ResearchPad - methods-manuscript https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[MALDI-TOF MS-based analysis of dried seed proteins immobilized on filter paper]]> https://www.researchpad.co/article/elastic_article_10895 Matrix-assisted laser-desorption and ionization time-of-flight mass spectroscopy (MALDI-TOF MS) is commonly used for the characterization of protein-containing biological samples. For this, we have previously developed sample-preparation methods that can be used for discrimination between Impatiens species and also between regional biotypes of Himalayan balsam (Impatiens glandulifera), initially using leaf samples and, more recently, using seed material. In the current article, we have developed a further MALDI-TOF MS-based method that can be used with seeds that uses only simple equipment and minimally hazardous reagents prior to storing and/or shipping dried seed proteins immobilized on filter paper for MALDI-TOF MS analysis. We have investigated I. glandulifera regional-biotype seeds originating from four different sites within the UK for which the parent plants differ in their susceptibility to the biological control agent Puccinia komarovii var. glanduliferae. Using a combination of time-course comparisons and principal-component analysis, we have demonstrated good MALDI-TOF MS spectral conservation, even after storage for 1 month at 35°C, of dried seed-protein samples immobilized on filter paper. This method may provide a further useful tool for the matching of biological control agents optimally to susceptible (regional) target-plant biotypes, and for seed characterization and/or identification in general.

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<![CDATA[MALDI-TOF MS protein fingerprinting of mixed samples]]> https://www.researchpad.co/article/elastic_article_10893 Analytical techniques currently available for the characterization of mixtures of microorganisms are generally based on next-generation sequencing. Motivated to develop practical and less-expensive methods for characterizing such mixtures, we propose, as an alternative or complement, the use of matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is capable of high-resolution discrimination between species and even between biotypes within species. Potential approaches employing this technique for such characterization are discussed along with impediments to their successful employment. As a consequence, our rationale has been to capitalize on the powerful algorithms currently available for spectral comparison. Following this rationale, the first priority is to ensure the generation of MALDI-TOF MS spectra from mixtures of microorganisms that contain manageable peak complexities and that can be handled by the existing spectral comparison algorithms, preferably with the option to archive and re-run sample preparations and to pipette replicates of these onto MALDI-TOF MS sample plates. The second priority is to ensure that database entry is comparably facile to sample preparation so that large databases of known microorganism mixture MALDI-TOF MS spectra could be readily prepared for comparison with the spectra of unknown mixtures. In this article, we address the above priorities and generate illustrative MALDI-TOF MS spectra to demonstrate the utility of this approach. In addition, we investigate methods aimed at chemically modulating the peak complexity of the obtained MALDI-TOF MS spectra.

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<![CDATA[Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems]]> https://www.researchpad.co/article/elastic_article_10891 Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how “self-avoiding molecular recognition systems” (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer–primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.

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<![CDATA[Plasma or serum? A qualitative study on rodents and humans using high-throughput microRNA sequencing for circulating biomarkers]]> https://www.researchpad.co/article/elastic_article_10890 microRNAs are small non-coding RNAs gaining interest for their potential roles as reliable biomarkers for the diagnosis and therapeutics of numerous pathologies, ranging from cancer to neurodegenerative or psychiatric disorders. Indeed, microRNAs are present in various accessible biofluids, including peripheral blood, and specific dysregulation of their expression may be associated with these different pathological conditions. microRNAs can be isolated from plasma or serum for sequencing with commercial kits. However, these two biofluids might exhibit some differences in their microRNA contents, due notably to the coagulation process occurring during serum collection. It remains unclear from previous studies and commercial recommendations which blood fraction is preferable. Because of the small amount of circulating microRNAs in a given blood volume, this question appears crucial for qualitative and quantitative optimization of microRNA profiling, especially in animal models used for investigating the pathophysiological relevancy of this approach. We therefore evaluated the efficiency of RNA isolation and microRNA levels from plasma and sera isolated from rats and humans, with a widely used extraction kit (QIAGEN miRNeasy), and assessed microRNA quality and quantity with high-throughput sequencing. Fewer reads with length corresponding to non-miRNAs sequences were observed in plasma than in serum, both from rats and humans. Moreover, rat plasma produced twice as many aligned reads compared to sera, as well as more aligned reads corresponding to microRNAs (84.6% against 38.7%), differences that were not find in human samples. Our results, therefore, clearly indicate that plasma should be preferred for miRNA investigations, particularly for translational studies.

