MFEprimer-3.0: quality control for PCR primers.

Quality control (QC) for lab-designed primers is crucial for the success of a polymerase chain reaction (PCR). Here, we present MFEprimer-3.0, a functional primer quality control program for checking non-specific amplicons, dimers, hairpins and other parameters. The new features of the current version include: (i) more sensitive binding site search using the updated k-mer algorithm that allows mismatches within the k-mer, except for the first base at the 3' end. The binding sites of each primer with a stable 3' end are listed in the output; (ii) new algorithms for rapidly identifying self-dimers, cross-dimers and hairpins; (iii) the command-line version, which has an added option of JSON output to enhance the versatility of MFEprimer by acting as a QC step in the 'primer design → quality control → redesign' pipeline; (iv) a function for checking whether the binding sites contain single nucleotide polymorphisms (SNPs), which will affect the consistency of binding efficiency among different samples. In summary, MFEprimer-3.0 is updated with the well-tested PCR primer QC program and it can be integrated into various PCR primer design applications as a QC module. The MFEprimer-3.0 server is freely accessible without any login requirement at: https://mfeprimer3.igenetech.com/ and https://www.mfeprimer.com/. The source code for the command-line version is available upon request.

sRNAPrimerDB: comprehensive primer design and search web service for small non-coding RNAs.

MOTIVATION: Small non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), play key roles in many biological processes. However, only a few tools can be used to develop the optimal primer or probe design for the expression profile of small ncRNAs. Here, we developed sRNAPrimerDB, the first automated primer designing and query web service for small ncRNAs. RESULTS: The primer online designing module of sRNAPrimerDB is composed of primer design algorithms and quality evaluation of the polymerase chain reaction (PCR) primer. Five types of primers, namely, generic or specific reverse transcription primers, specific PCR primers pairs, TaqMan probe, double-hairpin probe and hybridization probe for different small ncRNA detection methods, can be designed and searched using this service. The quality of PCR primers is further evaluated using melting temperature, primer dimer, hairpin structure and specificity. Moreover, the sequence and size of each amplicon are also provided for the subsequent experiment verification. At present, 531 306 and 2 941 669 primer pairs exist across 223 species for miRNAs and piRNAs, respectively, according to sRNAPrimerDB. Several primers designed by sRNAPrimerDB are further successfully validated by subsequent experiments. AVAILABILITY AND IMPLEMENTATION: sRNAPrimerDB is a valuable platform that can be used to detect small ncRNAs. This module can be publicly accessible at http://www.srnaprimerdb.com or http://123.57.239.141. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

[A consensus on the standardization of the next generation sequencing process for the diagnosis of genetic diseases (2) - Sample collection, processing and detection].

With high accuracy and precision, next generation sequencing (NGS) has provided a powerful tool for clinical testing of genetic diseases. To follow a standardized experimental procedure is the prerequisite to obtain stable, reliable, and effective NGS data for the assistance of diagnosis and/or screening of genetic diseases. At a conference of genetic testing industry held in Shanghai, May 2019, physicians engaged in the diagnosis and treatment of genetic diseases, experts engaged in clinical laboratory testing of genetic diseases and experts from third-party genetic testing companies have fully discussed the standardization of NGS procedures for the testing of genetic diseases. Experts from different backgrounds have provided opinions for the operation and implementation of NGS testing procedures including sample collection, reception, preservation, library construction, sequencing and data quality control. Based on the discussion, a consensus on the standardization of the testing procedures in NGS laboratories is developed with the aim to standardize NGS testing and accelerate implementation of NGS in clinical settings across China.

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia.

Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.

MFEprimer-2.0: a fast thermodynamics-based program for checking PCR primer specificity.

Evaluating the specificity of polymerase chain reaction (PCR) primers is an essential step in PCR primer design. The MFEprimer-2.0 server allows users to check primer specificity against genomic DNA and messenger RNA/complementary DNA sequence databases quickly and easily. MFEprimer-2.0 uses a k-mer index algorithm to accelerate the search process for primer binding sites and uses thermodynamics to evaluate binding stability between each primer and its DNA template. Several important characteristics, such as the sequence, melting temperature and size of each amplicon, either specific or non-specific, are reported on the results page. Based on these characteristics and the user-friendly output, users can readily draw conclusions about the specificity of PCR primers. Analyses for degenerate primers and multiple PCR primers are also supported in MFEprimer-2.0. In addition, the databases supported by MFEprimer-2.0 are comprehensive, and custom databases can also be supported on request. The MFEprimer-2.0 server does not require a login and is freely available at http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0. More over, the MFEprimer-2.0 command-line version and local server version are open source and can be downloaded at https://github.com/quwubin/MFEprimer/wiki/Manual/.

