scholarly journals Resolving the dark matter of ABCA4 for 1,054 Stargardt disease probands through integrated genomics and transcriptomics

2019 ◽  
Author(s):  
Mubeen Khan ◽  
Stéphanie S. Cornelis ◽  
Marta del Pozo-Valero ◽  
Laura Whelan ◽  
Esmee H. Runhart ◽  
...  
Keyword(s):  
Data Storage ◽  
Single Molecule ◽  
Large Scale ◽  
Cost Effective ◽  
Chromosome 1 ◽  
Stargardt Disease ◽  
Model Study ◽  
Integrated Genomics ◽  
Intronic Variants

ABSTRACTMissing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.

scholarly journals 3D DNA structural barcode copying and random access

2020 ◽  
Author(s):  
Filip Bošković ◽  
Alexander Ohmann ◽  
Ulrich F. Keyser ◽  
Kaikai Chen
Keyword(s):  
Data Storage ◽  
Single Molecule ◽  
Self Assembly ◽  
Large Scale ◽  
Three Dimensional ◽  
Random Access ◽  
Scale Production ◽  
Digital Information ◽  
Dna Nanostructures ◽  
Large Scale Production

AbstractThree-dimensional (3D) DNA nanostructures built via DNA self-assembly have established recent applications in multiplexed biosensing and storing digital information. However, a key challenge is that 3D DNA structures are not easily copied which is of vital importance for their large-scale production and for access to desired molecules by target-specific amplification. Here, we build 3D DNA structural barcodes and demonstrate the copying and random access of the barcodes from a library of molecules using a modified polymerase chain reaction (PCR). The 3D barcodes were assembled by annealing a single-stranded DNA scaffold with complementary short oligonucleotides containing 3D protrusions at defined locations. DNA nicks in these structures are ligated to facilitate barcode copying using PCR. To randomly access a target from a library of barcodes, we employ a non-complementary end in the DNA construct that serves as a barcode-specific primer template. Readout of the 3D DNA structural barcodes was performed with nanopore measurements. Our study provides a roadmap for convenient production of large quantities of self-assembled 3D DNA nanostructures. In addition, this strategy offers access to specific targets, a crucial capability for multiplexed single-molecule sensing and for DNA data storage.

scholarly journals Mixed Culture of Bacterial Cell for Large Scale DNA Storage

2020 ◽  
Author(s):  
Min Hao ◽  
Hongyan Qiao ◽  
Yanmin Gao ◽  
Zhaoguan Wang ◽  
Xin Qiao ◽  
...  
Keyword(s):  
Mixed Culture ◽  
Data Storage ◽  
Bacterial Cell ◽  
Living Cell ◽  
Large Scale ◽  
Digital Data ◽  
Human Society ◽  
Bacterial Cells ◽  
Digital Information

AbstractDNA emerged as novel material for mass data storage, the serious problem human society is facing. Taking advantage of current synthesis capacity, massive oligo pool demonstrated its high-potential in data storage in test tube. Herein, mixed culture of bacterial cells carrying mass oligo pool that was assembled in a high copy plasmid was presented as a stable material for large scale data storage. Living cells data storage was fabricated by a multiple-steps process, assembly, transformation and mixed culture. The underlying principle was explored by deep bioinformatic analysis. Although homology assembly showed sequence context dependent bias but the massive digital information oligos in mixed culture were constant over multiple successive passaging. In pushing the limitation, over ten thousand distinct oligos, totally 2304 Kbps encoding 445 KB digital data including texts and images, were stored in bacterial cell, the largest archival data storage in living cell reported so far. The mixed culture of living cell data storage opens up a new approach to simply bridge the in vitro and in vivo storage system with combined advantage of both storage capability and economical information propagation.

Cost‐effective molecular inversion probe‐based ABCA4 sequencing reveals deep‐intronic variants in Stargardt disease

2019 ◽  
Vol 40 (10) ◽  
pp. 1749-1759 ◽  
Author(s):  
Mubeen Khan ◽  
Stéphanie S. Cornelis ◽  
Muhammad Imran Khan ◽  
Duaa Elmelik ◽  
Eline Manders ◽  
...  
Keyword(s):  
Cost Effective ◽  
Stargardt Disease ◽  
Molecular Inversion Probe ◽  
Intronic Variants

Prevention of Toxic Effects of Mycotoxins by Means of Nonnutritive Adsorbent Compounds

