ResearchPad - genomics-and-bioinformatics https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[SSD - a free software for designing multimeric mono-, bi- and trivalent shRNAs]]> https://www.researchpad.co/article/elastic_article_12490 RNA interference (RNAi) is a powerful gene silencing technology, widely used in analyses of reverse genetics, development of therapeutic strategies and generation of biotechnological products. Here we present a free software tool for the rational design of RNAi effectors, named siRNA and shRNA designer (SSD). SSD incorporates our previously developed software Strand Analysis to construct template DNAs amenable for the large scale production of mono-, bi- and trivalent multimeric shRNAs, via in vitro rolling circle transcription. We tested SSD by creating a trivalent multimeric shRNA against the vitellogenin gene of Apis mellifera. RT-qPCR analysis revealed that our molecule promoted a decrease in more than 50% of the target mRNA, in a dose-dependent manner, when compared to the control group. Thus, SSD software allows the easy design of multimeric shRNAs, for single or multiple simultaneous knockdowns, which is especially interesting for studies involving large amounts of double-stranded molecules.

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<![CDATA[Metabarcoding reveals that a non-nutritive sweetener and sucrose yield similar gut microbiota patterns in Wistar rats]]> https://www.researchpad.co/article/elastic_article_12460 The effects of non-nutritive sweeteners (NNS) on the gut microbiota are an area of increasing research interest due to their potential influence on weight gain, insulin resistance, and inflammation. Studies have shown that mice and rats fed saccharin develop weight gain and metabolic alterations, possibly related to changes in gut microbiota. Here, we hypothesized that chronic exposure to a commercial NNS would change the gut microbiota composition in Wistar rats when compared to sucrose exposure. To test this hypothesis, Wistar rats were fed either NNS- or sucrose-supplemented yogurt for 17 weeks alongside standard chow (ad libitum). The gut microbiome was assessed by 16S rDNA deep sequencing. Assembly and quantification were conducted using the Brazilian Microbiome Project pipeline for Ion Torrent data with modifications. Statistical analyses were performed in the R software environment. We found that chronic feeding of a commercial NNS-sweetened yogurt to Wistar rats, within the recommended dose range, did not significantly modify gut microbiota composition in comparison to sucrose-sweetened yogurt. Our findings do not support the hypothesis that moderate exposure to NNS is associated with changes in gut microbiota pattern compared to sucrose, at least in this experimental model.

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<![CDATA[Methodological differences can affect sequencing depth with a possible impact on the accuracy of genetic diagnosis]]> https://www.researchpad.co/article/elastic_article_12440 For a better interpretation of variants, evidence-based databases, such as ClinVar, compile data on the presumed relationships between variants and phenotypes. In this study, we aimed to analyze the pattern of sequencing depth in variants from whole-exome sequencing data in the 1000 Genomes project phase 3, focusing on the variants present in the ClinVar database that were predicted to affect protein-coding regions. We demonstrate that the distribution of the sequencing depth varies across different sequencing centers (pair-wise comparison, p < 0.001). Most importantly, we found that the distribution pattern of sequencing depth is specific to each facility, making it possible to correctly assign 96.9% of the samples to their sequencing center. Thus, indicating the presence of a systematic bias, related to the methods used in the different facilities, which generates significant variations in breadth and depth in whole-exome sequencing data in clinically relevant regions. Our results show that methodological differences, leading to significant heterogeneity in sequencing depth, may potentially influence the accuracy of genetic diagnosis. Furthermore, our findings highlight how it is still challenging to integrate results from different sequencing centers, which may also have an impact on genomic research.

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<![CDATA[Boundaries in metagenomic screenings using <i>lac</i>Zα-based vectors]]> https://www.researchpad.co/article/elastic_article_12436 Metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from a soil sample by using the lacZα -based plasmid pSEVA232. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye for detection of both enzymes, as previously reported in literature. Although we effectively identified positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZ gene present in the original vector. Analyses of the thermodynamic stability of mRNA secondary structures indicated that recovering of positive clones was probably due to higher expression levels of the chimeric lacZα-genes in respect to the original from empty vector. We concluded that this method has a higher tendency for recovery false positive clones, when used in combination with a lacZα-based vector. As these vectors are massively used in functional metagenomic screenings, we highlight the importance of reporting boundaries in established metagenomic screenings methodologies.

