The human leukocyte antigen (HLA) class I and class II loci are the most 
polymorphic genes in the human genome; distinguishing the thousands of HLA 
alleles is challenging. Next generation sequencing of exonic amplicons with the 
454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides 
high resolution, high throughput HLA genotyping for eight class I and class II 
loci. HLA typing of potential donors for unrelated bone marrow donor registries 
typically uses a subset of these loci at high sample throughput and low cost per 
sample. The Fluidigm Access Array System enables the incorporation of 48 
different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples 
with up to 48 different primer pairs in a microfluidic device generating 2304 
parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are 
used. During genomic PCR, in this 4-primer system, the outer set of primers 
containing the MID and the 454 adaptor sequences are incorporated into an 
amplicon generated by the inner HLA target-specific primers each containing a 
common sequence tag at the 5' end of the forward and reverse primers. Pools of 
the resulting amplicons are used for emulsion PCR and clonal sequencing on the 
454 Life Sciences GS FLX System, followed by genotyping with Conexio software. 
We have genotyped 192 samples with 100% concordance to known genotypes using 8 
primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 
3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per 
amplicon was obtained. We have also genotyped 96 samples at high resolution (14 
primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 
3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 
sequence reads per amplicon.