Rapid Genotyping Research Articles

Linkage map construction using limited parental genotypic information

Genetic linkage maps based on single nucleotide polymorphisms (SNPs) represent an essential tool for a variety of genomic analyses. Today, next-generation sequencing (NGS) enables rapid genotyping of different mapping populations based on thousands of SNPs and the construction of highly saturated linkage maps. Nevertheless, missing data in the genotyping of the parental lines creates a bottleneck that determines the number of SNPs that can be used for the linkage map. As a proof of concept, a highly saturated genetic linkage map was constructed using the imputed genotypic data of a recombinant inbred line (RIL) population and the limited genotypic information of its parental lines. Two ABH genotype files were created from a pseudo-parental genotypic data set that includes all the SNPs present in the RIL population. In the first ABH file pseudo-parental 1 was considered parental A, while in the second pseudo-parental 1 was considered parental B. These two duplicate ABH genotype files were merged by chromosome and subjected to linkage map analysis. Since the ABH data were duplicated, two mirrored linkage groups were generated per chromosome. The correct linkage map was identified and selected based on the partial genotypic data of the parental lines. This strategy was effective for constructing a highly saturated linkage map of 33,421 SNPs based on the genotyping of 205 RILs and a limited number of 100 SNPs present in the parental lines. This strategy enables the use of all the NGS SNP data obtained from a low-coverage sequencing experiment in the mapping population.

Integration of Nucleic Acid Amplification, Detection, and Melting Curve Analysis for Rapid Genotyping of Antimicrobial Resistance

To prevent the overuse of antibiotics and reduce the spread of antimicrobial resistance (AMR), we developed an integrated genotypic test to identify target AMR genes and mutations with minimal user operation. The integrated AMR sensor consists of a reaction chamber built on a total internal reflection (TIR)-coupled DNA microarray, a temperature management unit, and a compact fluorescence reader. By executing a programmed temperature profile, the automated system can perform asymmetric polymerase chain reactions (PCR) to amplify multiple target genes, microarray hybridizations to detect the amplicons, and melting curve analysis (MCA) to identify resistance mutations. Eight AMR genes selected from <i>Acinetobacter baumannii</i>, <i>Klebsiella pneumonia</i>, <i>Escherichia coli</i>, <i>Campylobacter coli</i>, and <i>Campylobacter jejuni</i> were amplified using the asymmetric PCR and subsequently detected using the TIR-coupled microarray. The point mutation in the quinolone resistance-determining region of the gyrA gene of <i>Campylobacter jejuni</i> was further studied by performing MCA on the TIR-coupled microarray. The benefits of integrated and rapid assay, compact and automated instrument, and multiplexed analysis would facilitate point-of-care antimicrobial susceptibility testing for bacterial infections.

Development and Application of a High-Resolution Melting Analysis with Unlabeled Probes for the Screening of Short-Tailed Sheep TBXT Heterozygotes.

Simple SummaryTBXT (c.333G > C; c.334G > T) has been identified as a molecular genetic marker in short-tailed sheep. This paper describes a high-resolution melting (HRM) analysis using unlabeled probes and asymmetric PCR for the detection of genetic variants of TBXT in short-tailed sheep populations. The detection results of this method are consistent with those of Sanger sequencing and can help farmers with marker-assisted breeding.The short-tailed phenotype has long been considered one of the best traits for population genetic improvement in sheep breeding. In short-tailed sheep, not only is tail fat eliminated but also the pubic area is exposed due to the lack of a tail covering, giving them an advantage in reproduction. Recent studies have shown that two linked mutations in sheep TBXT at nucleotides 333 and 334 are associated with the short-tailed phenotype. In the population of short-tailed sheep, several heterozygous mutants of this gene are found. In our research, we used high-resolution melting (HRM) to identify homozygous and heterozygous genotypes in a flock of short-tailed sheep and compared the results with those of Sanger sequencing, which were identical. This demonstrates that our established HRM method, a rapid and inexpensive genotyping method, can be used to identify homozygous and heterozygous individuals in short-tailed sheep flocks.

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein.

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

Diversity of SAT2 foot-and-mouth disease virus in Sudan: implication for diagnosis and control.

