Construction Of Genomic Library Research Articles

Significant enhancement of the thermal stability and catalytic efficiency of transglutaminase in Streptomyces mobaraensis engineered through the novel S. mobaraensis genomic mutant library construction method GHR/Sml.

Herein, we developed a novel Streptomyces mobaraensis genomic mutant library construction method, GHR/Sml, to directly and significantly enhance the thermal stability and catalytic efficiency of TGase in the genome of S. mobaraensis. First, 13 key amino acid residues and their mutations for enhanced thermal stability were identified using error-prone PCR and site-directed mutagenesis. Then, the GHR/Sml method was developed to construct a TGase genomic mutant library with 13 mutations. Positive mutants S23Y/Y24N/S250R, S23Y/Y24N/S303K, S23Y/Y24N/K294L, S23Y/Y24N/S199A/R208L, S23Y/Y24N, and S250R were obtained from 1500 total mutants; their half-life values at 50°C were increased by 9.3-, 9.5-, 8.7-, 9.0-, 6.9-, and 4.8-fold compared with that of TGLD, respectively. Furthermore, the kcat/Km of mutant S23Y/Y24N/S250R increased by 1.25-fold over that of TGLD. The activity of S23Y/Y24N/S250R reached 65.34U/mL in a 1000-L fermenter, which was the highest activity reported. This novel GHR/Sml method is of great significance for systematically improving properties of additional enzymes in the genome of S. mobaraensis.

Distortion of Population Statistics due to the Use of Different Methodological Approaches to the Construction of Genomic DNA Libraries.

Several different methods of DNA library preparation for paleogenetic studies are now available. However, the chemical reactions underlying each of them can affect the primary sequence of ancient DNA (aDNA) in the libraries and taint the results of a statistical analysis. In this paper, we compare the results of a sequencing of the aDNA libraries of a Bronze Age sample from burials of the Caucasian burial ground Klady, prepared using three different approaches: (1) shotgun sequencing, (2) strategies for selecting target genomic regions, and (3) strategies for selecting target genomic regions, including DNA pre-treatment with a mixture of uracil-DNA glycosylase (UDG) and endonuclease VIII. The impact of the studied approaches to genomic library preparation on the results of a secondary analysis of the statistical data, namely F4 statistics, ADMIXTURE, and principal component analysis (PCA), was analyzed. It was shown that preparation of genomic libraries without the use of UDG can result in distorted statistical data due to postmortem chemical modifications of the aDNA. This distortion can be alleviated by analyzing only the single nucleotide polymorphisms caused by transversions in the genome.

Combining methods for non-invasive fecal DNA enables whole genome and metagenomic analyses in wildlife biology.

Non-invasive biological samples benefit studies that investigate rare, elusive, endangered, or dangerous species. Integrating genomic techniques that use non-invasive biological sampling with advances in computational approaches can benefit and inform wildlife conservation and management. Here, we used non-invasive fecal DNA samples to generate low- to medium-coverage genomes (e.g., >90% of the complete nuclear genome at six X-fold coverage) and metagenomic sequences, combining widely available and accessible DNA collection cards with commonly used DNA extraction and library building approaches. DNA preservation cards are easy to transport and can be stored non-refrigerated, avoiding cumbersome or costly sample methods. The genomic library construction and shotgun sequencing approach did not require enrichment or targeted DNA amplification. The utility and potential of the data generated was demonstrated through genome scale and metagenomic analyses of zoo and free-ranging African savanna elephants (Loxodonta africana). Fecal samples collected from free-ranging individuals contained an average of 12.41% (5.54-21.65%) endogenous elephant DNA. Clustering of these elephants with others from the same geographic region was demonstrated by a principal component analysis of genetic variation using nuclear genome-wide SNPs. Metagenomic analyses identified taxa that included Loxodonta, green plants, fungi, arthropods, bacteria, viruses and archaea, showcasing the utility of this approach for addressing complementary questions based on host-associated DNA, e.g., pathogen and parasite identification. The molecular and bioinformatic analyses presented here contributes towards the expansion and application of genomic techniques to conservation science and practice.

Effect of feeding raw potato starch on the composition dynamics of the piglet intestinal microbiome.

