Noninvasive diagnostics by sequencing 5-hydroxymethylated cell-free dna

Abstract

Provided herein is a method of sequencing hydroxymethyated cell-free DNA. In some embodiments, the method comprises adding an affinity tag to only hydroxymethyated DNA molecules in a sample of cfDNA, enriching for the DNA molecules that are tagged with the affinity tag; and sequencing the enriched DNA molecules.

What is claimed is: 
     
         1 . A method for identifying sources of DNA in a cell-free sample, comprising:
 (a) providing a sample that comprises a pool of cell-free DNA molecules that (i) are from a plurality of different sources, (ii) are adapter-ligated, (iii) comprise one or more hydroxymethylcytosines modified to contain a capture tag, and (iv) contain molecular barcodes to indicate their source;   (b) amplifying the cell-free DNA molecules to provide a population of amplification products;   (c) sequencing the amplification products to produce a plurality of sequence reads; and   (d) identifying the sources of the cell-free DNA molecules from the molecular barcodes observed in the sequence reads.   
     
     
         2 . The method of  claim 1 , wherein the capture tag comprises a biotin moiety. 
     
     
         3 . The method of  claim 1 , wherein the sample is enriched in DNA molecules comprising one or more hydroxymethylcytosines that are modified to contain the capture tag, such that the sample comprises an enriched composition. 
     
     
         4 . The method of  claim 3 , wherein at least 80% of the DNA molecules in the enriched composition comprise one or more hydroxymethylcytosines that are modified to contain the capture tag. 
     
     
         5 . The method of  claim 4 , wherein at least 90% of the DNA molecules in the enriched composition comprise one or more hydroxymethylcytosines that are modified to contain the capture tag. 
     
     
         6 . The method of  claim 5 , wherein the DNA molecules are linked to a solid support via the capture tag, and step (b) is carried out while the DNA molecules are linked to the solid support.