Segmentation-free inference of cell types from in situ transcriptomics data

Recent advances in the fields of genome editing, whole-genome sequencing, single-cell RNA sequencing, and in situ spatial transcriptomics have enabled the cost-efficient production of high-throughput big data. However, the lack of dedicated bioinformatics algorithms to analyze such data has been a big hurdle. In this thesis, several novel bioinformatics tools applicable to each field are presented. First, a series of web-based tools for genome editing are presented: Cpf1-Database, Cas-Analyzer, web-based Digenome-seq software, BE-Designer/Analyzer. These tools have been developed to guide researchers to easily use genome editing systems, using Cas9 or Cpf1, by providing an easily accessible web-based interface. Second, the development of two bioinformatics pipelines are described: a small variant calling pipeline to process tumor genome sequencing data without a matched control, and a pipeline to pre-process single-cell RNA sequencing data. Third, a novel segmentation-free algorithm to call cell-types from in situ transcriptomics data, namely Spot-based Spatial cell-type Analysis by Multidimensional mRNA density estimation (SSAM) is presented. Recent advances of in situ spatial transcriptomics techniques, such as multiplexed fluorescence in situ hybridization or in situ/intact tissue sequencing have enabled the discovery of spatial heterogeneity of cell types at the tissue level. However, cell type calling methods are often limited by cell segmentation algorithms due to various imaging problems. SSAM circumvents these problems by estimating spatial gene expressions as a density estimation of the mRNA in a spatial context and identifying de novo cell-types and their spatial organization without the need to segment cells. Optionally, SSAM can be guided by external sources of cell-type information, integrating them in a spatial context. In this thesis, SSAM is demonstrated with three different mouse brain tissues imaged by different imaging techniques: the somatosensory cortex (SSp) imaged by osmFISH; the hypothalamic preoptic region (POA) by MERFISH; and the visual cortex (VISp) by multiplexed smFISH. SSAM can produce similar results compared to segmentation-based methods and outperforms them when cell segmentation is the limiting factor. In summary, the bioinformatics tools presented in this thesis overcome major obstacles that would normally hinder effective analysis: the web-based tools for genome editing have a wide user base due to their easy-to-use web-based interfaces; omics data analysis pipeline that enables fast analysis of omics data utilizing a compute cluster and facilitate hypothesis generation when lacking control tissue; and SSAM that enables the analysis of in situ spatial transcriptomics data without being limited by cell segmentation. All of the tools and pipelines described in this thesis are open-sourced and freely accessible for non-profit, research-purpose use

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.

CFD-based Design Optimization of Ducted Hydrokinetic Turbines

Hydrokinetic turbines extract kinetic energy from moving water to generate renewable electricity, thus contributing to sustainable energy production and reducing reliance on fossil fuels. It has been hypothesized that a duct can accelerate and condition the fluid flow passing the turbine blades, improving the overall energy extraction efficiency. However, no substantial evidence has been provided so far for hydrokinetic turbines. To investigate this problem, we perform a CFD-based optimization study with a blade-resolved Reynolds-averaged Navier--Stokes (RANS) solver to explore the design of a ducted hydrokinetic turbine that maximizes the efficiency of energy extraction. To handle the high-dimensional design space of the blade and duct geometry, we use a gradient-based optimization approach where the gradients are computed using the adjoint method. The final design is re-evaluated through higher-fidelity unsteady RANS (URANS) simulations. Our optimized ducted turbine achieves an efficiency of about 54% over a range of operating conditions, higher than the typical 46% efficiency of unducted turbines such as the well-known Bahaj model

Web-based design and analysis tools for CRISPR base editing

Background: As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed. Results: We present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (&lt; 1 GB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/be-designer/ and http://www.rgenome.net/be-analyzer /, respectively. Conclusion: We develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors

The tropical rain belts with an annual cycle and a continent model intercomparison project: TRACMIP

