scholarly journals Knockout Sudoku, a method for rapidly curating gene disruption collections

2016 ◽  
Author(s):  
Isao A Anzai ◽  
Lev Shaket ◽  
Oluwakemi Adesina ◽  
Michael Baym ◽  
Buz Barstow
Keyword(s):  
Genetic Screening ◽  
Gene Disruption ◽  
Insertion Mutant ◽  
Inference Algorithm ◽  
High Quality ◽  
Microbial Genomes ◽  
Transposon Insertion ◽  
Wide Range ◽  
Combinatorial Pooling ◽  
Generation Sequencing

Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of approximately $10,000. The method uses manual 4-dimensional combinatorial pooling, next-generation sequencing and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. Here we present a protocol for the generation, combinatorial pooling and annotation of highly oversampled progenitor collections and their subsequent algorithmically guided condensation and curation into high-quality collections suitable for rapid genetic screening and gene discovery.

scholarly journals Knockout Sudoku, a method for rapidly curating gene disruption collections

2016 ◽  
Author(s):  
Isao A Anzai ◽  
Lev Shaket ◽  
Oluwakemi Adesina ◽  
Michael Baym ◽  
Buz Barstow
Keyword(s):  
Genetic Screening ◽  
Gene Disruption ◽  
Insertion Mutant ◽  
Inference Algorithm ◽  
High Quality ◽  
Microbial Genomes ◽  
Transposon Insertion ◽  
Wide Range ◽  
Combinatorial Pooling ◽  
Generation Sequencing

Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of approximately $10,000. The method uses manual 4-dimensional combinatorial pooling, next-generation sequencing and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. Here we present a protocol for the generation, combinatorial pooling and annotation of highly oversampled progenitor collections and their subsequent algorithmically guided condensation and curation into high-quality collections suitable for rapid genetic screening and gene discovery.

Reconstructing single genomes from complex microbial communities

2016 ◽  
Vol 58 (3) ◽  
Author(s):  
Dongwan D. Kang ◽  
Edward M. Rubin ◽  
Zhong Wang
Keyword(s):  
Microbial Communities ◽  
Environmental Samples ◽  
Research Community ◽  
Functional Metagenomics ◽  
High Quality ◽  
Microbial Genomes ◽  
Sequencing Technologies ◽  
Approaches To Study ◽  
Generation Sequencing ◽  
Pathway Level

AbstractHigh throughput next generation sequencing technologies have enabled cultivation-independent approaches to study microbial communities in environmental samples. To date much of functional metagenomics has been limited to the gene or pathway level. Recent breakthroughs in metagenome binning have made it feasible to reconstruct high quality, individual microbial genomes from complex communities with thousands of species. In this review we aim to compare several automated metagenome binning software tools for their performance, and provide a practical guide for the metagenomics research community to carry out successful binning analyses.

AMPCOLOY 570

Alloy Digest ◽  
1980 ◽  
Vol 29 (3) ◽  
Keyword(s):  
Corrosion Resistance ◽  
Cast Iron ◽  
Iron Alloy ◽  
Heat Treating ◽  
Nickel Aluminum ◽  
High Quality ◽  
Glass Making ◽  
Temperature Performance ◽  
Wide Range ◽  
Cobalt Iron

Abstract AMPCOLOY 570 is a cast copper-nickel-aluminum-cobalt-iron alloy specially developed for applications involving severe stresses and high temperatures, such as glass-making molds and plate-glass rolls. It is significantly superior to cast iron which has been commonly used for glass-making molds. Good foundry techniques will yield high-quality castings of Ampcoloy 570 in a wide range of section sizes. This datasheet provides information on composition, physical properties, hardness, elasticity, and tensile properties. It also includes information on high temperature performance and corrosion resistance as well as casting, heat treating, machining, and joining. Filing Code: Cu-392. Producer or source: Ampco Metal Inc..

scholarly journals The Double Mutation DSG2-p.S363X and TBX20-p.D278X Is Associated with Left Ventricular Non-Compaction Cardiomyopathy: Case Report

2021 ◽  
Vol 22 (13) ◽  
pp. 6775
Author(s):  
Roman Myasnikov ◽  
Andreas Brodehl ◽  
Alexey Meshkov ◽  
Olga Kulikova ◽  
Anna Kiseleva ◽  
...  
Keyword(s):  
Genetic Screening ◽  
Ventricular Dysfunction ◽  
Layer Structure ◽  
Left Ventricular ◽  
Cell Transfection ◽  
Nonsense Mutations ◽  
Double Mutation ◽  
T Box ◽  
Generation Sequencing

