Amplidiff: an optimized amplicon sequencing approach to estimating lineage abundances in viral metagenomes.

BACKGROUND: Metagenomic profiling algorithms commonly rely on genomic differences between lineages, strains, or species to infer the relative abundances of sequences present in a sample. This observation plays an important role in the analysis of diverse microbial communities, where targeted sequencing of 16S and 18S rRNA, both well-known hypervariable genomic regions, have led to insights into microbial diversity and the discovery of novel organisms. However, the variable nature of discriminatory regions can also act as a double-edged sword, as the sought-after variability can make it difficult to design primers for their amplification through PCR. Moreover, the most variable regions are not necessarily the most informative regions for the purpose of differentiation; one should focus on regions that maximize the number of lineages that can be distinguished. RESULTS: Here we present AmpliDiff, a computational tool that simultaneously finds highly discriminatory genomic regions in viral genomes of a single species, as well as primers allowing for the amplification of these regions. We show that regions and primers found by AmpliDiff can be used to accurately estimate relative abundances of SARS-CoV-2 lineages, for example in wastewater sequencing data. We obtain errors that are comparable with using whole genome information to estimate relative abundances. Furthermore, our results show that AmpliDiff is robust against incomplete input data and that primers designed by AmpliDiff also bind to genomes sampled months after the primers were selected. CONCLUSIONS: With AmpliDiff we provide an effective, cost-efficient alternative to whole genome sequencing for estimating lineage abundances in viral metagenomes.

ExonSurfer: a web-tool to design primers at exon-exon junctions.

BACKGROUND: Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design. RESULTS: ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets. CONCLUSION: ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

UPrimer: A Clade-Specific Primer Design Program Based on Nested-PCR Strategy and Its Applications in Amplicon Capture Phylogenomics.

Amplicon capture is a promising target sequence capture approach for phylogenomic analyses, and the design of clade-specific nuclear protein-coding locus (NPCL) amplification primers is crucial for its successful application. In this study, we developed a primer design program called UPrimer that can quickly design clade-specific NPCL amplification primers based on genome data, without requiring manual intervention. Unlike other available primer design programs, UPrimer uses a nested-PCR strategy that greatly improves the amplification success rate of the designed primers. We examined all available metazoan genome data deposited in NCBI and developed NPCL primer sets for 21 metazoan groups with UPrimer, covering a wide range of taxa, including arthropods, mollusks, cnidarians, echinoderms, and vertebrates. On average, each clade-specific NPCL primer set comprises ∼1,000 NPCLs. PCR amplification tests were performed in 6 metazoan groups, and the developed primers showed a PCR success rate exceeding 95%. Furthermore, we demonstrated a phylogenetic case study in Lepidoptera, showing how NPCL primers can be used for phylogenomic analyses with amplicon capture. Our results indicated that using 100 NPCL probes recovered robust high-level phylogenetic relationships among butterflies, highlighting the utility of the newly designed NPCL primer sets for phylogenetic studies. We anticipate that the automated tool UPrimer and the developed NPCL primer sets for 21 metazoan groups will enable researchers to obtain phylogenomic data more efficiently and cost-effectively and accelerate the resolution of various parts of the Tree of Life.

Multiobjective optimization-driven primer design mechanism: towards user-specified parameters of PCR primer.

Primers are critical for polymerase chain reaction (PCR) and influence PCR experimental outcomes. Designing numerous combinations of forward and reverse primers involves various primer constraints, posing a computational challenge. Most PCR primer design methods limit parameters because the available algorithms use general fitness functions. This study designed new fitness functions based on user-specified parameters and used the functions in a primer design approach based on the multiobjective particle swarm optimization (MOPSO) algorithm to address the challenge of primer design with user-specified parameters. Multicriteria evaluation was conducted simultaneously based on primer constraints. The fitness functions were evaluated using 7425 DNA sequences and compared with a predominant primer design approach based on optimization algorithms. Each DNA sequence was run 100 times to calculate the difference between the user-specified parameters and primer constraint values. The algorithms based on fitness functions with user-specified parameters outperformed the algorithms based on general fitness functions for 11 primer constraints. Moreover, MOPSO exhibited superior implementation in all experiments. Practical gel electrophoresis was conducted to verify the PCR experiments and established that MOPSO effectively designs primers based on user-specified parameters.

