Due to cost and time constraints most high-resolution 4-digit HLA genotyping methods employ sequence-based typing (SBT) or differential probe hybridization techniques primarily focused on the PBR consisting of exons 2 and 3 (546 bp total) for class I, and exon 2 (~270 bp) for class II loci.
We recently introduced a sequence and structure-based scoring method that employs sequence profile profile comparisons, but is anchored by structural alignments and showed that the functional information associated with the top-scoring hits found by the method agreed well with expert annotations published in the literature (Standley et al., 2008b).
Several methods employ this strategy, including genotyping-by-sequencing (GBS; Elshire et al. 2011 ) and RAD-Seq (Hohenlohe et al. 2010 ).
Most of these methods employ recombinant endoproteases that recognize a specific sequence.
It is important to note that the quality of genome sequences can vary depending on the depth of sequence coverage, combinations of sequence methods employed, heterozygosity and complexity of the genome in addition to annotation inputs and methodology, are among the most influential factors.
Although the primary advantage of structure-based prediction methods over the commonly employed sequence-based methods is their applicability to essentially any MHC allotype, this has not yet been convincingly demonstrated.
Details of the polymerase chain reaction (PCR) amplification and sequencing methods employed are given in Beckenbach (2011).
It is not clear whether these perceptibly corrupted versions of the genes represent biologically meaningful isoforms, mistakes by the splicing machinery that are of no consequence, or artifacts of the cloning and sequencing methods employed.
With the aids from comparative methods employing similarities among multiple genomic sequences and gene structure conservations, ab initio gene-finding programs could provide high enough specificities and outperform those purely gene prediction methods [ 19- 21].
The HLA genotyping methods mainly applied today are PCR-SSOP, such as the Luminex commercial methodology [ 25, 26], and SBT by the Sanger method employing capillary sequencing based on chain-termination reactions [ 14, 27].
