Searching for a Needle in a Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-Length Glutenin Genes in a Big Cereal Genome

Sequencing and epigenetic profiling of target genes in plants are important tasks with various applications ranging from marker design for plant breeding to the study of gene expression regulation. This is particularly interesting for plants with big genome size for which whole-genome sequencing can be time-consuming and costly. In this study, we asked whether recently proposed Cas9-targeted nanopore sequencing (nCATS) is efficient for target gene sequencing for plant species with big genome size. We applied nCATS to sequence the full-length glutenin genes (Glu-1Ax, Glu-1Bx and Glu-1By) and their promoters in hexaploid triticale (X Triticosecale, AABBRR, genome size is 24 Gb). We showed that while the target gene enrichment per se was quite high for the three glutenin genes (up to 645×), the sequencing depth that was achieved from two MinION flowcells was relatively low (5–17×). However, this sequencing depth was sufficient for various tasks including detection of InDels and single-nucleotide variations (SNPs), read phasing and methylation profiling. Using nCATS, we uncovered SNP and InDel variation of full-length glutenin genes providing useful information for marker design and deciphering of variation of individual Glu-1By alleles. Moreover, we demonstrated that glutenin genes possess a ‘gene-body’ methylation epigenetic profile with hypermethylated CDS part and hypomethylated promoter region. The obtained information raised an interesting question on the role of gene-body methylation in glutenin gene expression regulation. Taken together, our work disclosures the potential of the nCATS approach for sequencing of target genes in plants with big genome size.

A Comparative Study of Automatic Localization Algorithms for Spherical Markers within 3D MRI Data

Localization of features and structures in images is an important task in medical image-processing. Characteristic structures and features are used in diagnostics and surgery planning for spatial adjustments of the volumetric data, including image registration or localization of bone-anchors and fiducials. Since this task is highly recurrent, a fast, reliable and automated approach without human interaction and parameter adjustment is of high interest. In this paper we propose and compare four image processing pipelines, including algorithms for automatic detection and localization of spherical features within 3D MRI data. We developed a convolution based method as well as algorithms based on connected-components labeling and analysis and the circular Hough-transform. A blob detection related approach, analyzing the Hessian determinant, was examined. Furthermore, we introduce a novel spherical MRI-marker design. In combination with the proposed algorithms and pipelines, this allows the detection and spatial localization, including the direction, of fiducials and bone-anchors.

A Review of the Applications of Biochemicals Interference in Medical Imaging with the Synthesis of Contrast Nanoparticles and Marker Design (PROBE)

One of the main focuses in diagnostic medicine is molecular imaging. Nanoparticles allow us to image different components of a high-contrast molecule efficiently. In this study, various factors that should be considered when synthesizing contrast nanoparticles are discussed. Some of the most important examples are highlighted. This research has been done theoretically and in terms of content analysis method (Content Analysis) by searching for keywords nanotechnology, molecular imaging, drug delivery methods, gene therapy, and contrast nanoparticles in google, PubMed, Science direct, scholar, and Scopus websites. After the search, ten articles were selected from the obtained articles, and this article was written based on them. Most of the report’s nanoparticles produce new contrast agents, especially molecular imaging and cellular processes detection. Have been taken. The advantages of using these nanoparticles are the ability to produce high contrast, ease of integration of multiple properties, long circulation time in the blood, and the ability to carry high-volume materials (such as drugs). The basics and nanoparticle production methods have grown and expanded over the years, so more complex examples of nanoscale contrast agents such as paramagnetic particles, macrophages with quantum dots, quantum dots, machines that can make matter Atomic and molecular parts have been reported. MRI of microemulsions has also been used to examine blood vessels and deliver medication. The use of contrast nanoparticles provides more details about the processes of a disease and its effects. The fabrication of these materials has improved dramatically over the past decade by creating various functions in them. However, there are many areas for biocompatibility, efficacy, specificity, and diagnosis of further diseases. In general, contrast nanoparticles can be synthesized for a specific function, with specific properties to perform a specific program.

