Degenerate Primer Design Research Articles

A novel heat-inducible plasmid-driven T7 system for enhancing carbonic anhydrase in engineered Escherichia coli strains

Over the past decades, the heat-inducible (HI) promoter, derived from cI857 phage system has been widely utilized. However, the developments of alternative temperature-controlled promoters are limited. In this study, we explored a novel HI promoter through PCR amplification using the degenerate primer design. BLAST analysis identified this sequence as a partial plsB on Escherichia coli chromosome. Antisense RNAs targeting distinct regions of HI promoter were applied to examine the mechanism. The fluorescence of super-folder green fluorescent protein (sfGFP) driven by the HI promoter showed 5.5-fold increase from 30 to 42 °C, whereas the truncated HI yielded a trace amount of sfGFP. Among various E. coli strains, BL21 exhibited the highest fluorescence, reaching 3976 a.u. at 42 °C. Subsequently, the novel HI promoter was employed to drive T7RNA polymerase within a plasmid-driven T7 (PDT7) plasmid, serving its capability to express sfGFP and carbonic anhydrases (CA), respectively. The maximum intensity of sfGFP reached 38,702 a.u., and CA activity surged to 14286 WAU/mL in W3110 among other strains. Finally, the highest CA activity was 27,521 WAU/mL at pH 9. The promising results from the novel heat-inducible promoter-driven T7RNAP, incorporating the T7 terminator as HI-PDT7-TT, demonstrate potential for expressing more heterogeneous proteins across various chassis in the future.

A tool to automatically design multiplex PCR primer pairs for specific targets using diverse templates

Multiplex PCR is an increasingly popular method for identifying species, investigating environmental diversity, and conducting phylogenetic analysis. The complexity and increasing availability of diverse templates necessitate a highly automated approach to design degenerate primer pairs for specific targets with multiple sequences. Existing tools for degenerate primer design suffer from poor maintenance, semi-automation, low adaptability, and low tolerance for gaps. We developed PMPrimer, a Python-based tool for automated design and evaluation of multiplex PCR primer pairs for specific targets using diverse templates. PMPrimer automatically designs optimal multiplex PCR primer pairs using a statistical-based template filter; performs multiple sequence alignment, conserved region identification, and primer design; and evaluates the primers based on template coverage, taxon specificity, and target specificity. PMPrimer identifies conserved regions using Shannon’s entropy method, tolerates gaps using a haplotype-based method, and evaluates multiplex PCR primer pairs based on template coverage and taxon specificity. We tested PMPrimer using datasets with diverse levels of conservation, sizes, and applications, including tuf genes of Staphylococci, hsp65 genes of Mycobacteriaceae, and 16S ribosomal RNA genes of Archaea. PMPrimer showed outstanding performance compared with existing tools and experimental validated primers. PMPrimer is available as a Python package at https://github.com/AGIScuipeng/PMPrimer.

A Degenerate Primer Design Method Based on Ant Colony Optimization

For the existed biological degenerate primer design problem, a solution method based on an ant colony optimization algorithm is proposed, in which an optimization model considers primer coverage as well as degeneracy while allowing for a small number of base mismatches. Using the construction graph model, the algorithm makes full use of the dynamic change information during the iterative optimal path optimization process, strengthens the explored new path pheromone, and selects the primers that meet the constraints. The results of DNA template sequence matching experiments show that the degenerate primers designed by the proposed algorithm outperform existing approaches.

Optimized Design of Degenerate Primers for PCR Based on DNA or Protein Sequence Comparisons.

PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in three different applications, including the use of specific determinants at the 3'-end, to (i) improve PCR efficiency with related sequences for members of a protein family by complete degeneration at a core box of conserved genetic information at the 3'-end with the reduction of degeneration at the 5'-end, (ii) optimize specificity of allelic discrimination of closely related DNA sequences of orthologous by 5'-end fully degenerate primers, and (iii) increase the PCR efficiency of primers by targeting DNA sequences belonging to specific phylogenetic groups, within a large and diverse gene family, allowing the use of multiplex/degenerate PCR.

EntroPhylo: An entropy-based tool to select phylogenetic informative regions and primer design.

