Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

摘要: Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType assay provide very (VHR) typing, by addition 8 primer pairs original 14, 11 loci. These additional amplicons help resolve common well-documented alleles exclude commonly found null in ambiguity strings. Simplification workflow reduce initial preparation effort using early pooling or Fluidigm Access Array™ is also described. Performance VHR was evaluated on 28 well characterized cell lines Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance 98.4%; 1.6% had no assignment. Of concordant calls, 53% were unambiguous. To further assess assay, 59 clinical samples genotyped results compared unambiguous allele assignments obtained prior sequence-based typing supplemented with SSO and/or SSP. 98.7% 58.2% as calls; 1.3% could not be assigned. Our show that amplicon-based robust can replace current methodology. Together enhancements, it has potential even higher typing.