1025-LB

• Published in: Human immunology Vol. 74; no. 5; p. 498
• Main Authors: Moonsamy, Priscilla V, Williams, Timothy, Bonella, Persia, Holcomb, Cherie L, Hillman, Grantland, Turenchalk, Gregory S, Blake, Lisbeth A, Simen, Birgitte B, Goodridge, Damian, Hamilton, Amy, May, Andrew P, Erlich, Henry A
• Format: Journal Article
• Language: English
• Published: 01.05.2013
• Subjects: Allergy and Immunology
• ISSN: 0198-8859
• DOI: 10.1016/j.humimm.2012.11.058

Aim Next generation sequencing of HLA exonic amplicons with the 454 Life Sciences GS FLX System and Conexio Assign TM -ATF software provides high resolution, high throughput HLA genotyping for 8 class I and class II loci (Bentley et al., 20091 , Holcomb et al., 20112 ). HLA typing of potential donors for Unrelated Bone Marrow Donor Registries requires using a subset of these loci at high sample throughput and low cost per sample. To maximize the throughput, we have incorporated the Fluidigm® Access Array TM system to simplify amplicon library preparation and allow the efficient introduction of MIDs (Multiplex Identifiers) or barcodes. Methods For this application, a streamlined amplicon sequencing workflow employing many different Multiplex Identifiers (MIDs) enables multiplexed sequencing of a large number of samples, allowing researchers to meet these cost targets. The Fluidigm® 48 x 48 Access Array TM system and the “4 primer” approach provides an efficient way to achieve parallel semi-automated genomic PCRs with simultaneous incorporation of 48 different MIDs corresponding to 48 genomic DNA samples, and up to 48 different primer pairs, making possible 2,304 reactions in one amplification run. Minimal volumes (calculated to be about 45% less cost per run) of reagents are used in this micro- fluidics-based system. During genomic PCR, the outer set of primers containing the MIDs and the 454 adaptor sequences are incorporated into the amplicons generated by the inner set of HLA specific primers containing a complementary “universal” Fluidigm tag. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 GS FLX System, as well as genotyping with Conexio Assign TM -ATF software. Results Using the Access Array system, we have successfully (100% concordance with known genotypes) genotyped 192 samples with 8 primer pairs (covering exons 2 and 3 in HLA-A, B, C and Exon 2 in DRB1, DRB3/4/5 and DQB1) using 96 MIDs per region in a single GS FLX run on a 2 region PicoTiterPlate™ (PTP) and 96 samples using 48 MIDs per region in a 4 region PTP. An average of 166 and 137 sequence reads per amplicon were recovered respectively. We have also genotyped, in a single run, 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 175 sequence reads per amplicon at 100% concordance. The system reduces the overall time for the entire process (192 samples) from 8 days for manual processing to about 4 days with 1 FTE.