We present a novel entropy-based computational tool that selects phylogenetic informative genomic regions associated with degenerate primer design. This tool identifies proper phylogenetic markers and proposes suitable degenerate primers to amplify and sequence them. The algorithm calculates the entropy value per site, and the selected region is used for primer design. In order to evaluate the tool, sequences of bovine papillomavirus L1 gene were obtained. Once the molecular region was selected, the primers were designed by the software and used in a PCR reaction for viral detection. Three positive samples were tested with four different concentrations, and it was possible to detect the virus in all samples. The results show the applicability of a tool that can select informative regions for phylogenetic analysis and design primers to amplify and sequence these regions, becoming relevant for several studies focusing on pathogen detection, as well as phylogenetic and genetics studies of populations.

In silico design and validation of a highly degenerate primer pair: a systematic approach

BackgroundThe techniques of amplifying genetic materials have enabled the extensive study of several biological activities outside the biological milieu of living systems. More recently, this approach has been extended to amplify population of genes, from evolutionarily related gene family for detection and evaluation of microbial consortial with several unique potentialities (e.g., enzymatic degradability). Conceivably, primer mixtures containing substitutions of different bases at specific sites (degenerate primers) have enabled the amplification of these genes in PCR reaction. However, the degenerate primer design problem (DPD) is a constraint to designing this kind of primer. To date, different algorithms now exist to solve various versions of DPD problem, many of which, only few addresses and satisfy the criteria to design primers that can extensively cover high through-put sequences while striking the balance between specificity and efficiency. The highly degenerate primer (HYDEN) design software program primarily addresses this variant of DPD problem termed “maximum coverage-degenerate primer design (MC-DPD)” and its heuristics have been substantiated for optimal efficiency from significant successes in PCR. In spite of the premium presented for designing degenerate primers, literature search has indicated relatively little use of its heuristics. This has been thought to result from the complexity of the program since it is run only by command-line, hence limiting its accessibility. To solve this problem, researchers have optionally considered the manual design of degenerate primers or design through software programs that provides accessibility through a graphical user interface (GUI). Realizing this, we have attempted in this study to provide a user-friendly approach for researchers with little or no background in bioinformatics to design degenerate primers using HYDEN ResultsVirtual Tests of our designed degenerate primer pair through in silico PCR substantiated the correspondence between efficiency and coverage with the target sequences as pre-defined by the initial HYDEN output, thereby validating the potentials of HYDEN to effectively solve the MC-DPD problem. Additionally, the designed primer-pair mechanistically amplified all sequences used as a positive control with no amplification observed in the negative controls. ConclusionIn this study, we provided a turnkey protocol to simplify the design of degenerate primers using the heuristics of the HYDEN software program.

ARDEP, a Rapid Degenerate Primer Design Pipeline Based on k-mers for Amplicon Microbiome Studies.

The survey of microbial diversity in various environments has relied upon the widespread use of well-evaluated amplification primers for taxonomic marker genes (e.g., prokaryotic 16S and fungal ITS). However, it is urgent to develop a fast and accurate bioinformatic program to design primers for microbial functional genes to explore more mechanisms in the microbial community. Here, we provide a rapid degenerate primer design pipeline (ARDEP) based on the k-mer algorithm, which can bypass the time-consuming step of sequence alignment to greatly reduce run times while ensuring accuracy. In addition, we developed an open-access platform for the implementation of primer design projects that could also calculate the amplification product length, GC content, Annealing Temperature (Tm), and ΔG of primer self-folding, and identify covered species and functional groups. Using this new platform, we designed primers for several functional genes in the nitrogen cycle, including napA and amoA. Our newly designed primers achieved higher coverage than the commonly used primers for all tested genes. The program and the associated platform that applied the k-mer algorithm could greatly enhance the design and evaluation of primers for environmental microbiome studies.

Human T-cell receptor V gene segment of alpha and beta families: A revised primer design strategy.