Like other East African countries, Sudan experienced circulation of more than one topotype of SAT2 foot-and-mouth disease virus (FMDV). In Sudan, topotype XIII of SAT2 virus was recorded in 1977 and 2008 and topotype VII in 2007, 2010, 2013, 2014 and 2017. This work evaluated the impact of such diversity on diagnosis and control. After one or three doses of a vaccine derived from a Sudanese SAT2 virus of topotype VII originated in 2010, heterologous neutralizing antibody titres with Sudanese SAT2 viruses in 2008 were ≤ 1.2 log 10, not consistent with likely protection. Simultaneously, homologous titres were 1.65 (after one dose) or 1.95 and 2.55 log10 (after 3 doses). When r1 values between the vaccine virus and the SAT2 viruses isolated in 2008, whilst topotype XIII was circulating, were derived, values (≈ 0.00) suggested similarly poor antigenic relationship and unlikely cross protection. Concurrently, SAT2 positive field sera from Sudan in 2016 were not unvaryingly identified by virus neutralization tests (VNT) employing SAT2 viruses from 2010 and 2008. Proportions of positive sera by SAT2 virus from 2010 were always higher than those by viruses from 2008; consistent with the more frequent and recent circulation of topotype VII prior to 2016. Proportions by SAT2 virus from 2010 were 0.68 (± 0.1) in one location (n = 72), 0.39 (± 0.1) in another one (n = 94) and 0.52 (± 0.1) in the whole test group (n = 166). Corresponding values by viruses of 2008 were 0.53 (± 0.1), 0.27 (± 0.1) and 0.38 (± 0.1). In the whole test group, differences were statistically significant (p = .02339). Like post-vaccination sera, field sera (natural immunity) showed no considerable cross neutralization between topotype VII and presumably XIII; almost 45% (43/96) of SAT2 positive field sera were positive to one topotype but not to the other. Experimental and surveillance findings emphasized the implication of SAT2 diversity in Sudan. It is concluded that it is difficult to control SAT2 infection in Sudan using a monovalent vaccine. Beside a prophylactic vaccine from topotype VII, stockpiling of antigens from topotype XIII and enhanced virological surveillance with rapid genotyping and matching studies are necessary approaches. When more frequent circulation of more than one SAT2 topotype occurs, retrospective diagnosis by serological surveys could be problematic or imprecise.

Dissection of the practical soybean breeding pipeline by developing ZDX1, a high-throughput functional array

Key messageWe developed the ZDX1 high-throughput functional soybean array for high accuracy evaluation and selection of both parents and progeny, which can greatly accelerate soybean breeding.Microarray technology facilitates rapid, accurate, and economical genotyping. Here, using resequencing data from 2214 representative soybean accessions, we developed the high-throughput functional array ZDX1, containing 158,959 SNPs, covering 90.92% of soybean genes and sites related to important traits. By application of the array, a total of 817 accessions were genotyped, including three subpopulations of candidate parental lines, parental lines and their progeny from practical breeding. The fixed SNPs were identified in progeny, indicating artificial selection during the breeding process. By identifying functional sites of target traits, novel soybean cyst nematode-resistant progeny and maturity-related novel sources were identified by allele combinations, demonstrating that functional sites provide an efficient method for the rapid screening of desirable traits or gene sources. Notably, we found that the breeding index (BI) was a good indicator for progeny selection. Superior progeny were derived from the combination of distantly related parents, with at least one parent having a higher BI. Furthermore, new combinations based on good performance were proposed for further breeding after excluding redundant and closely related parents. Genomic best linear unbiased prediction (GBLUP) analysis was the best analysis method and achieved the highest accuracy in predicting four traits when comparing SNPs in genic regions rather than whole genomic or intergenic SNPs. The prediction accuracy was improved by 32.1% by using progeny to expand the training population. Collectively, a versatile assay demonstrated that the functional ZDX1 array provided efficient information for the design and optimization of a breeding pipeline for accelerated soybean breeding.

Visualized Genotyping from "Sample to Results" Within 25 Minutes by Coupling Recombinase Polymerase Amplification (RPA) With Allele-Specific Invasive Reaction Assisted Gold Nanoparticle Probes Assembling.

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from "samples to results" within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.

High-Resolution Melting PCR as Rapid Genotyping Tool for Brucella Species.