Left ventricular non-compaction cardiomyopathy (LVNC) is a rare heart disease, with or without left ventricular dysfunction, which is characterized by a two-layer structure of the myocardium and an increased number of trabeculae. The study of familial forms of LVNC is helpful for risk prediction and genetic counseling of relatives. Here, we present a family consisting of three members with LVNC. Using a next-generation sequencing approach a combination of two (likely) pathogenic nonsense mutations DSG2-p.S363X and TBX20-p.D278X was identified in all three patients. TBX20 encodes the cardiac T-box transcription factor 20. DSG2 encodes desmoglein–2, which is part of the cardiac desmosomes and belongs to the cadherin family. Since the identified nonsense variant (DSG2-p.S363X) is localized in the extracellular domain of DSG2, we performed in vitro cell transfection experiments. These experiments revealed the absence of truncated DSG2 at the plasma membrane, supporting the pathogenic relevance of DSG2-p.S363X. In conclusion, we suggest that in the future, these findings might be helpful for genetic screening and counseling of patients with LVNC.

scholarly journals Re-Evaluation of the Podosphaera tridactyla Species Complex in Australia

2021 ◽  
Vol 7 (3) ◽  
pp. 171
Author(s):  
Reannon L. Smith ◽  
Tom W. May ◽  
Jatinder Kaur ◽  
Tim I. Sawbridge ◽  
Ross C. Mann ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Powdery Mildew ◽  
Species Complex ◽  
Taxonomic Revision ◽  
Stone Fruit ◽  
Prunus Species ◽  
Wide Range ◽  
Unidentified Species ◽  
The Rose ◽  
Generation Sequencing

The Podosphaera tridactyla species complex is highly variable morphologically and causes powdery mildew on a wide range of Prunus species, including stone fruit. A taxonomic revision of the Po. tridactyla species complex in 2020 identified 12 species, seven of which were newly characterised. In order to clarify which species of this complex are present in Australia, next generation sequencing was used to isolate the fungal ITS+28S and host matK chloroplast gene regions from 56 powdery mildew specimens of stone fruit and ornamental Prunus species accessioned as Po. tridactyla or Oidium sp. in Australian reference collections. The specimens were collected in Australia, Switzerland, Italy and Korea and were collected from 1953 to 2018. Host species were confirmed using matK phylogenetic analysis, which identified that four had been misidentified as Prunus but were actually Malusprunifolia. Podosphaera species were identified using ITS+28S phylogenetic analysis, recognising three Podosphaera species on stone fruit and related ornamental Prunus hosts in Australia. These were Po.pannosa, the rose powdery mildew, and two species in the Po. tridactyla species complex: Po. ampla, which was the predominant species, and a previously unidentified species from peach, which we describe here as Po. cunningtonii.

scholarly journals Next-Generation Sequencing Analysis of the Tineola bisselliella Larval Gut Transcriptome Reveals Candidate Enzymes for Keratin Digestion

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1113
Author(s):  
Michael Schwabe ◽  
Sven Griep ◽  
Henrike Schmidtberg ◽  
Rudy Plarre ◽  
Alexander Goesmann ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Disulfide Bonds ◽  
Differentially Expressed ◽  
Intestinal Microbiome ◽  
Sequencing Analysis ◽  
High Quality ◽  
Transcriptomic Data ◽  
Next Generation Sequencing Analysis ◽  
Generation Sequencing ◽  
Tineola Bisselliella

The clothes moth Tineola bisselliella is one of a few insects that can digest keratin, leading to the destruction of clothing, textiles and artwork. The mechanism of keratin digestion is not yet fully understood, partly reflecting the lack of publicly available genomic and transcriptomic data. Here we present a high-quality gut transcriptome of T. bisselliella generated from larvae reared on keratin-rich and keratin-free diets. The overall transcriptome consists of 428,221 contigs that were functionally annotated and screened for candidate enzymes involved in keratin utilization. As a mechanism for keratin digestion, we identified cysteine synthases, cystathionine β-synthases and cystathionine γ-lyases. These enzymes release hydrogen sulfite, which may reduce the disulfide bonds in keratin. The dataset also included 27 differentially expressed contigs with trypsin domains, among which 20 were associated with keratin feeding. Finally, we identified seven collagenases that were upregulated on the keratin-rich diet. In addition to this enzymatic repertoire potentially involved in breaking down keratin, our analysis of poly(A)-enriched and poly(A)-depleted transcripts suggested that T. bisselliella larvae possess an unstable intestinal microbiome that may nevertheless contribute to keratin digestion.

