Detection Of Genetic Variants Research Articles

Whole-Genome Alignment: Methods, Challenges, and Future Directions

Whole-genome alignment (WGA) is a critical process in comparative genomics, facilitating the detection of genetic variants and aiding our understanding of evolution. This paper offers a detailed overview and categorization of WGA techniques, encompassing suffix tree-based, hash-based, anchor-based, and graph-based methods. It elaborates on the algorithmic properties of these tools, focusing on performance and methodological aspects. This paper underscores the latest progress in WGA, emphasizing the increasing capacity to manage the growing intricacy and volume of genomic data. However, the field still grapples with computational and biological hurdles affecting the precision and speed of WGA. We explore these challenges and potential future solutions. This paper aims to provide a comprehensive resource for researchers, deepening our understanding of WGA tools and their applications, constraints, and prospects.

Comprehensive genomic profiling of pulmonary spindle cell carcinoma using tissue and plasma samples: insights from a real-world cohort analysis.

Pulmonary spindle cell carcinoma (PSCC) is a rare and aggressive non-small cell lung cancer (NSCLC) subtype with a dismal prognosis. The molecular characteristics of PSCC are largely unknown due to its rarity, which limits the diagnosis and treatment of this historically poorly characterized malignancy. We present comprehensive genomic profiling results of baseline tumor samples from 22 patients histologically diagnosed with PSCC, representing the largest cohort to date. Somatic genetic variant detection was compared between paired plasma samples and primary tumors from 13 patients within our cohort. The associations among genomic features, treatment, and prognosis were also analyzed in representative patient cases. TP53 (54.5%), TERT (36.4%), CDKN2A (27.3%), and MET (22.7%) were most frequently mutated. Notably, 81.8% of patients had actionable targets in their baseline tumors, including MET (22.7%), ERBB2 (13.6%), EGFR (9.1%), KRAS (9.1%), ALK (9.1%), and ROS1 (4.5%). The median tumor mutation burden (TMB) for PSCC tumors was 5.5 mutations per megabase (muts/Mb). TMB-high tumors (>10 muts/Mb) exhibited a significantly higher mutation frequency in genes such as KRAS, ARID2, FOXL2, and LRP1B, as well as within the DNA mismatch repair pathway. The detection rates for single nucleotide variants and structural variants were comparable between matched tumor and plasma samples, with 48.6% of genetic variants being mutually identified in both sample types. Additionally, a patient with a high mutation load and positive PD-L1 expression demonstrated a 7-month survival benefit from chemoimmunotherapy. Furthermore, a patient with an ALK-rearranged tumor achieved a remarkable 3-year progression-free survival following crizotinib treatment. Overall, our findings deepen the understanding of the complex genomic landscape of PSCC, revealing actionable targets amenable to tailored treatment of this poorly characterized malignancy.

Abstract LB287: VariLink: A rapid, high-resolution proximity ligation method for the detection of structural variants and chromatin topology features in cancer

Abstract Despite numerous technological advances, including the widespread adoption of next-generation sequencing, many clinically relevant cancer driver mutations go undetected. The reason for this gap in understanding is two-fold: 1) Structural variants (SVs) are particularly challenging to detect using shotgun or long read-based approaches, particularly in heterogenous samples; and 2) It is increasingly accepted that epigenetic dysregulation may be a driver of disease state through mechanisms such as enhancer hijacking or changes to 3D genome organization. To address this gap in understanding, we developed VariLink: a dual-purpose proximity ligation library protocol that yields high-quality, high-resolution, unbiased Hi-C libraries capable of sensitive detection of structural variants and chromatin topological features from a single data set. Importantly, the VariLink protocol can be performed in under 8 hours—less than half of the time required for conventional Hi-C protocols. In the current study, we demonstrate the performance of VariLink for the detection of variants within the primary sequence (such as SVs, SNVs/Indels, and CNVs) in addition to high-resolution reporting of 3D chromatin conformation. We performed benchmarking analyses for both genetic variant detection and chromatin topology in two well-characterized lymphoblast cell lines (GM12878 and K562). We demonstrate the capability of VariLink data to detect interchromosomal translocations with 10-fold higher sensitivity over shotgun. Furthermore, using hybridization capture to detect the BCR-ABL1 fusion in K562 cells, we show that fewer than 100k read pairs is sufficient to detect this fusion with high confidence in VariLink libraries. We find that the shotgun-like, uniform coverage of VariLink data also enables detection of SNVs/Indels with improved sensitivity and precision over conventional Hi-C methods. Importantly, we demonstrate that VariLink libraries maintain topological features consistent with biologically relevant topologically-associated domains (TADs) and chromatin loops, providing insight into novel epigenetic cancer drivers. VariLink enables higher-resolution assessments of chromatin topology over conventional multi-RE-based Hi-C approaches, as demonstrated by 50% more TADs and loops called at 5-kb resolution. Taken together, our data demonstrate that VariLink is a next-generation Hi-C method that enables sensitive detection of clinically relevant structural variants and high-resolution chromatin topology in a single, rapid workflow. Citation Format: Jonathan Torchia, Mital Bhakta, Cory Padilla, Meredith L. Carpenter, Philip Uren, Lisa Munding. VariLink: A rapid, high-resolution proximity ligation method for the detection of structural variants and chromatin topology features in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB287.