VCF2CAPS–A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

Rapture facilitates inexpensive and high-throughput parent-based tagging in salmonids

Accurate methods for tracking individuals are crucial to the success of fisheries and aquaculture management. Management of migratory salmonid populations, which are important for the health of many economies, ecosystems, and indigenous cultures, is particularly dependent on data gathered from tagged fish. However, the physical tagging methods currently used have many challenges including cost, variable marker retention, and information limited to tagged individuals. Genetic tracking methods combat many of the problems associated with physical tags, but have their own challenges including high cost, potentially difficult marker design, and incompatibility of markers across species. Here we show the feasibility of a new genotyping method for parent-based tagging (PBT), where individuals are tracked through the inherent genetic relationships with their parents. We found that Rapture sequencing, a combination of restriction-site associated DNA and capture sequencing, provides sufficient data for parentage assignment. Additionally, the same capture bait set, which targets specific restriction-site associated DNA loci, can be used for both Rainbow Trout Oncorhynchus mykiss and Chinook Salmon Oncorhynchus tshawytscha. We input 248 single nucleotide polymorphisms from 1,121 samples to parentage assignment software and compared parent-offspring relationships of the spawning pairs recorded in a hatchery. Interestingly, our results suggest sperm contamination during hatchery spawning occurred in the production of 14% of offspring, further confirming the need for genetic tagging in accurately tracking individuals. PBT with Rapture successfully assigned progeny to parents with a 98.86% accuracy with sufficient genetic data. Cost for this pilot study was approximately $3 USD per sample. As costs vary based on the number of markers used and individuals sequenced, we expect that when implemented at a large-scale, per sample costs could be further decreased. We conclude that Rapture PBT provides a cost-effective and accurate alternative to the physical coded wire tags, and other genetic-based methods.

Quality Assessment of Additively Manufactured Fiducial Markers to Support Augmented Reality-Based Part Inspection

This paper proposes an augmented reality (AR) framework and tool on smartphones as an alternative to conventional inspection for AM parts. The framework attempts to introduce the rapid inspection potential of smartphone based AR within manufacturing by leveraging the manufacturing capability of additive manufacturing (AM) to integrate markers onto AM parts. The key step from this framework that is explored in this paper is the design and quality assessment of AM markers for marker registration. As part of the marker design and quality assessment objectives, this research conducts an evaluation on the effects of different AM processes on the quality of augmentation achieved from AM fiducial markers. Furthermore, it evaluates the minimum fiducial pattern size that on integration onto AM parts will be viable for augmentation. The results suggest that the AM process and the size of the fiducial pattern play a significant role in determining the quality of the AM markers. The paper concludes by stating that dual material extrusion AM markers provide the highest number of detectable features and therefore the highest quality of AM markers, and the smallest viable fiducial pattern for Cybercode/QR code marker can be sized at 19 × 19mm2.

Introduction

Providing an introduction to the planning process for the Waste Isolation Pilot Project and the marker design that continues to be the basis of nuclear waste repository proposals in the United States, including for Yucca Mountain, this chapter lays the groundwork for consideration of the contradictions between opinions produced through expert consultation and the expertise of archaeologists. US government efforts described enlisted a variety of “experts” to propose alternative futures, identify models for communication over long spans of time, and assess the likely durability of proposed designs for a marker over nuclear waste repositories. To understand these expert reports, this chapter introduces the concept of an anthropology of common sense as a way to understand how government experts understood the archaeological sites that they offered as models.

Rosetta Stones and Cuneiform Tablets

This chapter examines the role in the marker design of buried objects intended to be inscribed with messages. Modeled on works like the Rosetta Stone, the stele of Hammurabi, and cuneiform tablets, this part of the design turned the marker system into an artificial archaeological site. Debates are explored about how meanings are effectively communicated, with an emphasis on parallel languages as securing transmission of meaning countered by historical evidence of English literary texts that rapidly became difficult to read. The chapter explores how Rosetta Stone came to be treated as a metaphor for a key to decipherment that would have been familiar to the expert consultants. Archaeological context demonstrates that the models used never actually were aimed to communicate into long-term futures. An interlude after the chapter explores the experts’ understanding of how Land Art worked, and how the artists creating such works understood them.

Serpent Mound

This chapter explores the second major element of the marker design, which called for a massive earthen berm, supported by citing mounds of the US Midwest, including Monk’s Mound at Cahokia and the Great Serpent Mound. It explores how the engineering knowledge needed to construct these mounds is underestimated by the markers’ experts, and how the archaeological sites treated as simple actually have complex histories of development, including repair and changes. It relates the dismissive treatment of this indigenous technology to earlier commentaries that questioned the creation of earthworks by Native Americans. It explores the concept of common sense and the kinds of expert opinion that were represented in the history of developing proposals for markers, and the special role given to meaning in identifying appropriate archaeological models to use. It is followed by an interlude considering Robert Smithson’s Spiral Jetty as a model for a monumental earthwork subject to entropy.