Sequence-based Typing Research Articles

Development of an ARMS multiplex real-time PCR assay for the detection of HLA-B*13:01 genotype by detecting highly specific SNPs.

HLA-B*13:01 was strongly associated with Dapsone Hypersensitivity Syndrome (DHS). This study aimed to develop and validate a rapid and economical method for HLA-B*13:01 genotyping. Two tubes multiplex real-time PCR detection system comprising amplification refractory mutation system primers and TaqMan probes was established for HLA-B*13:01 genotyping. Sequence-based typing was applied to validate the accuracy of the assay. The accuracy of the assay was 100% for HLA-B*13:01 genotyping. The detection limit of the new method was 0.025 ng DNA. The positive rate of HLA-B*13:01 in the Bouyei (20%, n = 50) populations was significantly higher than that in the Uighur population (4%, n = 100), Han (4.5%, n = 200), and Tibetan (1%, n = 100) ( P < 0.05). The proposed method is rapid and reliable for HLA-B*13:01 screening in a clinical setting.

Multilocus Sequence Typing of Emerging Azole-Resistant Candida auris Isolates from Critically Ill Patients in a New York City Medical Center

Abstract Candida auris has developed into a slow-moving global pandemic since its emergence in 2009, particularly as a source of nosocomial infection amongst immunocompromised and critically-ill patients. According to CDC data, C. auris emergence has accelerated in the US during the COVID-19 pandemic and has garnered particular attention for its high level of resistance to multiple classes of antifungals. C. auris has been categorized into five genetically diverse clades, each with unique antifungal drug resistance patterns. We have observed a sharp increase in cases identified at our tertiary medical center in New York City over the past three years. In this study, we designed a set of C. auris-specific oligonucleotides to determine the clonality of isolates through Multilocus Sequence Typing (MLST) of four genetic loci known to have a high level of divergence amongst Candida species. From 2020 through 2022 we identified 22 patients with C. auris bloodstream infection at our institution by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and performed MLST on isolates that were available from 19 patients. We assessed four genetic loci: ITS1/ITS2, D1/D2, RPB1 and RPB2; ERG11 was included to assess for mutations associated with fluconazole resistance. We found that the number of blood stream infections increased from 2 cases in 2020 to 12 in 2022. Our MLST results showed that all C. auris isolates from blood and body fluids belong to CDC-defined Clade 1c, which carries the ERG11 gene mutation resulting the amino acid substitution K143R in 14-α-Demethylase associated with fluconazole resistance. These isolates also demonstrated fluconazole resistance by in vitro testing (19/19), amphotericin resistance was present in 44% of isolates with available data (7/16), and all isolates were susceptible to micafungin (19/19). Of 19 patients with blood cultures positive for C. auris, 13 (68%) were in a critical care setting at time of collection, 17 (89%) had been in an ICU during the same admission, and 8 (42%) expired within 3 months of C. auris positivity. MLST is considered a relatively fast and inexpensive method of sequence-based strain typing and has recently been applied to assessing the clonality of C. auris isolates. We show that the use of C. auris-specific oligonucleotides for sequencing, rather than those derived from other Candida species, produces high quality data for MLST, as well as the detection of specific mutations involved in antifungal resistance, such as ERG11. Further assessment is needed by the gold standard in sequence typing, Whole Genome Sequencing (WGS), to confirm our analysis of clonality here. MLST may therefore represent a useful tool in understanding the interrelatedness of invasive C. auris infections in hospitalized patients as well as inform clinical decision-making around infection control and empiric antifungal therapy.

Allele-specific long-range sequencing as a method for ABO haplotyping in clinical blood group diagnosis and immunohematology research.

Safe transfusion therapy requires accurate testing of blood donors and recipients to determine their ABO blood group compatibility. Genotyping does not always clarify serological blood typing discrepancies and conventional PCR methods are not suitable to identify ABO haplotypes. Therefore, an allele-specific long-range sequencing-based typing method was established. Study samples (n = 100) and six patient samples were ABO phenotyped and screened for specific single nucleotide polymorphisms (SNP) in the ABO gene. Based on identified heterozygous SNPs in intron 1 (12897G>A), 2 (437C>T) or 4 (102C>A, 1511G>T) both ABO alleles were investigated separately using a high-fidelity long-range PCR system and Sanger sequencing. The alleles were correlated to the ABO phenotypes determined. Direct sequencing of allelic PCR products up to 6743 bases has been successful in discriminating common combinations of the ABO*A1.01, ABO*A2.01, ABO*B.01, ABO*O.01.01, ABO*O.01.02 and ABO*O.02.01 alleles. 10 out of 64 haplotypes were found to be not previously described. The uncommon ABO*AW.31.01 and the unusual O alleles ABO*O.05 and ABO*O.02.03 alleles were detected in patient samples, resolving the initial inconclusive serologic ABO typing results. This method is an effective tool for analyzing ABO haplotypes. Applicable for ABO molecular diagnostics and immunohematology research it may help to improve pre-transfusion blood type testing.