The application of human TCR in cancer immunotherapy has gained momentum with developments in tumor killing strategies using endogenous adaptive immune responses. The successful coverage of a diverse TCR repertoire is mainly attributed to the primer design of the human TCR V genes. Here, we present a refined primer design strategy of the human TCR V gene by clustering V gene sequence homolog for degenerate primer design based on the data from IMGT. The primers designed were analyzed and the PCR efficiency of each primer set was optimized. A total of 112 alpha and 160 beta sequences were aligned and clustered using a phylogram yielding 32 and 27 V gene primers for the alpha and beta family. The new primer set was able to provide 93.75% and 95.63% coverage for the alpha and beta family, respectively. A semi-qualitative approach using the designed primer set was able to provide a relative view of the TCR V gene diversity in different populations. Taken together, the new primers provide a more comprehensive coverage of the TCR gene diversity for improved TCR library generation and TCR V gene analysis studies.

Identification of Two Genotypes of Argas persicus and Associated Rickettsia-Specific Genes from Different Regions of Inner Mongolia

Ticks are important vectors and reservoirs of several zoonotic pathogens. Recently, both known and unknown tick-borne pathogens have emerged and re-emerged, causing unpredictable epidemics. In this study, 211 soft tick samples were collected from Tongliao and Alxa in Inner Mongolia, China. Tick species were identified by morphological and molecular biological analyses. Morphological analysis showed that there was no significant difference in surface features between ticks from the 2 areas. Cloning by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene showed that all ticks belonged to the species Argas persicus. Analysis using Genetyx software indicated that there was a limited degree of diversity between ticks from the 2 areas. Three base changes were detected in the 16S rRNA gene. We constructed phylogenetic trees using MEGA 6.0 software and showed that the ticks from the 2 areas might have evolved independently from species in other geographical areas. To assess the presence of Rickettsia, Streptococcus suis, and Staphylococcus aureus pathogens in tick samples, over 100 16S rRNA sequences belonging to these 3 pathogens were obtained from GenBank. We used CLC Sequence Viewer 7.0 to determine conserved sequences for the design of degenerate primers. Using standard PCR, we detected Rickettsia-specific genes, including 16S rRNA, 17KD, and ompB, in gDNA samples of ticks from Alxa. This study has laid a foundation for future studies on the biodiversity of ticks and for a new pathogen information database of ticks in local areas.

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Real-Time Polymerase Chain Reaction (Real-Time PCR).

Baculoviridae is a highly diverse family of rod-shaped viruses with double-stranded DNA. To date, almost 100 species have had their complete genomic sequences deposited in the GenBank database, a quarter of which comprises granuloviruses (GVs). Many of the genomes are sequenced using next-generation sequencing, which is currently considered the best method for characterizing new species, but it is time-consuming and expensive. Baculoviruses form a safe alternative to overused chemical pesticides and therefore there is a constant need for identifying new species that can be active components of novel biological insecticides. In this study, we have described a fast and reliable method for the detection of new and differentiation of previously analyzed granulovirus species based on a real-time polymerase chain reaction (PCR) technique with melting point curve analysis. The sequences of highly conserved baculovirus genes, such as granulin and late expression factors 8 and 9 (lef-8 and lef-9), derived from GVs available to date have been analyzed and used for degenerate primer design. The developed method was tested on a representative group of eight betabaculoviruses with comparisons of melting temperatures to allow for quick and preliminary granulovirus detection. The proposed real-time PCR procedure may be a very useful tool as an easily accessible screening method in a majority of laboratories.

Base-By-Base Version 3: New Comparative Tools for Large Virus Genomes.

Base-By-Base is a comprehensive tool for the creation and editing of multiple sequence alignments that is coded in Java and runs on multiple platforms. It can be used with gene and protein sequences as well as with large viral genomes, which themselves can contain gene annotations. This report describes new features added to Base-By-Base over the last 7 years. The two most significant additions are: (1) The recoding and inclusion of “consensus-degenerate hybrid oligonucleotide primers” (CODEHOP), a popular tool for the design of degenerate primers from a multiple sequence alignment of proteins; and (2) the ability to perform fuzzy searches within the columns of sequence data in multiple sequence alignments to determine the distribution of sequence variants among the sequences. The intuitive interface focuses on the presentation of results in easily understood visualizations and providing the ability to annotate the sequences in a multiple alignment with analytic and user data.

A Diverse Range of Human Gut Bacteria Have the Potential To Metabolize the Dietary Component Gallic Acid.

