the computer system to perform the method of  claim 2 .