scholarly journals Simple and cost-effective SNP genotyping method for discriminating subpopulations of the fish pathogen, Nocardia seriolae

2021 ◽  
Author(s):  
Cuong Thanh Le ◽  
Erin P Prince ◽  
Derek S Sarovich ◽  
Thu Thi Anh Nguyen ◽  
Hung VuKhac ◽  
...  
Keyword(s):  
Low Cost ◽  
Bacterial Pathogen ◽  
Cost Effective ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Putative Origin ◽  
Nocardia Seriolae ◽  
Mutation Assay ◽  
Economic And Social Impacts ◽  
Pathogen Surveillance

Nocardia seriolae has caused significant fish losses in Asia and the Americas in recent decades, including in Vietnam, which has witnessed devastating economic and social impacts due to this bacterial pathogen. Surveillance strategies are urgently needed to mitigate N. seriolae dissemination in Vietnamese aquaculture and mariculture industries. Whole-genome sequencing (WGS) offers the highest level of resolution to discriminate closely related strains and to determine their putative origin and transmission routes. However, WGS is impractical for epidemiological investigations and pathogen surveillance due to its time-consuming and costly nature, putting this technology out-of-reach for many industry end-users. To overcome this issue, we targeted two previously characterised, phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. Using the mismatch amplification mutation assay (MAMA) format, we developed assays that genotype strains based on differences in amplicon melting temperature (melt-MAMA) and size (agarose-MAMA). Our MAMA assays accurately genotyped strains both from culture and fish tissues at low cost, using either real-time (~AUD$1/per sample) or conventional (~AUD$0.50/per sample) PCR instrumentation. Our novel assays provide a rapid, reproducible, and cost-effective tool for routine genotyping of this pathogen, allowing faster identification and treatment of nocardiosis-effected permit fish within Vietnamese aquaculture/mariculture facilities, an essential step in mitigating N. seriolae-associated losses.

scholarly journals Genotyping Single-Nucleotide Polymorphisms by the Invader Assay with Dual-Color Fluorescence Polarization Detection

2001 ◽  
Vol 47 (8) ◽  
pp. 1373-1377 ◽  
Author(s):  
Tony M Hsu ◽  
Scott M Law ◽  
Shenghui Duan ◽  
Bruce P Neri ◽  
Pui-Yan Kwok
Keyword(s):  
Fluorescence Polarization ◽  
Cost Effective ◽  
Snp Markers ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Clear Cut ◽  
Pcr Product ◽  
Invader Assay ◽  
Pcr Products ◽  
Incorporation Assay

Abstract Background: The PCR-Invader® assay is a robust, homogeneous assay that has been shown to be highly sensitive and specific in genotyping single-nucleotide polymorphism (SNP) markers. In this study, we introduce two changes to improve the assay: (a) we streamline the PCR-Invader method by assaying both alleles for each SNP in one reaction; and (b) we reduce the cost of the method by adopting fluorescence polarization (FP) as the detection method. Methods: PCR product was incubated with Invader oligonucleotide and two primary probes at 93 °C for 5 min. Signal probes corresponding to the cleaved flaps of the primary probes [labeled with fluorescein and 6-carboxytetramethylrhodamine (TAMRA) dye] and Cleavase® VIII enzyme (a flap endonuclease) were then added to the mixture. This reaction mixture was incubated at 63 °C for 5 min. FP measurements were made with a fluorescence plate reader. Results: Eighty-eight individuals were genotyped across a panel of 10 SNPs, using PCR product as template, for a total of 880 genotypes. An average “no call” rate of 3.2% was observed after first round of experiments. PCR products were remade in those samples that failed to produce any genotype in the first round, and all gave clear-cut genotypes. When the genotypes determined by the PCR-Invader assay and template-directed dye-terminator incorporation assay with FP were compared, they were in 100% concordance for all SNP markers and experiments. Conclusions: The improvements introduced in this study make PCR-Invader assay simpler and more cost-effective, and therefore more suitable for high-throughput genotyping.

scholarly journals MassARRAY-based single nucleotide polymorphism analysis in breast cancer of north Indian population

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Divya Bakshi ◽  
Ashna Nagpal ◽  
Varun Sharma ◽  
Indu Sharma ◽  
Ruchi Shah ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Mutations ◽  
Cost Effective ◽  
P Value ◽  
Polymorphism Analysis ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Cost Effective Method ◽  
Common Genetic Variants ◽  
Underlying Causes