The human gut microbiota contains a broad variety of bacteria that possess functional genes, with resultant metabolites that affect human physiology and therefore health. Dietary gallates are phenolic components that are present in many foods and beverages and are regarded as having health-promoting attributes. However, the potential for metabolism of these phenolic compounds by the human microbiota remains largely unknown. The emergence of high-throughput sequencing (HTS) technologies allows this issue to be addressed. In this study, HTS was used to assess the incidence of gallate-decarboxylating bacteria within the gut microbiota of healthy individuals for whom bacterial diversity was previously determined to be high. This process was facilitated by the design and application of degenerate PCR primers to amplify a region encoding the catalytic C subunit of gallate decarboxylase (LpdC) from total metagenomic DNA extracted from human fecal samples. HTS resulted in the generation of a total of 3,261,967 sequence reads and revealed that the primary gallate-decarboxylating microbial phyla in the intestinal microbiota were Firmicutes (74.6%), Proteobacteria (17.6%), and Actinobacteria (7.8%). These reads corresponded to 53 genera, i.e., 47% of the bacterial genera detected previously in these samples. Among these genera, Anaerostipes and Klebsiella accounted for the majority of reads (40%). The usefulness of the HTS-lpdC method was demonstrated by the production of pyrogallol from gallic acid, as expected for functional gallate decarboxylases, among representative strains belonging to species identified in the human gut microbiota by this method.IMPORTANCE Despite the increasing wealth of sequencing data, the health contributions of many bacteria found in the human gut microbiota have yet to be elucidated. This study applies a novel experimental approach to predict the ability of gut microbes to carry out a specific metabolic activity, i.e., gallate metabolism. The study showed that, while gallate-decarboxylating bacteria represented 47% of the bacterial genera detected previously in the same human fecal samples, no gallate decarboxylase homologs were identified from representatives of Bacteroidetes The presence of functional gallate decarboxylases was demonstrated in representative Proteobacteria and Firmicutes strains from the human microbiota, an observation that could be of considerable relevance to the in vivo production of pyrogallol, a physiologically important bioactive compound.

芒果F3'5'H基因的克隆及其表达分析

黄酮-3',5'-羟基化酶(flavonoid-3',5'-hydroxylase, F3'5'H)是合成飞燕草色素苷的关键酶基因，缺乏F3'5'H的植物不能形成蓝色花，故又被称为“蓝色基因”。本研究根据已经报道的F3'5'H基因的序列设计兼并引物，采用3'RACE和5'RACE方法，克隆得到了一个全长cDNA序列为1598 bp的芒果果实F3'5'H基因。该基因开放阅读框为1539 bp，编码512个氨基酸，分子重量为57.20 KD。对基因组扩增得到了1633 bp长度的片段，分析发现该基因含有一个内含子。通过系统发育分析发现该基因编码的蛋白与石斛、马鞭草等植物具有较近的亲缘关系。对不同芒果品种的F3'5'H基因的表达进行分析发现：红色的贵妃品种中表达量较高，而绿色的桂七品种中表达量较低。 Flavonoid-3',5'-hydroxylase (F3'5'H), was the key enzyme gene for synthesizing delphinidin glycosides; due to the lack of F3'5'H, plants cannot form a blue flower, which was also known as the “blue gene”. According to the design of degenerate primers of F3’5’H gene sequence reported, full- length cDNA sequence of F3'5'H gene of mango fruit was obtained with 3'RACE and 5'RACE method. The full-length cDNA sequence of F3'5'H was 1598 bp, whose open reading frame of the gene was 1539 bp, encoding 512 amino acids, with the molecular weight of 57.20 KD. The 1633 bp length fragment was amplified from genome and analysis, which contained one intron, in 909 - 1003 bp. It was found that the gene encoding for the protein had close relationship with dendrobium, verbena plant through phylogenetic analysis. Expression of F3'5'H gene in different mango varieties showed: the high expression of the “Guifei” varieties, and the expression of green Guiqi varieties with low volume.

CEMAsuite: open source degenerate PCR primer design.

The codon-equivalent multiple alignment suite begins conservational analysis for polymerase chain reaction primer design at the protein level, allowing the user to design consensus primers capable of detecting homologous coding sequences even when low-to-moderate sequence information is available. This package also condenses the wealth of information associated with multiple sequence alignments and presents them in an intuitive manner, allowing the user to quickly and effectively address degenerate primer design considerations. https://sourceforge.net/projects/cemasuite/. benton@lsu.edu or cemasuite@gmail.com Supplementary data are available at Bioinformatics online.