A reliable and quick method for screening alternative splicing variants for low-abundance genes.

Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS events, such as expressed sequence tags (EST), microarrays and RNA-seq. However, EST has limitations in identifying low-abundance genes, while microarray and RNA-seq are high-throughput technologies, and PCR-based technology is needed for validation. To overcome the limitations of EST and shortcomings of high-throughput technologies, we established a method to identify AS events, especially for low-abundance genes, by reverse transcription (RT) PCR with gene-specific primers (GSPs) followed by nested PCR. This process includes two major steps: 1) the use of GSPs to amplify as long as the specific gene segment and 2) multiple rounds of nested PCR to screen the AS and confirm the unknown splicing variants. With this method, we successfully identified three new splicing variants, namely, GenBank Accession No. HM623886 for the bdnf gene (GenBank GeneID: 12064), GenBank Accession No. JF417977 for the trkc gene (GenBank GeneID: 18213) and GenBank Accession No. HM623888 for the glb-18 gene (GenBank GeneID: 172485). In addition to its reliability and simplicity, the method is also cost-effective and labor-intensive. In conclusion, we developed an RT-nested PCR method using gene-specific primers to efficiently identify known and novel AS variants. This approach overcomes the limitations of existing methods for detecting rare transcripts. By enabling the discovery of new isoforms, especially for low-abundance genes, this technique can aid research into aberrant splicing in disease. Future studies can apply this method to uncover AS variants involved in cancer, neurodegeneration, and other splicing-related disorders.

GLB-13 is associated with oxidative stress resistance in Caenorhabditis elegans.

Globins constitute a superfamily of heme-binding proteins that is widely present in many species. There are 33 putative globins in the genome of Caenorhabditis elegans, where glb-13 is a homolog of neuroglobin (Ngb) based on sequence analysis and specific expression in neurons. Here we examined whether glb-13 as well as Ngb is also associated with resistance to reactive oxygen species (ROS) induced by paraquat. Our results showed that the mRNA level of glb-13 was significantly upregulated after paraquat exposure. Expression of a green fluorescent protein (GFP) reporter gene directed by the glb-13 promoter was increased by paraquat exposure. The mutant C. elegans strain glb-13(tm2825) was sensitive to paraquat-induced oxidative stress. Overexpression of human Ngb (hNgb) in C. elegans neuronal cells can rescue the paraquat sensitive phenotype of the mutant strain. glb-13 mutation or hNgb overexpression did not affect the expression of antioxidant enzymes such as superoxide dismutase (SOD). To examine the ROS-scavenging capabilities of hNgb and glb-13, we further examined the level of ROS in glb-13 mutant and hNgb transgenic (hNgb-Tg) worms. There was no statistical difference in ROS levels in the untreated controls; however in paraquat-treated worms, the ROS level was statistically repressed in the hNgb-Tg relative to enhanced green fluorescent protein (EGFP)-Tg worms or wildtype animals. Additionally, the ROS level of glb-13 mutant was statistically higher than the wildtype animals. Furthermore, hNgb overexpression diminished the ROS level of glb-13 mutant. In conclusion, hNgb can rescue the ROS sensitive phenotype of the glb-13 mutant strain. The protein GLB-13 seems to have an hNgb-like function, suggesting the importance of the globin protein family in maintaining the homeostasis of ROS signals. Our data provided evidence for the first time that glb-13 is associated with the resistance against oxidative stress-induced toxicity.

Genome-wide computational identification of bicistronic mRNA in humans.

Mammalian bicistronic mRNA is a recently discovered mammalian gene structure. Several reported cases of mammalian bicistronic mRNA indicated that genes of this structure play roles in some important biological processes. However, a genome-wide computational identification of bicistronic mRNA in mammalian genome, such as human genome, is still lacking. Here we used a comparative genomics approach to identify the frequency of human bicistronic mRNA. We then validated the result by using a new support vector machine (SVM) model. We identified 43 human bicistronic mRNAs in 30 distinct genes. Our literature analysis shows that our method recovered 100 % (6/6) of the previously known bicistronic mRNAs which had been experimentally confirmed by other groups. Our graph theory-based analysis and GO analysis indicated that human bicistronic mRNAs are prone to produce different yet closely functionally related proteins. In addition, we also described and analyzed three different mechanisms of ORF fusion. Our method of identifying bicistronic mRNAs in human genome provides a model for the computational identification of characteristic gene structures in mammalian genomes. We anticipate that our data will facilitate further molecular characterization and functional study of human bicistronic mRNA.

