Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization.

The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (216 = 65,536 or 39 = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ.

Pgltools: a genomic arithmetic tool suite for manipulation of Hi-C peak and other chromatin interaction data.

BACKGROUND: Genomic interaction studies use next-generation sequencing (NGS) to examine the interactions between two loci on the genome, with subsequent bioinformatics analyses typically including annotation, intersection, and merging of data from multiple experiments. While many file types and analysis tools exist for storing and manipulating single locus NGS data, there is currently no file standard or analysis tool suite for manipulating and storing paired-genomic-loci: the data type resulting from "genomic interaction" studies. As genomic interaction sequencing data are becoming prevalent, a standard file format and tools for working with these data conveniently and efficiently are needed. RESULTS: This article details a file standard and novel software tool suite for working with paired-genomic-loci data. We present the paired-genomic-loci (PGL) file standard for genomic-interactions data, and the accompanying analysis tool suite "pgltools": a cross platform, pypy compatible python package available both as an easy-to-use UNIX package, and as a python module, for integration into pipelines of paired-genomic-loci analyses. CONCLUSIONS: Pgltools is a freely available, open source tool suite for manipulating paired-genomic-loci data. Source code, an in-depth manual, and a tutorial are available publicly at www.github.com/billgreenwald/pgltools , and a python module of the operations can be installed from PyPI via the PyGLtools module.

FEATnotator: A tool for integrated annotation of sequence features and variation, facilitating interpretation in genomics experiments.

As approaches are sought for more efficient and democratized uses of non-model and expanded model genomics references, ease of integration of genomic feature datasets is especially desirable in multidisciplinary research communities. Valuable conclusions are often missed or slowed when researchers refer experimental results to a single reference sequence that lacks integrated pan-genomic and multi-experiment data in accessible formats. Association of genomic positional information, such as results from an expansive variety of next-generation sequencing experiments, with annotated reference features such as genes or predicted protein binding sites, provides the context essential for conclusions and ongoing research. When the experimental system includes polymorphic genomic inputs, rapid calculation of gene structural and protein translational effects of sequence variation from the reference can be invaluable. Here we present FEATnotator, a lightweight, fast and easy to use open source software program that integrates and reports overlap and proximity in genomic information from any user-defined datasets including those from next generation sequencing applications. We illustrate use of the tool by summarizing whole genome sequence variation of a widely used natural isolate of Arabidopsis thaliana in the context of gene models of the reference accession. Previous discovery of a protein coding deletion influencing root development is replicated rapidly. Appropriate even in investigations of a single gene or genic regions such as QTL, comprehensive reports provided by FEATnotator better prepare researchers for interpretation of their experimental results. The tool is available for download at http://featnotator.sourceforge.net.

Genomic loci associated with leaf abscission contribute to machine picking and environmental adaptability in upland cotton (Gossypium hirsutum L.).

INTRODUCTION: Defoliation by applying defoliants before machine picking is an important agricultural practice that enhances harvesting efficiency and leads to increased raw cotton purity. However, the fundamental characteristics of leaf abscission and the underlying genetic basis in cotton are not clearly understood. OBJECTIVES: In this study, we aimed to (1) reveal the phenotypic variations in cotton leaf abscission, (2) discover the whole-genome differentiation sweeps and genetic loci related to defoliation, (3) identify and verify the functions of key candidate genes associated with defoliation, and (4) explore the relationship between haplotype frequency of loci and environmental adaptability. METHODS: Four defoliation-related traits of 383 re-sequenced Gossypium hirsutum accessions were investigated in four environments. The genome-wide association study (GWAS), linkage disequilibrium (LD) interval genotyping and functional identification were conducted. Finally, the haplotype variation related to environmental adaptability and defoliation traits was revealed. RESULTS: Our findings revealed the fundamental phenotypic variations of defoliation traits in cotton. We showed that defoliant significantly increased the defoliation rate without incurring yield and fiber quality penalties. The strong correlations between defoliation traits and growth period traits were observed. A genome-wide association study of defoliation traits identified 174 significant SNPs. Two loci (RDR7 on A02 and RDR13 on A13) that significantly associated with the relative defoliation rate were described, and key candidate genes GhLRR and GhCYCD3;1, encoding a leucine-rich repeat (LRR) family protein and D3-type cell cyclin 1 protein respectively, were functional verified by expression pattern analysis and gene silencing. We found that combining of two favorable haplotypes (HapRDR7 and HapRDR13) improved sensitivity to defoliant. The favorable haplotype frequency generally increased in high latitudes in China, enabling adaptation to the local environment. CONCLUSION: Our findings lay an important foundation for the potentially broad application of leveraging key genetic loci in breeding machine-pickable cotton.