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<![CDATA[Identification of LincRNA from <i>Dermatophagoides farinae</i> (Acari: Pyroglyphidae) for Potential Allergen-Related Targets]]> https://www.researchpad.co/article/elastic_article_12423 Long noncoding RNAs (lncRNAs), especially their important subclass of long intergenic noncoding RNAs (lincRNAs), have been identified in some insects. They play important roles in the regulation of biological processes, such as immune response or cell differentiation and as possible evolutionary precursors for protein coding genes. House dust mites (HDMs) are recognized as allergenic mites because allergens are found in their feces and bodies. Dermatophagoides farinae is one of the most important pyroglyphid mites because of its abundance in the household. To determine if lincRNAs can regulate allergen presentation in HDMs, we analyzed RNA-seq data for HDMs. We identified 11 lincRNAs that are related to mRNAs coding for allergens in HDMs. Using qRT-PCR, we amplified 10 lincRNAs and their putative target allergen-encoding mRNAs, confirming expression of these lincRNAs and allergen genes. The results suggest that lincRNAs might be involved in the regulation of allergen production in HDMs and might represent potential acaricidal candidates to inhibit mite allergen production.

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<![CDATA[Transcriptional regulators and regulatory pathways involved in prostate gland adaptation to a hypoandrogen environment]]> https://www.researchpad.co/article/elastic_article_12401 Anti-androgen therapies, including orchiectomy, are effective at promoting prostate cancer remission, but are followed by progression to the more aggressive castration-resistant prostate cancer (CRPC). Castration promotes gland and tumor shrinkage. However, prostate adaptation to androgen deprivation involves striking parallel events, all requiring changes in gene expression. We hypothesized that transcription factors (TF) and other transcription-related genes are needed to orchestrate those changes. In this work, downstream analysis using bioinformatic tools and published microarray data allowed us to identify sixty transcriptional regulators (including 10 TF) and to integrate their function in physiologically relevant networks. Functional associations revealed a connection between Arnt, Bhlhe41 and Dbp circadian rhythm genes with the Ar circuitry and a small gene network centered in Pex14, which might indicate a previously unanticipated metabolic shift. We have also identified human homologs and mapped the corresponding genes to human chromosome regions commonly affected in prostate cancer, with particular attention to the PTEN/HHEX/MXI1 cluster at 10q23-25 (frequently deleted in PCa) and to MAPK1 at 22q11.21 (delete in intermediate risk but not in high risk PCa). Twenty genes were found mutated or with copy number alterations in at least five percent of three cancer cohorts and six of them (PHOX2A, NFYC, EST2, EIF2S1, SSRP1 and PARP1) associated with impacted patient survival. These changes are specific to the adaptation to the hypoandrogen environment and seem important for the progression to CRPC when mutated.

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<![CDATA[A comparison between SOLiD 5500XLand Ion Torrent PGM-derived miRNA expression profiles in two breast cell lines]]> https://www.researchpad.co/article/elastic_article_10869 Next-generation sequencing (NGS) platforms allow the analysis of hundreds of millions of molecules in a single sequencing run, revolutionizing many research areas. NGS-based microRNA studies enable expression quantification in unprecedented scale without the limitations of closed-platforms. Yet, whereas a massive amount of data produced by these platforms is available, comparisons of quantification/discovery capabilities between platforms are still lacking. Here we compare two NGS-platforms: SOLiD and PGM, by evaluating their microRNA identification/quantification capabilities using two breast-derived cell-lines. A high expression correlation (R2 > 0.9) was achieved, encompassing 97% of the miRNAs, and the few discrepancies in miRNA counts were attributable to molecules that have very low expression. Quantification divergences indicative of artefactual representation were seen for 14 miRNAs (higher in SOLiD-reads) and another 10 miRNAs more abundant in PGM-data. An inspection of these revealed an increased and statistically significant count of uracyls and uracyl-stretches for PGM-enriched miRNAs, compared to SOLiD and to the miRBase. In parallel, adenines and adenine-stretches were enriched for SOLiDderived miRNA reads. We conclude that, whereas both platforms are overall consistent and can be used interchangeably for microRNA expression studies, particular sequence features appear to be indicative of specific platform bias, and their presence in microRNAs should be considered for database-analyses.

