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1. WO2020118046 - QUANTIFYING FOREIGN DNA IN LOW-VOLUME BLOOD SAMPLES USING SNP PROFILING

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CLAIMS

What is Claimed is:

1. A method of selectively amplifying short DNA fragments in a DNA sample that comprises both long and short DNA fragments, the method comprising:

(a) ligating a universal adaptor oligonucleotide to each end of the long and short DNA fragments, thereby generating adaptor-modified long and short DNA fragments,

(b) selectively amplifying the adaptor-modified short DNA fragments by performing

PCR with an extension time of between about 1 second and about 15 seconds and using oligonucleotide primers that hybridize to the universal adaptor, thereby generating amplified short DNA fragments, and

(c) performing size selection to isolate the amplified short DNA fragments.

2. The method of claim 1, wherein the short DNA fragments have a length between about 50 nucleotides and 400 nucleotides.

3. The method of claim 1-2, wherein the PCR in step (b) is performed with an annealing time of between about 1 second and about 30 seconds.

4. The method of any one of claims 1-3, wherein the DNA sample comprises cell-free DNA (cfDNA).

5. The method of claim 4, wherein the short DNA fragments comprise cell-free DNA (cfDNA).

6. The method of any one of claims 1-5, wherein the DNA sample comprises DNA extracted from total blood.

7. The method of any one of claims 1-5, wherein the DNA sample is extracted from a buccal swab or urine.

8. The method of any one of claims 1-7, wherein, prior to step (a), the long and short DNA fragments are subjected to end-repair.

9. The method of any one of claims 1-8, wherein, prior to step (b), the adaptor-modified long and short DNA fragments are column purified.

10. The method of any one of claims 1-9, wherein the universal adaptors comprise, from 5’ to 3’, a region that is complementary to the oligonucleotide primers and a region that is not complementary to the oligonucleotide primers.

11. The method of any one of claims 1-10, wherein the size selection of step (c) comprises gel electrophoresis purification or beads-based purification.

12. The method of any one of claims 1-11, further comprising (d) sequencing the amplified short DNA fragments.

13. The method of claim 12, wherein the sequencing in step (d) is next-generation sequencing.

14. The method of claim 13, wherein the next-generation sequencing is paired-end sequencing or single-read sequencing.

15. The method of claim 14, further comprising (e) enriching the amplified short DNA fragment sequences by (1) aligning the sequences to a reference genome to determine the amplicon length and (2) removing any sequences with an amplicon length greater than 400 nucleotides.

16. A method of analyzing single nucleotide polymorphisms (SNPs) in a DNA sample, the method comprising

(a) hybridizing the DNA sample to a mixture of hybrid-capture probes, wherein at least 80% of the hybrid-capture probes correspond, independently, to a genomic region having a SNP with a population minor allele frequency of greater than 25%, wherein each genomic region:

(1) occurs no more than 10 times in the genome;

(2) has a GC content of between about 0.25 and about 0.75; and

(3) does not contain any string of a single base that is longer than 4 nucleotides,

thereby generating capture probe-bound DNA;

(b) isolating the hybrid-capture probe-bound DNA;

(c) ligating a universal adaptor oligonucleotide to each end of the hybrid-capture probe-bound DNA;

(d) amplifying the hybrid-capture probe-bound DNA using primers that hybridize to the adaptor sequences, thereby generating amplified DNA; and

(e) sequencing the amplified DNA.

17. The method of claim 16, wherein each genomic region comprises the 80 nucleotides surrounding the SNP.

18. The method of claim 17, wherein each genomic region is unique in the genome.

19. The method of any one of claims 16-18, wherein the method analyzes between about 500 and about 1,000,000 SNPs.

20. The method of any one of claims 16-19, wherein the DNA sample is amplified prior to step (a), thereby generating an amplified double- stranded DNA sample.

21. The method of claim 20, wherein the DNA sample is amplified according to the method of any one of claims 1-15.

22. The method of claim 20 or 21, wherein the amplified DNA sample comprises DNA fragments having a length of between about 50 nucleotides and about 400 nucleotides.

23. The method of claim 20, wherein the amplified double- stranded DNA sample is denatured prior to step (a), thereby generating an amplified single-stranded DNA sample.

24. The method of claim 23, wherein the amplified double- stranded DNA sample is denatured by heating the amplified double-stranded DNA sample at a temperature of at least 80°C for at least 2 minutes.

25. The method of claim 23, wherein the amplified double- stranded DNA sample is denatured by chemical denaturation.

26. The method of claim 25, wherein the chemical denaturation comprises incubating the amplified double- stranded DNA sample with sodium hydroxide.

27. The method of claim 23, wherein the amplified double- stranded DNA sample is denatured by enzymatic denaturation.

28. The method of any one of claims 16-27, wherein the sequencing in step (d) is next- generation sequencing.

29. The method of claim 28, wherein the next-generation sequencing is paired-end sequencing.

30. The method of claim 28, wherein the next-generation sequencing is single-read sequencing.

31. The method of any one of claims 16-30, wherein the isolating in step (b) comprises solid-phase capture of the hybrid-capture probe-bound DNA.

