Non-invasive diagnosis of graft rejection in organ transplant patients

A method of preparing a cell-free nucleic acid fraction for analyzing transplant-derived cell-free nucleic acids in a biological sample from a solid organ transplant recipient, the method comprising:

• (a) providing a biological sample from the solid organ transplant recipient after the solid organ transplant recipient has received a solid organ transplant from a solid organ transplant donor;
• (b) isolating a fraction from the biological sample from the solid organ transplant recipient to obtain an isolated fraction, wherein the isolated fraction comprises cell-free nucleic acids from the solid organ transplant;
• (c) extracting nucleic acids from the isolated fraction to obtain extracted nucleic acids, wherein the extracted nucleic acids comprise cell-free nucleic acids from the solid organ transplant;
• (d) generating prepared extracted solid organ transplant-derived cell-free nucleic acids by preparing the extracted nucleic acids for a high-throughput sequencing reaction (HTS) or a digital polymerase chain reaction (dPCR);
• and
• (e) analyzing transplant-derived cell-free nucleic acids by detecting and quantifying a homozygous or a heterozygous single nucleotide polymorphism (SNP) from the solid organ transplant donor amongst the transplant-derived cell-free nucleic acids using a high-throughput sequencing reaction (HTS) or a digital polymerase chain reaction (dPCR).