genotyping assay major histocompatibility typing assay PMC:2628004 Genotyping assays should ideally identify which part of the genome the information is about. We do not currently have a good way to do this. That information should be added later. PMID:27516207 A genotyping assay in which the alleles of genes encoding for major histocompatibility complex molecules are determined. MHC typing, HLA typing IEDB Blood samples from Holstein-Friesian heifer calves were used to isolate white blood cell (WBC) pellets, from which total RNA was extracted using Tri-reagent and cDNA generated using a Reverse Transcription Kit, both_ according to the manufacturers? instructions. An alignment of sequences of known bovine MHCI gene cDNAs, as presented in the IPD-MHC database (May 2014), was used to design a series of 3_ (for) and 5_ (rev) pan-MHCI primers. cDNA from individual animals was subject to PCR amplification in two separate reactions using either the For1/Rev2 or the For3/Rev1 primer pairs. For each sample primers using a unique combination of MID tags were used to allow subsequent de-multiplexing of the sequence data. PCRs were conducted using the Phusion High-Fidelity PCR kit. Following amplification, 5?_l of PCR products from each sample were pooled, purified using Agencourt AMPure XP Beads and run on a 1.5?% agarose gel. Bands of the appropriate size_ were extracted and purified using the Qiagen Gel extraction kit. Libraries were submitted to Edinburgh Genomics where after standard quality control procedures they underwent 300 base paired-end sequencing on an Illumina MiSeq v3. Data were separated into reads generated from For1Rev2 and For3Rev1 primer pairs, generating separate datasets for up to 192 samples. Within each sample, reads were clustered into unique variants which were subsequently compared using BLAST against a database of the previously identified bovine MHCI sequences. This process identified >140 novel classical MHCI genes and defined 62 novel MHCI haplotypes, dramatically expanding the known bovine MHCI repertoire. major histocompatibility typing assay metadata complete