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<![CDATA[Comparative analysis of the complete chloroplast genomes from six Neotropical species of Myrteae (Myrtaceae)]]> https://www.researchpad.co/article/elastic_article_6282 Myrteae is the largest and most diverse tribe within Myrtaceae and represents the majority of its diversity in the Neotropics. Members of Myrteae hold ecological importance in tropical biomes for the provision of food sources for many animal species. Thus, due to its several roles, a growing interest has been addressed to this group. In this study, we report the sequencing and de novo assembly of the complete chloroplast (cp) genomes of six Myrteae species: Eugenia brasiliensis, E. pyriformis, E. nitida, Myrcianthes pungens, Plinia edulis and Psidium cattleianum. We characterized genome structure, gene content, and identified SSRs to detect variation within Neotropical Myrteae. The six newly sequenced plastomes exhibit a typical quadripartite structure, gene content and organization highly conserved among Myrtaceae species. Some differences in genome length, protein-coding genes and non-coding regions were found. Besides, IR boundaries present structural changes among species. Increased sequence diversity was observed in some intergenic regions, suggesting their suitability for investigating intraand interspecific genetic diversity in populational studies. These data also contribute to the improvement of taxa sampling in further phylogenetic investigations to understand Myrtaceae evolution.

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<![CDATA[Mitochondrial genomes of genus <i>Atta</i> (Formicidae: Myrmicinae) reveal high gene organization and giant intergenic spacers]]> https://www.researchpad.co/article/Nd060d5f4-5c74-462f-a3db-79e9e3b91524 The ants of the genus Atta are considered important pests to agriculture in the Americas, although Atta species are also important contributors to ecosystem functions in the various habitats in which they occur. The aim of this study was to assemble four complete mitochondrial genomes of the genus Atta, construct the phylogenomic tree, and analyze the gene content, order, and organization. The mitogenomes of A. colombica, A. opaciceps, A. texana, and A. sexdens rubropilosa comprise 18,392, 19,257, 19,709, and 19,748 bp, respectively. The four Atta mitogenomes showed the charactistics typical of those of insects, with 13 protein-coding genes, 22 tRNAs, and 2 rRNAs, with genes displayed in the conventional order. Analysis for intergenic spacer regions showed that Atta intergenic spacers are larger than those of the outgroups. Phylogenomic analyses using partial cytochrome oxidase I gene sequences showed similar topologies to previous phylogenetic analyses, with high clade support values. We conclude that Atta mitogenomes are characterized by high conservation in gene order and have giant intergenic spacers in the genus Atta.

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<![CDATA[Araucaria angustifolia chloroplast genome sequence and its relation to other Araucariaceae]]> https://www.researchpad.co/article/Nceba5e53-1c97-4f92-bd63-160bda088ec1

Abstract

Araucaria angustifolia is endemic to southern Brazil. Known as Brazilian pine, A. angustifolia is the only native conifer species with economic and social relevance in this country. Due to massive exploitation, it has suffered a significant population decline and currently is classified as critically endangered. This encouraged the scientific community to investigate genetic features in Brazilian pine to increase resources for management and preservation. In this work, RNA-Seq data was used to determine the complete nucleotide sequence of the A. angustifolia chloroplast genome (cpDNA). The cpDNA is 146,203 bp in length and contains 122 genes, including 80 protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes. Coding regions comprise 45.02%, 4.96% correspond to rRNAs and tRNAs, and 50.02% of the genome encompasses non-coding regions. Genes found in the inverted repeat (IR) are present as single copy, with exception of the rrn5 and trnI-CAU loci. The typical LSC, SSC, IRa and IRb organization reported in several land-plant groups is not present in A. angustifolia cpDNA. Phylogenetic analyses using Bayesian and Maximum Likelihood methods clustered A. angustifolia in the Araucariaceae family, with A. heterophylla and A. columnaris as congeneric species. The screening of A. angustifolia cpDNA reveled 100 SSRs, 14 of them corresponding to tetrapolymer loci.

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