Raw potato starch (RPS) is resistant to digestion, escapes absorption, and is metabolized by intestinal microflora in the large intestine and acts as their energy source. In this study, we compared the effect of different concentrations of RPS on the intestinal bacterial community of weaned piglets. Male weaned piglets (25-days-old, 7.03±0.49 kg) were either fed a corn/soybean-based control diet (CON, n = 6) or two treatment diets supplemented with 5% RPS (RPS5, n = 4) or 10% RPS (RPS10, n = 4) for 20 days and their fecal samples were collected. The day 0 and 20 samples were analyzed using a 16S rRNA gene sequencing technology, followed by total genomic DNA extraction, library construction, and high-throughput sequencing. After statistical analysis, five phyla and 45 genera accounting for over 0.5% of the reads in any of the three groups were further analyzed. Furthermore, short-chain fatty acids (SCFAs) in the day 20 fecal samples were analyzed using gas chromatography. Significant changes were not observed in the bacterial composition at the phylum level even after 20 d post feeding (dpf); however, the abundance of Intestinimonas and Barnesiella decreased in both RPS treatment groups compared to the CON group. Consumption of 5% RPS increased the abundance of Roseburia (p<0.05) and decreased the abundance of Clostridium (p<0.01) and Mediterraneibacter (p< 0.05). In contrast, consumption of 10% RPS increased the abundance of Olsenella (p<0.05) and decreased the abundance of Campylobacter (p<0.05), Kineothrix (p<0.05), Paraprevotella (p<0.05), and Vallitalea (p<0.05). Additionally, acetate (p<0.01), butyrate (p<0.05), valerate (p = 0.01), and total SCFAs (p = 0.01) were upregulated in the RPS5 treatment group. Feeding 5% RPS altered bacterial community composition and promoted gut health in weaned piglets. Thus, resistant starch as a feed additive may prevent diarrhea in piglets during weaning.

Genomic DNA Extraction: The Critical Stage in Plant Molecular Analysis Activities

&lt;p&gt;DNA extraction is a crucial stage in plant molecular analysis activities that determine the success of further downstream analysis stages. Molecular analysis activities, including Polymerase Chain Reaction (PCR), restriction digestion, DNA hybridisation, genomic library construction, and DNA sequencing, mostly require DNA with high quality and enough quantity. Some plants contain high levels of polysaccharides, proteins, and secondary metabolites, such as polyphenols, tannins, and alkaloids, which can affect the purity of the isolated DNA. The presence of these contaminants can inhibit the activity of several enzymes, such as DNA polymerase and restriction enzymes, so that DNA could not be amplified or digested. Various DNA extraction methods have been developed, ranging from manual method, manual commercial kits, and robotic techniques. Each method has its advantages and disadvantages since the selection of the right method needs to be determined carefully in order to obtain the best results. Some modifications at certain stages in DNA extraction process need to be conducted to minimize the number of DNA contaminants. Several rapid DNA extraction methods nowadays have also been developed to save times, labors, and costs. In addition, the rapid DNA extraction method could also reduce the use of some hazardous chemical compounds, so that the method is not only giving economic benefits, but also good for human health.&lt;/p&gt;

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform.

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

Use of magnetic beads in Next-Generation Sequencing in the Construction of Low Cost 16S rRNA Gene Genomic Libraries

This work aimed to test the new Mag-Bind® TotalPure magnetic beads in the construction of a genomic library of corn and sugarcane endophytes. The technique Next-Generation Sequencing (NGS) is very expensive and for that, one of the most expensive reagents are the magnetics beads that serve to purify the amplified DNA and separate by size depending on their proportion, as shown in the Illumina protocol. We observed that the use of these beads, which are five times cheaper and have the same capacity and quality as those on the market, are good for a satisfactory study of metagenomics and microbiomes. We conclude that the use of omega brand beads has an excellent result in the construction of a genomic library for NGS.

Construction of Genomic Library and Screening of Edwardsiella tarda Immunogenic Proteins for Their Protective Efficacy Against Edwardsiellosis.

Edwardsiella tarda is a severe aquaculture pathogen that can infect many hosts including humans, animals, and fish. Timely diagnosis and treatment are crucial for the control of edwardsiellosis in the aqua industry. By using rabbit polyclonal antibody, an expression gene library of virulent Edwardsiella tarda strain ED-BDU 1 isolated in south India was constructed and screened. The identified immune expressive proteins were characterized, and the corresponding coding sequences were cloned, expressed, and the purified recombinant proteins were used as antigens. The identified immunoreactive proteins namely HflC, HflK, and YhcI were studied for their immune protective potential in vivo by challenge experiments. The protective efficacy of HflC, HflK, and YhcI showed that the clearance of Edwardsiella from the host with ~ 60% survivability. Further, the immunoreactive proteins induce a strong immune response upon infection and elicit the significant production of IL-10, IFN-γ, Th1, and Th2 mediated mRNA expression and were therefore effective in vaccine production for edwardsiellosis.