GetPrimers: A generalized PCR-based genetic targeting primer designer enabling easy and standardized targeted gene modification across multiple systems.

Genetic targeting (e.g., gene knockout and tagging) based on polymerase chain reaction (PCR) is a simple yet powerful approach for studying gene functions. Although originally developed in classic budding and fission yeast models, the same principle applies to other eukaryotic systems with efficient homologous recombination. One-step PCR-based genetic targeting is conventionally used but the sizes of the homologous arms that it generates for recombination-mediated genetic targeting are usually limited. Alternatively, gene targeting can also be performed via fusion PCR, which can create homologous arms that are orders of magnitude larger, therefore substantially increasing the efficiency of recombination-mediated genetic targeting. Here, we present GetPrimers (https://www.evomicslab.org/app/getprimers/), a generalized computational framework and web tool to assist automatic targeting and verification primer design for both one-step PCR-based and fusion PCR-based genetic targeting experiments. Moreover, GetPrimers by design runs for any given genetic background of any species with full genome scalability. Therefore, GetPrimers is capable of empowering high-throughput functional genomic assays at multipopulation and multispecies levels. Comprehensive experimental validations have been performed for targeting and verification primers designed by GetPrimers across multiple organism systems and experimental setups. We anticipate GetPrimers to become a highly useful and popular tool to facilitate easy and standardized gene modification across multiple systems.

Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR.

The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). 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 ARGs' biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments.

Development of alternative diagnosis of HH1, HH3, HH5 and HCD fertility haplotypes and subfertility syndrome in cattle.

The increasing prevalence of hereditary anomalies in Holstein cattle populations presents a pressing issue, leading to concerns such as embryonic mortality and the birth of non-viable offspring. This study addresses the urgency of managing harmful genetic mutations in Holstein cattle by developing alternative diagnostic methods. The research aims to devise effective means to diagnose fertility haplotypes HH1, HH3, HH5, HCD and BY and subfertility syndrome in cattle. To achieve this goal, a range of molecular genetic techniques were employed, including Tetra-Primer ARMS-PCR methods, PCR-RFLP analysis and allele-specific PCR. These methods facilitated the identification of heterozygous carriers of various fertility haplotypes and subfertility syndrome in Holstein cows and servicing bulls. The study reveals the prevalence of these genetic defects within the Republic of Kazakhstan's cattle population. HH1, HH3, HH5, HCD and BY fertility haplotypes were found to have occurrence rates ranging from 1.4% to 16.6%, with subfertility syndrome detected in 4.5% of Simmental bulls. The practical significance of this research lies in its contribution to genetic monitoring and management strategies for Holstein cattle populations. By introducing affordable, rapid and accurate diagnostic methods, such as the T-ARMS-PCR, the study provides a valuable tool for controlling and mitigating the spread of harmful genetic mutations, ultimately improving the overall genetic health and productivity of Holstein cattle in the region. This research addresses a critical need in the cattle breeding industry and underscores the importance of genetic monitoring to ensure the long-term viability and sustainability of Holstein cattle populations.

Dig-up Primers: A Pipeline for Identification of Polymorphic Microsatellites Loci within Assemblies of Related Species.

Simple sequence repeats (SSRs) have become one of the most popular molecular markers and are used in numerous fields, including conservation genetics, population genetic studies, and genetic mapping. Advances in next-generation sequencing technology and the growing amount of genomic data are driving the development of bioinformatics tools for SSR marker design. These tools work with different combinations of input data, which can be raw reads or assemblies, and with one or more input datasets. We present here a new strategy and implementation of a simple standalone pipeline that utilizes more than one assembly for the in silico design of PCR primers for microsatellite loci in more than one species. Primers are tested in silico to determine if they are polymorphic, eliminating the need to test time-consuming cross-species amplification in the laboratory. The end result is a set of markers that are in silico polymorphic in all analyzed species and have great potential for the identification of interspecies hybrids. The efficiency of the tool is demonstrated using two examples at different taxonomic levels and with different numbers of input assemblies to generate promising, high-quality SSR markers.

primerJinn: a tool for rationally designing multiplex PCR primer sets for amplicon sequencing and performing in silico PCR.