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<![CDATA[Generation of <sup>34</sup>S-substituted protein-bound [4Fe-4S] clusters using <sup>34</sup>S-L-cysteine]]> https://www.researchpad.co/article/elastic_article_10888 The ability to specifically label the sulphide ions of protein-bound iron–sulphur (FeS) clusters with 34S isotope greatly facilitates structure–function studies. In particular, it provides insight when using either spectroscopic techniques that probe cluster-associated vibrations, or non-denaturing mass spectrometry, where the ∼+2 Da average increase per sulphide enables unambiguous assignment of the FeS cluster and, where relevant, its conversion/degradation products. Here, we employ a thermostable homologue of the O-acetyl-l-serine sulfhydrylase CysK to generate 34S-substituted l-cysteine and subsequently use it as a substrate for the l-cysteine desulfurase NifS to gradually supply 34S2− for in vitro FeS cluster assembly in an otherwise standard cluster reconstitution protocol.

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<![CDATA[Enzyme-linked oligonucleotide hybridization assay for direct oligo measurement in blood]]> https://www.researchpad.co/article/elastic_article_10886 Small oligonucleotides (oligos) are increasingly being utilized as diagnostics or treatments for disease. An impediment to broader use is the ability to readily measure oligos in biological fluids. Here, we describe a very straightforward assay with detection in the sub-picomole range that does not require extraction from serum/plasma or polymerization chain reaction amplification. As a result, there are no losses or errors due to sample handling, and the assay can be used to measure oligos modified in a variety of ways that increase therapeutic efficacy. The enzyme-linked oligonucleotide hybridization assay (ELOHA) is based on competition with a detection oligo for hybridization to a capture oligo covalently linked to a solid substrate. The versatility of ELOHAs is demonstrated by application to the measurement of three oligos, including two morpholino-oligos with 3′-octaguanidine derivatization for efficient cell uptake. The third oligo is unmodified and has a DNA sequence equivalent to miR93. The assays have sensitivity as low as 0.28 pmol/sample reaction at 50% hybridization. Adding to clinical utility is the need for only a simple 96-well absorbance plate reader and the finding that neither EDTA nor heparin interferes with detection.

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<![CDATA[Using SimulATe to model the effects of antibiotic selective pressure on the dynamics of pathogenic bacterial populations]]> https://www.researchpad.co/article/elastic_article_10885 Antibiotics are notable weapons in fighting bacteria. Nowadays, however, the effectiveness of antibiotics is severely hindered by the increasing levels of antibiotic resistances in pathogenic bacterial populations, which can persist due to the selective pressure caused by antibiotic exposure. Arguably, the main cause of antibiotic resistances endurance in nature is antibiotic misuse, such as via overusing, inappropriate prescribing as well as the uncontrolled use in agriculture and livestock. There is also a lack of knowledge on appropriate antibiotic usage by the general public. Public scientific literacy and more research on therapeutic practices are fundamental to tackle this problem. Here, we present SimulATe a software which allows the simulation of the effects of antibiotic therapies on bacterial populations during human infections. This software can be used to develop students’ scientific literacy, using infections and antibiotic treatments as context to engage students in scientific practices, and discussions on antibiotic treatment onset and duration or on its use in immunosuppressed or critically ill individuals. SimulATe’s features also allow it to be used for research purposes allowing the simulation of real scenarios and exploration of their outcomes across the parameters’ landscape.

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<![CDATA[A method for filamentous fungal growth and sample preparation aimed at more consistent MALDI-TOF MS spectra despite variations in growth rates and/or incubation times]]> https://www.researchpad.co/article/elastic_article_10884 Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry can be used for the characterization and identification of filamentous fungi, for which it is desirable to have a means of growth in which the resulting spectra remain as consistent as possible over time. To this end, we initially opted for growth in oil-overlaid small-volume liquid culture, using a medium (Czapek Dox) not containing significant amount of proteins or peptides, and with protein extraction from the entire culture volume. For both 3-week and 10-day time courses, however, we observed marked spectral changes over growth time, along with lower peak richness compared to agar-plate controls. Guided by the above, we next employed a more nutrient-rich MALDI-TOF MS-compatible liquid-culture medium, now used without an oil overlay. For a 10-day time course, we again observed marked spectral changes over growth time, along with lower peak richness compared to agar-plate controls. Finally, we opted for a method employing filter-paper-supported growth in the same MALDI-TOF MS-compatible rich medium within sealed 1.5 ml Eppendorf tubes, again with protein extraction from the entire culture volume. Using this final method, while we observed significant spectral changes between 2 days and 3 days, from 3 days to 10 days the spectra remained very consistent, with comparable peak richness to agar-plate controls. This method gave slightly better identifications and lower spectral variance compared to agar-plate controls, and the use of this method for the construction of growth-time-point-specific databases for fungal identification is discussed.