Brucella sp. are the causative agents of brucellosis. One of the main characteristics of the Brucella genus concerns its very high genetic homogeneity. To date, classical bacteriology typing is still considered as the gold standard assay for direct diagnosis of Brucella. Molecular approaches are routinely used for the identification of Brucella at the genus level. However, genotyping is more complex, and to date, no method exists to quickly assign a strain into species and biovar levels, and new approaches are required. Next generation sequencing (NGS) opened a new era into the diagnosis of bacterial diseases. In this study, we designed a high-resolution melting (HRM) method for the rapid screening of DNA and direct assignment into one of the 12 species of the Brucella genus. This method is based on 17 relevant single nucleotide polymorphisms (SNPs), identified and selected from a whole genome SNP (wgSNP) analysis based on 988 genomes (complete and drafts). These markers were tested against the collection of the European Reference Laboratory (EU-RL) for brucellosis (1440 DNAs extracted from Brucella strains). The results confirmed the reliability of the panel of 17 SNP markers, allowing the differentiation of each species of Brucella together with biovars 1, 2, and 3 of B. suis and vaccine strain Rev1 (B. melitensis) within 3 h, which is a considerable gain of time for brucellosis diagnosis. Therefore, this genotyping tool provides a new and quick alternative for Brucella identification based on SNPs with the HRM-PCR assay.

A new insight into the SNP genotyping using high-resolution melting method after the correlation analysis of the SNPs with WSSV-resistant traits

Procambarus clarkii is an important freshwater cultured crayfish in China. With the gradual development of its aquaculture industry, research on white spot disease, which is harmful to healthy culture of P. clarkii, increases gradually. The prophenoloxidase (proPO) system is an important part of crayfish's innate immunity and plays a role in virus resistance. In this study, based on the early discovery of three SNP sites in the intron of proPO gene, the linkage disequilibrium and haplotype were analyzed for the SNPs, and it was found that there was a strong linkage disequilibrium relationship among them. Through the analysis on association between the haplotypes and genotype of each SNP site with the WSSV-resistant traits, the detection of the SNP_7081 genotype was considered as the most convenient and efficient way for WSSV-resistant group selection. Furtherly, the high-resolution melting curve (HRM), which is a rapid and economic genotyping method, was chosen to establish for SNP_7081 site genotyping. The 68 bp target fragment with 27.94% GC content was amplified and melting curve analysis were performed. However, the appearance of false negatives which led to unable automatically grouped although the melting curves of genotypes CC, C>T and T>C were obviously different, and could be treated as standard to manually genotype the samples with an accuracy rate of 97.61%. The low GC content which correlated with the Tm value, was confirmed as the reason for the false negatives by the assay about the recombinant plasmid PMD18-T-SNP_7081 constructed with 45.24% GC content. Eventually, the adaptor primers were used to increase the GC content of the target fragment, and a modified HRM method for genotyping SNP_7081 site that could group automatically was established, which could provide a new insight for the HRM method to genotype SNPs.

Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti.

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

High-Throughput Adaptable SARS-CoV-2 Screening for Rapid Identification of Dominant and Emerging Regional Variants.

ObjectivesEmerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant strains can be associated with increased transmissibility, more severe disease, and reduced effectiveness of treatments. To improve the availability of regional variant surveillance, we describe a variant genotyping system that is rapid, accurate, adaptable, and able to detect new low-level variants built with existing hospital infrastructure.MethodsWe used a tiered high-throughput SARS-CoV-2 screening program to characterize variants in a supraregional health system over 76 days. Combining targeted reverse transcription–polymerase chain reaction (RT-PCR) and selective sequencing, we screened SARS-CoV-2 reactive samples from all hospitals within our health care system for genotyping dominant and emerging variants.ResultsThe median turnaround for genotyping was 2 days using the high-throughput RT-PCR–based screen, allowing us to rapidly characterize the emerging Delta variant. In our population, the Delta variant is associated with a lower cycle threshold value, lower age at infection, and increased vaccine-breakthrough cases. Detection of low-level and potentially emerging variants highlights the utility of a tiered approach.ConclusionsThese findings underscore the need for fast, low-cost, high-throughput monitoring of regional viral sequences as the pandemic unfolds and the emergence of SARS-CoV-2 variants increases. Combining RT-PCR–based screening with selective sequencing allows for rapid genotyping of variants and dynamic system improvement.