Genetic variant detection in a South African haemophilia B population.

Haemophilia B is characterised by a deficiency of factor IX (FIX) protein due to genetic variants in the FIX gene (F9). Genetic testing may have a vital role in effectively managing haemophilia B. However, in many developing countries, comprehensive genetic variant detection is unavailable. This study aimed to address the lack of genetic data in our country by conducting genetic variant detection on people affected by haemophilia B in our region. Twenty-one participants were screened with a direct Sanger sequencing method to identify variants in the F9 gene. The identified variants were then compared to previously published variants and/or to a reference database. A total of ten F9 genetic changes were detected, with five of them being novel. These identified variants were distributed across different domains of the FIX protein. Only one participant had a history of inhibitor formation against FIX replacement therapy. Notably, this participant had two distinct genetic changes present adjacent to each other. Thus, we hypothesise that the presence of multiple variants within the same functional region of the gene may increase the risk for inhibitor development. The discovery of novel pathogenic variations in the F9 gene highlights the importance of genetic analysis in specific geographical regions. The possible link between a complex variant and inhibitor formation illustrates the potential role that genetic screening has as a pre-treatment tool in predicting treatment reactions and outcomes.

The growing detection of genetic variants of uncertain significance in amyotrophic lateral sclerosis: What is their significance for the disease etiopathogenesis?

The growing detection of genetic variants of uncertain significance in amyotrophic lateral sclerosis: What is their significance for the disease etiopathogenesis?

Genome-Wide Association Studies (GWAS) Approaches for the Detection of Genetic Variants Associated with Antibiotic Resistance: A Systematic Review.

Antibiotic resistance is a significant threat to public health worldwide. Genome-wide association studies (GWAS) have emerged as a powerful tool to identify genetic variants associated with this antibiotic resistance. By analyzing large datasets of bacterial genomes, GWAS can provide valuable insights into the resistance mechanisms and facilitate the discovery of new drug targets. The present study aimed to undertake a systematic review of different GWAS approaches used for detecting genetic variants associated with antibiotic resistance. We comprehensively searched the PubMed and Scopus databases to identify relevant studies published from 2013 to February 2023. A total of 40 studies met our inclusion criteria. These studies explored a wide range of bacterial species, antibiotics, and study designs. Notably, most of the studies were centered around human pathogens such as Mycobacterium tuberculosis, Escherichia coli, Neisseria gonorrhoeae, and Staphylococcus aureus. The review seeks to explore the several GWAS approaches utilized to investigate the genetic mechanisms associated with antibiotic resistance. Furthermore, it examines the contributions of GWAS approaches in identifying resistance-associated genetic variants through binary and continuous phenotypes. Overall, GWAS holds great potential to enhance our understanding of bacterial resistance and improve strategies to combat infectious diseases.

