11-OR: VERY HIGH RESOLUTION HLA GENOTYPING WITH THE 454 LIFE SCIENCES GS FLX SYSTEM: SIMPLIFICATION OF WORKFLOW USING FUSION PRIMERS OR A FOUR PRIMER SYSTEM

摘要: Aim We have previously reported the development of very high resolution (VHR) HLA genotyping, designed to resolve majority both “common and well documented alleles” null alleles using 454 GS FLX Sequencing System Conexio genotyping software. Amplicon library construction was performed 22 PCR fusion primers, containing genome target specific sequence with sequencing adaptors. The VHR assay but required extensive handling individual amplicons. To simplify workflow, we prepared amplicon libraries by three alternative procedures. Methods amplicons were generated from 10 cell lines for: HLA-A/B exons 1-5, HLA-C 1-7 some intronic class I regions; DPA1, DPB1, DQA1 exon 2; DQB1, DRB 2 3. Amplification pairs primers (14 primer in GType plates 454, plus 8 a third plate) Multiplex Identifiers (MIDs), or each containing, at 5′end, universal which matched 3′ end set that contained MIDs Amplicons derived purified, quantified, diluted standard concentration, then pooled OR prior other operations. four system on Fluidigm Access Array™ handled latter procedure. Pools sequenced genotyped Results All procedures resulted comparable (concordance up >98%). Pooling immediately following genomic gave reduction number pipetting steps necessary produce an ∼80% ∼90% system, respectively. Conclusions early preparation procedure greatly simplifies workflow for as 4 system. Holcomb: Roche: Employee. Hoglund: Williams: Grant Research. Erlich: