Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing

Abstract

BackgroundHLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.ResultsWe developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.ConclusionsIn this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1514-4) contains supplementary material, which is available to authorized users.

Single molecule real‐time DNA sequencing of HLA genes at ultra‐high resolution from 126 International HLA and Immunogenetics Workshop cell lines

“…There are several next generation sequencing‐based strategies being applied to HLA typing . Recently, a capture oligonucleotide probe and Illumina sequencing‐based study carried out KIR and HLA class I typing on 97 IHW cell lines .…”

“…There are several next generation sequencing-based strategies being applied to HLA typing. [34][35][36][37][38] Recently, a capture oligonucleotide probe and Illumina sequencingbased study carried out KIR and HLA class I typing on 97 IHW cell lines. 39 Of the 126 cell lines we typed with SMRT sequencing, 80 were also typed in the probe-Illumina study, but the results were not totally consistent between the two methods.…”

Evaluation of the AllType kit for HLA typing using the Ion Torrent S5 XL platform

“…The use of multiplex identifiers allows samples to be tagged and pooled, increasing throughput and decreasing costs. One of the first platforms used for HLA typing was the 454/GS FLX sequencer (Roche), which has been progressively supplanted by Illumina and Ion Torrent technologies . Advanced technologies are also used, such as Pacific Biosciences's single‐molecule real‐time sequencing and Oxford Nanopore single‐cell DNA template strand sequencing …”

“…One of the first platforms used for HLA typing was the 454/GS FLX sequencer (Roche), [13][14][15] which has been progressively supplanted by Illumina [16][17][18][19][20] and Ion Torrent technologies. [21][22][23][24] Advanced technologies are also used, such as Pacific Biosciences's single-molecule real-time sequencing [25][26][27][28][29][30] and Oxford Nanopore single-cell DNA template strand sequencing. [31][32][33] We recently implemented an NGS method for HLA typing using the AllType NGS kit from One Lambda and Thermo Fisher Scientific reagents on the Ion S5 XL platform.…”

Next-Generation Sequencing — An Overview of the History, Tools, and “Omic” Applications

“…On the other hand, there was little difference between the Junior Roche and the Ion PGM platforms for "in phase" sequence genotyping of HLA loci, and either platform could be used with excellent results [16]. In this case, the lower cost of reagents and a slightly quicker turnaround time favored the Ion PGM platform [97].…”