Non-invasive early detection of solid organ transplant rejection by quantitative analysis of mixtures by deep sequencing of HLA gene amplicons using next generation systems

A method of generating sequence information from a solid organ graft recipient, the method comprising:

• (a) providing a blood sample from the recipient;
• (b) generating HLA allele sequence at the loci DPB1 Exon 2, DRB1 Exon 2, DRB3 Exon 2, DRB4 Exon 2, DRB5 Exon 2, and DOB1 Exon 2 by a method comprising:
• i. contacting nucleic acid from the blood sample with a forward primer and reverse primer that have an HLA-hybridizing sequence consisting of SEQ ID NO:
• 1 and 7 for DPB 1 Exon 2, a forward primer and reverse primer that have an HLA-hybridizing sequence consisting of SEQ ID NO:
• 2 and 11 for DRB1 Exon 2, DRB3 Exon 2, DRB4 Exon2, and DRB5 Exon 2, and a forward primer and reverse primer that have an HLA-hybridizing sequence consisting of SEQ ID NO:
• 4 and 10 for DQB1 Exon 2, and amplifying to produce HLA amplicons;
• ii. performing clonal sequencing to determine the sequence of the HLA amplicons obtained in step (i);
• iii. among the sequences determined in step (ii), identifying at least one recipient HLA allele and at least one non-recipient HLA allele at the same locus.