Rapid Genotyping of 32 Insertion/Deletion Panel for Human Identification Using Fluorogenic Probes-Based Multiplex Real-Time PCR

Rapid Genotyping of 32 Insertion/Deletion Panel for Human Identification Using Fluorogenic Probes-Based Multiplex Real-Time PCR

Development of a microwave-based extraction for forensic biological samples

In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples.

Rapid Genotyping of Sars-Cov-2 Variants with a Duplex-Targeting Lateral Flow Strip by Crispr-Cas 12a/13a in Pam Independent Manner

Rapid Genotyping of Sars-Cov-2 Variants with a Duplex-Targeting Lateral Flow Strip by Crispr-Cas 12a/13a in Pam Independent Manner

Ultra-rapid real-time polymerase chain reaction assay for genotyping of the NT5E gene in Japanese black beef using a mobile PCR device, PCR 1100.

The concentration of inosine 5'-monophosphate (IMP) in beef is an important factor contributing to beef palatability. A previous study suggested that single nucleotide polymorphisms (SNPs) in the ecto-5'-nucleotidase (NT5E) gene strongly affect the concentration of IMP under postmortem conditions by regulating NT5E enzymatic activity in beef. Genotyping of the NT5E gene is performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or real-time PCR assay. However, these conventional laboratory assays require large installed instruments. They also involve complicated procedures and are time-consuming. Here, the PCR primers and probes for the NT5E gene (rs42508588 SNP) were designed and synthesized, and we examined the rapid genotyping of the NT5E gene using a PicoGene PCR 1100 mobile PCR device. The results showed that this system enabled rapid amplification of each allele at approximately 19.4s per cycle, with a total run time of 13 min 36 s. This device is portable and does not require a power supply, which facilitates its use not only in specific laboratories but also in meat production farms and distribution stages of beef.

Rapid Molecular Determination of Sex for Western North American Chub (Gila spp.)

AbstractGathering sex data is frequently a complicated matter for fish species that lack sexually dimorphic traits. Secondary sexual characteristics are cryptic or nonexistent in many minnows (Cyprinidae) during early life stages or outside of the breeding season, which may require lethal methods for determining sex. This complicates many conservation actions and population genetic studies, including (1) the development of hatchery broodstocks for threatened and endangered species, (2) sex ratio determination in wild populations, (3) the influence of sex ratio on the effective population size estimates, and (4) limitations on the implementation of genetic tagging and parentage assignment in wild populations. Here, we remedy this issue for four western North American chub species (Gila spp.) through restriction‐site‐associated DNA sequencing of Bonytail G. elegans, which identified a single locus indicating an XY sex determination system and no candidate loci consistent with a WZ system. This locus cross‐amplified in three additional Gila species (Chihuahua Chub G. nigrescens, Virgin Chub G. seminuda, and White River Chub G. jordani), although species‐specific differences necessitated the development of three novel primer/probe sets. Real‐time PCR with hydrolysis probe assays was used to genotype sex‐linked single‐nucleotide polymorphisms for each species. The measurement of endpoint fluorescence in a real‐time PCR thermal cycler allowed for rapid and accurate sex genotyping of these species, with accuracy &gt;97% relative to phenotypic sex identification when three PCR replicates were employed. These assays provided high precision, with unanimous results across &gt;87% of the replicates and a majority consensus genotype for 98.6% of the samples. The locus identified herein was conserved across all of the Gila species that we tested (n = 6) and other western North American cyprinids; however, sex‐linked polymorphisms were found only in the four species that are named above. Consequently, this locus has promise for the identification and development of sex‐linked assays for other Gila and closely related cyprinids.

Programmable endonuclease combined with isothermal polymerase amplification to selectively enrich for rare mutant allele fractions

Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells. Recently, programmable endonucleases such as clustered regularly interspaced short palindromic repeats (CRISPR) associated protein (Cas) and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions, enabling their detection with deep next generation sequencing (NGS). However, the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage. To overcome these shortcomings, we conceived a new assay (Programmable Enzyme-Assisted Selective Exponential Amplification, PASEA) that combines the cleavage of wild type alleles with concurrent polymerase amplification. While PASEA increases the numbers of both wild type and mutant alleles, the numbers of mutant alleles increase at much greater rates, allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles. By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification, we converted samples with 0.01% somatic mutant allele fractions (MAFs) to products with 70% MAFs in a single step within 20 min, enabling inexpensive, rapid genotyping with such as Sanger sequencers. Furthermore, PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes. Real-time PASEA’ performance was proved equivalent to clinical amplification refractory mutation system (ARMS)-PCR and NGS when testing over hundred cancer patients’ samples. This strategy has the potential to reduce the cost and time of cancer screening and genotyping, and to enable targeted therapies in resource-limited settings.