Development of low-cost in-house tetra-ARMS-PCR assay for the screening of five CBS mutations found in Pakistani homocystinuria patients

Background Classical homocystinuria is an inborn amino acid metabolism disorder resulting from mutations in the Cystathionine-β-Synthase (CBS) gene. These mutations lead to elevated homocysteine and methionine levels and reduced cysteine levels in the blood. Typically, diagnosis occurs after patients display symptoms, and various lab methods confirm it. DNA sequencing is the best option for early detection of genetic variants in asymptomatic suspected individuals. Unfortunately, its high cost can hinder its use, especially in low-income countries like Pakistan. Objective Aim of this study was to devise a robust low-cost diagnostic/screening assay based on Tetra-ARMS-PCR for five prevalent genetic variants found in Pakistani classical homocystinuria patients. Materials and methods In the current study, T-ARMS-PCR assays were developed for five mutations (c.975G > C, c.770C > T, c.752T > C, c.1039 + 1G > T, c.451 + 1GG > TA), which were characterized previously in classical homocystinuria patients. These low-cost T-ARMS-PCR assays were then used to screen the affected individuals and their family members to identify their genotypes for pathogenic variations in the asymptomatic patients and carriers in their respective families. Results The outcomes were entirely consistent with those obtained from Sanger DNA sequencing, confirming the sensitivity, specificity, and reliability of the T-ARMS-PCR assay for detecting CBS mutations. Conclusion T-ARMS-PCR has wide applications for low-income countries for the screening and early diagnosis of asymptomatic patients and carriers in the homocystinuria affected families as well as other inherited diseases.

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.

Weighted multiple testing procedures in genome-wide association studies.

Multiple testing procedures controlling the false discovery rate (FDR) are increasingly used in the context of genome wide association studies (GWAS), and weighted multiple testing procedures that incorporate covariate information are efficient to improve the power to detect associations. In this work, we evaluate some recent weighted multiple testing procedures in the specific context of GWAS through a simulation study. We also present a new efficient procedure called wBHa that prioritizes the detection of genetic variants with low minor allele frequencies while maximizing the overall detection power. The results indicate good performance of our procedure compared to other weighted multiple testing procedures. In particular, in all simulated settings, wBHa tends to outperform other procedures in detecting rare variants while maintaining good overall power. The use of the different procedures is illustrated with a real dataset.

Accuracy of a simplified sequencing and bioinformatics platform on genetic variant detection in patients with prostate cancer.

e17061 Background: Consensus guidelines suggest that genetic testing is clinically indicated for prostate cancer (PCa) patients who meet certain family history, stage and histopathological grade criteria. Current next generation sequencing (NGS) diagnostics rely on capital and maintenance intensive sequencers which require highly trained technical personnel to operate. This may restrict availability of testing where specimen transport or access to personnel/capital is limited. We sought to compare results obtained using a simpler sequencer coupled with a bioinformatics platform which requires minimal maintenance and capital cost with those from more traditional NGS equipment. Methods: We compared results in detection of 10 genes recommended by NCCN PCa guidelines (ATM, BRCA1, BRCA2, CHEK2, MLH1, MSH2, MSH6, PALB2, PSM2, and HOXB13) between the Oxford Nanopore Technologies MinION to the Illumina NextSeq sequencer. All samples were sequenced in a depth greater than 50x. Bioinformatics for the Illumina sequencer was performed using Illumina’s TruSight Hereditary Cancer panel. As the MinION reads long DNA fragments, and cancer diagnostics is focused on short exon sequencing, we used the CorrSeq Dx by Foresee Genomics to create long DNA molecules consisting of several tandem repeats of short DNA fragments separated by sequences recognized by automated bioinformatics software. Statistical analysis was performed using Cohen’s kappa test. This IRB-exempt study was conducted in accordance with the Declaration of Helsinki. Results: We detected using 380 variants in 13 patients on the Illumina platform and 379 (3 false negatives, 2 false positives) variants on the same patients using the MinION sequencer with CorrSeq Dx assay. The positive predictive value and sensitivity for the simplified platform was 99.5% (375/(375+2)) and 99.2% (375/(375+3)), respectively. The demonstrated accuracy of the simplified process was 99.9% (Cohen's kappa > 0.99). Conclusions: Thisl data suggests that a simpler sequencing solution with appropriate bioinformatics could allow clinical laboratories with limited technical and capital resources to perform germline and possibly somatic testing in-house; use of this technology may also serve to reduce costs for labs currently performing these tests. While some labs or clinics can outsource their NGS diagnostics if such commercial options are available, these options may not be possible where transport of specimens is logistically difficult or if access to technology is limited. A simpler process for NGS testing could improve access to guideline-based care in both remote and economically disadvantaged areas, resulting in better treatment outcomes as well as increased global healthcare equity. Further testing of this technology in prostate cancer as well as other disease states is warranted to fully understand its potential impact on patient care.

