Methods for determination of transplant rejection

A method of preparing a composition of amplified DNA derived from a blood, plasma, serum or urine sample of a transplant recipient useful for determination of transplant rejection, comprising:

• (a) extracting cell-free DNA from the blood, plasma, serum or urine sample of the transplant recipient, wherein the extracted cell-free DNA comprises donor-derived cell-free DNA and recipient-derived cell-free DNA;
• (b) preparing a composition of amplified DNA by performing targeted amplification of the extracted DNA at 200-50,000 target loci in a single reaction volume, and sequencing the amplified DNA by high-throughput sequencing to obtain sequencing reads and quantifying the amount of donor-derived cell-free DNA and the amount of Torque teno virus (TTV) based on the sequencing reads;
• and
• (c) determining whether a combination of the amount of donor-derived cell-free DNA or a function thereof and the amount of TTV or a function thereof exceeds a cutoff threshold indicating transplant rejection,
• wherein the blood, plasma, serum or urine sample is obtained from the transplant recipient less than 18 months post-transplantation.

A method of administering immunosuppressive therapy in a transplant recipient, comprising:

• (a) measuring the amount of Torque teno virus (TTV) in a blood, plasma, serum, or urine sample of the transplant recipient;
• (b) measuring the amount of donor-derived cell-free DNA in a blood, plasma, serum, or urine sample of the transplant recipient;
• and
• (c) titrating the dosage of an immunosuppressive therapy according to the amount of TTV or a function thereof and the amount of donor-derived cell-free DNA or a function thereof,
• wherein the blood, plasma, serum or urine sample is obtained from the transplant recipient less than 18 months post-transplantation.