Evaluation of a Rapid Automated Next Generation Sequencing Assay for Precision Medicine in Acute Myeloid Leukemia

Background:In the last decade there have been significant advances in diagnosing and classifying adult acute myeloid leukemia (AML) based on genomic profiling, enabling risk-stratification and targeted therapies. In 2017 the US FDA approved the first gene mutation targeted therapies for AML with multiple additional targeted therapies since approved or in development. Given the typical acuity of AML at initial presentation however and the current turnaround time for next-generation sequencing (NGS) assays, most patients will start definitive initial therapy before all potentially targetable mutations are known. There is, therefore, a significant need for a fast molecular genotyping test to determine eligibility for personalized therapy in AML.The NCI Myeloid Assay (NMA) is a comprehensive targeted NGS assay on the Ion Torrent Genexus System, a fully automated platform that provides a rapid turnaround time from specimen receipt to clinical reporting. NMA utilizes Thermo Fisher Scientific's Oncomine Myeloid Assay GX and appears ideally suited for use in upcoming AML targeted therapy trials but has yet to be extensively tested in a cohort of AML patient diagnostic samples and compared to a standard targeted “myeloid panel” NGS assay platform (s-NGS).Methods:DNA samples (n=173) extracted from pretreatment bone marrow and/or peripheral blood of adult patients (n=112) diagnosed with de novo AML or high-risk myelodysplastic syndrome (MDS), were blindly tested in parallel using the NMA and s-NGS assays. For the NMA assay, 27.75ng of DNA was put into the Genexus System. All runs, controls, and samples were first analyzed for sequencing quality using established quality control (QC) metrics to assess pass/fail status. For all samples that passed QC metrics, variant results generated by the Ion Torrent Genexus pipeline were manually reviewed prior to being called true positive variants. For the s-NGS, using the ArcherDx Myeloid VariantPlex assay, a DNA input of 50ng was used for library preparation on a dual pre- and post-PCR separated automated liquid-handling workflow. Resulting libraries were sequencing on the Novaseq 6000 (Illumina) and the data analyzed using the Archer Analysis software and filtered as previously described (PMID: 34258102). Results from the two assays were compared for mutations with a variant allele fraction (VAF) >5% occurring in genes of interest in small molecule targeted clinical trials including: FLT3, IDH1, IDH2, JAK2, KIT, NPM1, NRAS, KRAS, and TP53. For FLT3-ITD comparison, the presence or absence of a call by the assay was used.Results:Utilizing a 5% VAF reporting threshold, a total of 171 and 174 variants were detected by NMA and s-NGS assays, respectively. A high rate of concordance was observed between the assays, with NMA detecting 96% of s-NGS variants and s-NGS detecting 95% of NMA variants. The VAF of detected single nucleotide variants was highly correlated (r=0.9848, P<0.0001, Figure 1A). NPM1 mutation VAF values trended lower by s-NGS compared to NMA.We investigated the discordant calls (n=15 total in 11 patients). One patient was correctly identified as having an NRAS p.Gly12 mutation by both approaches, but the resulting mutation was incorrectly annotated by the s-NGS pipeline. Samples from two patients (including one with both blood and marrow tested) were correctly identified as being FLT3 tyrosine kinase domain mutated by both sequencing approaches, although only the major of two missense variants identified by s-NGS was reported by the NMA pipeline. None of these patients, however, would be misclassified. The remaining 11 discordant calls were false negatives (including 6 variants detected by s-NGS but not by NMA). All of these “edge case” variants were detectable by lowering the VAF reporting threshold below 5% (Figure 1B).Conclusions:NMA is an automated sample-to-results workflow that can identify myeloid disorder-associated genomic variants in less than 48 hours from library preparation to clinical reporting. We show that NMA is highly concordant with a standard DNA NGS assay for detecting mutations within recurrently mutated AML genes. Accurate rapid genotyping is required for assignment to initial treatment with targeted therapy, and this technology may be a valuable tool for upcoming clinical trials for patients with myeloid malignancies. [Display omitted] DisclosuresZhang: Thermo Fisher Scientific: Current Employment. Sedova: Thermo Fisher Scientific: Current Employment. Huang: Thermo Fisher Scientific: Current Employment. Mittal: Thermo Fisher Scientific: Current Employment. Hatch: Thermo Fisher Scientific: Current Employment. Ni: Thermo Fisher Scientific: Current Employment. Kaznadzey: Thermo Fisher Scientific: Current Employment. Sadis: Thermo Fisher Scientific: Current Employment. Smith: Thermo Fisher Scientific: Current Employment. Williams: Illumina: Other: CRADA. Hourigan: Sellas: Research Funding.