LBA02-10 ACCURACY OF A SIMPLIFIED SEQUENCING AND BIOINFORMATICS PLATFORM ON GENETIC VARIANT DETECTION IN PROSTATE CANCER PATIENTS

You have accessJournal of UrologyCME1 Apr 2023LBA02-10 ACCURACY OF A SIMPLIFIED SEQUENCING AND BIOINFORMATICS PLATFORM ON GENETIC VARIANT DETECTION IN PROSTATE CANCER PATIENTS Deepak Kapoor, Jonathan Anderson, and Ann Anderson Deepak KapoorDeepak Kapoor More articles by this author , Jonathan AndersonJonathan Anderson More articles by this author , and Ann AndersonAnn Anderson More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003361.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Consensus guidelines suggest that genetic testing is clinically indicated for prostate cancer (PCa) patients who meet certain family history, stage and histopathological grade criteria. Current next generation sequencing (NGS) diagnostics rely on capital and maintenance intensive sequencers and require highly trained technical personnel to operate. This may restrict availability of testing where specimen transport or access to personnel/capital is limited. We sought to compare a simpler sequencer coupled with a bioinformatics platform which requires minimal maintenance and capital cost with traditional NGS equipment. METHODS: We compared results in detection of 10 genes recommended by NCCN PCa guidelines (ATM, BRCA1, BRCA2, CHEK2, MLH1, MSH2, MSH6, PALB2, PSM2, and HOXB13) between the Oxford Nanopore Technologies MinION to the Illumina NextSeq sequencer. All samples were sequenced in a depth greater than 50x. Bioinformatics for the Illumina sequencer was Illumina’s TruSight™ Hereditary Cancer panel. As the MinION reads long DNA fragments, and cancer diagnostics is focused on short exon sequencing, we used the CorrSeq Dx™ by Foresee Genomics to create long DNA molecules consisting of several tandem repeats of short DNA fragments separated by sequences recognized by the automated bioinformatics software. Statistical analysis was performed using Cohen’s kappa test. This IRB-exempt study was conducted in accordance with the Declaration of Helsinki. RESULTS: We detected using 380 variants in 13 patients on the Illumina platform and 379 (3 false negatives, 2 false positives) variants on the same patients using the MinION sequencer with CorrSeq Dx assay. The positive predictive value and sensitivity for the simplified platform was 99.5% (375/(375+2)) and 99.2% (375/(375+3)), respectively. The demonstrated accuracy of the simplified process was 99.9% (Cohen's kappa >0.99). CONCLUSIONS: Our data suggests that this application could allow smaller clinical laboratories which are currently outsourcing their NGS diagnostics to perform genetic testing in-house. Use of a simpler process for NGS testing could improve access to guideline-based care in remote and economically disadvantaged areas, resulting in better treatment outcomes as well as increased global healthcare equity. Further testing of this technology is warranted to further assess its impact on patient care. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1190 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Deepak Kapoor More articles by this author Jonathan Anderson More articles by this author Ann Anderson More articles by this author Expand All Advertisement PDF downloadLoading ...

Validation of a New PCR-Based Screening Method for Prevention of Serratia marcescens Outbreaks in the Neonatal Intensive Care Unit

Background: Serratia marcescens may cause severe nosocomial infections, mostly in very low birth weight infants. Since S. marcescens exhibits by far the highest adjusted incidence rate for horizontal transmission, it can cause complex outbreak situations in neonatal intensive care units. Objective: The aim of this study was to establish a fast and highly sensitive colonization screening for prompt cohorting and barrier nursing strategies. Methods: A probe-based duplex PCR assay targeting the 16S rRNA gene of S. marcescens was developed and validated by using 36 reference strains, 14 S. marcescens outbreak- and nonoutbreak isolates, defined by epidemiological linkage and molecular typing, and applied in 1,347 clinical specimens from 505 patients. Results and Conclusions: The novel PCR assay proved to be highly specific and had an in vitro sensitivity of 100 gene copies per reaction (∼15 bacteria). It showed a similar (in laryngeal/tracheal specimens) or even higher (in rectal/stoma swabs) in vivo sensitivity in comparison to routine microbial culture and was much quicker (<24 h vs. 2 days). By combining different oligonucleotide primers, there was robust detection of genetic variants of S. marcescens strains. PCR inhibition was low (1.6%) and observed with rectal swabs only. Cohort analysis illustrated applicability of the PCR assay as a quick tool to prevent outbreak scenarios by allowing rapid decisions on cohorting and barrier nursing. In summary, this novel molecular screening for colonization by S. marcescens is specific, highly sensitive, and substantially accelerates detection.