Abstract Background Breast Cancer (BC) is associated with inherited gene mutations. High throughput genotyping of BC samples has led to the identification and characterization of biomarkers for the diagnosis of BC. The most common genetic variants studied are SNPs (Single Nucleotide Polymorphisms) that determine susceptibility to an array of diseases thus serving as a potential tool for identifying the underlying causes of breast carcinogenesis. Methods SNP genotyping employing the Agena MassARRAY offers a robust, sensitive, cost-effective method to assess multiple SNPs and samples simultaneously. In this present study, we analyzed 15 SNPs of 14 genes in 550 samples (150 cases and 400 controls). We identified four SNPs of genes TCF21, SLC19A1, DCC, and ERCC1 showing significant association with BC in the population under study. Results The SNPs were rs12190287 (TCF21) having OR 1.713 (1.08–2.716 at 95% CI) p-value 0.022 (dominant), rs1051266 (SLC19A1) having OR 3.461 (2.136–5.609 at 95% CI) p-value 0.000000466 (dominant), rs2229080 (DCC) having OR 0.6867 (0.5123–0.9205 at 95% CI) p-value 0.0116 (allelic) and rs2298881 (ERCC1) having OR 0.669 (0.46–0.973 at 95% CI), p-value 0.035 (additive) respectively. The in-silico analysis was further used to fortify the above findings. Conclusion It is further anticipated that the variants should be evaluated in other population groups that may aid in understanding the genetic complexity and bridge the missing heritability.

scholarly journals Novel Hairpin-Shaped Primer Assay To Study the Association of the −44 Single-Nucleotide Polymorphism of the DEFB1 Gene with Early-Onset Periodontal Disease

2004 ◽  
Vol 11 (4) ◽  
pp. 766-769 ◽  
Author(s):  
Michele Boniotto ◽  
Manzour Hernando Hazbón ◽  
William James Jordan ◽  
Greig Patrick Lennon ◽  
Joyce Eskdale ◽  
...  
Keyword(s):  
Periodontal Disease ◽  
Real Time ◽  
Early Onset ◽  
Pcr Amplification ◽  
Cost Effective ◽  
Similar Distribution ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Specific Allele

ABSTRACT A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The −44 C→G transversion in the DEFB1 gene (which encodes human β-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the −44 SNP. We used an HP assay to study the distribution of the −44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the −44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.

scholarly journals LongPhase: an ultra-fast chromosome-scale phasing algorithm for small and large variants

2021 ◽  
Author(s):  
Jyun-Hong Lin ◽  
Liang-Chi Chen ◽  
Shu-Qi Yu ◽  
Yao-Ting Huang
Keyword(s):  
Variant Calling ◽  
Cost Effective ◽  
Nucleotide Polymorphisms ◽  
Structural Variations ◽  
Single Nucleotide ◽  
Chromosome Conformation ◽  
Long Reads ◽  
Cost Effective Approach ◽  
Long Read ◽  
Microbial Strains

AbstractLong-read phasing has been used for reconstructing diploid genomes, improving variant calling, and resolving microbial strains in metagenomics. However, the phasing blocks of existing methods are broken by large Structural Variations (SVs), and the efficiency is unsatisfactory for population-scale phasing. This paper presents an ultra-fast algorithm, LongPhase, which can simultaneously phase single nucleotide polymorphisms (SNPs) and SVs of a human genome in ∼10-20 minutes, 10x faster than the state-of-the-art WhatsHap and Margin. In particular, LongPhase produces much larger phased blocks at almost chromosome level with only long reads (N50=26Mbp). We demonstrate that LongPhase combined with Nanopore is a cost-effective approach for providing chromosome-scale phasing without the need for additional trios, chromosome-conformation, and single-cell strand-seq data.

scholarly journals Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

2021 ◽  
Author(s):  
Jane Mellor ◽  
Phillip Lorenz ◽  
Anna Lamstaes ◽  
Harry J Fischl ◽  
Shidong Xi ◽  
...  
Keyword(s):  
Low Cost ◽  
Cost Effective ◽  
Pulse Labelling ◽  
Single Nucleotide ◽  
Functional Regions ◽  
Nascent Rna ◽  
Nucleotide Resolution ◽  
Nascent Transcripts ◽  
The Impact ◽  
Single Nucleotide Resolution

Genomes are pervasively transcribed leading to stable and unstable transcripts that define functional regions of genomes and contribute to cellular phenotypes. Defining comprehensive nascent transcriptomes is pivotal to understand gene regulation, disease processes, and the impact of extracellular signals on cells. However, currently employed methods are laborious, technically challenging and costly. We developed single-nucleotide resolution 4sU-sequencing (SNU-Seq), involving pulse labelling, biotinylation and direct isolation of nascent transcripts. Artificial poly-(A)-tailing of the 3' most nucleotide of nascent transcripts ensures oligo-d(T) primer-based library preparation and sequencing using commercial 3' RNA-Seq kits. We show that SNU-Seq is a cost-effective new method generating even read profiles across transcription units. We used SNU-Seq to identify transcription elongation parameters, to map usage of polyadenylation (PAS) sites and novel enhancers. Remarkably, 4sU labelled nascent RNA accumulates short ~100nt transcripts that map to the 5' end of genes. We show that isolation of these short nascent RNA and sequencing the 5' and 3' ends using size-selected SNU-Seq (ssSNU-Seq) provides highly sensitive annotations of mapped and novel TSSs, promoter-proximal pause/termination sites. Thus, SNU-seq and ssSNU-seq combined yield comprehensive transcriptomics data at low cost with high spatial and temporal resolution.

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