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<![CDATA[Discrimination between Asian populations of the parasitoid wasp <i>Ganaspis</i> cf. <i>brasiliensis</i> using a simple MALDI-TOF MS-based method for use with insects]]> https://www.researchpad.co/article/elastic_article_8858 The fruit fly Drosophila suzukii has recently become an invasive pest insect of significant economic impact in Europe and the USA. In contrast to other Drosophila species, D. suzukii is able to infest intact fruit by means of a saw-like ovipositor, which allows females to deposit eggs beneath the skin of the fruit. Classical biological control using the parasitoid wasp Ganaspis cf. brasiliensis is currently being researched as an environmentally sustainable option for the control of D. suzukii. In particular, the host specificity of this parasitoid has been assessed for populations from different regions in China and Japan. In order to study the relationship between the differences in specificity and molecular variations, we have adapted a matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based method, originally developed for use with plant material, to discriminate between example populations of G. cf. brasiliensis. We have employed a combination of principal component analysis and blind-tested comparison between reference sample MALDI-TOF MS spectra and test sample spectra to discriminate, on the basis of the acid-soluble insect protein spectra generated, between four populations of G. cf. brasiliensis (originally collected from Tokyo and Hasuike in Japan and Dali and Ximing in China). MALDI-TOF MS analysis is able to discriminate with 100% accuracy between populations G. cf. brasiliensis. The Chinese populations were observed to be similar, but the Tokyo population is slightly different and the Hasuike population is significantly different from the other populations. The Tokyo population appears more closely related to the Chinese populations than the Hasuike population, even though both originate from Japan.

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<![CDATA[Genome editing in the nematode <i>Caenorhabditis briggsae</i> using the CRISPR/Cas9 system]]> https://www.researchpad.co/article/N74099e0f-492b-4ab2-b5c1-42be553f7b96 The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.

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<![CDATA[Process for an efficient lentiviral cell transduction]]> https://www.researchpad.co/article/N93655598-5dc3-4a95-bd88-650e319eab69 The combination of lentiviruses with techniques such as CRISPR-Cas9 has resulted in efficient and precise processes for targeted genome modification. An often-limiting aspect, however, is the efficiency of cell transduction. Low efficiencies with particular cell types and/or the high complexity of lentiviral libraries can cause insufficient representation. Here, we present a protocol that yielded substantial increases in transduction efficiency in various cell lines in comparison to several other procedures.

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<![CDATA[A bioinformatic pipeline to analyze ChIP-exo datasets]]> https://www.researchpad.co/article/N5e123f09-0388-47c9-ab5a-755eb83a5ad3 The decrease of sequencing cost in the recent years has made genome-wide studies of transcription factor (TF) binding through chromatin immunoprecipitation methods like ChIP-seq and chromatin immunoprecipitation with lambda exonuclease (ChIP-exo) more accessible to a broader group of users. Especially with ChIP-exo, it is now possible to map TF binding sites in more detail and with less noise than previously possible. These improvements came at the cost of making the analysis of the data more challenging, which is further complicated by the fact that to this date no complete pipeline is publicly available. Here we present a workflow developed specifically for ChIP-exo data and demonstrate its capabilities for data analysis. The pipeline, which is completely publicly available on GitHub, includes all necessary analytical steps to obtain a high confidence list of TF targets starting from raw sequencing reads. During the pipeline development, we emphasized the inclusion of different quality control measurements and we show how to use these so users can have confidence in their obtained results.

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<![CDATA[Target enrichment from a DNA mixture by oligoribonucleotide interference-PCR (ORNi-PCR)]]> https://www.researchpad.co/article/Na291302f-20b5-427d-9f8a-0af670da0c81 Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method that suppresses PCR amplification of target DNA in an ORN-specific manner. In this study, we examined whether ORNi-PCR can be used to enrich desirable DNA sequences from a DNA mixture by suppressing undesirable DNA amplification. ORNi-PCR enriched edited DNA sequences from a mixture of genomic DNA subjected to genome editing. ORNi-PCR enabled more efficient analysis of the types of insertion/deletion mutations introduced by genome editing. In addition, ORNi-PCR reduced the detection of 16S ribosomal RNA (16S rRNA) genes in 16S rRNA gene-based microbiome profiling, which might permit a more detailed assessment of populations of other 16S rRNA genes. Enrichment of desirable DNA sequences by ORNi-PCR may be useful in molecular biology, medical diagnosis, and other fields.

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<![CDATA[Practical genotyping by single-nucleotide primer extension]]> https://www.researchpad.co/article/N4f2795a4-f879-4a90-905e-1051cdd270be Genome-wide association studies bring into focus specific genetic variants of particular interest for which validation is often sought in large numbers of study subjects. Practical alternative methods are limiting for the application of genotyping few variants in many samples. A common scenario is the need to genotype a study population at a specific high-value single nucleotide polymorphism (SNP) or insertion-deletion (indel). Not all such variants, however, will be amenable to assay by a given approach. We have adapted a single-nucleotide primer extension (SNuPE) method that may be tailored to genotype a required variant, and implemented it as a useful general laboratory protocol. We demonstrate reliable application for production-scale genotyping, successfully converting 87% of SNPs and indels for assay with an estimated error rate of 0.003. Our implementation of the SNuPE genotyping assay is a viable addition to existing alternative methods; it is readily customizable, scalable, and uses standard reagents and a laboratory plate reader.

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<![CDATA[Leveraging modern DNA assembly techniques for rapid, markerless genome modification]]> https://www.researchpad.co/article/N69caf8b9-134d-4c95-9d58-5498d9ce7328 The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the Escherichia coli genome named FAST-GE for Fast Assembly-mediated Scarless Targeted Genome Editing. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both E. coli B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.

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<![CDATA[A comprehensive comparison and overview of R packages for calculating sample entropy]]> https://www.researchpad.co/article/Ncd697ef4-26e5-4ac3-bfb1-f11ad655d373

Abstract

Sample entropy is a powerful tool for analyzing the complexity and irregularity of physiology signals which may be associated with human health. Nevertheless, the sophistication of its calculation hinders its universal application. As of today, the R language provides multiple open-source packages for calculating sample entropy. All of which, however, are designed for different scenarios. Therefore, when searching for a proper package, the investigators would be confused on the parameter setting and selection of algorithms. To ease their selection, we have explored the functions of five existing R packages for calculating sample entropy and have compared their computing capability in several dimensions. We used four published datasets on respiratory and heart rate to study their input parameters, types of entropy, and program running time. In summary, NonlinearTseries and CGManalyzer can provide the analysis of sample entropy with different embedding dimensions and similarity thresholds. CGManalyzer is a good choice for calculating multiscale sample entropy of physiological signal because it not only shows sample entropy of all scales simultaneously but also provides various visualization plots. MSMVSampEn is the only package that can calculate multivariate multiscale entropies. In terms of computing time, NonlinearTseries, CGManalyzer, and MSMVSampEn run significantly faster than the other two packages. Moreover, we identify the issues in MVMSampEn package. This article provides guidelines for researchers to find a suitable R package for their analysis and applications using sample entropy.

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<![CDATA[Absolute quantitation of poly(R)-3-hydroxybutyric acid using spectrofluorometry in recombinant Escherichia coli]]> https://www.researchpad.co/article/N69109f93-c528-4535-ab89-f70fe6defbe5

Abstract

Poly(R)-3-hydroxybutyric acid (PHB) is a biodegradable natural polymer produced by microorganisms and plants under nitrogen deprivation and physiological stress. Metabolic engineering and synthetic biology approaches are underway to develop strains that can produce PHB and its co-polymers. One of the major limitations to the scaling and success of strain development for biosynthesis of PHB is the absence of fast, accurate, quantitative and scalable methods to estimate PHB in polymer producing cells. In this study, a Nile red-based spectrofluorometric method is developed for absolute quantitation of PHB in recombinant Escherichia coli. The method is a modification of an existing Nile red-based method currently only used for relative quantitation. The two added steps of sonication and ethanol extraction increase the dynamic range of the assay and limit of detection/quantitation. Sonication of PHB standards provides uniform distribution of surface area to volume ratios. This ensures reproducibility and accuracy (lower %relative error) of quantitative staining of granules by Nile red even in a higher dynamic concentration range of 125–1000 µg/ml. Ethanolic extraction of the PHB bound Nile red allows higher recovery and accurate absolute quantitation. To reproduce high recovery and ensure accuracy and precision of the analytical method directly using cells, a protein digestion step was added. This accounted for fluorescence from over-expressed protein and resulted in screening of nonproducers of PHB amongst samples. Thus, the method developed is rapid, accurate, and reproducible, requires low sample volumes and processing compared to other conventional methods. This method is scalable to other PHA’s and diverse plastics.

