HLA genotyping by next-generation sequencing of complementary DNA

Abstract

BackgroundGenotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA.MethodsEach HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database.ResultsThe genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology.ConclusionThe performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4300-7) contains supplementary material, which is available to authorized users.

Capturing Differential Allele-Level Expression and Genotypes of All Classical HLA Loci and Haplotypes by a New Capture RNA-Seq Method

“…In order to test our RNA sequencing method before applying it to disease and transplantation research, it was considered important to first understand comprehensively the normal pattern of RNA levels expressed by each allele using healthy individuals within the same ethnic group. In this way, the differences in RNA expression levels (sequence read numbers) could be compared more reliably among the HLA alleles at the different HLA loci as described previously (43).…”

“…New RNA quantitative techniques based on RNA-sequencing (RNA-Seq) have emerged recently (41), and genotyping, mapping the expression quantitative trait locus, and analyzing allele-specific expression from public RNA-Seq data are promising new development (42). In addition, a computational pipeline to accurately estimate expression for HLA genes based on RNA-Seq was developed for both locus-level and allele-level estimates (43).…”

Adapting Cord Blood Collection and Banking Standard Operating Procedures for HLA-Homozygous Induced Pluripotent Stem Cells Production and Banking for Clinical Application

“…However, the introduction of Next-Generation Sequencing (NGS) in the determination of HLA haplotypes has united high throughput with high resolution. New developments allow for HLA determination at very high resolution of big numbers of samples simultaneously, for a low price [29,30]. Therefore, given the advantages and universalisation of NGS for HLA typing, this is probably the method of choice for HLA confirmation of the homozygous before being selected for reprogramming and inclusion in the iPSC bank.…”

“…The clinical utility of an iPSC line could be compromised if some information or documents are not present in the file. The International Stem Cell Banking Initiative (ISCBI) published a list of typical information that might be required in a cell line history [30]. In any case, the final expansion and stock production, quality control and storage should be made with GMP-compliant processes in clinical-grade facilities.…”

Evaluation of Ion Torrent sequencing technology for rapid clinical human leucocyte antigen typing

“…Flows of dNTPs are sequentially washed over the microwells and each time a dNTP is incorporated during DNA synthesis, a hydrogen ion is released causing a pH change in the reaction buffer. These changes in pH are recorded allowing the sequence to be determined (Barone et al, ; Segawa, Kukita, & Kato, ).…”