Unique molecular identifier enhanced hla genotyping and transcript quantitation using nanopore technology

A method for genotyping a donor cell, tissue, and/or organ meant for transplantation into a recipient and/or for genotyping the recipient thereof, the method comprising:

• (a) providing a donor cell, tissue, and/or organ meant for transplantation into a recipient and/or a cell, tissue, and/or organ from the recipient thereof that comprises mRNA derived from an HLA gene product;
• (b) reverse transcribing the mRNA to produce a pool of cDNAs, wherein the pool of cDNAs comprises a plurality of HLA-specific cDNAs;
• (c) amplifying the cDNAs that result from reverse transcription of HLA-specific mRNAs to produce a pool of HLA-specific cDNAs, wherein:
• (i) the HLA-specific mRNAs are transcription products of one or more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, MICA, and MICB loci, optionally for each of at least HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQA1, and HLA-DQB1;
• (ii) the amplifying comprises an enrichment step and an amplification step, wherein the amplification step is performed subsequent to the enrichment step;
• and
• (iii) the amplification employs HLA-specific primers that comprise unique molecular identifiers (UMIs) such that the HLA-specific cDNAs are each uniquely coded;
• and
• (d) determining the sequence of each HLA-specific cDNAs,
• whereby the donor cell, tissue, and/or organ meant for transplantation into a recipient and/or the recipient thereof is genotyped.