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<![CDATA[CD-tagging-MS2: detecting allelic expression of endogenous mRNAs and their protein products in single cells]]> https://www.researchpad.co/article/N083d3fb1-0647-4a2d-8341-259bf883a680

Abstract

Discriminating between the mRNA and protein outputs of each of the alleles of an endogenous gene in intact cells, is a difficult task. To examine endogenous transcripts originating from a specific allele, we applied Central Dogma tagging (CD-tagging), which is based on a tag insertion into an endogenous gene by creation of a new exon. Previously, CD-tagging was used to tag endogenous proteins. Here we developed a CD-tagging-MS2 approach in which two tags were inserted in tandem; a fluorescent protein tag in conjunction with the mRNA MS2 tag used for tagging mRNAs in cells. A cell clone library of CD-tagged-MS2 genes was generated, and protein and mRNA distributions were examined and characterized in single cells. Taking advantage of having one allele tagged, we demonstrate how the transcriptional activity of all alleles, tagged and untagged, can be identified using single molecule RNA fluorescence in situ hybridization (smFISH). Allele-specific mRNA expression and localization were quantified under normal and stress conditions. The latter generate cytoplasmic stress granules (SGs) that can store mRNAs, and the distribution of the mRNAs within and outside of the SGs was measured. Altogether, CD-tagging-MS2 is a robust and inexpensive approach for direct simultaneous detection of an endogenous mRNA and its translated protein product in the same cell.

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<![CDATA[A highly sensitive trap vector system for isolating reporter cells and identification of responsive genes]]> https://www.researchpad.co/article/N4f753506-7154-4bba-956c-13cfcff46435

Abstract

We devised a versatile vector system for efficient isolation of reporter cells responding to a certain condition of interest. This system combines nontoxic GAL4-UAS and piggyBac transposon systems, allowing application to mammalian cells and improved expression of a fluorescent reporter protein for cell sorting. Case studies under conditions of c-MYC gene induction or endoplasmic reticulum (ER) stress with thapsigargin on mouse or human cell lines confirmed easy and efficient isolation of responsive reporter cells. Sequence analyses of the integrated loci of the thapsigargin-responsive clones identified responsive genes including BiP and OSBPL9. OSBPL9 is a novel ER stress-responsive gene and we confirmed that endogenous mRNA expression of OSBPL9 is upregulated by thapsigargin, and is repressed by IRE1α inhibitors, 4μ8C and toyocamycin, but not significantly by a PERK inhibitor, GSK2656157. These results demonstrate that this approach can be used to discover novel genes regulated by any stimuli without the need for microarray analysis, and that it can concomitantly produce reporter cells without identification of stimuli-responsive promoter/enhancer elements. Therefore, this system has a variety of benefits for basic and clinical research.

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<![CDATA[PrimerPooler: automated primer pooling to prepare library for targeted sequencing ]]> https://www.researchpad.co/article/N031d3c85-30cb-46cc-a5f5-b413ef24b7cc

Abstract

Targeted next-generation sequencing based on PCR amplification involves pooling of hundreds to thousands of primers, for preamplification and subsequent parallel single/multiplex PCR. It is often necessary to allocate the set of primers into subpools, a common issue being potential cross-hybridization. For smaller numbers of primers, pool division can be done manually using trial and error to minimize potential hybridization, but this becomes inefficient and time consuming with increasing numbers of primer pairs. We developed PrimerPooler that automates swapping of primer pairs between any user-defined number of subpools to obtain combinations with low-potential interactions. PrimerPooler performs inter-/intra-primer hybridization analysis to identify the adverse interactions, as well as simultaneous mapping of all primers onto a genome sequence in a single run without requiring a prior index of the genome. This allows detection of overlapping primer pairs and allocation of these primer pairs into separate subpools where tiling approaches are used. Using PrimerPooler, 1153 primer pairs were assigned to three preamplification pools (388, 389 and 376 primer pairs each), then 144 subpools of six- to nine-plex PCR for Fluidigm Access Array PCR, followed by Illumina MiSeq sequencing. With optimized experimental protocols, an average of 3269 reads was achieved for the targeted regions, with 95% of targets covered by at least 50 reads, the minimal depth of reads for confident variant calling. PrimerPooler provides a fast and highly efficient stratification of primer pairs for targeted enrichment, thus ensuring representative amplification of the targeted sequences. PrimerPooler is also able to analyse degenerate primers, and is thus also useful for microbiological identification and related target sequencing.

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