HLA genotyping by next-generation sequencing is now widely performed. We aimed at evaluating the performance of the One Lambda AllType kit using Thermo Fisher Scientific reagents on the Ion S5 XL platform. Reads were analyzed using the TypeStream Visual software. We performed 15 runs between April and September 2018 to type DNA at the HLA-A/B/C/DRB1/3/4/5/DQA1/DQB1/DPA1/DPB1 loci from 340 samples and 15 positive controls. We observed only seven (0.1%) critical mistakes among the 6009 alleles typed, corresponding to two allele dropouts, one false heterozygous typing assignment, and four phasing abnormalities. Among the 1793 presumably new alleles detected by the analysis software, 11 displayed exon mismatches, of which nine were confirmed as new alleles and two had been described previously. Intron mismatches were observed among the remaining presumably new alleles, of which 371 were considered as probably new, and 1411 were rejected for at least one sequence feature such as homopolymers (n = 1206), nucleotide doublet repeats (n = 26), low read depth ( 20%, n = 79), or phasing abnormalities (n = 7). A comparison of the AllType results with those obtained using other methods at the second-field resolution level showed 99.5% (1497/1504) concordance for the HLA-A/B/C/DRB1/DQB1/DPB1 loci. Similar agreement was observed between the HLA-C or HLA-DRB3/4/5 results and common linkage disequilibrium, with 96.6% (657/680) and 97.2% (530/545) concordance, respectively. Therefore, the AllType kit used with the Ion S5 XL platform displayed satisfactory performance for HLA typing in current clinical practice.

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

Recently, HLA epitopes on donor HLA molecules have been shown to be important in the success of solid organ transplantation. However, these epitopes can only be defined using high-resolution typing results of which are often not available prior to deceased donor allocation. The ability to perform high-resolution typing at all HLA loci for deceased organ donor allocation prior to transplantation would have major clinical benefits, in particular for highly sensitised recipients. We, therefore, developed a rapid high-resolution next generation sequencing (NGS) HLA typing (ONT-Rapid HR HLA) method for on-call deceased donor allocation using the AllType 11 loci single-tube assay (OneLambda Inc), modified in-house to reduce polymerase chain reaction amplification time, and the Oxford Nanopore single-molecule sequencing platform on the Flongle flow cell. The ONT-Rapid HR HLA method was validated on 42 samples previously typed by current on-call sequence-specific oligonucleotide (HistoSpot) and NGS methods (AllType/Ion Torrent). High-resolution typing obtained using the ONT-Rapid HR HLA typing method was 100% concordant with both the current SSO and NGS methods, and in some cases, obtained higher resolution than either of the current methods. The rapid ONT-Rapid HR HLA typing method was able to obtain these typing results at all loci in 4 to 4.5 hours. The novel ONT-Rapid HR HLA typing method is the first reported NGS HLA typing method utilised for deceased donor allocation. The ability to provide high-resolution HLA typing on deceased donors before implantation will in the future allow improvements in matching, which will ultimately provide clinical benefits to patients.

Rapid high-resolution HLA genotyping by MinION Oxford nanopore sequencing for deceased donor organ allocation.

New challenges, new opportunities: Next generation sequencing and its place in the advancement of HLA typing.

SARS-CoV-2 is spreading worldwide with continuously evolving variants, some of which occur in the Spike protein and appear to increase viral transmissibility. However, variants that cause severe COVID-19 or lead to other breakthroughs have not been well characterized. To discover such viral variants, we assembled a cohort of 683 COVID-19 patients; 388 inpatients ("cases") and 295 outpatients ("controls") from April to August 2020 using electronically captured COVID test request forms and sequenced their viral genomes. To improve the analytical power, we accessed 7137 viral sequences in Washington State to filter out viral single nucleotide variants (SNVs) that did not have significant expansions over the collection period. Applying this filter led to the identification of 53 SNVs that were statistically significant, of which 13 SNVs each had 3 or more variant copies in the discovery cohort. Correlating these selected SNVs with case/control status, eight SNVs were found to significantly associate with inpatient status (q-values < 0.01). Using temporal synchrony, we identified a four SNV-haplotype (t19839-g28881-g28882-g28883) that was significantly associated with case/control status (Fisher's exact p = 2.84 × 10-11). This haplotype appeared in April 2020, peaked in June, and persisted into January 2021. The association was replicated (OR = 5.46, p-value = 4.71 × 10-12) in an independent cohort of 964 COVID-19 patients (June 1, 2020 to March 31, 2021). The haplotype included a synonymous change N73N in endoRNase, and three non-synonymous changes coding residues R203K, R203S and G204R in the nucleocapsid protein. This discovery points to the potential functional role of the nucleocapsid protein in triggering "cytokine storms" and severe COVID-19 that led to hospitalization. The study further emphasizes a need for tracking and analyzing viral sequences in correlations with clinical status. 

/pdf/mutations-in-viral-nucleocapsid-protein-and-endornase-are-2fkdu96z.pdf

Mutations in viral nucleocapsid protein and endoRNase are discovered to associate with COVID19 hospitalization risk

This review highlights literature pertinent to chimerism analysis in the context of hematopoietic cell transplantation (HCT). We also conducted a survey of testing practices of program members of CIBMTR worldwide. Questions included testing methods, time points, specimen type, cell lineage tested and testing indications. Recent literature suggests that detection of low level mixed chimerism has a clinical utility in predicting relapse. There is also increasing recognition of HLA loss relapse to potentially guide rescue decisions in cases of relapse. These developments coincide with wider access to high sensitivity next generation sequencing (NGS) in clinical laboratories. Our survey revealed a heterogeneity in practices as well as in findings and conclusions of published studies. Although the most commonly used method is STR, studies support more sensitive methods such as NGS, especially for predicting relapse. There is no conclusive evidence to support testing chimerism in BM over PB, particularly when using a high sensitivity testing method. Periodic monitoring of chimerism especially in diagnoses with a high risk of relapse is advantageous. Lineage specific chimerism is more sensitive than whole blood in predicting impending relapse. Further studies that critically assess how to utilize chimerism testing results will inform evidence based clinical management decisions. 

Chimerism analysis for clinicians: a review of the literature and worldwide practices

Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the "gold standard." However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/tan.13619

Comparison of sequence-specific oligonucleotide probe vs next generation sequencing for HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single-pass high-resolution HLA typing in support of solid organ and hematopoietic cell transplant programs.

Crystal Y. Usenko

Papers

Comparison of sequence-specific oligonucleotide probe vs next generation sequencing for HLA-A, B, C, DRB1, DRB3/B4/B5, DQA1, DQB1, DPA1, and DPB1 typing: Toward single-pass high-resolution HLA typing in support of solid organ and hematopoietic cell transplant programs.