A low-cost pipeline for soil microbiome profiling.

Common bottlenecks in environmental and crop microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low‐cost genomic DNA extraction method for soil samples. We also present an Illumina‐compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non‐commercial extraction method by Zou et al. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad‐leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column‐based DNA extraction with extensive washing, delivered the cleanest genomic DNA, for example, best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. Here, we present a low‐cost genomic DNA extraction method for soil samples that can be coupled to an Illumina‐compatible simple two‐step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high‐quality genomic DNA at a fraction of the cost of commercial kits and enables cost‐effective, large‐scale amplicon sequencing projects. Notably, our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, and Dovetail genomics.

Growth of Saccharomyces cerevisiae and Preparation of DNA.

This protocol describes methods for isolation of total DNA from a strain of Sacchromyces cerevisiae carrying a recombinant yeast artificial chromosome (YAC). This method is appropriate for preparing DNA that will be subjected to regular agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, polymerase chain reaction (PCR), or other methods that do not require intact high-molecular-weight DNA. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. Drop dialysis should be used to exchange buffers. The expected yield from a 10-mL culture is 2-4 µg of yeast DNA.

Data on the Draft Genome Sequence of Bacillus sp. Strain AN2 Isolated from Agricultural Soil in Brazil

Aims: This research aims to report the genome sequence of the Bacillus sp. strain AN2, isolated from agricultural soil from Rio de Janeiro, Brazil.&#x0D; Place and Duration of Study: Laboratory of Environmental Biotechnology, Western Rio Janeiro State University in Brazil, between January 2019 and December 2019.&#x0D; Methodology: High-quality genomic DNA was extracted using a GenElute Bacterial Genomic DNA kit. The Nextera XT DNA Library Prep Kit was used for genomic library construction. Paired-end sequence reads were generated by an Illumina MiSeq instrument with the 600 cycles MiSeq Reagent Kit v3. Sequence data were assembled with A5-MiSeq pipeline software and the contigs were annotated by Rapid Annotation using Subsystems Technology (RAST).&#x0D; Results: The obtained genome sequence of Bacillus AN2 included 21 contigs with a calculated size of 3,681,081 bp in length. The G + C content for the draft genome is 41.4%. A total of 3824 coding sequences (CDS) were predicted and encoded at least 88 tRNAs.

Construction of a multicopy genomic DNA library and its application for suppression analysis.

Suppression analysis is used for the identification of new genes and genetic interactions when there is a notable phenotype available for genetic selection or screening. A random genomic DNA library constructed on a multi-copy plasmid is a useful tool for suppression analysis when one expects that an overdose of a few genes will suppress the phenotype. These libraries have been successfully used to determine the function of a gene by revealing genes whose functions are related to the gene of interest. They have also been used to identify the targets of chemical or biological agents by increasing the number of unaffected target gene products in a cell. In this article, I will discuss important considerations for constructing multicopy genomic DNA libraries. The protocol provided in this paper should be a useful guide for constructing genomic DNA libraries in many bacterial species for which multi-copy plasmids are available.

Focused Size Selection of Cell-Free DNA Samples for Liquid Biopsy Applications that Rely on Next-Generation Sequencing

Genomic analysis of circulating, cell-free DNA (cfDNA) is being used extensively for molecular diagnostics. Many approaches rely on the construction of cfDNA genomic libraries, targeted retrieval of specific genomic regions and analysis by next-generation DNA sequencing. Several steps during sample preparation require isolation of DNA fragments within a particular size range. In this Benchmark article, two related methods for size-selective DNA fragment enrichment are described.

Quorum Quenching Enzyme APTM01, an Acylhomoserine-Lactone Acylase from Marine Bacterium of Pseudoalteromonas tetraodonis Strain MQS005.

Quorum sensing is a system of stimuli and response correlated to population density and involves in pathogen infection, colonization, and pathogenesis. Quorum quenching enzymes as quorum sensing inhibitors have been identified in a number of bacteria and been used to control by triggering the pathogenic phenotype. The marine bacteria of Pseudoalteromonas had wide activity of degrading AHLs as a type of signal molecule associated with quorum sensing. We screened many Pseudoalteromonas strains in large scale to explore genes of quorum quenching enzymes from the China seas by whole-genome sequencing rather than genomic library construction. Nine target strains were obtained and an acylases gene APTM01 from the strain MQS005 belonging to PvdQ type on sub-branch in phylogenetic tree. And the heterogenous host containing the vector with target gene could degrade C10-HSL, C12-HSL and OC12-HSL. The obtained AHL acylase gene would be a candidate quorum quenching gene to apply in some fields. We identified that the strains of Pseudoalteromonas have wide AHL-degrading ability depending on quorum quenching. The strains would be a resource to explore new quorum quenching enzymes.

