scholarly journals A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Michelle Spoto ◽  
Changhui Guan ◽  
Elizabeth Fleming ◽  
Julia Oh
Keyword(s):  
Gene Function ◽  
Large Scale ◽  
Essential Gene ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Model Organisms ◽  
Design Parameters ◽  
Bacterial Genomes ◽  
Wide Range ◽  
User Friendly

ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species. IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

scholarly journals A universal, genome-wide guide finder for CRISPR/Cas9 targeting in microbial genomes

2017 ◽  
Author(s):  
Michelle Spoto ◽  
Elizabeth Fleming ◽  
Julia Oh
Keyword(s):  
Large Scale ◽  
Essential Gene ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Design Parameters ◽  
Bacterial Genomes ◽  
Microbial Genomes ◽  
Genome Wide ◽  
Cas9 Protein ◽  
User Friendly

AbstractBackgroundThe CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression or activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, no pan-bacterial, genome-wide tools exist for guide discovery. We have created Guide Finder: a customizable, user-friendly program that can design guides for any annotated bacterial genome.ResultsGuide Finder designs guides from NGG PAM sites for any number of genes using an annotated genome and fasta file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, a feature unique to Guide Finder. Guide Finder has been tested on a variety of diverse bacterial genomes, on average finding guides for 95% of genes. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species.ConclusionsThrough the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies for a variety of bacterial species.

scholarly journals Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Matthew R. Olm ◽  
Alexander Crits-Christoph ◽  
Spencer Diamond ◽  
Adi Lavy ◽  
Paula B. Matheus Carnevali ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Ribosomal Proteins ◽  
Large Scale ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Species Discrimination ◽  
Bacterial Genomes ◽  
Discrimination Power

ABSTRACT Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination. IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.

scholarly journals SCAPP: an algorithm for improved plasmid assembly in metagenomes

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
David Pellow ◽  
Alvah Zorea ◽  
Maraike Probst ◽  
Ori Furman ◽  
Arik Segal ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Bacterial Genome ◽  
Biological Knowledge ◽  
Assessment Procedure ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Human Gut ◽  
Double Stranded Dna ◽  
Wide Range ◽  
Python Package

Abstract Background Metagenomic sequencing has led to the identification and assembly of many new bacterial genome sequences. These bacteria often contain plasmids: usually small, circular double-stranded DNA molecules that may transfer across bacterial species and confer antibiotic resistance. These plasmids are generally less studied and understood than their bacterial hosts. Part of the reason for this is insufficient computational tools enabling the analysis of plasmids in metagenomic samples. Results We developed SCAPP (Sequence Contents-Aware Plasmid Peeler)—an algorithm and tool to assemble plasmid sequences from metagenomic sequencing. SCAPP builds on some key ideas from the Recycler algorithm while improving plasmid assemblies by integrating biological knowledge about plasmids. We compared the performance of SCAPP to Recycler and metaplasmidSPAdes on simulated metagenomes, real human gut microbiome samples, and a human gut plasmidome dataset that we generated. We also created plasmidome and metagenome data from the same cow rumen sample and used the parallel sequencing data to create a novel assessment procedure. Overall, SCAPP outperformed Recycler and metaplasmidSPAdes across this wide range of datasets. Conclusions SCAPP is an easy to use Python package that enables the assembly of full plasmid sequences from metagenomic samples. It outperformed existing metagenomic plasmid assemblers in most cases and assembled novel and clinically relevant plasmids in samples we generated such as a human gut plasmidome. SCAPP is open-source software available from: https://github.com/Shamir-Lab/SCAPP.

scholarly journals Influence of pipeline geometry on hydrodynamics and heat transfer processes by an example of a ship steam generator

2021 ◽  
Vol 2088 (1) ◽  
pp. 012033
Author(s):  
O V Mitrofanova ◽  
A V Fedorinov
Keyword(s):  
Nuclear Power ◽  
Large Scale ◽  
Complex Geometry ◽  
Swirling Flow ◽  
Thermal Power ◽  
Design Parameters ◽  
Nuclear Power Installation ◽  
Wide Range ◽  
Nuclear Power Installations ◽  
Power Installations

Abstract The theoretical and computational analysis proposed in this work is aimed at identifying the features of thermal and hydrodynamic processes carried out in the steam-generating channels of the ship type water-moderated nuclear power installations. It is shown that the complex geometry of the thermohydraulic tract curvilinear channels of the steam generating system has a significant effect on the efficiency of the transport nuclear power installation. In addition to the formation of large-scale vortex structures and swirling flow in the pipeline, the phenomenon of the swirling flow crisis is revealed, under which the low-frequency component of the acoustic spectrum is enhanced. The scientific and applied significance of the proposed research is associated with the need to ensure a wide range of operational changes in efficient and safe operation power modes of icebreaker nuclear power installations. The research, aimed at developing the principles of physical and mathematical modeling of complex vortex flows, is necessary to optimize the design parameters of the thermal power equipment elements of new generation ship nuclear power installations in order to ensure increased safety and reliability of their operation.