BACKGROUND: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers. RESULTS: We developed primerJinn, a tool that designs a set of multiplex primers and allows for the in silico PCR evaluation of primer sets against numerous input genomes. We used primerJinn to create a multiplex PCR for the sequencing of drug resistance-conferring gene regions from Mycobacterium tuberculosis, which were then successfully sequenced. CONCLUSIONS: primerJinn provides a user-friendly, efficient, and accurate method for designing multiplex PCR primers for targeted sequencing and performing in silico PCR. It can be used for various applications in molecular biology and bioinformatics research, including the design of assays for amplifying and sequencing drug-resistance-conferring regions in important pathogens.

CircPrime: a web-based platform for design of specific circular RNA primers.

BACKGROUND: Circular RNAs (circRNAs) are covalently closed-loop RNAs with critical regulatory roles in cells. Tens of thousands of circRNAs have been unveiled due to the recent advances in high throughput RNA sequencing technologies and bioinformatic tools development. At the same time, polymerase chain reaction (PCR) cross-validation for circRNAs predicted by bioinformatic tools remains an essential part of any circRNA study before publication. RESULTS: Here, we present the CircPrime web-based platform, providing a user-friendly solution for DNA primer design and thermocycling conditions for circRNA identification with routine PCR methods. CONCLUSIONS: User-friendly CircPrime web platform ( http://circprime.elgene.net/ ) works with outputs of the most popular bioinformatic predictors of circRNAs to design specific circular RNA primers. CircPrime works with circRNA coordinates and any reference genome from the National Center for Biotechnology Information database).

V-primer: software for the efficient design of genome-wide InDel and SNP markers from multi-sample variant call format (VCF) genotyping data.

DNA markers are indispensable tools in genetics and genomics research as well as in crop breeding, particularly for marker-assisted selection. Recent advances in next-generation sequencing technology have made it easier to obtain genome sequences for various crop species, enabling the large-scale identification of DNA polymorphisms among varieties, which in turn has made DNA marker design more accessible. However, existing primer design software is not suitable for designing many types of genome-wide DNA markers from next-generation sequencing data. Here, we describe the development of V-primer, high-throughput software for designing insertion/deletion, cleaved amplified polymorphic sequence, and single-nucleotide polymorphism (SNP) markers. We validated the applicability of these markers in different crops. In addition, we performed multiplex PCR targeted amplicon sequencing using SNP markers designed with V-primer. Our results demonstrate that V-primer facilitates the efficient and accurate design of primers and is thus a useful tool for genetics, genomics, and crop breeding. V-primer is freely available at https://github.com/ncod3/vprimer.

Identification of fish species through tRNA-based primer design.

BACKGROUND: The correct establishment of the barcode classification system for fish can facilitate biotaxonomists to distinguish fish species, and it can help the government to verify the authenticity of the ingredients of fish products or identify unknown fish related samples. The Cytochrome c oxidation I (COI) gene sequence in the mitochondria of each species possesses unique characteristics, which has been widely used as barcodes in identifying species in recent years. Instead of using COI gene sequences for primer design, flanking tRNA segments of COI genes from 2618 complete fish mitochondrial genomes were analyzed to discover suitable primers for fish classification at taxonomic family level. The minimal number of primer sets is designed to effectively distinguish various clustered groups of fish species for identification applications. Sequence alignment analysis and cross tRNA segment comparisons were applied to check and ensure the primers for each cluster group are exclusive. RESULTS: Two approaches were applied to improve primer design and re-cluster fish species. The results have shown that exclusive primers for 2618 fish species were successfully discovered through in silico analysis. In addition, we applied sequence alignment analysis to confirm that each pair of primers can successfully identify all collected fish species at the taxonomic family levels. CONCLUSIONS: This study provided a practical strategy to discover unique primers for each fishery species and a comprehensive list of exclusive primers for extracting COI barcode sequences of all known fishery species. Various applications of verification of fish products or identification of unknown fish species could be effectively achieved.

rprimer: an R/bioconductor package for design of degenerate oligos for sequence variable viruses.