1996 ◽  
Vol 59 (6) ◽  
pp. 631-641 ◽  
Author(s):  
ANTONIO-JAVIER RAMOS ◽  
JOHANA FINK-GREMMELS ◽  
ENRIQUE HERNÁNDEZ
Keyword(s):  
Large Scale ◽  
Cost Effective ◽  
Toxic Effects ◽  
Animal Products ◽  
Recent Approach ◽  
Calcium Aluminosilicate ◽  
Exchange Resins ◽  
Biological Methods

Mycotoxins comprise a family of fungal toxins, many of which have been implicated as chemical progenitors of toxicity in man and animals. The most thoroughly studied are the aflatoxins. A variety of physical, chemical, and biological methods to counteract the mycotoxin problem have been reported, but large-scale, practical, and cost-effective methods for detoxifying mycotoxin-containing feedstuffs are currently not available. The most recent approach to the problem has been the addition to the animal's diet of nonnutritive sorbents that sequester mycotoxins and reduce their gastrointestinal absorption, avoiding their toxic effects on livestock and toxin carryover into animal products. This review comments on the in vitro efficacy of several of the adsorbents assayed, and their in vivo applications in a range of animals will be discussed. The sorbents reviewed are activated charcoal, bentonite, zeolite, hydrated sodium calcium aluminosilicate (HSCAS) and a wide variety of clays and synthetic ion-exchange resins.

Cell-based models for mycotoxin screening and toxicity evaluation: an update

2014 ◽  
Vol 7 (2) ◽  
pp. 153-166 ◽  
Author(s):  
F. Cheli ◽  
E. Fusi ◽  
A. Baldi
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Mycotoxin Research ◽  
Cost Effective ◽  
Screening Tests ◽  
Test Model ◽  
Related Factors ◽  
Technical Problems ◽  
Third Dimension

This review presents the applications of cell-based models in mycotoxin research, with a focus on models for mycotoxin screening and cytotoxicity evaluation. Various cell-based models, cell and cell culture condition related factors, toxicity endpoints and culture systems as well as predictive value of cell-based bioassays are reviewed. Advantages, drawbacks and technical problems regarding set up and validation of consistent, robust, reproducible and high-throughput cell-based models are discussed. Various cell-based models have been developed and used as screening tests for mycotoxins but the data obtained are difficult to compare. However, the results highlight the potential of cell-based models as promising in vitro platforms for the initial screening and cytotoxicity evaluation of mycotoxins and as a significant analytical approach in mycotoxin research before any animal or human clinical studies. To develop cell-based models as powerful high-throughput laboratory platforms for the analysis of large numbers of samples, there are mainly two fundamental requirements that should be met, i.e. the availability of easy-to-use and, if possible, automated cell platforms and the possibility to obtain reproducible results that are comparable between laboratories. The transition from a research model to a test model still needs optimisation, standardisation, and validation of analytical protocols. The validation of a cell-based bioassay is a complex process, as several critical points, such as the choice of the cellular model, the assay procedures, and the appropriate use and interpretation of the results, must be strictly defined to ensure more consistency in the results. The development of cell-based models exploring the third dimension together with automation and miniaturisation will bring cellular platforms to a level appropriate for cost-effective and large-scale analysis in the field of mycotoxin research.

scholarly journals Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4

2021 ◽  
Vol 22 (9) ◽  
pp. 4621
Author(s):  
Tomasz Z. Tomkiewicz ◽  
Nuria Suárez-Herrera ◽  
Frans P. M. Cremers ◽  
Rob W. J. Collin ◽  
Alejandro Garanto
Keyword(s):  
Antisense Oligonucleotide ◽  
Mrna Splicing ◽  
Stargardt Disease ◽  
Aberrant Splicing ◽  
Suitable Candidate ◽  
Pathogenic Variants ◽  
Splice System ◽  
Splicing Defects ◽  
Intronic Variants

The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.

scholarly journals Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein

2020 ◽  
Vol 16 (11) ◽  
pp. e1008293
Author(s):  
Dylan Girodat ◽  
Avik K. Pati ◽  
Daniel S. Terry ◽  
Scott C. Blanchard ◽  
Karissa Y. Sanbonmatsu
Keyword(s):  
Time Scales ◽  
Single Molecule ◽  
Conformational Changes ◽  
Time Resolution ◽  
Large Scale ◽  
Md Simulations ◽  
Orientation Factor ◽  
Ribonucleoprotein Complexes ◽  
Fluorescence Measurements

Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.