32. The method of claim 31, wherein the solid-phase capture of the hybrid-capture probe- bound DNA comprises incubating the hybrid-capture probe-bound DNA with strep tavidin-coated beads.

33. The method of claim 31, wherein the isolating in step (b) further comprises separating, washing, and releasing the hybrid-capture probe-bound DNA.

34. The method of claim 33, wherein separating comprises magnetic separation or centrifugation.

35. The method of claim 33, wherein releasing comprises heating the captured hybrid- capture probe-bound DNA at at least 80°C for at least 2 minutes.

36. The method of claim 33, wherein the hybrid-capture probes further comprise an enzyme recognition moiety.

37. The method of claim 36, wherein the enzyme recognition moiety is a deoxyuridine.

38. The method of claim 36, wherein releasing comprises performing enzymatic cleavage of the enzyme recognition moiety.

39. The method of claim 37, wherein releasing comprises incubating the captured hybrid- capture probe-bound DNA with a USER enzyme.

40. The method of any one of claims 16-39, wherein the DNA sample comprises cell-free DNA (cfDNA).

41. The method of claim 40, wherein the cfDNA is amplified prior to step (a).

42. The method of any one of claims 16-41, wherein the hybrid-capture probes are biotinylated.

43. The method of any one of claims 16-41, wherein the hybrid-capture probes are hybridized to a biotinylated oligonucleotide.

44. A method of determining the number of unique cfDNA fragments in a sample containing less than 4 ng of cfDNA and/or correcting errors from amplification and sequencing, the method comprising:

(a) amplifying the cfDNA fragments;

(b) sequencing the amplified cfDNA fragments using paired-end next-generation sequencing;

(c) aligning the sequences to a reference genome, and determining the start and end position of each sequenced cfDNA fragment;

(d) separating the sequences by the genomic loci they are aligned to, and calling the fragment sequence based on majority vote of all the sequencing reads with the same start and end positions; and

(e) counting the number of unique start and end positions from among the sequenced cfDNA fragments, thereby determining the number of cfDNA fragments at each genomic locus of interest corresponding to each different genotype in the sample.

45. The method of claim 44, wherein the start and end positions are determined by next- generation sequencing paired-end reads.

46. A method of determining the number of unique cfDNA fragments in a sample containing more than 4 ng of cfDNA and/or correcting errors from amplification and sequencing, the method comprising:

(a) ligating an adaptor nucleic acid to each end of each cfDNA fragment, wherein the adaptor nucleic acid comprises a degenerate sequence;

(b) amplifying the adaptor- ligated cfDNA fragments;

(c) sequencing the amplified cfDNA fragments using paired-end next-generation sequencing;

(d) aligning the sequences to a reference genome, and determining the start and end position of each sequenced cfDNA fragment;

(e) separating the sequences by the genomic loci they are aligned to, and calling the fragment sequence based on majority vote of all the sequencing reads with the same combined start and end positions and degenerate sequences; and

(f) counting the number of unique combined start and end positions and degenerate sequences from among the sequenced cfDNA fragments, thereby determining the number of cfDNA fragments at each genomic locus of interest corresponding to each different genotype in the sample.

47. The method of claim 46, wherein the start and end positions are determined by next- generation sequencing paired-end reads.

48. A method of monitoring organ transplant rejection by SNP profiling, the method comprising:

(a) extracting cell-free DNA (cfDNA) and genomic DNA (gDNA) from a DNA sample obtained from an organ transplant recipient;

(b) selectively amplifying short fragments of cell-free DNA using the method of any one of claims 1-15;

(c) obtaining sequence reads for at least 500 single nucleotide polymorphisms (SNPs) in the amplified cell-free DNA using the method of any one of claims 16-43; and

(d) quantifying a fraction of the organ transplant donor-derived cell-free DNA versus the DNA of the organ recipient.

49. The method of claim 48, wherein the cfDNA and gDNA are extracted from whole blood.

50. The method of claim 49, wherein the cfDNA and gDNA are extracted from a low- volume of whole blood.

51. The method of claim 49, wherein the extraction in step (a) further comprises plasma separation.

52. The method of claim 49, wherein the whole blood is venous blood.

53. The method of claim 49, wherein the whole blood is obtained from a finger-stick.

54. The method of claim 48, wherein the cfDNA and gDNA are extracted from a buccal swab.

55. The method of any one of claims 48-54, wherein step (c) comprises analyzing between 500 and about 1,000,000 SNPs.

56. The method of any one of claims 48-55, wherein step (d) comprises:

(1) removing sequencing reads that comprise undetermined bases; and

(2) determining the number of unique DNA fragments for each SNP locus and each genotype.

57. The method of claim 56, wherein determining the number of unique DNA fragments for each SNP locus and each genotype comprises performing the method of any one of claims 44-47.

58. The method of any one of claims 48-57, wherein the at least 500 SNPs consists of SNPs for which the organ transplant recipient is homozygous.

59. The method of any one of claims 48-58, wherein the at least 500 SNPs consists of

SNPs for which the organ transplant recipient and the organ donor are not identical.

60. The method of any one of claims 48-59, wherein if the fraction of the short fragments of cell-free DNA that correspond to the genomic DNA of the organ transplant donor is above a normal range or increases over time, then the organ transplant recipient is considered to be rejecting the transplanted organ.