BACKGROUND: This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. A multiple DNA sequence alignment is used as input data, while the outputs consist of comprehensive tables (data frames) and dashboard-like plots. The workflow can be run directly from the R console or through a graphical user interface (Shiny application). Here, rprimer is demonstrated and evaluated by using it to design two norovirus genogroup I (GI) assays: one RT-qPCR assay for quantitative detection and one RT­PCR assay for Sanger sequencing and polymerase-capsid based genotyping. RESULTS: The assays generated were evaluated using stool samples testing positive for norovirus GI. The RT-qPCR assay accurately amplified and quantified all samples and showed comparable performance to a widely-used standardised assay, while the RT-PCR assay resulted in successful sequencing and genotyping of all samples. Merits and limitations of the package were identified through comparison with three similar freely available software packages. Several features were comparable across the different tools, but important advantages of rprimer were its speed, flexibility in oligo design and capacity for visualisation. CONCLUSIONS: An R/Bioconductor package, rprimer, was developed and shown to be successful in designing primers and probes for quantitative detection and genotyping of a sequence-variable virus. The package provides an efficient, flexible and visual approach to degenerate oligo design, and can therefore assist in virus research and method development.

Pangenomes-identified singletons for designing specific primers to identify bacterial strains in a plant growth-promoting consortium.

BACKGROUND: The use of plant growth-promoting microorganisms represents a sustainable way to increase agricultural yields and plant health. Thus, the identification and tracking of these microorganisms are determinants for validating their positive effects on crops. Pangenomes allow the identification of singletons that can be used to design specific primers for the detection of the studied strains. OBJECTIVE: This study aimed to establish a strategy based on the use of whole-genome sequencing and pangenomes for designing and validating primer sets for detecting Bacillus cabrialesii TE3T, Priestia megaterium TRQ8, and Bacillus paralicheniformis TRQ65, a promising beneficial bacterial consortium for wheat. METHODS AND RESULTS: The identification of singletons of TE3T, TRQ8, and TRQ65 was performed by pangenomes using the Kbase platform and subsequently analyzed using BLAST®. The identified DNA regions were used for primer design in AlleleID version 7. Primers were validated by multiplex PCR using pure template DNA from each studied strain, combinations of two or three DNA from these strains, and DNA from agricultural soil samples enriched (and not) with the bacterial consortium. Here, we report the first design of primers capable of detecting and identifying the beneficial strains TE3T, TRQ8, and TRQ65. CONCLUSIONS: The use of pangenomes allowed the distinction of unique sequences that enables the design of primers for specific identification of the studied bacterial strains. This strategy can be widely used for the design of primer sets to detect other strains of interest for combating biopiracy, and commercial protection of biological products, among other applications.

Novel primers for 16S rRNA gene-based archaeal and bacterial community analysis in oceanic trench sediments.

High-throughput sequencing of the 16S ribosomal RNA (16S rRNA) gene has been successfully applied to explore the microbial structure and dynamics in various environments. The distinctive microbial communities in oceanic trench sediments are expected because of the extremely high pressure and V-shape topology that caused the isolation from the other marine sediments. However, they have only been primarily targeted using 'universal' primers that provide variable performances for different environments. It is necessary to design specific primers to improve the detection resolution of unique microbial groups in oceanic trenches. Here, we designed one pair of bacterial and two pairs of archaeal specific primers based on 16S rRNA gene full-length sequences that truly come from trench sediment and tested their performances in 30 oceanic trench sediment samples. An in silico analysis showed that the V3-V4 hypervariable region was the most informative and representative for oceanic trench microbial groups. Compared with the 'universal' primers, 46 bacterial families were only detected by newly designed primer B344F/B749R, and eight archaeal families were only detected by the newly designed primer A306F/A713R which covered the one or two orders of magnitude more ASVs (amplicon sequence variants) (1,470,216) in the tested total 30 samples. Moreover, A306F/A713R had the largest number of observed ASVs suggesting its better performance in discovering more archaeal species which were easily ignored in universal primer-based experiments for oceanic trench sediments. The novel primers designed in this research could be a better option to access the unique microbial communities in extreme oceanic trench sediments.Key points• Defining V3-V4 as the most adequate hypervariable region for archaea and bacteria from oceanic trench sediments.• Three sets of bacterial and archaeal primers appear validity and advantage in revealing the real trench microbial communities.• The novel primers provide a better option to specifically detect the unique microbial communities in extreme oceanic trench sediments.

A Pipeline for the Development of Microsatellite Markers using Next Generation Sequencing Data.

