ACKR1 Alleles at 5.6 kb in a Well-Characterized Renewable US Food and Drug Administration (FDA) Reference Panel for Standardization of Blood Group Genotyping.
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ABSTRACT:
SUBMITTER: Srivastava K
PROVIDER: S-EPMC7538814 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic because of a disease state or recent transfusions. In addition, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well-characterized renewable reference panel for standardization of blood group genotyping was developed. The panel consists of genomic DNA lyophilized and stored in glass vials. Genomic DNA was extracted in bulk from immortalized lymphoblastoid cell lines, generated by Epstein-Barr virus transformation of peripheral blood lymphocytes harvested from volunteer blood donors. The panel was validated by an international collaborative study involving 28 laboratories that tested each DNA panel member for 41 polymorphisms associated with 17 blood group systems. Overall, analysis of genotyping results showed >98% agreement with the expected outcomes, demonstrating suitability of the material for use as reference. Highest levels of discordance were observed for the genes CR1, CD55, BSG, and RHD. Although limited, observed inconsistencies and procedural limitations reinforce the importance of reference reagents to standardize and harmonize results. Results of stability and accelerated degradation studies support the suitability of this panel for use as reference reagent for blood group genotyping assay development and standardization.
| S-EPMC7648275 | biostudies-literature
Project description:BACKGROUND:In all Streptococcus mutans strains, 5-13% carry a 5.6-kb plasmid. Despite its frequency, little is known about its mediated functions with most of the information coming from a single study focussing on plasmid pUA140. OBJECTIVE:Here, we describe the sequence and genetic organization of two S. mutans 5.6-kb plasmids, pDC09 and pNC101. RESULTS:Based on PicoGreen dsDNA quantification and Real-Time quantitative PCR (RTQ-PCR), the plasmid copy number was found to range between 10 and 74, depending on the strain tested. In contrast to literature, we identified six instead of five open reading frames (ORFs). While the putative gene products of ORF1 (as a Rep-protein) and ORF2 (as a Mob-protein) could be confirmed as being identical to those from pUA140, the functions of ORF3 (unknown) and ORF 4 (possibly AtpE homologue) could not be further revealed. However, the product of ORF5 showed a fairly high identity (38-50%) and structural similarity (58-74%) to RelE of Streptococcus pneumoniae, Streptococcus equi, and Streptococcus downei. In addition, we identified a functionally corresponding ORF6 encoding a protein with 61-68% identity (81-86% similarity) to the S. equi and S. downei antitoxin of the RelB family. RelE and RelB together form a plasmid-encoded toxin-antitoxin (TA) system, RelBE(plas). Despite its rather limited sequence similarity with chromosomal TA systems in S. mutans (RelBE(chro), MazEF, HicBA), we found similar tertiary structures applying I-Tasser protein prediction analysis. CONCLUSION:Type II-toxins, as the plasmid-encoded RelE, are RNA endonucleases. Depending on their mRNA cleavage activity, they might 1) kill every plasmid-free progeny, thereby stabilizing plasmid transfer at the expense of the host and/or 2) help S. mutans enter a dormant state and survive unfavourable environmental conditions. Whilst a function in plasmid stabilization has been confirmed, a function in persistence under nutritional stress, tested here by inducing amino acid starvation, could not be demonstrated so far.
| S-EPMC3547324 | biostudies-literature
Project description:BackgroundClinically effective and safe genotyping relies on correct reference sequences, often represented by haplotypes. The 1000 Genomes Project recorded individual genotypes across 26 different populations and, using computerized genotype phasing, reported haplotype data. In contrast, we identified long reference sequences by analyzing the homozygous genomic regions in this online database, a concept that has rarely been reported since next generation sequencing data became available.Study design and methodsPhased genotype data for a 80.6 kb region of chromosome 1 was downloaded for all 2,504 unrelated individuals of the 1000 Genome Project Phase 3 cohort. The data was centered on the ACKR1 gene and bordered by the CADM3 and FCER1A genes. Individuals with heterozygosity at a single site or with complete homozygosity allowed unambiguous assignment of an ACKR1 haplotype. A computer algorithm was developed for extracting these haplotypes from the 1000 Genome Project in an automated fashion. A manual analysis validated the data extracted by the algorithm.ResultsWe confirmed 902 ACKR1 haplotypes of varying lengths, the longest at 80,584 nucleotides and shortest at 1,901 nucleotides. The combined length of haplotype sequences comprised 19,895,388 nucleotides with a median of 16,014 nucleotides. Based on our approach, all haplotypes can be considered experimentally confirmed and not affected by the known errors of computerized genotype phasing.ConclusionsTracts of homozygosity can provide definitive reference sequences for any gene. They are particularly useful when observed in unrelated individuals of large scale sequence databases. As a proof of principle, we explored the 1000 Genomes Project database for ACKR1 gene data and mined long haplotypes. These haplotypes are useful for high throughput analysis with next generation sequencing. Our approach is scalable, using automated bioinformatics tools, and can be applied to any gene.
| S-EPMC8150616 | biostudies-literature
Project description:Background:In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods:After long-range PCR amplification spanning the whole ACKR1 gene locus (?2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results:High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion:Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a "longitudinal" overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.