scholarly journals Deconstructing Gastrulation at the Single Cell Level

2021 ◽  
Author(s):  
Tomer Stern ◽  
Sebastian J Streichan ◽  
Stanislav Y Shvartsman ◽  
Eric F Wieschaus
Keyword(s):  
Large Scale ◽  
Drosophila Embryo ◽  
Cell Segmentation ◽  
Model Organisms ◽  
Specific Cell ◽  
Cell Behaviors ◽  
Animal Development ◽  
Wide Range ◽  
Cell Groups ◽  
Epithelial Sheets

Gastrulation movements in all animal embryos start with regulated deformations of patterned epithelial sheets. Current studies of gastrulation use a wide range of model organisms and emphasize either large-scale tissue processes or dynamics of individual cells and cell groups. Here we take a step towards bridging these complementary strategies and deconstruct early stages of gastrulation in the entire Drosophila embryo, where transcriptional patterns in the blastoderm give rise to region-specific cell behaviors. Our approach relies on an integrated computational framework for cell segmentation and tracking and on efficient algorithms for event detection. Our results reveal how thousands of cell shape changes, divisions, and intercalations drive large-scale deformations of the patterned blastoderm, setting the stage for systems-level dissection of a pivotal step in animal development.

scholarly journals Bactopia: a flexible pipeline for complete analysis of bacterial genomes

2020 ◽  
Author(s):  
Robert A. Petit ◽  
Timothy D. Read
Keyword(s):  
Standard Procedure ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Complete Analysis ◽  
Comparative Genomic ◽  
Bacterial Genomes ◽  
Analysis Pipeline ◽  
Genomic Analyses ◽  
Conserved Genes ◽  
Downstream Analysis

AbstractSequencing of bacterial genomes using Illumina technology has become such a standard procedure that often data are generated faster than can be conveniently analyzed. We created a new series of pipelines called Bactopia, built using Nextflow workflow software, to provide efficient comparative genomic analyses for bacterial species or genera. Bactopia consists of a dataset setup step (Bactopia Datasets; BaDs) where a series of customizable datasets are created for the species of interest; the Bactopia Analysis Pipeline (BaAP), which performs quality control, genome assembly and several other functions based on the available datasets and outputs the processed data to a structured directory format; and a series of Bactopia Tools (BaTs) that perform specific post-processing on some or all of the processed data. BaTs include pan-genome analysis, computing average nucleotide identity between samples, extracting and profiling the 16S genes and taxonomic classification using highly conserved genes. It is expected that the number of BaTs will increase to fill specific applications in the future. As a demonstration, we performed an analysis of 1,664 public Lactobacillus genomes, focusing on L. crispatus, a species that is a common part of the human vaginal microbiome. Bactopia is an open source system that can scale from projects as small as one bacterial genome to thousands that allows for great flexibility in choosing comparison datasets and options for downstream analysis. Bactopia code can be accessed at https://www.github.com/bactopia/bactopia.

scholarly journals T4SP Database 2.0: An Improved Database for Type IV Secretion Systems in Bacterial Genomes with New Online Analysis Tools

2016 ◽  
Vol 2016 ◽  
pp. 1-4 ◽  
Author(s):  
Na Han ◽  
Weiwen Yu ◽  
Yujun Qiang ◽  
Wen Zhang
Keyword(s):  
Recipient Cell ◽  
Bacterial Species ◽  
Genetic Material ◽  
Type Iv Secretion ◽  
Bacterial Strains ◽  
Secretion Systems ◽  
Bacterial Genomes ◽  
Type Iv ◽  
Type Iv Secretion Systems ◽  
User Friendly

Type IV secretion system (T4SS) can mediate the passage of macromolecules across cellular membranes and is essential for virulent and genetic material exchange among bacterial species. The Type IV Secretion Project 2.0 (T4SP 2.0) database is an improved and extended version of the platform released in 2013 aimed at assisting with the detection of Type IV secretion systems (T4SS) in bacterial genomes. This advanced version provides users with web server tools for detecting the existence and variations of T4SS genes online. The new interface for the genome browser provides a user-friendly access to the most complete and accurate resource of T4SS gene information (e.g., gene number, name, type, position, sequence, related articles, and quick links to other webs). Currently, this online database includes T4SS information of 5239 bacterial strains.Conclusions. T4SS is one of the most versatile secretion systems necessary for the virulence and survival of bacteria and the secretion of protein and/or DNA substrates from a donor to a recipient cell. This database on virB/D genes of the T4SS system will help scientists worldwide to improve their knowledge on secretion systems and also identify potential pathogenic mechanisms of various microbial species.

