Non-invasive determination of tissue source of cell-free dna

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.

What is claimed is: 
     
         1 . A method of analyzing a biological sample from a subject, the method comprising:
 (a) obtaining sequence reads for cell-free DNA molecules from the biological sample of the subject, wherein the sequence reads include methylation statuses for the cell-free DNA molecules at single nucleotide resolution, and wherein the sequence reads comprise at least 100,000 sequence reads;   (b) analyzing, including aligning to a reference genome, the at least 1000,000 sequence reads to determine a methylation profile for a plurality of sites based on the methylation statuses for the plurality of sites; and   (c) determining a tissue source for at least a portion of the cell-free DNA molecules from the biological sample based, at least in part, on the methylation profile.   
     
     
         2 . The method of  claim 1 , wherein determining the tissue source comprises:
 comparing the methylation profile to one or more reference methylation profiles.   
     
     
         3 . The method of  claim 2 , wherein at least one of the one or more reference methylation profiles is determined from methylation statuses of one or more reference sample obtained from another subject known to have cancer. 
     
     
         4 . The method of  claim 3 , wherein at least another one of the one or more reference methylation profiles is obtained from methylation statuses of at least one other sample obtained from a healthy subject. 
     
     
         5 . The method of  claim 2 , wherein the comparison of the methylation profile to the one or more reference methylation profiles detects changes in methylation status of CpG islands. 
     
     
         6 . The method of  claim 1 , wherein the methylation profile comprises a pattern of the cell-free DNA molecules that are methylated at the plurality of sites, wherein the plurality of sites includes at least 20,000 sites. 
     
     
         7 . The method of  claim 1 , wherein at least a portion of the cell-free DNA molecules are cancer-derived molecules, the method further comprising determining a type of cancer of the subject based, at least in part, on the methylation profile. 
     
     
         8 . The method of  claim 7 , wherein the type of cancer is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, prostate cancer, nasopharyngeal cancer, gastric cancer, testicular cancer, skin cancer, cancer affecting the nervous system, bone cancer, ovarian cancer, liver cancer, hematologic malignancies, pancreatic cancer, endometriocarcinoma, and kidney cancer. 
     
     
         9 . The method of  claim 1 , further comprising sequencing the cell-free DNA molecules to obtain the sequence reads. 
     
     
         10 . The method of  claim 9 , wherein the sequencing comprises methylation-aware sequencing. 
     
     
         11 . The method of  claim 10 , wherein the methylation-aware sequencing comprises bisulfate sequencing. 
     
     
         12 . The method of  claim 10 , further comprising enriching the cell-free DNA molecules before the sequencing, and wherein the enriching comprises use of hybridization probes, polymerase chain reaction amplification, or solid phase hybridization. 
     
     
         13 . The method of  claim 1 , wherein the plurality of sites comprise one or more CpG sites. 
     
     
         14 . The method of  claim 13 , wherein the one or more CpG sites comprise a plurality of CpG sites that are organized into one or more CpG islands. 
     
     
         15 . The method of  claim 14 , wherein the determining the methylation profile for the plurality of sites comprises, for each CpG island of a plurality of CpG islands, determining a number of sequence reads showing methylation at the CpG sites in the CpG island. 
     
     
         16 . The method of  claim 1 , wherein the determining the methylation profile for the plurality of sites comprises, for each site of the plurality of sites, determining a total number of sequence reads at the plurality of sites. 
     
     
         17 . The method of  claim 1 , wherein the analyzing further comprises determining locations of the cell-free DNA molecules in a genome. 
     
     
         18 . The method of  claim 1 , wherein the determining the methylation profile comprises determining a number of sequence reads of the cell-free DNA molecules showing methylation at sites in a genomic region. 
     
     
         19 . The method of  claim 1 , wherein the biological sample is selected from a group consisting of blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, plural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage, fluid, and stool. 
     
     
         20 . A non-transitory computer-readable medium comprising instructions that, upon execution by one or more computer processors of a computer system, causes the computer system to perform a method, the method comprising:
 (a) obtaining sequence reads for cell-free DNA molecules from a biological sample of a subject, wherein the sequence reads include methylation statuses for the cell-free DNA molecules at single nucleotide resolution, and wherein the sequence reads comprise at least 100,000 sequence reads;   (b) analyzing, including aligning to a reference genome, the at least 1000,000 sequence reads to determine a methylation profile for a plurality of sites based on the methylation statuses for the plurality of sites; and   (c) determining a tissue source for at least a portion of the cell-free DNA molecules from the biological sample based, at least in part, on the methylation profile.