A Novel Approach for Transcription Factor Analysis Using SELEX with High-Throughput Sequencing (TFAST)

A broad diversity of microorganisms can be found in soil, where they are essential for nutrient cycling and energy transfer. Recent high-throughput sequencing methods have greatly advanced our knowledge about how soil, climate and vegetation variables structure the composition of microbial communities in many world regions. However, we are lacking information from several regions in the world, e.g. Middle-East. We have collected soil from 19 different habitat types for studying the diversity and composition of soil microbial communities (both fungi and bacteria) in Qatar and determining which edaphic parameters exert the strongest influences on these communities. Preliminary results indicate that in overall bacteria are more abundant in soil than fungi and few sites have notably higher abundance of these microbes. In addition, we have detected some soil patameters, which tend to have reduced the overall fungal abundance and enhanced the presence of arbuscular mycorrhizal fungi and N-fixing bacteria. More detailed information on the diversity and composition of soil microbial communities is expected from the high-throughput sequenced data.

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ATAC-seq with unique molecular identifiers improves quantification and footprinting

Communications Biology ◽

10.1038/s42003-020-01403-4 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Tao Zhu ◽

Keyan Liao ◽

Rongfang Zhou ◽

Chunjiao Xia ◽

Weibo Xie

Keyword(s):

Transcription Factor ◽

High Throughput ◽

High Throughput Sequencing ◽

Pcr Amplification ◽

Chromatin Accessibility ◽

Link Type ◽

Pcr Duplicates ◽

Identify Transcription Factor ◽

Accessible Chromatin ◽

Accurate Quantification

AbstractATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq.

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A novel approach for characterisation of conformational allergen epitopes combining phage display and high-throughput sequencing

Clinical and Translational Allergy ◽

10.1186/2045-7022-4-s2-p27 ◽

2014 ◽

Vol 4 (S2) ◽

Author(s):

Anders Christiansen ◽

Christian Skjodt Hansen ◽

Jens Vindahl Kringelum ◽

Ole Lund ◽

Katrine Lindholm Bogh ◽

...

Keyword(s):

Phage Display ◽

High Throughput ◽

High Throughput Sequencing ◽

Novel Approach

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NPARS—A Novel Approach to Address Accuracy and Reproducibility in Genomic Data Science

Frontiers in Big Data ◽

10.3389/fdata.2021.725095 ◽

2021 ◽

Vol 4 ◽

Author(s):

Li Ma ◽

Erich A. Peterson ◽

Ik Jae Shin ◽

Jason Muesse ◽

Katy Marino ◽

...

Keyword(s):

High Throughput ◽

Quantitative Methods ◽

Data Science ◽

High Throughput Sequencing ◽

Genomic Data ◽

Scientific Data ◽

Genomic Research ◽

Data Sets ◽

Novel Approach ◽

Genomic Studies

Background: Accuracy and reproducibility are vital in science and presents a significant challenge in the emerging discipline of data science, especially when the data are scientifically complex and massive in size. Further complicating matters, in the field of genomic-based science high-throughput sequencing technologies generate considerable amounts of data that needs to be stored, manipulated, and analyzed using a plethora of software tools. Researchers are rarely able to reproduce published genomic studies.Results: Presented is a novel approach which facilitates accuracy and reproducibility for large genomic research data sets. All data needed is loaded into a portable local database, which serves as an interface for well-known software frameworks. These include python-based Jupyter Notebooks and the use of RStudio projects and R markdown. All software is encapsulated using Docker containers and managed by Git, simplifying software configuration management.Conclusion: Accuracy and reproducibility in science is of a paramount importance. For the biomedical sciences, advances in high throughput technologies, molecular biology and quantitative methods are providing unprecedented insights into disease mechanisms. With these insights come the associated challenge of scientific data that is complex and massive in size. This makes collaboration, verification, validation, and reproducibility of findings difficult. To address these challenges the NGS post-pipeline accuracy and reproducibility system (NPARS) was developed. NPARS is a robust software infrastructure and methodology that can encapsulate data, code, and reporting for large genomic studies. This paper demonstrates the successful use of NPARS on large and complex genomic data sets across different computational platforms.

