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

Abstract

The development of techniques to define the human leucocyte antigen (HLA) region has proven to be challenging due to its high level of polymorphism. Within a clinical laboratory, a technique for high-resolution HLA typing, which is rapid and cost effective is essential. NGS has provided a rapid, high-resolution HLA typing solution, which has reduced the number of HLA ambiguities seen with other typing methods. In this study, the One Lambda NXType NGS kit was tested on the Ion Torrent PGM platform. A total of 362 registry donors from four ethnic populations (Europeans, South Asians, Africans and Chinese) were NGS HLA typed across 9-loci (HLA-A, -B, -C, -DRB1,-DRB345 -DQB1 and -DPB1). Concordance rates of 91%-98% were obtained (for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) when compared to historical PCR-SSO HLA types, and the identification of uncommon alleles such as A*24:07:01 and C*04:82 were observed. A turnaround time of four days was achieved for typing 44 samples. However, some limitations were observed; primer locations did not allow all ambiguities to be resolved for HLA Class II where Exon I and IV amplification are needed (HLA-DRB1*04:07:01/04:92, HLA-DRB1*09:01:02/*09:21 and HLA-DRB1*12:01:01/*12:10). This study has demonstrated high-resolution typing by NGS can be achieved in an acceptable turnaround time for a clinical laboratory; however, the Ion Torrent workflow has some technical limitations that should be addressed.

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

“…Notably, repeat testing of these DNA samples using another NGS method at the CHRU laboratory revealed the same type of phasing error. Moreover, a previous study using the original One Lambda NXType kit showed lower concordance (93%) than that reported in prior studies using older typing techniques, especially for the HLA-DPB1 locus, 24 suggesting that the algorithms for read alignments at the HLA-DPB1 locus are complex and require improvement.…”

“…To the best of our knowledge, this study is the first to analyze the performance of the AllType kit using the Ion S5 XL sequencer. Previous studies have used Ion Torrent technology for NGS HLA typing using in‐house or commercial reagents (One Lambda NXType NGS kit, which preceded the AllType kit) for library preparation and the Ion PGM system for sequencing. The major advantage of the AllType kit over the NXType kit is that all class I and II loci investigated can be amplified using a single PCR.…”

“…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.…”

Characterization of the novel HLA‐A*11:280 allele by next‐generation sequencing in a Chinese cord blood donor

“…HLA plays a significant role in immunity and disease susceptibility and displays extensive polymorphism . Next‐generation sequencing (NGS) has become a major technology in HLA genotyping and it provided a rapid, high‐resolution HLA genotyping solution, which has reduced the number of HLA ambiguities seen when compared with other genotyping methods . The NGS method has improved the capability and precision of HLA genotyping and now many HLA novel alleles are being identified by NGS methods.…”