GPUDePiCt: A Parallel Implementation of a Clustering Algorithm for Computing Degenerate Primers on Graphics Processing Units.

In order to make multiple copies of a target sequence in the laboratory, the technique of Polymerase Chain Reaction (PCR) requires the design of "primers", which are short fragments of nucleotides complementary to the flanking regions of the target sequence. If the same primer is to amplify multiple closely related target sequences, then it is necessary to make the primers "degenerate", which would allow it to hybridize to target sequences with a limited amount of variability that may have been caused by mutations. However, the PCR technique can only allow a limited amount of degeneracy, and therefore the design of degenerate primers requires the identification of reasonably well-conserved regions in the input sequences. We take an existing algorithm for designing degenerate primers that is based on clustering and parallelize it in a web-accessible software package GPUDePiCt, using a shared memory model and the computing power of Graphics Processing Units (GPUs). We test our implementation on large sets of aligned sequences from the human genome and show a multi-fold speedup for clustering using our hybrid GPU/CPU implementation over a pure CPU approach for these sequences, which consist of more than 7,500 nucleotides. We also demonstrate that this speedup is consistent over larger numbers and longer lengths of aligned sequences.

Multiplex degenerate primer design for targeted whole genome amplification of many viral genomes.

Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions. This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.

DegePrime, a program for degenerate primer design for broad-taxonomic-range PCR in microbial ecology studies.

The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.

Family-Specific Degenerate Primer Design: A Tool to Design Consensus Degenerated Oligonucleotides

Designing degenerate PCR primers for templates of unknown nucleotide sequence may be a very difficult task. In this paper, we present a new method to design degenerate primers, implemented in family-specific degenerate primer design (FAS-DPD) computer software, for which the starting point is a multiple alignment of related amino acids or nucleotide sequences. To assess their efficiency, four different genome collections were used, covering a wide range of genomic lengths: Arenavirus (10 × 104 nucleotides), Baculovirus (0.9 × 105 to 1.8 × 105 bp), Lactobacillus sp. (1 × 106 to 2 × 106 bp), and Pseudomonas sp. (4 × 106 to 7 × 106 bp). In each case, FAS-DPD designed primers were tested computationally to measure specificity. Designed primers for Arenavirus and Baculovirus were tested experimentally. The method presented here is useful for designing degenerate primers on collections of related protein sequences, allowing detection of new family members.

Metagenomic and whole-genome analysis reveals new lineages of gokushoviruses and biogeographic separation in the sea

Much remains to be learned about single-stranded (ss) DNA viruses in natural systems, and the evolutionary relationships among them. One of the eight recognized families of ssDNA viruses is the Microviridae, a group of viruses infecting bacteria. In this study we used metagenomic analysis, genome assembly, and amplicon sequencing of purified ssDNA to show that bacteriophages belonging to the subfamily Gokushovirinae within the Microviridae are genetically diverse and widespread members of marine microbial communities. Metagenomic analysis of coastal samples from the Gulf of Mexico (GOM) and British Columbia, Canada, revealed numerous sequences belonging to gokushoviruses and allowed the assembly of five putative genomes with an organization similar to chlamydiamicroviruses. Fragment recruitment to these genomes from different metagenomic data sets is consistent with gokushovirus genotypes being restricted to specific oceanic regions. Conservation among the assembled genomes allowed the design of degenerate primers that target an 800 bp fragment from the gene encoding the major capsid protein. Sequences could be amplified from coastal temperate and subtropical waters, but not from samples collected from the Arctic Ocean, or freshwater lakes. Phylogenetic analysis revealed that most sequences were distantly related to those from cultured representatives. Moreover, the sequences fell into at least seven distinct evolutionary groups, most of which were represented by one of the assembled metagenomes. Our results greatly expand the known sequence space for gokushoviruses, and reveal biogeographic separation and new evolutionary lineages of gokushoviruses in the oceans.

Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing

BackgroundIn a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally.ResultsWe have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute’s (JCVI) high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates.ConclusionsAugmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus sequence that encapsulates the allelic variation of the targeted population and is a key step prior to designing degenerate primers is also formally described.