Genetic algorithm based image reconstruction applying the digital holography process with the Discrete Orthonormal Stockwell Transform technique for diagnosis of COVID-19.

World Health Organization has described the real-time reverse transcription-polymerase chain reaction test method for the diagnosis of the novel coronavirus disease (COVID-19). However, the limited number of test kits, the long-term results of the tests, the high probability of the disease spreading during the test and imaging without focused images necessitate the use of alternative diagnostic methods such as chest X-ray (CXR) imaging. The storage of data obtained for the diagnosis of the disease also poses a major problem. This causes misdiagnosis and delays treatment. In this work, we propose a hybrid 3D reconstruction method of CXR images (CXRI) to detect coronavirus pneumonia and prevent misdiagnosis on CXRI. We used the digital holography technique (DHT) for obtaining a priori information of CXRI stored in created digital hologram (CDH). In this way, the elimination of the storage problem that requires high space was revealed. In addition, Discrete Orthonormal S-Transform (DOST) is applied to the reconstructed CDH image obtained by using DHT. This method is called CDH_DHT_DOST. A multiresolution spatial-frequency representation of the lung images that belong to healthy people and diseased people with the COVID-19 virus is obtained by using the CDH_DHT_DOST. Moreover, the genetic algorithm (GA) is adopted for the reconstruction process for optimization of the CDH image and then DOST is applied. This hybrid method is called CDH_GA_DOST. Finally, we compare the results obtained from CDH_DHT_DOST and CDH_GA_DOST. The results show the feasibility of reconstructing CXRI with CDH_GA_DOST. The proposed method holds promises to meet demands for the detection of the COVID-19 virus.

Evaluation of CRISPR gene-editing tools in zebrafish.

BACKGROUND: Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create 'knockout' models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies. METHODS: To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression. RESULTS: We compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (< 1%). To characterize if commonly used 'mock' CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the 'mock' injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation. CONCLUSION: Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.

Diverse Molecular Mechanisms Contribute to Differential Expression of Human Duplicated Genes.

Emerging evidence links genes within human-specific segmental duplications (HSDs) to traits and diseases unique to our species. Strikingly, despite being nearly identical by sequence (>98.5%), paralogous HSD genes are differentially expressed across human cell and tissue types, though the underlying mechanisms have not been examined. We compared cross-tissue mRNA levels of 75 HSD genes from 30 families between humans and chimpanzees and found expression patterns consistent with relaxed selection on or neofunctionalization of derived paralogs. In general, ancestral paralogs exhibited greatest expression conservation with chimpanzee orthologs, though exceptions suggest certain derived paralogs may retain or supplant ancestral functions. Concordantly, analysis of long-read isoform sequencing data sets from diverse human tissues and cell lines found that about half of derived paralogs exhibited globally lower expression. To understand mechanisms underlying these differences, we leveraged data from human lymphoblastoid cell lines (LCLs) and found no relationship between paralogous expression divergence and post-transcriptional regulation, sequence divergence, or copy-number variation. Considering cis-regulation, we reanalyzed ENCODE data and recovered hundreds of previously unidentified candidate CREs in HSDs. We also generated large-insert ChIP-sequencing data for active chromatin features in an LCL to better distinguish paralogous regions. Some duplicated CREs were sufficient to drive differential reporter activity, suggesting they may contribute to divergent cis-regulation of paralogous genes. This work provides evidence that cis-regulatory divergence contributes to novel expression patterns of recent gene duplicates in humans.

Assessment of Autism Zebrafish Mutant Models Using a High-Throughput Larval Phenotyping Platform.

In recent years, zebrafish have become commonly used as a model for studying human traits and disorders. Their small size, high fecundity, and rapid development allow for more high-throughput experiments compared to other vertebrate models. Given that zebrafish share >70% gene homologs with humans and their genomes can be readily edited using highly efficient CRISPR methods, we are now able to rapidly generate mutations impacting practically any gene of interest. Unfortunately, our ability to phenotype mutant larvae has not kept pace. To address this challenge, we have developed a protocol that obtains multiple phenotypic measurements from individual zebrafish larvae in an automated and parallel fashion, including morphological features (i.e., body length, eye area, and head size) and movement/behavior. By assaying wild-type zebrafish in a variety of conditions, we determined optimal parameters that avoid significant developmental defects or physical damage; these include morphological imaging of larvae at two time points [3 days post fertilization (dpf) and 5 dpf] coupled with motion tracking of behavior at 5 dpf. As a proof-of-principle, we tested our approach on two novel CRISPR-generated mutant zebrafish lines carrying predicted null-alleles of syngap1b and slc7a5, orthologs to two human genes implicated in autism-spectrum disorder, intellectual disability, and epilepsy. Using our optimized high-throughput phenotyping protocol, we recapitulated previously published results from mouse and zebrafish models of these candidate genes. In summary, we describe a rapid parallel pipeline to characterize morphological and behavioral features of individual larvae in a robust and consistent fashion, thereby improving our ability to better identify genes important in human traits and disorders.