emiRIT: A text-mining based resource for microRNA information

2020 ◽  
Author(s):  
Debarati Roychowdhury ◽  
Samir Gupta ◽  
Xihan Qin ◽  
Cecilia N. Arighi ◽  
K. Vijay-Shanker
Keyword(s):  
Text Mining ◽  
Information Needs ◽  
Large Scale ◽  
Biological Process ◽  
Essential Gene ◽  
Mirna Gene ◽  
Easy Access ◽  
Context Specific ◽  
Biological Entities ◽  
User Friendly

AbstractMotivationmicroRNAs (miRNAs) are essential gene regulators and their dysregulation often leads to diseases. Easy access to miRNA information is crucial for interpreting generated experimental data, connecting facts across publications, and developing new hypotheses built on previous knowledge. Here, we present emiRIT, a text mining-based resource, which presents miRNA information mined from the literature through a user-friendly interface.ResultsWe collected 149,233 miRNA-PubMed ID pairs from Medline between January 1997 to May 2020. emiRIT currently contains miRNA-gene regulation (60,491 relations); miRNA-disease (cancer) (12,300 relations); miRNA-biological process and pathways (23,390 relations); and circulatory miRNAs in extracellular locations (3,782 relations). Biological entities and their relation to miRNAs were extracted from Medline abstracts using publicly available and in-house developed text mining tools, and the entities were normalized to facilitate querying and integration. We built a database and an interface to store and access the integrated data, respectively.ConclusionWe provide an up-to-date and user-friendly resource to facilitate access to comprehensive miRNA information from the literature on a large-scale, enabling users to navigate through different roles of miRNA and examine them in a context specific to their information needs. To assess our resource’s information coverage, in the absence of gold standards, we have conducted two case studies focusing on the target and differential expression information of miRNAs in the context of diseases. Database URL: https://research.bioinformatics.udel.edu/emirit/

Dynamic Performance Analysis of Large-Scale Packed Bed Truncated Conical Thermal Energy Storage

2019 ◽  
Author(s):  
Shobhana Singh ◽  
Kim Sørensen
Keyword(s):  
Energy Storage ◽  
Large Scale ◽  
Storage System ◽  
Transient Behavior ◽  
Packed Bed ◽  
Energy Storage System ◽  
Heat Transfer Fluid ◽  
Design Parameters ◽  
Wide Range ◽  
Dynamic Performance Analysis

Abstract In the present paper, a high-temperature packed bed energy storage system of volume 175,000m3 is numerically investigated. The system is a underground packed bed of truncated conical shape, which comprises of rocks as a storage medium and air as a heat transfer fluid. A one-dimensional, two-phase model is developed to simulate the transient behavior of the storage. The developed model is used to conduct a parametric study with a wide range of design parameters to investigate the change in performance during both charging and discharging operation. Results show that the model satisfactorily predicts the dynamic behavior, and the truncated conical shaped storage with a rock diameter of 3cm, insulation thickness up to 0.6m and charging-discharging rate of 553kg/s leads to lower thermal losses and higher energy efficiencies. The paper provides useful insight into the transient performance and efficiency of a large-scale packed bed energy storage system within the range of parameters investigated.

Effect of Design Parameters on the Performance of Thermal Energy Storage Systems

Solar Energy ◽  
2004 ◽  
Author(s):  
Gregor P. Henze
Keyword(s):  
Energy Storage ◽  
Thermal Energy ◽  
Thermal Energy Storage ◽  
Large Scale ◽  
Control Strategies ◽  
Storage System ◽  
Operating Cost ◽  
Energy Storage System ◽  
Design Parameters ◽  
Wide Range

This paper describes simulation-based results of a large-scale investigation of a commercial cooling plant including a thermal energy storage system. A cooling plant with an ice-on-coil system with external melt and a reciprocating compressor operating in a large office building was analyzed under four different control strategies. Optimal control as the strategy that minimizes the total operating cost (demand and energy charges) served as a benchmark to assess the performance of the three conventional controls. However, all control strategies depend on properly selected design parameters. The storage and chiller capacities as the primary design parameters were varied over a wide range and the dependence of the system’s cost saving performance on these parameters was evaluated.

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