Comparative epigenomics reveals the impact of ruminant-specific regulatory elements on complex traits.

Insights into the genetic basis of complex traits and disease in both human and livestock species have been achieved over the past decade through detection of genetic variants in genome-wide association studies(GWAS). A majority of such variants were found located in noncoding genomic regions, and though the involvement of numerous regulatory elements (REs) has been predicted across multiple tissues in domesticated animals, their evolutionary conservation and effects on complex traits have not been fully elucidated, particularly in ruminants. Here, we systematically analyzed 137 epigenomic and transcriptomic datasets of six mammals, including cattle, sheep, goats, pigs, mice, and humans, and then integrated them with large-scale GWAS of complex traits. Using 40 ChIP-seq datasets of H3K4me3 and H3K27ac, we detected 68,479, 58,562, 63,273, 97,244, 111,881, and 87,049 REs in the liver of cattle, sheep, goats, pigs, humans and mice, respectively. We then systematically characterized the dynamic functional landscapes of these REs by integrating multi-omics datasets, including gene expression, chromatin accessibility, and DNA methylation. We identified a core set (n = 6359) of ruminant-specific REs that are involved in liver development, metabolism, and immune processes. Genes with more complex cis-REs exhibited higher gene expression levels and stronger conservation across species. Furthermore, we integrated expression quantitative trait loci (eQTLs) and GWAS from 44 and 52 complex traits/diseases in cattle and humans, respectively. These results demonstrated that REs with different degrees of evolutionary conservation across species exhibited distinct enrichments for GWAS signals of complex traits. We systematically annotated genome-wide functional REs in liver across six mammals and demonstrated the evolution of REs and their associations with transcriptional output and conservation. Detecting lineage-specific REs allows us to decipher the evolutionary and genetic basis of complex phenotypes in livestock and humans, which may benefit the discovery of potential biomedical models for functional variants and genes of specific human diseases.

DdRADseq-mediated detection of genetic variants in sugarcane.

The article presents an optimization of the key parameters for the identification of SNPs in sugarcane using a GBS protocol based on two Illumina NextSeq and NovaSeq platforms. Sugarcane (Saccharum sp.), a world-wide known feedstock for sugar production, bioethanol, and energy, has an extremely complex genome, being highly polyploid and aneuploid. A double-digestion restriction site-associated DNA sequencing protocol (ddRADseq) was tested in four commercial sugarcane hybrids and one high-fibre biotype for the detection of single nucleotide polymorphisms (SNPs). In this work we tested two Illumina sequencing platforms, read size (70 vs. 150bp), different sequencing coverage per individual (medium and high coverage), and single-reads versus paired-end reads. We also explored different variant calling strategies (with and without reference genome) and filtering schemes [combining two minor allele frequencies (MAFs) with three depth of coverage thresholds]. For the discovery of a large number of novel SNPs in sugarcane, we recommend longer size and paired-end reads, medium sequencing coverage per individual and Illumina platform NovaSeq6000 for a cost-effective approach, and filter parameters of lower MAF and higher depth coverages thresholds. Although the de novo analysis retrieved more SNPs, the reference-based method allows downstream characterization of variants. For the two best performing matrices, the number of SNPs per chromosome correlated positively with chromosome length, demonstrating the presence of variants throughout the genome. Multivariate comparisons, with both matrices, showed closer relationships among commercial hybrids than with the high-fibre biotype. Functional analysis of the SNPs demonstrated that more than half of them landed within regulatory regions, whereas the other half affected coding, intergenic and intronic regions. Allelic distances values were lower than 0.07 when analysing two replicated genotypes, confirming the protocol robustness.

Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis.