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ATAC-seq with unique molecular identifiers improves quantification and footprinting

10.1101/2020.10.22.351478 ◽

2020 ◽

Author(s):

Tao Zhu ◽

Keyan Liao ◽

Rongfang Zhou ◽

Chunjiao Xia ◽

Weibo Xie

Keyword(s):

Transcription Factor ◽

High Throughput ◽

High Throughput Sequencing ◽

Pcr Amplification ◽

Chromatin Accessibility ◽

Link Type ◽

Pcr Duplicates ◽

Identify Transcription Factor ◽

Accessible Chromatin ◽

Accurate Quantification

AbstractATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces an increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. In our study, UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq, which helps identify an additional 50% or more of footprints. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq.

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One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

BioMed Research International ◽

10.1155/2015/703213 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Emanuele Sasso ◽

Rolando Paciello ◽

Francesco D’Auria ◽

Gennaro Riccio ◽

Guendalina Froechlich ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Phage Display ◽

High Throughput ◽

High Throughput Sequencing ◽

Hepatitis C Virus Infection ◽

Novel Approach ◽

Phage Display Libraries ◽

Junction Protein

Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.

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AtrR Is an Essential Determinant of Azole Resistance in Aspergillus fumigatus

mBio ◽

10.1128/mbio.02563-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 5

Author(s):

Sanjoy Paul ◽

Mark Stamnes ◽

Grace Heredge Thomas ◽

Hong Liu ◽

Daisuke Hagiwara ◽

...

Keyword(s):

Transcription Factor ◽

Aspergillus Fumigatus ◽

High Throughput ◽

Transcriptional Control ◽

High Throughput Sequencing ◽

Target Genes ◽

Azole Resistance ◽

Promoter Regions ◽

Mobility Shift ◽

Content Type

ABSTRACT Aspergillosis associated with azole-resistant Aspergillus fumigatus has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the cyp51A gene that encodes the target of azole drugs, lanosterol α-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of cyp51A and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of cyp51A as well as that of an important membrane transporter protein gene called abcG1. In this work, we characterize two different mutant alleles of atrR, either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of atrR for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the abcG1 and cyp51A promoters. The ATRE in cyp51A was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis. IMPORTANCE Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Infections associated with A. fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene cyp51A as an important contributor to resistance in A. fumigatus. Here, we demonstrate that the transcription factor AtrR binds to a region in the cyp51A promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of A. fumigatus.

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Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative yeast two hybrid-high throughput sequencing approach

10.1101/2021.05.11.443545 ◽

2021 ◽

Author(s):

Ana Lechuga ◽

Cédric Lood ◽

Mónica Berjón-Otero ◽

Alicia Del Prado ◽

Jeroen Wagemans ◽

...

Keyword(s):

High Throughput ◽

Protein Interactions ◽

High Throughput Sequencing ◽

Genomic Library ◽

Protein Protein Interactions ◽

Yeast Two Hybrid ◽

Protein Protein Interaction ◽

Novel Approach ◽

Diverse Groups ◽

Two Hybrid

Bacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35- Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35’s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35- B. thuringiensis interaction at the molecular level.

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clusterSeq: methods for identifying co-expression in high-throughput sequencing data

10.1101/188581 ◽

2017 ◽

Cited By ~ 1

Author(s):

Thomas J. Hardcastle ◽

Irene Papatheodorou

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Structural Elements ◽

Sequencing Data ◽

Experimental Conditions ◽

Functional Relationships ◽

Link Type ◽

High Throughput Sequencing Data ◽

Novel Approach ◽

Significant Step

ABSTRACTSummary:Identifying gene co-expression is a significant step in understanding functional relationships between genes. Existing methods primarily depend on analyses of correlation between pairs of genes; however, this neglects structural elements between experimental conditions. We present a novel approach to identifying clusters of co-expressed genes that incorporates these structures.Availability:The methods are released on Bioconductor as the clusterSeq package (https://bioconductor.org/packages/release/bioc/html/clusterSeq.html).Contact: [email protected]

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Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid–High Throughput Sequencing Approach

International Journal of Molecular Sciences ◽

10.3390/ijms222011105 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11105

Author(s):

Ana Lechuga ◽

Cédric Lood ◽

Mónica Berjón-Otero ◽

Alicia del Prado ◽

Jeroen Wagemans ◽

...

Keyword(s):

High Throughput ◽

Protein Interactions ◽

High Throughput Sequencing ◽

Genomic Library ◽

Structural Protein ◽

Protein Protein Interactions ◽

Yeast Two Hybrid ◽

Novel Approach ◽

Host Membrane ◽

Two Hybrid

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein–protein interactions (PPIs) network for a tectivirus–host system by studying the Bam35–Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35′s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait–prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus–host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host–phage system from the other reported host–phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35–B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus–host models.

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