A Lasso regression model for the construction of microRNA-target regulatory networks.

MOTIVATION: MicroRNAs have recently emerged as a major class of regulatory molecules involved in a broad range of biological processes and complex diseases. Construction of miRNA-target regulatory networks can provide useful information for the study and diagnosis of complex diseases. Many sequence-based and evolutionary information-based methods have been developed to identify miRNA-mRNA targeting relationships. However, as the amount of available miRNA and gene expression data grows, a more statistical and systematic method combining sequence-based binding predictions and expression-based correlation data becomes necessary for the accurate identification of miRNA-mRNA pairs. RESULTS: We propose a Lasso regression model for the identification of miRNA-mRNA targeting relationships that combines sequence-based prediction information, miRNA co-regulation, RISC availability and miRNA/mRNA abundance data. By comparing this modelling approach with two other known methods applied to three different datasets, we found that the Lasso regression model has considerable advantages in both sensitivity and specificity. The regression coefficients in the model can be used to determine the true regulatory efficacies in tissues and was demonstrated using the miRNA target site type data. Finally, by constructing the miRNA regulatory networks in two stages of prostate cancer (PCa), we found the several significant miRNA-hubbed network modules associated with PCa metastasis. In conclusion, the Lasso regression model is a robust and informative tool for constructing the miRNA regulatory networks for diagnosis and treatment of complex diseases. AVAILABILITY: The R program for predicting miRNA-mRNA targeting relationships using the Lasso regression model is freely available, along with the described datasets and resulting regulatory network, at http://biocompute.bmi.ac.cn/CZlab/alarmnet/. The source code is open for modification and application to other miRNA/mRNA expression datasets. CONTACT: zhangcg@bmi.ac.cn SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

VizPrimer: a web server for visualized PCR primer design based on known gene structure.

SUMMARY: The visualization of gene structure plays an important role in polymerase chain reaction (PCR) primer design, especially for eukaryotic genes with a number of splice variants that users need to distinguish between via PCR. Here, we describe a visualized web server for primer design named VizPrimer. It utilizes the new information technology (IT) tools, HTML5 to display gene structure and JavaScript to interact with the users. In VizPrimer, the users can focus their attention on the gene structure and primer design strategy, without wasting time calculating the exon positions of splice variants or manually configuring complicated parameters. In addition, VizPrimer is also suitable for the design of PCR primers for amplifying open reading frames and detecting single nucleotide polymorphisms (SNPs). AVAILABILITY: VizPrimer is freely available at http://biocompute.bmi.ac.cn/CZlab/VizPrimer/. The web server supported browsers: Chrome (≥5.0), Firefox (≥3.0), Safari (≥4.0) and Opera (≥10.0). CONTACT: zhangcg@bmi.ac.cn; yangyi528@vip.sina.com.

PCR Primer Design for the Rapidly Evolving SARS-CoV-2 Genome.

Real-time quantitative PCR is currently the most widely used method for the human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identification. Due to the rapid evolution of the SARS-CoV-2 genome, novel mutations on the primer binding sites will cause the failure of PCR. Therefore, in addition to a well-designed primer set, these primers need to be updated and evaluated regularly to ensure that the rapidly evolving genome primers can be amplified. In this protocol, (1) we firstly use assembled genome sequences in the SARS-CoV-2 database to identify and characterize indels and point mutations; (2) design primers skipping the sites of mutations; (3) check the coverage of the primers with the daily update SARS-CoV-2 database; (4) redesign them if novel mutations found in the primer binding sites. Although this protocol takes SARS-CoV-2 as an example, it is suitable for other species that have genomes accumulating mutations over time.

Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens.