| S-EPMC7036534 | biostudies-literature
Project description:BackgroundVariants in numerous genes are thought to affect the success or failure of cancer chemotherapy. Interindividual variability can result from genes involved in drug metabolism and transport, drug targets (receptors, enzymes, etc), and proteins relevant to cell survival (e.g., cell cycle, DNA repair, and apoptosis). The purpose of the current study is to establish a flexible, cost-effective, high-throughput genotyping platform for candidate genes involved in chemoresistance and -sensitivity, and treatment outcomes.MethodsWe have adopted SNPlex for genotyping 432 single nucleotide polymorphisms (SNPs) in 160 candidate genes implicated in response to anticancer chemotherapy.ResultsThe genotyping panels were applied to 39 patients with chronic lymphocytic leukemia undergoing flavopiridol chemotherapy, and 90 patients with colorectal cancer. 408 SNPs (94%) produced successful genotyping results. Additional genotyping methods were established for polymorphisms undetectable by SNPlex, including multiplexed SNaPshot for CYP2D6 SNPs, and PCR amplification with fluorescently labeled primers for the UGT1A1 promoter (TA)nTAA repeat polymorphism.ConclusionThis genotyping panel is useful for supporting clinical anticancer drug trials to identify polymorphisms that contribute to interindividual variability in drug response. Availability of population genetic data across multiple studies has the potential to yield genetic biomarkers for optimizing anticancer therapy.
| S-EPMC2442111 | biostudies-literature
Project description:We discuss a decision-theoretic approach to building a panel-based, preemptive genotyping program. The method is based on findings that a large percentage of patients are prescribed medications that are known to have pharmacogenetic associations, and over time, a substantial proportion are prescribed additional such medication. Preemptive genotyping facilitates genotype-guided therapy at the time medications are prescribed; panel-based testing allows providers to reuse previously collected genetic data when a new indication arises. Because it is cost-prohibitive to conduct panel-based genotyping on all patients, we describe a three-step approach to identify patients with the highest anticipated benefit. First, we construct prediction models to estimate the risk of being prescribed one of the target medications using readily available clinical data. Second, we use literature-based estimates of adverse event rates, variant allele frequencies, secular death rates, and costs to construct a discrete event simulation that estimates the expected benefit of having an individual's genetic data in the electronic health record after an indication has occurred. Finally, we combine medication prescription risk with expected benefit of genotyping once a medication is indicated to calculate the expected benefit of preemptive genotyping. For each patient-clinic visit, we calculate this expected benefit across a range of medications and select patients with the highest expected benefit overall. We build a proof of concept implementation using a cohort of patients from a single academic medical center observed from July 2010 through December 2012. We then apply the results of our modeling strategy to show the extent to which we can improve clinical and economic outcomes in a cohort observed from January 2013 through December 2015.
| S-EPMC6699004 | biostudies-literature
Project description:We present a new efficient screening tool for detection of S-alleles in apple. The protocol using general and multiplexed primers for PCR reaction and fragment detection on an automatized capillary DNA sequencer exposed a higher number of alleles than any previous studies. Analysis of alleles is made on basis of three individual fragment sizes making the allele interpretation highly accurate. The method was employed to genotype 432 Malus accessions and exposed 25 different S-alleles in a selection of Malus domestica cultivars of mainly Danish origin (402 accessions) as well as a selection of other Malus species (30 accessions). The allele S3 (28 %) was the most common among the Danish cultivars followed by S1 and S7 (both 27 %). The alleles S36 and S40 not previously reported from M. domestica were found in 6 and 17 cultivars, respectively. Complete allelic composition was found in 91 % of the 369 diploid accessions and in 86 % of the 63 triploids concerned. We further identified a relatively high frequency of S33 and S34, which has not been considered by most previous studies. The protocol presented here is easy to adopt and saves both time and work effort compared to previous methods. The robustness is illustrated by the great accuracy and a high number of S-alleles presented.
| S-EPMC4760992 | biostudies-literature
Project description:Powerful and simple, RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish, and can be used to target multiple genes simultaneously. One of the challenges associated with the large scale application of this technique to zebrafish is the lack of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing - a robust and cost-effective method to genotype multiple targets in a single reaction. Our approach can identify indels in up to four targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis-based sequencing equipment.
| S-EPMC7035419 | biostudies-literature
Project description: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 18 months, 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.
| S-EPMC10611506 | biostudies-literature
Project description:BackgroundSequence information generated from next generation sequencing is often computationally phased using haplotype-phasing algorithms. Utilizing experimentally derived allele or haplotype information improves this prediction, as routinely used in HLA typing. We recently established a large dataset of long ERMAP alleles, which code for protein variants in the Scianna blood group system. We propose the phylogeny of this set of 48 alleles and identify evolutionary steps to derive the observed alleles.MethodsThe nucleotide sequence of > 21 kb each was used for all physically confirmed 48 ERMAP alleles that we previously published. Full-length sequences were aligned and variant sites were extracted manually. The Bayesian coalescent algorithm implemented in BEAST v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny.ResultsThe phylogenetic analysis allowed us to identify the evolutionary relationships among the 48 ERMAP alleles, predict 4243 potential ancestral alleles and calculate a posterior probability for each of these unobserved alleles. Some of them coincide with observed alleles that are extant in the population.ConclusionsOur proposed strategy places known alleles in a phylogenetic framework, allowing us to describe as-yet-undiscovered alleles. In this new approach, which relies heavily on the accuracy of the alleles used for the phylogenetic analysis, an expanded set of predicted alleles can be used to infer alleles when large genotype data are analyzed, as typically generated by high-throughput sequencing. The alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of next generation sequencing data.
| S-EPMC6371619 | biostudies-literature