Novel ‘Bacteriospray’ Method Facilitates the Functional Screening of Metagenomic Libraries for Antimicrobial Activity

Metagenomic techniques, notably the cloning of environmental DNA (eDNA) into surrogate hosts, have given access to the genome of uncultured bacteria. However, the determination of gene functions based on DNA sequences alone remains a significant challenge. The functional screening of metagenomic libraries represents an interesting approach in the discovery of microbial metabolites. We describe here an optimized screening approach that facilitates the identification of new antimicrobials among large metagenomic libraries. Notably, we report a detailed genomic library construction protocol using Escherichia coli DH10B as a surrogate host, and demonstrate how vector/genomic DNA dephosphorylation, ligase inactivation, dialysis of the ligation product and vector/genomic DNA ratio greatly influence clone recovery. Furthermore, we describe the use of an airbrush device to screen E. coli metagenomic libraries for their antibacterial activity against Staphylococcus aureus, a method we called bacteriospray. This bacterial spraying tool greatly facilitates and improves the functional screening of large genomic libraries, as it conveniently allows the production of a thinner and more uniform layer of target bacteria compared to the commonly used overlay method, resulting in the screening of 5–10 times more clones per agar plate. Using the Burkholderia thailandensis E264 genomic DNA as a proof of concept, four clones out of 70,000 inhibited the growth of S. aureus and were found to each contain a DNA insert. Analysis of these chromosomic fragments revealed genomic regions never previously reported to be responsible for the production of antimicrobials, nor predicted by bioinformatics tools.

Development and characterization of Novel Microsatellite markers in great snakehead, Channa marulia ( )

The species-specific microsatellite markers in the great snakehead, Channa marulia, were mined from the genome, through an enriched genomic library construction. Total 27 microsatellite loci exhibited polymorphism. The repeat containing sequences were annotated to ensure the genetic neutrality of the loci. Nineteen, consistently scorable loci were validated, and used to estimate the genetic indices in n = 67 individuals sampled from three Indian rivers (Godavari, Mahanadi and Teesta). The polymorphism information content (PIC) values ranged from 0.366 to 0.831, while heterozygosities varied from 0.077 to 0.818. Bayesian model-based clustering grouped the samples into three discrete populations with evidences of little genetic admixture. The results confirmed a set of 19 microsatellite markers, as promising molecular tool, for estimating genetic diversity studies in C. marulia. Successful cross-transferability was observed in three congener species viz. C. punctata, C. gachua, and C. striata. The results can be useful in conservation and management of C. marulia.

Targeted next generation sequencing identified a high frequency genetic mutated profile in wood smoke exposure-related lung adenocarcinoma patients.

BackgroundWood smoke exposure (WSE) has been associated with an increased risk of lung cancer development. WSE has been related with high frequency of EGFR mutations and low frequency of KRAS mutations. The aim of this study was to evaluate large scale genomic alterations in lung adenocarcinomas associated with WSE using targeted next generation sequencing.MethodsDNA multi-targeted sequencing was performed in 42 fresh-frozen samples of advanced lung adenocarcinomas. The TruSeQ Cancer Panel (Illumina) was used for genomic library construction and sequencing assays.ResultsWSE rate was higher in women (p=0.037) and non-smokers (p=0.001). WSE correlated with mutations in the genes SMARCB1 (p=0.002), Ataxia telangiectasia mutated (p=0.004), Kinase Insert Domain Receptor (p=0.006), and were borderline significant in RET and EGFR exon. Genomic alterations significantly co-occurred in the tumor suppressor gene ATM with the following genes: SMARCB1, EGFR exon 7, RET and KDR. Clinical factors associated with poor prognosis were ECOG ≥ 2 (p= 0.014), mutations in KDR (p= 0.004) and APC genes (p < 0.001).ConclusionsLung adenocarcinoma patients with WSE showed a distinctive mutated profile for the SMARCB1, ATM, EGFR exon 7, RET and KDR genes. ECOG status and KDR gene mutations were significantly associated with poor prognosis.

The use of chloroplast genome sequences to solve phylogenetic incongruences in Polystachya Hook (Orchidaceae Juss).

