Methods of monitoring immunosuppressive therapies in a transplant recipient

A method of quantifying an amount of DNA molecules from transplant donor-derived cell-free DNA in a transplant recipient of an organ transplant from multiple transplant donors without consideration of genotype information from the transplant recipient or each donor, the method comprising:

• a) providing cell-free DNA from a sample obtained from the transplant recipient, comprising DNA molecules from the transplant donors and DNA molecules from the recipient;
• b) amplifying, in a targeted manner from the cell-free DNA, each single nucleotide polymorphism (SNP) in a panel of two or more SNPs;
• c) sequencing the amplified SNPs to obtain homozygous or heterozygous allele distribution patterns of the SNPs in the panel;
• and
• d) quantifying, without consideration of genotype information from each transplant donor or recipient, an amount of DNA molecules from the transplant donor-derived cell-free DNA by assaying variance in homozygous or heterozygous allele distribution patterns of the SNPs in the panel as compared to expected homozygous or heterozygous allele distribution patterns, wherein an amount of DNA molecules from the transplant donor-derived cell-free DNA above a suitable threshold is indicative of transplant rejection or the risk thereof, and wherein an amount of DNA molecules from the transplant donor-derived cell-free DNA below a suitable threshold is indicative of transplant tolerance, and wherein individual genotyping of each donor and the recipient across the genome as a whole or portions thereof to determine which allele of each SNP in the panel belongs to each donor and the recipient is not performed.

A kit for quantifying an amount of transplant donor-derived cell-free DNA in a transplant recipient of an organ transplant from multiple transplant donors without consideration of prior genotype information from the transplant recipient or each donor, comprising:

• primers, reagents, controls for targeted single nucleotide polymorphism (SNP) amplification and sequencing, instructions for use, and instructions for accessing and using software for quantifying an amount of transplant donor-derived cell-free DNA in the transplant recipient without consideration of prior genotype information from the transplant recipient or each donor, wherein quantifying the amount of transplant donor-derived cell-free DNA comprises:
• obtaining measurements of a panel of single nucleotide polymorphisms (SNPs) that were amplified, in a targeted manner, from cell-free DNA derived from a sample from the organ transplant recipient following transplantation and sequenced to provide homozygous or heterozygous allele distribution patterns of the SNPs in the panel;
• and
• quantifying, without consideration of genotype information from each transplant donor or recipient, the amount of transplant donor-derived cell-free DNA by assaying variance in homozygous or heterozygous allele distribution patterns of the SNPs in the panel as compared to expected homozygous or heterozygous allele distribution patterns, wherein an amount of transplant donor-derived cell-free DNA above a suitable threshold is indicative of transplant rejection or the risk thereof, and wherein an amount of transplant donor-derived cell-free DNA below a suitable threshold is indicative of transplant tolerance, and wherein individual genotyping of each donor and the recipient across the genome as a whole or portions thereof to determine which allele of each SNP in the panel belongs to each donor and the recipient is not performed.