scholarly journals Next-generation sequencing in the clinical genetic screening of patients with pheochromocytoma and paraganglioma

2013 ◽  
Vol 2 (2) ◽  
pp. 104-111 ◽  
Author(s):  
Joakim Crona ◽  
Alberto Delgado Verdugo ◽  
Dan Granberg ◽  
Staffan Welin ◽  
Peter Stålberg ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genetic Screening ◽  
Bioinformatics Analysis ◽  
Cost Effective ◽  
Susceptibility Genes ◽  
Next Generation ◽  
Clinical Genetic ◽  
Cost Effective Method ◽  
Agilent Sureselect ◽  
Generation Sequencing

BackgroundRecent findings have shown that up to 60% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are caused by germline or somatic mutations in one of the 11 hitherto known susceptibility genes: SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, HIF2A (EPAS1), RET, NF1, TMEM127 and MAX. This list of genes is constantly growing and the 11 genes together consist of 144 exons. A genetic screening test is extensively time consuming and expensive. Hence, we introduce next-generation sequencing (NGS) as a time-efficient and cost-effective alternative.MethodsTumour lesions from three patients with apparently sporadic PCC were subjected to whole exome sequencing utilizing Agilent Sureselect target enrichment system and Illumina Hi seq platform. Bioinformatics analysis was performed in-house using commercially available software. Variants in PCC and PGL susceptibility genes were identified.ResultsWe have identified 16 unique genetic variants in PCC susceptibility loci in three different PCC, spending less than a 30-min hands-on, in-house time. Two patients had one unique variant each that was classified as probably and possibly pathogenic: NF1 Arg304Ter and RET Tyr791Phe. The RET variant was verified by Sanger sequencing.ConclusionsNGS can serve as a fast and cost-effective method in the clinical genetic screening of PCC. The bioinformatics analysis may be performed without expert skills. We identified process optimization, characterization of unknown variants and determination of additive effects of multiple variants as key issues to be addressed by future studies.

Guiding Principals: Middle-Manager Coaching and Human-Capital Reform

2020 ◽  
Vol 122 (10) ◽  
pp. 1-50
Author(s):  
Susan Bush-Mecenas ◽  
Julie A. Marsh ◽  
Katharine O. Strunk
Keyword(s):  
Professional Development ◽  
Human Capital ◽  
Policy Implementation ◽  
Survey Data ◽  
Empirical Work ◽  
Think Aloud ◽  
High Quality ◽  
Wide Range ◽  
Role Of Principal

Background/Context School leaders are central to state and district human-capital reforms (HCRs), yet they are rarely equipped with the skills to implement new evaluation, professional development, and personnel data systems. Although districts increasingly offer principals coaching and training, there has been limited empirical work on how these supports influence principals’ HCR-related practices. Purpose Drawing on a two-year, mixed-methods study in the Los Angeles Unified School District (LAUSD), this article examines the role of principal supervisors in HCRs. We ask: What role did principal supervisors (Instructional Directors [IDs]) play in the implementation of human-capital reforms? What did high-quality coaching on the part of IDs look like in this context? Research Design Our two-part analysis draws upon survey and interview data. First, we conducted descriptive analyses and significance testing using principal and ID survey data to examine the correlations among principals’ ratings of ID coaching quality, ID coaching practices, and principals’ implementation of HCRs. Second, we conducted in-depth interviews, using a think-aloud protocol, with two sets of IDs—those consistently highly-rated and those with mixed ratings—who were identified using principals’ reports of coaching quality. Following interview coding, we created various case-ordered metamatrix displays to analyze our qualitative data in order to identify patterns in coaching strategy and approach across IDs, content, and contexts. Findings First, our survey data indicate that receiving high-quality coaching from IDs is correlated with stronger principal support for and implementation of HCRs. Our survey findings further illustrate that IDs support a wide range of principals’ HCR activities. Second, our think-aloud interviews with case IDs demonstrate that coaching strategy and approach vary between consistently highly-rated and mixed-rated coaches: Consistently highly-rated IDs emphasize the importance of engaging in, or defining HCR problems as, joint work alongside principals, while mixed-rated IDs often emphasize the use of tools to guide principal improvement. We find that, on the whole, the consistently highly-rated IDs in our sample employ a nondirective approach to coaching more often than mixed-rated coaches. Conclusions These findings contribute to a growing literature on the crucial role of principal supervisors as coaches to improve principals’ instructional leadership and policy implementation. While exploratory, this study offers the first steps toward building greater evidence of the connections between high-quality coaching and policy implementation, and it may have implications for the design and implementation of professional development for principal supervisors and the selection and placement of supervisors with principals.

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