High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ system for simplified amplicon library preparation

Abstract

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.

A multilocus molecular phylogeny for the avian genus Liocichla (Passeriformes: Leiothrichidae: Liocichla)

“…Oligonucleotide tags are commonly used to facilitate the addition of adapters and barcodes to amplicons for next-generation sequencing [37]. In our study the addition of common 5′ tags allowed for CS1 and CS2 to be utilized as sequencing primers for all amplicons across all loci.…”

Diagnostic approach for FSHD revisited: SMCHD1 mutations cause FSHD2 and act as modifiers of disease severity in FSHD1

“…28 For target enrichment, the Access Array System of Fluidigm (South San Francisco, CA, USA) was used that allows parallel amplification of 48 target regions for 48 samples in one single PCR setup. 29 For sequencing of SMCHD1, up to 24 patients were processed in parallel to obtain a coverage of 420x for all exons (except for exon 1). NGS data were evaluated with the software GensearchNGS (PhenoSystems, Lillois Witterzée, Belgium) using the reference sequence GRCh37 (hg19) and SMCHD1 transcript NM_015295.2.…”

Comparative Analyses of Low, Medium and High-Resolution HLA Typing Technologies for Human Populations

“…The limitation we noticed in 454 typing is that the short reads mapped to more than 2 alleles per gene in most of our samples (90%). Also 454 data resulted in the ambiguity in haplotype phasing because we typed only few exons and missed the information from other exons and introns [9,11]. Among the second-generation NGS platforms, 454 carry more errors in especially around homo-polymorphic sequences [4].…”

“…454 sequencing was one of the first NGS platforms tested for HLA typing. It was considered advantageous due to availability of large-numbers of barcodes and relatively longer reads (upto 1 Kb) [11]. As the HLA genes (A, B, C, DRB1 and DQB1) are more than 5 Kb, the 454 or other short read sequencing was not able to resolve haplotypes of donors.…”