Pharmacogenetics studies provide clinically relevant information on the identified associations between genetic variants and individual variability in drug response, which, in turn, offers great promise for guiding personalized drug therapy and clinical trial design. However, there is a lack of information concerning the evidence-based clinical annotations of specific CYP2E1 genetic variants. To design and evaluate the next-generation sequencing-based method for full-length CYP2E1 gene polymorphism analysis. Seven gene-specific oligonucleotide primer pairs targeting overlapping CYP2E1 gene fragments spanning all nine gene exons with interleaving introns, untranslated (UTR) and intergenic regions were designed. Human DNA samples (n = 3) were used as a training set to check the primer performance and to optimize the PCR conditions. The effectiveness of the developed target amplification and sequencing protocol was evaluated using the test set comprising human DNA samples (n = 3) obtained from tuberculosis patients. Sequencing data analysis was performed on the Galaxy online-based platform. The sequencing data quality was sufficient for the detection of genetic variants dispersed throughout the CYP2E1 gene with a high degree of confidence in fully covered regions achieving optimal reading depth of the targeted fragment with high base call accuracy. Developed protocol can be applied in subpopulation-level association studies to determine whether single nucleotide variants (SNVs) or variant combinations from multiple regions of the CYP2E1 gene are of clinical significance.

Using genetic markers for detection and subtyping of the emerging Salmonella enterica subspecies enterica serotype Muenchen

Non-typhoidal Salmonella (NTS) poses a global threat to public health. Poultry, one of the main reservoirs of NTS, is usually not clinically affected by most NTS, yet the economic losses to the poultry industry due to control and mitigation efforts, and due to negative publicity can be tremendous.NTS strains are routinely characterized into serotypes in a time-consuming, labor-intensive multistep process that requires skilled personnel. Moreover, the discriminatory power of serotyping is limited compared to other subtyping methods. Whole-genome sequence data enable the identification of genetic variation within serotypes. However, sequencing is often limited by available resources, and analyzing and interpreting the genetic data may be time-consuming. Source tracing during epidemiological outbreak investigations requires rapid and efficient characterization of strains to control pathogen spread. Here we designed a multiplex polymerase chain reaction (PCR) assay for the detection of genetic variants of Salmonella Muenchen, a serotype that has emerged in Israel in the last 3 yr in both clinical human cases and different hosts. Test sensitivity of 99.21% and specificity of 94 to 100% were determined using in-silico PCR with a dataset of 18,282 NTS assemblies from 37 NTS serotypes. Similarly, test sensitivity of 100% and specificity of 96.2 to 100% were determined in-vitro with 120 NTS isolates of 52 serotypes. Moreover, the test enabled differentiation between the common sequence types of serotype Muenchen using both approaches. As opposed to traditional serotyping and other subtyping methods, the designed test allows for rapid and cost-efficient detection of the emerging S. Muenchen serotype and its variants in a single step. Future development of similar assays for other dominant serotypes may help reduce the time and cost required for detection and initial characterization of dominant NTS strains. Overall, these tests will be beneficial to both public health and for reducing of the economic losses to the poultry industry due to NTS infections.

Modified Taq DNA Polymerase for Allele-Specific Ultra-Sensitive Detection of Genetic Variants

Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.

Genome resequencing clarifies phylogeny and reveals patterns of selection in the toxicogenomics model Pimephales promelas.

BackgroundThe fathead minnow (Pimephales promelas) is a model species for toxicological research. A high-quality genome reference sequence is available, and genomic methods are increasingly used in toxicological studies of the species. However, phylogenetic relationships within the genus remain incompletely known and little population-genomic data are available for fathead minnow despite the potential effects of genetic background on toxicological responses. On the other hand, a wealth of extant samples is stored in museum collections that in principle allow fine-scale analysis of contemporary and historical genetic variation.MethodsHere we use short-read shotgun resequencing to investigate sequence variation among and within Pimephales species. At the genus level, our objectives were to resolve phylogenetic relationships and identify genes with signatures of positive diversifying selection. At the species level, our objective was to evaluate the utility of archived-sample resequencing for detecting selective sweeps within fathead minnow, applied to a population introduced to the San Juan River of the southwestern United States sometime prior to 1950.ResultsWe recovered well-supported but discordant phylogenetic topologies for nuclear and mitochondrial sequences that we hypothesize arose from mitochondrial transfer among species. The nuclear tree supported bluntnose minnow (P. notatus) as sister to fathead minnow, with the slim minnow (P. tenellus) and bullhead minnow (P. vigilax) more closely related to each other. Using multiple methods, we identified 11 genes that have diversified under positive selection within the genus. Within the San Juan River population, we identified selective-sweep regions overlapping several sets of related genes, including both genes that encode the giant sarcomere protein titin and the two genes encoding the MTORC1 complex, a key metabolic regulator. We also observed elevated polymorphism and reduced differentation among populations (FST) in genomic regions containing certain immune-gene clusters, similar to what has been reported in other taxa. Collectively, our data clarify evolutionary relationships and selective pressures within the genus and establish museum archives as a fruitful resource for characterizing genomic variation. We anticipate that large-scale resequencing will enable the detection of genetic variants associated with environmental toxicants such as heavy metals, high salinity, estrogens, and agrichemicals, which could be exploited as efficient biomarkers of exposure in natural populations.