This paper introduces the Tropical Rain belts with an Annual cycle and a Continent Model Intercomparison Project (TRACMIP). TRACMIP studies the dynamics of tropical rain belts and their response to past and future radiative forcings through simulations with 13 comprehensive and one simplified atmosphere models coupled to a slab ocean and driven by seasonally-varying insolation. Five idealised experiments, two with an aquaplanet setup and three with a setup with an idealized tropical continent, fill the space between prescribed-SST aquaplanet simulations and realistic simulations provided by CMIP5/6. The simulations reproduce key features of present-day climate and expected future climate change, including an annual-mean intertropical convergence zone (ITCZ) that is located north of the equator and Hadley cells and eddy-driven jets that are similar to present-day climate. Quadrupling CO2 leads to a northward ITCZ shift and preferential warming in Northern high-latitudes. The simulations show interesting CO2-induced changes in the seasonal excursion of the ITCZ and indicate a possible state-dependence of climate sensitivity. The inclusion of an idealized continent modulates both the control climate and the response to increased CO2; for example, it reduces the northward ITCZ shift associated with warming and, in some models, climate sensitivity. In response to eccentricity-driven orbital seasonal insolation changes, seasonal changes in oceanic rainfall are best characterized as a meridional dipole, while seasonal continental rainfall changes tend to be symmetric about the equator. This survey illustrates TRACMIP's potential to engender a deeper understanding of global and regional climate and to address questions on past and future climate

Smaller pineal gland is associated with rapid eye movement sleep behavior disorder in Alzheimers disease

Background To investigate the association between pineal gland volume and symptoms of rapid eye movement (REM) sleep behavior disorder (RBD) in Alzheimers disease (AD) patients without any feature of dementia with Lewy bodies. Methods We enrolled 296 community-dwelling probable AD patients who did not meet the diagnostic criteria for possible or probable dementia with Lewy bodies. Among them, 93 were amyloid beta (Aβ) positive on 18F-florbetaben amyloid brain positron emission tomography. We measured RBD symptoms using the REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) and defined probable RBD (pRBD) as the RBDSQ of 5 or higher. We manually segmented pineal gland on 3T structural T1-weighted brain magnetic resonance imaging. Results The participants with pRBD had smaller pineal parenchyma volume (VPP) than those without pRBD (p <  0.001). The smaller the VPP, the more severe the RBD symptoms (p <  0.001). VPP was inversely associated with risk of prevalent pRBD (odds ratio = 0.909, 95% confidence interval [CI] = 0.878–0.942, p <  0.001). Area under the receiver operator characteristic curve for pRBD of VPP was 0.80 (95% CI = 0.750–0.844, p <  0.0001). These results were not changed when we analyzed the 93 participants with Aβ-positive AD separately. Conclusions In AD patients, reduced pineal gland volume may be associated with RBD.This study was supported by the grants from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (grant no.HI09C1379 [A092077]) and the Institute for Information & Communications Technology Promotion (IITP) grant funded by the Korea government (MSIT) (2018-2-00861, Intelligent SW Technology Development for Medical Data Analysis)

RAS-pathway mutation patterns define epigenetic subclasses in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood characterized by mutations activating RAS signaling. Established clinical and genetic markers fail to fully recapitulate the clinical and biological heterogeneity of this disease. Here we report DNA methylome analysis and mutation profiling of 167 JMML samples. We identify three JMML subgroups with unique molecular and clinical characteristics. The high methylation group (HM) is characterized by somatic PTPN11 mutations and poor clinical outcome. The low methylation group is enriched for somatic NRAS and CBL mutations, as well as for Noonan patients, and has a good prognosis. The intermediate methylation group (IM) shows enrichment for monosomy 7 and somatic KRAS mutations. Hypermethylation is associated with repressed chromatin, genes regulated by RAS signaling, frequent co-occurrence of RAS pathway mutations and upregulation of DNMT1 and DNMT3B, suggesting a link between activation of the DNA methylation machinery and mutational patterns in JMML

gnudatalanguage/gdlde: v1.0.1

An IDE for GDL (GNU Data Language

gnudatalanguage/gdlde: v1.0.1

An IDE for GDL (GNU Data Language

Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites

We present Cas-Designer, a user-friendly program to aid researchers in choosing appropriate target sites in a gene of interest for type II CRISPR/Cas-derived RNA-guided endonucleases, which are now widely used for biomedical research and biotechnology. Cas-Designer rapidly provides the list of all possible guide RNA sequences in a given input DNA sequence and their potential off-target sites including bulge-type sites in a genome of choice. In addition, the program assigns an out-of-frame score to each target site to help users choose appropriate sites for gene knockout. Cas-Designer shows the results in an interactive table and provides user-friendly filter functions.135371sciescopu