Genetic diversity of multidrug resistant Salmonella enterica subsp. enterica serovar Brancaster isolated from chicken in Malaysia

Salmonella enterica subsp. enterica serovar Brancaster is a chicken-associated serovar that is increasingly reported in the Asian region, including Malaysia. The shortfall of conventional serovar identification for nomenclature alone is insufficient for studying the genetic and clonal relationships of Salmonella of the same serovar. Whereas DNA sequence-based typing method is useful to study the genetic diversity and relatedness of the highly diverse Salmonella. In this study, the genetic diversity, phenotypic antimicrobial resistance (AMR) profiles, and detection of selected AMR genes from ten representative Salmonella Brancaster isolates from chicken cloacal swabs and raw chicken meat were carried out. Based on multilocus sequence typing (MLST), all Salmonella Brancaster belonged to sequence type ST2133. Further analysis of the MLST sequencing data using concatenated nucleotide sequences of the seven housekeeping genes divided them into nine clusters, revealed heterogeneity and genetic diversity among the isolates. All isolates were multidrug-resistant (MDR), which confers resistance to three or more classes of antibiotics. Seven Salmonella Brancaster isolates exhibited phenotypic ACSSuT-type MDR profile, and two of them were also resistant towards ciprofloxacin. Salmonella Brancaster in the present study possessed at least one and up to six resistance genes, with the most prevalent being the ampicillin (blaTEM), tetracycline (tetA), chloramphenicol (floR), gentamicin (ant(3”)-Ia), streptomycin (strA), and sulfonamides (sul-2). Chickens are common reservoir for serovar Brancaster and the emergence, persistence, and dissemination of MDR Salmonella Brancaster possessed a public health risk. The authorities need to strengthen salmonellosis control management programme in poultry farms, abattoirs, and processing facilities.

Characterization of the novel HLA-DRB1*13:03:13 allele by sequencing-based typing.

HLA-DRB1*13:03:13 differs from HLA-DRB1*13:03:01:01 by one nucleotide substitution in codon 180 in exon 3.

Characterization of the novel HLA-DPA1*01:03:51 allele by sequencing-based typing.

HLA-DPA1*01:03:51 differs from HLA-DPA1*01:03:01:01 by one nucleotide substitution in codon 146 in exon 3.

Characterization of the novel HLA-DRB1*11:324 allele by sequencing-based typing.

HLA-DRB1*11:324 differs from HLA-DRB1*11:62:02 by one nucleotide substitution in codon 38 in exon 2.

Individual and population diversity of 20 representative olfactory receptor genes in pigs

Understanding the influence of genetic variations in olfactory receptor (OR) genes on the olfaction-influenced phenotypes such as behaviors, reproduction, and feeding is important in animal biology. However, our understanding of the complexity of the OR subgenome is limited. In this study, we analyzed 1120 typing results of 20 representative OR genes belonging to 13 OR families on 14 pig chromosomes from 56 individuals belonging to seven different breeds using a sequence-based OR typing method. We showed that the presence of copy number variations, conservation of locus-specific diversity, abundance of breed-specific alleles, presence of a loss-of-function allele, and low-level purifying selection in pig OR genes could be common characteristics of OR genes in mammals. The observed nucleotide sequence diversity of pig ORs was higher than that of dogs. To the best of our knowledge, this is the first report on the individual- or population-level characterization of a large number of OR family genes in livestock species.

Novel alleles in the era of next-generation sequencing-based HLA typing calls for standardization and policy.

Next-Generation Sequencing (NGS) has transformed clinical histocompatibility laboratories through its capacity to provide accurate, high-throughput, high-resolution typing of Human Leukocyte Antigen (HLA) genes, which is critical for transplant safety and success. As this technology becomes widely used for clinical genotyping, histocompatibility laboratories now have an increased capability to identify novel HLA alleles that previously would not be detected using traditional genotyping methods. Standard guidelines for the clinical verification and reporting of novelties in the era of NGS are greatly needed. Here, we describe the experience of a clinical histocompatibility laboratory's use of NGS for HLA genotyping and its management of novel alleles detected in an ethnically-diverse population of British Columbia, Canada. Over a period of 18months, 3,450 clinical samples collected for the purpose of solid organ or hematopoietic stem cell transplantation were sequenced using NGS. Overall, 29 unique novel alleles were identified at a rate of ∼1.6 per month. The majority of novelties (52%) were detected in the alpha chains of class II (HLA-DQA1 and -DPA1). Novelties were found in all 11 HLA classical genes except for HLA-DRB3, -DRB4, and -DQB1. All novelties were single nucleotide polymorphisms, where more than half led to an amino acid change, and one resulted in a premature stop codon. Missense mutations were evaluated for changes in their amino acid properties to assess the potential effect on the novel HLA protein. All novelties identified were confirmed independently at another accredited HLA laboratory using a different NGS assay and platform to ensure validity in the reporting of novelties. The novel alleles were submitted to the Immuno Polymorphism Database-Immunogenetics/HLA (IPD-IMGT/HLA) for official allele name designation and inclusion in future database releases. A nationwide survey involving all Canadian HLA laboratories confirmed the common occurrence of novel allele detection but identified a wide variability in the assessment and reporting of novelties. In summary, a considerable proportion of novel alleles were identified in routine clinical testing. We propose a framework for the standardization of policies on the clinical management of novel alleles and inclusion in proficiency testing programs in the era of NGS-based HLA genotyping.