Long-term artificial selection of Hanwoo (Korean) cattle left genetic signatures for the breeding traits and has altered the genomic structure

Indigenous Korean breeds such as Hanwoo (Korean) cattle have adapted to their local environment during the past 5000 years. In the 1980s, the National Genetic Improvement Program was established to develop a modern economic breed for beef production in Korea through artificial selection. This process is thought to have altered the genomic structure of breeding traits over time. The detection of genetic variants under selection could help to elucidate the genetic mechanism of artificial selection in modern cattle breeds. Indigenous Hanwoo cattle have adapted in response to local natural and artificial selection during a 40-year breeding program. We analyzed genomic changes in the selection signatures of an unselected population (USP; n = 362) and a selected population (KPN; n = 667) of Hanwoo cattle. Genomic changes due to long-term artificial selection were identified using a genome-wide integrated haplotype score (iHS) and a genome-wide association study (GWAS). Signatures of recent selection were detected as positive (piHS > 6) or negative (piHS < –6) iHS scores spanning more than 46 related genes in KPN cattle, but none in USP cattle. A region adjacent to the PLAG1 gene was found to be under strong selection for carcass weight. The GWAS results also showed a selection signature on BTA14, but none on BTA13. Pathway and quantitative trait locus analysis results identified candidate genes related to energy metabolism, feed efficiency, and reproductive traits in Hanwoo cattle. Strong selection significantly altered Hanwoo cattle genome structural properties such as linkage disequilibrium (LD) and haplotypes through causal mutation for target traits. Haplotype changes of genome structure which are changes of ancestral allele to derived alleles due to selection were clearly identified on BTA13 and BTA14; however, the structure of the LD block was not clearly observed except BTA14. Thus, selection based on EBVs would be working very well in Hanwoo cattle breeding program appears to have been highly successful.

Clinical Utility of Implementing a Frontline NGS-Based DNA and RNA Fusion Panel Test for Patients with Suspected Myeloid Malignancies.

The use of molecular genetic biomarkers is rapidly advancing to aid diagnosis, prognosis, and clinical management of hematological disorders. We have implemented a next-generation sequencing (NGS) assay for detection of genetic variants and fusions as a frontline test for patients suspected with myeloid malignancy. In this study, we summarize the findings and assess the clinical impact in the first 1613 patients tested. All patients were assessed using NGS based Oncomine Myeloid Research Assay (ThermoFisher) including 40 genes (17 full genes and 23 genes with clinically relevant "hotspot" regions), along with a panel of 29 fusion driver genes (including over fusion 600 partners). Among 1613 patients with suspected myeloid malignancy, 43% patients harbored at least one clinically relevant variant: 91% (90/100) in acute myeloid leukemia patients, 71.7% (160/223) in myelodysplastic syndrome (MDS), 77.5% (308/397) in myeloproliferative neoplasm (MPN), 83% (34/41) in MPN/MDS, and 100% (40/40) in chronic myeloid leukemia patients. Comparison of NGS and cytogenetics results revealed a high degree of concordance in gene fusion detection. Our findings demonstrate clinical utility and feasibility of integrating a NGS-based gene mutation and fusion testing assay as a frontline diagnostic test in a large reported cohort of patients with suspected myeloid malignancy, in a clinical laboratory setting. Overlap with cytogenetic test results provides opportunity for testing reduction and streamlining.