Background: Also known as Simple Sequence Repetitions (SSRs), microsatellites are profoundly informative molecular markers and powerful tools in genetics and ecology studies on plants. Objective: This research presents a workflow for developing microsatellite markers using genome skimming. Methods: The pipeline was proposed in several stages that must be performed sequentially: obtaining DNA sequences, identifying microsatellite regions, designing primers, and selecting candidate microsatellite regions to develop the markers. Our pipeline efficiency was analyzed using Illumina sequencing data from the non-model tree species Pterodon emarginatus Vog. Results: The pipeline revealed 4,382 microsatellite regions and drew 7,411 pairs of primers for P. emarginatus. However, a much larger number of microsatellite regions with the potential to develop markers were discovered from our pipeline. We selected 50 microsatellite regions with high potential for developing markers and organized 29 microsatellite regions in sets for multiplex PCR. Conclusion: The proposed pipeline is a powerful tool for fast and efficient development of microsatellite markers on a large scale in several species, especially nonmodel plant species.

New PCR-Based Assay for the Identification of Pectobacterium carotovorum Causing Potato Soft Rot.

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management because different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is Pectobacterium carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of Pectobacterium versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen Pectobacterium parmentieri. This should be useful for the routine diagnosis of potato soft rot.

CoVrimer: A tool for aligning SARS-CoV-2 primer sequences and selection of conserved/degenerate primers.

As mutations in SARS-CoV-2 virus accumulate rapidly, novel primers that amplify this virus sensitively and specifically are in demand. We have developed a webserver named CoVrimer by which users can search for and align existing or newly designed conserved/degenerate primer pair sequences against the viral genome and assess the mutation load of both primers and amplicons. CoVrimer uses mutation data obtained from an online platform established by NGDC-CNCB (12 May 2021) to identify genomic regions, either conserved or with low levels of mutations, from which potential primer pairs are designed and provided to the user for filtering based on generalized and SARS-CoV-2 specific parameters. Alignments of primers and probes can be visualized with respect to the reference genome, indicating variant details and the level of conservation. Consequently, CoVrimer is likely to help researchers with the challenges posed by viral evolution and is freely available at http://konulabapps.bilkent.edu.tr:3838/CoVrimer/.

Loop-Mediated Isothermal Amplification: From Theory to Practice.

Increasing the accuracy of pathogen identification and reducing the duration of analysis remain relevant for modern molecular diagnostics up to this day. In laboratory and clinical practice, detection of pathogens mostly relies on methods of nucleic acid amplification, among which the polymerase chain reaction (PCR) is considered the "gold standard." Nevertheless, in some cases, isothermal amplification methods act as an alternative to PCR diagnostics. Upon more than thirty years of the development of isothermal DNA synthesis, the appearance of loop-mediated isothermal amplification (LAMP) has enabled new directions of in-field diagnostics of bacterial and viral infections. This review examines the key characteristics of the LAMP method and corresponding features in practice. We discuss the structure of LAMP amplicons with single-stranded loops, which have the sites for primer annealing under isothermal conditions. The latest achievements in the modification of the LAMP method are analyzed, which allow considering it as a unique platform for creating the next-generation diagnostic assays.

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

BACKGROUND: Design of valid high-quality primers is essential for qPCR experiments. MRPrimer is a powerful pipeline based on MapReduce that combines both primer design for target sequences and homology tests on off-target sequences. It takes an entire sequence DB as input and returns all feasible and valid primer pairs existing in the DB. Due to the effectiveness of primers designed by MRPrimer in qPCR analysis, it has been widely used for developing many online design tools and building primer databases. However, the computational speed of MRPrimer is too slow to deal with the sizes of sequence DBs growing exponentially and thus must be improved. RESULTS: We develop a fast GPU-based pipeline for primer design (GPrimer) that takes the same input and returns the same output with MRPrimer. MRPrimer consists of a total of seven MapReduce steps, among which two steps are very time-consuming. GPrimer significantly improves the speed of those two steps by exploiting the computational power of GPUs. In particular, it designs data structures for coalesced memory access in GPU and workload balancing among GPU threads and copies the data structures between main memory and GPU memory in a streaming fashion. For human RefSeq DB, GPrimer achieves a speedup of 57 times for the entire steps and a speedup of 557 times for the most time-consuming step using a single machine of 4 GPUs, compared with MRPrimer running on a cluster of six machines. CONCLUSIONS: We propose a GPU-based pipeline for primer design that takes an entire sequence DB as input and returns all feasible and valid primer pairs existing in the DB at once without an additional step using BLAST-like tools. The software is available at https://github.com/qhtjrmin/GPrimer.git .