scholarly journals OptiDiff: structural variation detection from single optical mapping reads

2022 ◽  
Author(s):  
Mehmet Akdel ◽  
Dick de Ridder
Keyword(s):  
Single Molecule ◽  
Large Scale ◽  
Structural Variation ◽  
Optical Mapping ◽  
Cost Effective ◽  
Sequencing Data ◽  
Mapping Technology ◽  
Cost Effective Alternative ◽  
Algorithmic Approaches ◽  
Broad Interest

Detecting structural variation (SV) in eukaryotic genomes is of broad interest due to its often dramatic phenotypic effects, but remains a major, costly challenge based on DNA sequencing data. A cost-effective alternative in detecting large-scale SV has become available with advances in optical mapping technology. However, the algorithmic approaches to identifying SVs from optical mapping data are limited. Here, we propose a novel, open-source SV detection tool, OptiDiff, which employs a single molecule based approach to detect and classify homozygous and heterozygous SVs at coverages as low as 20x, showing better performance than the state of the art.

A Comprehensive Study on Commercial Applications of Cloud Computing

2020 ◽  
Vol 17 (9) ◽  
pp. 4411-4418
Author(s):  
S. Jagannatha ◽  
B. N. Tulasimala
Keyword(s):  
Cloud Computing ◽  
Data Storage ◽  
High Performance ◽  
Large Scale ◽  
Service Providers ◽  
Cost Effective ◽  
Computer Hardware ◽  
Computing Technology ◽  
Computing Systems ◽  
Computational Performance

In the world of information communication technology (ICT) the term Cloud Computing has been the buzz word. Cloud computing is changing its definition the way technocrats are using it according to the environment. Cloud computing as a definition remains very contentious. Definition is stated liable to a particular application with no unanimous definition, making it altogether elusive. In spite of this, it is this technology which is revolutionizing the traditional usage of computer hardware, software, data storage media, processing mechanism with more of benefits to the stake holders. In the past, the use of autonomous computers and the nodes that were interconnected forming the computer networks with shared software resources had minimized the cost on hardware and also on the software to certain extent. Thus evolutionary changes in computing technology over a few decades has brought in the platform and environment changes in machine architecture, operating system, network connectivity and application workload. This has made the commercial use of technology more predominant. Instead of centralized systems, parallel and distributed systems will be more preferred to solve computational problems in the business domain. These hardware are ideal to solve large-scale problems over internet. This computing model is data-intensive and networkcentric. Most of the organizations with ICT used to feel storing of huge data, maintaining, processing of the same and communication through internet for automating the entire process a challenge. In this paper we explore the growth of CC technology over several years. How high performance computing systems and high throughput computing systems enhance computational performance and also how cloud computing technology according to various experts, scientific community and also the service providers is going to be more cost effective through different dimensions of business aspects.

scholarly journals In Vitro Propagation of Easter Island Curcuma longa from Rhizome Explants Using Temporary Immersion System

Agronomy ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2121
Author(s):  
María José Marchant ◽  
Paula Molina ◽  
Miriam Montecinos ◽  
Leda Guzmán ◽  
Cristobal Balada ◽  
...  
Keyword(s):  
In Vitro Propagation ◽  
Large Scale ◽  
Curcuma Longa ◽  
Cost Effective ◽  
Rapa Nui ◽  
Temporary Immersion System ◽  
Term Survival ◽  
Temporary Immersion ◽  
Large Scale Cultivation

Curcuma longa (C. longa) is widely known for its medicinal properties. However, the potential overexploitation of this plant raises doubts about its long-term survival on Rapa Nui. Micropropagation using a temporary immersion system (TIS) could be the basis for developing a cost-effective and highly productive method of large-scale cultivation of this plant. Our objective was to develop and refine the in vitro multiplication system for mass propagation of C. longa, and thus help restore the fragile ecosystem of Rapa Nui. Three parameters were evaluated: number of explants per flask, flask capacity, and LEDs spectrum. For each parameter evaluated, four aspects were analyzed: fresh weight per plant, number of shoots, percentage of non-sprouting explants, and the proliferation rate. The use of 30 explants per two-liter flask results in more plants with high fresh biomass than other configurations. In addition, LEDs with a red:blue ratio of 2:1 provided the best lighting conditions for in vitro propagation and positively affected C. longa proliferation and rooting. Therefore, our results show that 30 explants per two-liter flask and an LED source with a red:blue ratio of 2:1 allow a higher number of C. longa plants to be obtained using TIS.

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