BACKGROUND: The Ahringer C. elegans RNAi feeding library prepared by cloning genomic DNA fragments has been widely used in genome-wide analysis of gene function. However, the library has not been thoroughly validated by direct sequencing, and there are potential errors, including: 1) mis-annotation (the clone with the retired gene name should be remapped to the actual target gene); 2) nonspecific PCR amplification; 3) cross-RNAi; 4) mis-operation such as sample loading error, etc. RESULTS: Here we performed a reliability analysis on the Ahringer C. elegans RNAi feeding library, which contains 16,256 bacterial strains, using a bioinformatics approach. Results demonstrated that most (98.3%) of the bacterial strains in the library are reliable. However, we also found that 2,851 (17.54%) bacterial strains need to be re-annotated even they are reliable. Most of these bacterial strains are the clones having the retired gene names. Besides, 28 strains are grouped into unreliable category and 226 strains are marginal because of probably expressing unrelated double-stranded RNAs (dsRNAs). The accuracy of the prediction was further confirmed by direct sequencing analysis of 496 bacterial strains. Finally, a freely accessible database named CelRNAi (http://biocompute.bmi.ac.cn/CelRNAi/) was developed as a valuable complement resource for the feeding RNAi library by providing the predicted information on all bacterial strains. Moreover, submission of the direct sequencing result or any other annotations for the bacterial strains to the database are allowed and will be integrated into the CelRNAi database to improve the accuracy of the library. In addition, we provide five candidate primer sets for each of the unreliable and marginal bacterial strains for users to construct an alternative vector for their own RNAi studies. CONCLUSIONS: Because of the potential unreliability of the Ahringer C. elegans RNAi feeding library, we strongly suggest the user examine the reliability information of the bacterial strains in the CelRNAi database before performing RNAi experiments, as well as the post-RNAi experiment analysis.

SCOPE2: A Platform for Sars-COv-2 Primer covErage Evaluation.

Currently, there is an increasing number and speed of SARS-CoV-2 mutation taking place around the world, posing a threat to promising public health and challenge to existing diagnostic tools. RT-PCR technology is recognized as the gold standard diagnosing methodology but has shown inaccuracy under some mutated SARS-CoV-2 circumstances. In this study, we developed a platform named SCOPE2 (Sars-COv-2 Primer covErage Evaluation) based on our previous publication. Testing by commonly-used SARS-COV-2 PCR primers, SCOPE2 is proved to effectively and efficiently assess the quality in terms of detection coverage, which may provide a practical tool for primer selection acceleration and primer design improvement.Clinical Relevance-This assists in single SARS-COV-2 Primer selection and suggestion of different SARS-COV-2 Primer combinations.

MPprimer: a program for reliable multiplex PCR primer design.

BACKGROUND: Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. RESULTS: A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2x to 5x plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20x, 20x, 20x, 14x, and 5x plex PCR reactions in five tubes to detect underlying exon deletions. CONCLUSIONS: MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

MFEprimer: multiple factor evaluation of the specificity of PCR primers.

SUMMARY: We developed a program named MFEprimer for evaluating the specificity of PCR primers based on multiple factors, including sequence similarity, stability at the 3'-end of the primer, melting temperature, GC content and number of binding sites between the primer and DNA templates. MFEprimer can help the user to select more suitable primers before running either standard or multiplex PCR reactions. The cDNA and genomic DNA databases of 10 widely used species, as well as user custom databases, were used as DNA templates for analyzing primers specificity. Furthermore, we maintained a Primer3Plus server with a modified Primer3Manager for one-stop primer design and specificity checking.

REMAS: a new regression model to identify alternative splicing events from exon array data.

BACKGROUND: Alternative splicing (AS) is an important regulatory mechanism for gene expression and protein diversity in eukaryotes. Previous studies have demonstrated that it can be causative for, or specific to splicing-related diseases. Understanding the regulation of AS will be helpful for diagnostic efforts and drug discoveries on those splicing-related diseases. As a novel exon-centric microarray platform, exon array enables a comprehensive analysis of AS by investigating the expression of known and predicted exons. Identifying of AS events from exon array has raised much attention, however, new and powerful algorithms for exon array data analysis are still absent till now. RESULTS: Here, we considered identifying of AS events in the framework of variable selection and developed a regression method for AS detection (REMAS). Firstly, features of alternatively spliced exons were scaled by reasonably defined variables. Secondly, we designed a hierarchical model which can represent gene structure and transcriptional influence to exons, and the lasso type penalties were introduced in calculation because of huge variable size. Thirdly, an iterative two-step algorithm was developed to select alternatively spliced genes and exons. To avoid negative effects introduced by small sample size, we ranked genes as parameters indicating their AS capabilities in an iterative manner. After that, both simulation and real data evaluation showed that REMAS could efficiently identify potential AS events, some of which had been validated by RT-PCR or supported by literature evidence. CONCLUSION: As a new lasso regression algorithm based on hierarchical model, REMAS has been demonstrated as a reliable and effective method to identify AS events from exon array data.

Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array.

BACKGROUND: The balance between endothelial cell survival and apoptosis during stress is an important cellular process for vessel integrity and vascular homeostasis, and it is also pivotal in angiogenesis during the development of many vascular diseases. However, the underlying molecular mechanisms remain largely unknown. Although both transcription and alternative splicing are important in regulating gene expression in endothelial cells under stress, the regulatory mechanisms underlying this state and their interactions have not yet been studied on a genome-wide basis. RESULTS: Human umbilical vein endothelial cells (HUVECs) were treated with cobalt chloride (CoCl2) both to mimic hypoxia and to induce cell apoptosis and alternative splicing responses. Cell apoptosis rate analysis indicated that HUVECs exposed to 300 microM CoCl2 for 24 hrs were initially counterbalancing apoptosis with cell survival. We therefore used the Affymetrix exon array system to determine genome-wide transcript- and exon-level differential expression. Other than 1583 differentially expressed transcripts, 342 alternatively spliced exons were detected and classified by different splicing types. Sixteen alternatively spliced exons were validated by RT-PCR. Furthermore, direct evidence for the ongoing balance between HUVEC survival and apoptosis was provided by Gene Ontology (GO) and protein function, as well as protein domain and pathway enrichment analyses of the differentially expressed transcripts. Importantly, a novel molecular module, in which the heat shock protein (HSP) families play a significant role, was found to be activated under mimicked hypoxia conditions. In addition, 46% of the transcripts containing stress-modulated exons were differentially expressed, indicating the possibility of combinatorial regulation of transcription and splicing. CONCLUSION: The exon array system effectively profiles gene expression and splicing on the genome-wide scale. Based on this approach, our data suggest that transcription and splicing not only regulate gene expression, but also carry out combinational regulation of the balance between survival and apoptosis of HUVECs under mimicked hypoxia conditions. Since cell survival following the apoptotic challenge is pivotal in angiogenesis during the development of many vascular diseases, our results may advance the knowledge of multilevel gene regulation in endothelial cells under physiological and pathological conditions.

CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif.

Designing efficient and specific CRISPR single-guide RNAs (sgRNAs) is vital for the successful application of CRISPR technology. Currently, a growing number of new RNA-guided endonucleases with a different protospacer adjacent motif (PAM) have been discovered, suggesting the necessity to develop a versatile tool for designing sgRNA to meet the requirement of different RNA-guided DNA endonucleases. Here, we report the development of a flexible sgRNA design program named "CRISPR-offinder". Support for user-defined PAM and sgRNA length was provided to increase the targeting range and specificity. Additionally, evaluation of on- and off-target scoring algorithms was integrated into the CRISPR-offinder. The CRISPR-offinder has provided the bench biologist a rapid and efficient tool for identification of high quality target sites, and it is freely available at https://sourceforge.net/projects/crispr-offinder-v1-2/ or http://www.biootools.com.

A MeSH-based text mining method for identifying novel prebiotics.

Prebiotics contribute to the well-being of their host by altering the composition of the gut microbiota. Discovering new prebiotics is a challenging and arduous task due to strict inclusion criteria; thus, highly limited numbers of prebiotic candidates have been identified. Notably, the large numbers of published studies may contain substantial information attached to various features of known prebiotics that can be used to predict new candidates. In this paper, we propose a medical subject headings (MeSH)-based text mining method for identifying new prebiotics with structured texts obtained from PubMed. We defined an optimal feature set for prebiotics prediction using a systematic feature-ranking algorithm with which a variety of carbohydrates can be accurately classified into different clusters in accordance with their chemical and biological attributes. The optimal feature set was used to separate positive prebiotics from other carbohydrates, and a cross-validation procedure was employed to assess the prediction accuracy of the model. Our method achieved a specificity of 0.876 and a sensitivity of 0.838. Finally, we identified a high-confidence list of candidates of prebiotics that are strongly supported by the literature. Our study demonstrates that text mining from high-volume biomedical literature is a promising approach in searching for new prebiotics.

Selecting specific PCR primers with MFEprimer.

Selecting specific primers is crucial for polymerase chain reaction (PCR). Nonspecific primers will bind to unintended genes and result in nonspecific amplicons. MFEprimer is a program for checking the specificity of PCR primers against the background DNA. In this chapter, we introduce: (1) the factors that affect the specificity of primers; (2) the principle of MFEprimer and its settings; (3) how to use the MFEprimer to examine the specificity of primers.