Nuclease-associated end signature analysis for cell-free nucleic acids

A method of estimating a fractional concentration of clinically-relevant DNA molecules in a biological sample of a subject, the method comprising:

• identifying that a first nuclease is differentially regulated in a target tissue type relative to at least one other tissue type of a plurality of tissue types, wherein the clinically-relevant DNA molecules are from the target tissue type;
• determining that the first nuclease preferentially cuts DNA into DNA molecules having a first sequence end signature relative to other sequence end signatures;
• analyzing a plurality of cell-free DNA molecules from the biological sample to obtain sequence reads, wherein the biological sample includes a mixture of cell-free DNA molecules from the plurality of tissue types, and wherein the sequence reads include ending sequences corresponding to ends of the plurality of the cell-free DNA molecules;
• identifying a first set of the sequence reads, wherein each sequence read of the first set of the sequence reads includes an ending sequence corresponding to the first sequence end signature;
• determining a first amount of the first set of the sequence reads;
• determining a first parameter using the first amount of the sequence reads;
• and
• estimating the fractional concentration of the clinically-relevant DNA molecules in the biological sample using the first parameter and one or more calibration values determined from one or more calibration samples whose fractional concentration of the clinically-relevant DNA molecules are known.

A method of determining a characteristic of a target tissue type, the method comprising:

• identifying that a first nuclease is differentially regulated in the target tissue type relative to at least one other tissue type of a plurality of tissue types;
• determining that the first nuclease preferentially cuts DNA into DNA molecules having a first sequence end signature relative to other sequence end signatures;
• analyzing a plurality of cell-free DNA molecules from a biological sample to obtain sequence reads, wherein the biological sample includes a mixture of cell-free DNA molecules from the plurality of tissue types, and wherein the sequence reads include ending sequences corresponding to ends of the plurality of cell-free DNA molecules;
• identifying a first set of the sequence reads, wherein each sequence read of the first set of the sequence reads includes an ending sequence corresponding to the first sequence end signature;
• determining a first amount of the first set of the sequence reads;
• determining a first parameter for the first amount of the sequence reads;
• and
• estimating a first value for the characteristic of the target tissue type using the first parameter and one or more calibration values determined from one or more calibration samples whose values for the characteristic are known.