BackgroundCurrent evidence suggests that for more robust estimates of species tree and divergence times, several unlinked genes are required. However, most phylogenetic trees for non-model organisms are based on single sequences or just a few regions, using traditional sequencing methods. Techniques for massive parallel sequencing or next generation sequencing (NGS) are an alternative to traditional methods that allow access to hundreds of DNA regions. Here we use this approach to resolve the phylogenetic incongruence found in Polystachya Hook. (Orchidaceae), a genus that stands out due to several interesting aspects, including cytological (polyploid and diploid species), evolutionary (reticulate evolution) and biogeographical (species widely distributed in the tropics and high endemism in Brazil). The genus has a notoriously complicated taxonomy, with several sections that are widely used but probably not monophyletic.MethodsWe generated the complete plastid genome of 40 individuals from one clade within the genus. The method consisted in construction of genomic libraries, hybridization to RNA probes designed from available sequences of a related species, and subsequent sequencing of the product. We also tested how well a smaller sample of the plastid genome would perform in phylogenetic inference in two ways: by duplicating a fast region and analyzing multiple copies of this dataset, and by sampling without replacement from all non-coding regions in our alignment. We further examined the phylogenetic implications of non-coding sequences that appear to have undergone hairpin inversions (reverse complemented sequences associated with small loops).ResultsWe retrieved 131,214 bp, including coding and non-coding regions of the plastid genome. The phylogeny was able to fully resolve the relationships among all species in the targeted clade with high support values. The first divergent species are represented by African accessions and the most recent ones are among Neotropical species.DiscussionOur results indicate that using the entire plastid genome is a better option than screening highly variable markers, especially when the expected tree is likely to contain many short branches. The phylogeny inferred is consistent with the proposed origin of the genus, showing a probable origin in Africa, with later dispersal into the Neotropics, as evidenced by a clade containing all Neotropical individuals. The multiple positions of Polystachya concreta (Jacq.) Garay & Sweet in the phylogeny are explained by allotetraploidy. Polystachya estrellensis Rchb.f. can be considered a genetically distinct species from P. concreta and P. foliosa (Lindl.) Rchb.f., but the delimitation of P. concreta remains uncertain. Our study shows that NGS provides a powerful tool for inferring relationships at low taxonomic levels, even in taxonomically challenging groups with short branches and intricate morphology.

Efficient construction of xenogeneic genomic libraries by circumventing restriction-modification systems that restrict methylated DNA

An efficient method to construct xenogeneic genomic libraries with low errors and bias by circumventing restriction-modification systems that restrict methylated DNA was developed. Un-methylated genomic DNA of Escherichia coli prepared by ϕ29 DNA polymerase was introduced to Corynebacterium glutamicum R after ligation with un-methylated vector plasmids.

The Diversity of Sequence and Chromosomal Distribution of New Transposable Element-Related Segments in the Rye Genome Revealed by FISH and Lineage Annotation.

Transposable elements (TEs) in plant genomes exhibit a great variety of structure, sequence content and copy number, making them important drivers for species diversity and genome evolution. Even though a genome-wide statistic summary of TEs in rye has been obtained using high-throughput DNA sequencing technology, the accurate diversity of TEs in rye, as well as their chromosomal distribution and evolution, remains elusive due to the repetitive sequence assembling problems and the high dynamic and nested nature of TEs. In this study, using genomic plasmid library construction combined with dot-blot hybridization and fluorescence in situ hybridization (FISH) analysis, we successfully isolated 70 unique FISH-positive TE-related sequences including 47 rye genome specific ones: 30 showed homology or partial homology with previously FISH characterized sequences and 40 have not been characterized. Among the 70 sequences, 48 sequences carried Ty3/gypsy-derived segments, 7 sequences carried Ty1/copia-derived segments and 15 sequences carried segments homologous with multiple TE families. 26 TE lineages were found in the 70 sequences, and among these lineages, Wilma was found in sequences dispersed in all chromosome regions except telomeric positions; Abiba was found in sequences predominantly located at pericentromeric and centromeric positions; Wis, Carmilla, and Inga were found in sequences displaying signals dispersed from distal regions toward pericentromeric positions; except DNA transposon lineages, all the other lineages were found in sequences displaying signals dispersed from proximal regions toward distal regions. A high percentage (21.4%) of chimeric sequences were identified in this study and their high abundance in rye genome suggested that new TEs might form through recombination and nested transposition. Our results also gave proofs that diverse TE lineages were arranged at centromeric and pericentromeric positions in rye, and lineages like Abiba might play a role in their structural organization and function. All these results might help in understanding the diversity and evolution of TEs in rye, as well as their driving forces in rye genome organization and evolution.