Contribution of the IdyllaTM System to Improving the Therapeutic Care of Patients with NSCLC through Early Screening of EGFR Mutations.

Epidermal growth factor receptor (EGFR) genotyping, a critical examen for the treatment decisions of patients with non-small cell lung cancer (NSCLC), is commonly assayed by next-generation sequencing (NGS), but this global approach takes time. To determine whether rapid EGFR genotyping tests by the IdyllaTM system guides earlier therapy decisions, EGFR mutations were assayed by both the IdyllaTM system and NGS in 223 patients with NSCLC in a bicentric prospective study. IdyllaTM demonstrated agreement with the NGS method in 187/194 cases (96.4%) and recovered 20 of the 26 (77%) EGFR mutations detected using NGS. Regarding the seven missed EGFR mutations, five were not detected by the IdyllaTM system, one was assayed in a sample with insufficient tumoral cells, and the last was in a sample not validated by the IdyllaTM system (a bone metastasis). IdyllaTM did not detect any false positives. The average time between EGFR genotyping results from IdyllaTM and the NGS method was 9.2 ± 2.2 working days (wd) (12.6 ± 4.0 calendar days (cd)). Subsequently, based on the IdyllaTM method, the timeframe from tumor sampling to the initiation of EGFR-TKI was 7.7 ± 1.2 wd (11.4 ± 3.1 cd), while it was 20.3 ± 6.7 wd (27.2 ± 8.3 cd) with the NGS method (p < 0.001). We thus demonstrated here that the IdyllaTM system contributes to improving the therapeutic care of patients with NSCLC by the early screening of EGFR mutations.

Development of a marker panel for genotyping of domestic soybean cultivars for genes controlling the duration of vegetation and response to photoperiod.

Soybean, Glycine max L., is one of the most important agricultural crops grown in a wide range of latitude. In this regard, in soybean breeding, it is necessary to pay attention to the set of genes that control the transition to the f lowering stage, which will make it possible to adapt genotypes to local growing conditions as accurately as possible. The possibilities of soybean breeding for this trait have now signif icantly expanded due to identif ication of the main genes (E1–E4, GmFT2a, GmFT5a) that control the processes of f lowering and maturation in soybean, depending on the day length. The aim of this work was to develop a panel of markers for these genes, which could be used for a rapid and eff icient genotyping of domestic soybean cultivars and selection of plant material based on sensitivity to photoperiod and the duration of vegetation. Combinations of 10 primers, both previously developed and our own, were tested to identify different alleles of the E1–E4, GmFT2a, and GmFT5a genes using 10 soybean cultivars from different maturity groups. As a result, 5 combinations of dominant and recessive alleles for the E1–E4 genes were identif ied: (1) e1-nl(e1-as)/ e2-ns/e3-tr(e3-fs)/e4; (2) e1-as/e2-ns/e3-tr/E4; (3) e1-as/e2-ns/E3-Ha/e4; (4) E1/e2-ns/e3-tr/E4; (5) e1-nl/e2-ns/E3-Ha/E4. The studied cultivars contained the most common alleles of the GmFT2a and GmFT5a genes, with the exception of the ‘Cassidi’ cultivar having a rare dominant allele GmFT5a-H4. The degree of earliness of cultivars positively correlated with the number of recessive genes E1–E4, which is consistent with the data of foreign authors on different sets of cultivars from Japan and North China. Thus, the developed panel of markers can be successfully used in the selection of soybean for earliness and sensitivity to photoperiod.