Telomere-to-telomere assembly of a complete human X chromosome.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

Identification of Structural Variation in Chimpanzees Using Optical Mapping and Nanopore Sequencing.

Recent efforts to comprehensively characterize great ape genetic diversity using short-read sequencing and single-nucleotide variants have led to important discoveries related to selection within species, demographic history, and lineage-specific traits. Structural variants (SVs), including deletions and inversions, comprise a larger proportion of genetic differences between and within species, making them an important yet understudied source of trait divergence. Here, we used a combination of long-read and -range sequencing approaches to characterize the structural variant landscape of two additional Pan troglodytes verus individuals, one of whom carries 13% admixture from Pan troglodytes troglodytes. We performed optical mapping of both individuals followed by nanopore sequencing of one individual. Filtering for larger variants (>10 kbp) and combined with genotyping of SVs using short-read data from the Great Ape Genome Project, we identified 425 deletions and 59 inversions, of which 88 and 36, respectively, were novel. Compared with gene expression in humans, we found a significant enrichment of chimpanzee genes with differential expression in lymphoblastoid cell lines and induced pluripotent stem cells, both within deletions and near inversion breakpoints. We examined chromatin-conformation maps from human and chimpanzee using these same cell types and observed alterations in genomic interactions at SV breakpoints. Finally, we focused on 56 genes impacted by SVs in >90% of chimpanzees and absent in humans and gorillas, which may contribute to chimpanzee-specific features. Sequencing a greater set of individuals from diverse subspecies will be critical to establish the complete landscape of genetic variation in chimpanzees.

Potential genetic biomarkers in the early diagnosis of Alzheimer disease: APOE and BIN1.

BACKGROUND/AIM: Alzheimer disease (AD) is triggered by interactions of multiple genetic and environmental factors. The APOE gene E4 allele is the best-known risk factor for AD, yet it represents a small ratio of genetic factors. According to genome-wide association studies, the BIN1 gene is the second important risk factor for AD, following the APOE gene. We aimed to identify a novel biomarker indicating susceptibility to AD by investigating APOE alleles and BIN1 gene polymorphisms in a Turkish population. MATERIALS AND METHODS: Fifty-three AD patients and 56 controls were included to examine polymorphism and allele frequency of the APOE and BIN1 genes. Genomic DNAs were isolated from whole blood by SDS/proteinase K treatment, phenol-chloroform extraction, and ethanol precipitation. RFLP was done for identification of polymorphisms in the APOE gene and allele-specific PCR was used for the BIN1 gene. RESULTS: Frequency of the APOE E4 allele was higher in the AD patient group, while the frequency of the E2 allele was higher in controls. The E4/E4 genotype was detected in the AD patient group, while this genotype was not observed in the controls. The frequencies of BIN1 alleles were similar in both groups. CONCLUSION: There was a strong association between AD and the APOE E4 allele, while no such relation was observed with BIN1 gene polymorphism.

The effects and interactions of APOE and APH-1A polymorphisms in Alzheimer disease.

BACKGROUND/AIM: Alzheimer disease (AD) is characterized by the accumulation of senile plaques composed of amyloid ß-peptide, which is derived from ß-amyloid precursor protein through degradation by ß-secretase and y-secretase complexes. One of the major components of y-secretase complex, anterior pharynx-defective-1 (APH-1), is responsible for the activity of the γ-secretase complex. In this study, we searched for not only the most known common genetic risk factor, APOE, but also the APH-1a gene polymorphism in AD patients in a Turkish population. MATERIALS AND METHODS: In this study, 49 AD patients and 45 healthy controls were included. The genetic polymorphisms and allele frequencies of APOE and APH-1a were investigated. Patients were evaluated for behavioral, cognitive, and functional domains by detailed neurocognitive tests, and comparison between the above-mentioned polymorphisms and disease severity was made. RESULTS: Although there was an increased tendency of the APO ε4 allele in the AD group, no statistically significant difference was detected either in APOE or APH-1a polymorphisms, not suggesting a strong susceptibility to the development of AD. CONCLUSION: While searching for the pathogenesis of AD in order to develop novel diagnostic as well as therapeutic approaches, analysis of other genes with a possible role in AD is warranted.