GPrimer: a fast GPU-based pipeline for primer design for qPCR experiments

Bae, Jeongmin; Jeon, Hajin; Kim, Min-Soo

doi:10.1186/s12859-021-04133-4

Cited by 7 publications

(3 citation statements)

References 31 publications

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“…These requirements could be addressed with the many primer‐designing tools available online (Markham & Zuker, 2005; Untergasser et al, 2012). Recent resources have been developed for improving oligo specificity, taking advantage of publicly available transcriptome (Arvidsson et al, 2008) or genome (Kim et al, 2017; Bae et al, 2021) datasets. However, these tools are intended to be used for organisms with well‐annotated genomes, and their application to non‐model species makes target sequence identification very cumbersome.…”

Section: Introductionmentioning

confidence: 99%

PABLOG: a Primer Analysis tool using a Bee‐Like approach on Orthologous Genes

Ferrigno,

Frazzetto,

Prjibelski

et al. 2024

Physiologia Plantarum

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RNA‐seq data is currently generated in numerous non‐model organisms that lack a reference genome. Nevertheless, the confirmation of gene expression levels using RT‐qPCR remains necessary, and the existing techniques do not seamlessly interface with the omics pipeline workflow. Developing primers for many targets by utilising orthologous genes can be a laborious, imprecise, and subjective process, particularly for plant species that are not commonly studied and do not have a known genome. We have developed a primer design tool, named PABLOG, that analyses the alignments generated from long or short RNA‐seq reads and a reference orthologous gene. PABLOG scans, much like a bee searching several flowers for pollen, and presents a sorted list of potential exon‐exon junction locations, ranked according to their reliability. Through computational analysis across the whole genomes of several non‐model species, we demonstrate that PABLOG performs more effectively than other methods in identifying exon‐exon junctions since it generates significantly fewer false‐positive results. Examination of candidate regions at the gene level, in conjunction with laboratory studies, shows that the suggested primers successfully amplified particular targets in non‐model plants without any presence of genomic contamination. Our tool includes a consensus sequence feature that enables the complete process of primer design, from aligning with the target gene to determining amplification parameters. The utility can be accessed via the GitHub repository located at: https://github.com/tools4plant-omics/PABLOG.

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Section: Introductionmentioning

confidence: 99%

PABLOG: a Primer Analysis tool using a Bee‐Like approach on Orthologous Genes

Ferrigno,

Frazzetto,

Prjibelski

et al. 2024

Physiologia Plantarum

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“…Therefore, to contribute to the control of ARB dissemination, the reliable determination by qPCR of the abundance of ARGs in primary sources of contamination is mandatory. In this regard, to obtain the highest accuracy of the abundance measure of a speci c target gene during qPCR assays, the proper design of the corresponding primers stands out as the most important factor [18,19]. Literature regarding quality primer pair design describes several signi cant properties to consider, mainly, the primer size, the percentage of guanine and cytosine, the lack of formation of secondary structures, and an adequate range of annealing temperatures [17,20,21].…”

Section: Introductionmentioning

confidence: 99%

Design and validation of primer sets for the detection and quantification of antibiotic resistance genes in environmental samples by quantitative PCR

Perez-Bou

González‐Martínez

Cabrera

et al. 2023

Preprint

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The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Hence, it is vital to dispose of molecular tools that allow proper monitoring of antibiotic resistant genes (ARGs) encoding resistances to these important therapeutic compounds. For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides), ampC, blaTEM, blaSHV, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARG’s biodiversity than those of several primers described to date. The adequate design and well performance of the new molecular tools were validated in vivo in six samples, retrieved from both natural and engineered environments. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), consistency across replicate experiments, and a wide dynamic range. The new methodology described here provide valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs in the environment by qPCR.

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“…Several primer design software packages can take a MSA as input (Perini et al 2020 ; Yoon and Leitner 2015 ), but these packages are not able to deal with massive alignment data. MRPrimerV (Kim et al 2017 ), a database containing PCR primer pairs for the detection of 1,818 RNA virus, cannot be used for SARS-CoV-2 diagnoses because it has not been updated since 2016, despite the recent update of the primer design engine from MapReduce to a GPU-based pipeline (Bae et al 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design

Jeong

Lee²,

Ko³

et al. 2022

Genes Genom

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Background As the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive. Objective To extract conserved sequences from SARS-CoV-2 genomes for the design of diagnostic PCR primers using a bioinformatics approach that can handle massive genomic sequences efficiently. Methods A total of 230,163 full-length viral genomes were retrieved from the NCBI SARS-CoV-2 Resources and GISAID EpiCoV database. This number was reduced to 14.11% following removal of 5′-/3′-untranslated regions and sequence dereplication. Fast, reference-based, multiple sequence alignments identified conserved sequences and specific primer sets were designed against these regions using a conventional tool. Primer sets chosen among the candidates were evaluated by in silico PCR and RT-qPCR. Results Out of 17 conserved sequences (totaling 4.3 kb), two primer sets targeting the nsp2 and ORF3a genes were picked that exhibited > 99.9% in silico amplification coverage against the original dataset (230,163 genomes) when a 5% mismatch between the primers and target was allowed. In addition, the primer sets successfully detected nine SARS-CoV-2 variant RNA samples (Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa) in experimental RT-qPCR validations. Conclusion In addition to the RdRp, E, N, and S genes that are targeted commonly, our approach can be used to identify novel primer targets in SARS-CoV-2 and should be a priority strategy in the event of novel SARS-CoV-2 variants or other pandemic outbreaks. Supplementary Information The online version contains supplementary material available at 10.1007/s13258-022-01264-7.

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GPrimer: a fast GPU-based pipeline for primer design for qPCR experiments

Cited by 7 publications

References 31 publications

PABLOG: a Primer Analysis tool using a Bee‐Like approach on Orthologous Genes

PABLOG: a Primer Analysis tool using a Bee‐Like approach on Orthologous Genes

Design and validation of primer sets for the detection and quantification of antibiotic resistance genes in environmental samples by quantitative PCR

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design

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