Characterization of the novel HLA-C*06:176:02 allele by sequencing-based typing.

HLA-C*06:176:02 differs from HLA-C*06:176:01 by two nucleotide substitutions in codons 236 and 237 in exon 4.

Characterization of the novel HLA-B*40:539 allele by sequencing-based typing.

HLA-B*40:539 differs from HLA-B*40:01:01 by one nucleotide substitution in codon 46 in exon 2.

Characterization of the novel HLA-A*33:237 allele by sequencing-based typing.

A novel HLA-A*33 allele, officially designated HLA-A*33:237, was identified by next-generation sequencing.

B-253 Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing

Abstract Background High-resolution human leukocyte antigen (HLA) typing is a crucial process in transplant matching. Sequencing-based typing (SBT) has been the gold standard method used in HLA-based clinical applications. Recently, next-generation sequencing (NGS) has been adopted in clinical laboratories for HLA typing. NGS HLA typing assays cover whole or near-whole target HLA genes, allowing the detection of genetic variants outside of key exons. Multiplex amplicon-based NGS is an efficient high-resolution HLA typing method with minimal ambiguity and a high throughput. Here, we report the performance and validation of NGS for HLA typing and compare the results obtained using NGS with those of SBT to identify any discrepancies between the 2 methods. Methods A total of 237 samples from 2019 to July 2022 were collected from Chang Gung Memorial Hospital, including 42 samples previously subjected to SBT sequencing for 4-digit HLA analysis. NGS was performed for 5 whole genes (HLA-A/B/C/DQA1/DPA1) and 3 near-whole genes (HLA-DRB1/DQB1/DPB1; excluding exon 1 and part of intron 1) by using the AllType NGS kit (One Lambda), Illumina MiSeq platform, and Type Steam Visual NGS analysis software (One Lambda). Results Our validation process comprised 34 NGS runs and 237 samples. In total, 42 samples were analyzed with an overall accuracy and precision of 100% and 100% reproducibility. The overall ambiguity rate was 0% in HLA-A/B/C compared with 56.9% for SBT. Two discrepant alleles of HLA-A and DRB1 were noted in NGS and Sanger sequencing. In accordance with additional SBT exon analyses, one of the results was corrected according to the NGS results. Conclusion NGS HLA typing is a high-throughput approach with high accuracy and precision and a low ambiguity and is promising for use in clinical HLA laboratories.

Characterization of the novel HLA-B*13:181 allele by sequencing-based typing.

HLA-B*13:181 differs from HLA-B*13:02:01:01 by one nucleotide substitution in codon 72 in exon 2.

The novel HLA-A*02:405 allele characterized by sequencing-based typing.

HLA-A*02:405 differs from HLA-A*02:06:01:01 by one nucleotide substitution in codon 161 in exon 3.

Characterization of the novel HLA-B*15:269 allele by sequencing-based typing.

HLA-B*15:269 differs from HLA-B*15:63 by two nucleotide substitutions in exon 3.

The novel HLA-A*26:236 allele identified by sequence-based typing.

The new allele A*26:236 differs from A*26:01:01:01 at position 340 (G>T) of exon 2.

Genomic full-length sequence of the HLA-B*13:64 allele was identified in a Chinese bone marrow donor.

Genomic full-length sequence of HLA-B*13:64 was identified in a Chinese individual by sequence-based typing.

Characterization of the novel HLA-DRB3*02:192 allele by sequencing-based typing.

HLA-DRB3*02:192 differs from DRB3*02:02:01:02 by one nucleotide substitution in codon 204 in exon 4.

Characterization of the novel HLA-DRB3*02:193 allele by sequencing-based typing.

HLA-DRB3*02:193 differs from DRB3*02:02:01:11 by one nucleotide substitution in exon 1, and intronic changes.