Non-invasive diagnosis of graft rejection in organ transplant patients

1. A method of quantifying kidney transplant-derived circulating cell-free deoxyribonucleic acids in a human kidney transplant recipient, said method comprising:

(a) providing a plasma sample from said human kidney transplant recipient, wherein said human kidney transplant recipient has received a kidney transplant from a kidney transplant donor, wherein said plasma sample from said human kidney transplant recipient comprises kidney transplant-derived circulating cell-free deoxyribonucleic acid and human kidney transplant recipient-derived circulating cell-free deoxyribonucleic acid;

(b) extracting circulating cell-free deoxyribonucleic acid from said plasma sample from said human kidney transplant recipient in order to obtain extracted circulating cell-free deoxyribonucleic acid, wherein said extracted circulating cell-free deoxyribonucleic acid comprises said kidney transplant-derived circulating cell-free deoxyribonucleic acid and human kidney transplant recipient-derived circulating cell-free deoxyribonucleic acid;

(c) performing a selective amplification of target deoxyribonucleic acid sequences, wherein said selective amplification of said target deoxyribonucleic acid sequences is of said extracted circulating cell-free deoxyribonucleic acid, wherein said selective amplification of said target deoxyribonucleic acid sequences amplifies a plurality of genomic regions comprising at least 1,000 single nucleotide polymorphisms, wherein said at least 1,000 single nucleotide polymorphisms comprise homozygous single nucleotide polymorphisms, heterozygous single nucleotide polymorphisms, or both homozygous single nucleotide polymorphisms and heterozygous single nucleotide polymorphisms, and wherein said selective amplification of said target deoxyribonucleic acid sequences is by polymerase chain reaction (PCR);

(d) performing a high throughput sequencing reaction, wherein said high throughput sequencing reaction comprises performing a sequencing-by-synthesis reaction on said selectively-amplified target deoxyribonucleic acid sequences from said extracted circulating cell-free deoxyribonucleic acid, wherein said sequencing-by-synthesis reaction has a sequencing error rate of less than 1.5%;

(e) providing sequences from said high throughput sequencing reaction, wherein said provided sequences from said high throughput sequencing reaction comprise said at least 1,000 single nucleotide polymorphisms; and

(f) quantifying an amount of said kidney transplant-derived circulating cell-free deoxyribonucleic acid in said plasma sample from said human kidney transplant recipient to obtain a quantified amount, wherein said quantifying said amount of said kidney transplant-derived circulating cell-free deoxyribonucleic acid in said plasma sample from said human kidney transplant recipient comprises using markers distinguishable between said human kidney transplant recipient and said kidney transplant donor, wherein said markers distinguishable between said human kidney transplant recipient and said kidney transplant donor comprises single nucleotide polymorphisms selected from said at least 1,000 single nucleotide polymorphisms identified in said provided sequences from said high throughput sequencing reaction, and wherein said quantified amount of said kidney transplant-derived circulating cell-free deoxyribonucleic acid in said plasma sample from said human kidney transplant recipient comprises at least 0.03% of the total circulating cell-free deoxyribonucleic acid from said plasma sample from said human kidney transplant recipient.

2. The method of claim 1 , wherein said at least 1,000 single nucleotide polymorphisms comprise single nucleotide polymorphisms that occur at an allele frequency greater than 1% of a population.

3. The method of claim 1 , wherein said at least 1,000 single nucleotide polymorphisms comprise homozygous single nucleotide polymorphisms.

4. The method of claim 1 , wherein said selective amplification of target deoxyribonucleic acid sequences amplifies a plurality of genomic regions comprising at least 5,000 single nucleotide polymorphisms.

5. The method of claim 4 , wherein said selective amplification of target deoxyribonucleic acid sequences amplifies a plurality of genomic regions comprising at least 10,000 single nucleotide polymorphisms.

6. The method of claim 1 , wherein said high throughput sequencing reaction comprises assigning a quality score to bases of said provided sequences.

7. The method of claim 1 , wherein said quantified amount of said kidney transplant-derived circulating cell-free deoxyribonucleic acid in said plasma sample from said human kidney transplant recipient using markers distinguishable between said human kidney transplant recipient and said kidney transplant donor comprises between 1.5% and 8% of said total circulating cell-free deoxyribonucleic acid from said plasma sample from said human kidney transplant recipient.

8. The method of claim 1 , wherein said quantified amount of said kidney transplant-derived circulating cell-free deoxyribonucleic acid in said plasma sample from said human kidney transplant recipient using markers distinguishable between said human kidney transplant recipient and said kidney transplant donor comprises at least 0.05% of said total circulating cell-free deoxyribonucleic acid from said plasma sample from said human kidney transplant recipient.

9. The method of claim 1 , wherein said quantified amount of said kidney transplant-derived circulating cell-free deoxyribonucleic acid in said plasma sample from said human kidney transplant recipient using markers distinguishable between said human kidney transplant recipient and said kidney transplant donor comprises at least 2% of said total circulating cell-free deoxyribonucleic acid from said plasma sample from said human kidney transplant recipient.

10. The method of claim 1 , wherein said quantified amount of said kidney transplant-derived circulating cell-free deoxyribonucleic acid in said plasma sample from said human kidney transplant recipient using markers distinguishable between said human kidney transplant recipient and said kidney transplant donor comprises between 0.03% and less than 8% of said total circulating cell-free deoxyribonucleic acid from said plasma sample from said human kidney transplant recipient.