**6. Conclusions**

Usually, only exons 2 and 3 of HLA class I genes and exon 2 of HLA class II genes are assessed for polymorphisms, while other parts of HLA genes are not, due to time and cost constraints. Many databases, such as dbMHC, AFND, IMGT/HLA, and others [12, 48, 49] keep a record of known HLA allelic sequences and track newly found polymorphisms. Most HLA allelic sequences are maintained there as CDS or partial exonic regions. This way, 8-digit HLA typing resolution, which is the ultimate goal, cannot be achieved.

To address this issue, complete genomic sequence analysis of all HLA exons, along with other important HLA regulatory sequences and also other significant genes, must be performed.

The high-resolution HLA typing of NGS is advantageous compared with the existing PCR/ SSO, PCR/SSP, and PCR/SBT techniques, but infers limitations that seem impossible to overcome, the most important being their incapability of sequencing long enough fragments, in order to confront without ambiguity the allele phasing issue [17].

This is important because HLA is a highly polymorphic region, therefore it is quite difficult to determine which variants are associated with the final phenotype, the latest resulting from the complete end-to-end haplotype, meaning all variants across the MHC and also other genomic loci [16, 40].

The above issues, of HLA ambiguity and phasing multiple short-read fragments, are resolved with third-generation sequencers able to analyze long-reads that cover entire intronic-exonic regions of whole genes. Further optimization and gradually reducing the cost and error rates of these sequencers will establish their dominance in the field of HLA typing.