CRISPR/Pepper-tDeg: A Live Imaging System Enables Non-Repetitive Genomic Locus Analysis with One Single-Guide RNA.

CRISPR-based genomic-imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal-to-noise ratio (SNR) at a non-repetitive genomic locus. Here, an efficient genomic-imaging system is proposed, termed CRISPR/Pepper-tDeg, by engineering the CRISPR sgRNA scaffolds with the degron-binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target-dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5-fold higher SNR than conventional CRISPR/MS2-MCP system using "always-on" fluorescent protein tag. Subsequently, CRISPR/Pepper-tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper-tDeg system is extended to non-repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non-repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic-imaging system in biological research, clinical diagnosis and therapy.

Genome-wide association study reveals genomic loci of sex differentiation and gonadal development in Plectropomus leopardus.

Introduction: Plectropomus leopardus, a commercially significant marine fish, is primarily found in the Western Pacific regions and along the coast of Southeast Asia. A thorough analysis of the molecular mechanisms involved in sex differentiation is crucial for gaining a comprehensive understanding of gonadal development and improving sex control breeding. However, the relevant fundamental studies of P. leopardus are relatively lacking. Methods: In this study, a genome-wide association study (GWAS) was conducted to investigate the genetic basis mechanism of sex differentiation and gonadal developmental traits in P. leopardus utilizing about 6,850,000 high-quality single-nucleotide polymorphisms (SNPs) derived from 168 individuals (including 126 females and 42 males) by the genome-wide efficient mixed-model association (GEMMA) algorithm. Results: The results of these single-trait GWASs showed that 46 SNP loci (-log10 p > 7) significantly associated with sex differentiation, and gonadal development traits were distributed in multiple different chromosomes, which suggested the analyzed traits were all complex traits under multi-locus control. A total of 1,838 potential candidate genes were obtained by considering a less-stringent threshold (-log10 p > 6) and ±100 kb regions surrounding the significant genomic loci. Moreover, 31 candidate genes were identified through a comprehensive analysis of significant GWAS peaks, gene ontology (GO) annotations, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, including taf7, ddx6, apoeb, sgk1, a2m, usf1, hsd3b7, dll4, xbp1, tet3, esr1, and gli3. These trait-associated genes have been shown to be involved in germline development, male sex differentiation, gonad morphogenesis, hormone receptor binding, oocyte development, male gonad development, steroidogenesis, estrogen-synthetic pathway, etc. Discussion: In the present study, multiple genomic loci of P. leopardus associated with sex differentiation and gonadal development traits were identified for the first time by using GWAS, providing a valuable resource for further research on the molecular genetic mechanism and sex control in P. leopardus. Our results also can contribute to understanding the genetic basis of the sex differentiation mechanism and gonadal development process in grouper fish.

A chemical method for genome- and proteome-wide enrichment and O-GlcNAcylation profiling of chromatin-associated proteins.

O-Linked ß-N-acetylglucosamine (O-GlcNAc), a versatile posttranslational modification (PTM), is found on many chromatin-associated proteins (CAPs), such as transcription factors and their cofactors (TFCs). O-GlcNAc turnover influences the dynamic interactions of CAPs with chromatin and thereby regulates gene expression. Therefore, both global profiling of O-GlcNAc chromatin-associated proteins (OCAPs) and genome-wide mapping of their DNA binding sites are invaluable for understanding the functions of OCAPs and the regulatory machinery of O-GlcNAcylation on gene transcription. However, it is difficult to conduct genome- and proteome-wide OCAP studies using the widely adopted chromatin immunoprecipitation (ChIP) method due to the lack of highly O-GlcNAc-specific panantibodies. Therefore, we developed a chemical enrichment method (AFT-OCAP) for simultaneously profiling OCAPs and mapping their binding DNA via mass spectrometry (MS) analysis and DNA sequencing. In our method, we developed an alkynyl-functionalized trimethylpiperidine (AFT) reagent to perform highly efficient chemical derivatizations of azide-labeled OCAP-DNA complexes. The reversible affinity between the immobilized anti-trimethylpiperidine antibody resin and AFT reagent leads to specific enrichment and efficient elution of the OCAP-DNA complexes for both MS identification and sequencing. Deep coverage of OCAPs was achieved from HeLa cells, including 1951 O-GlcNAc peptides from 1136 O-GlcNAc chromatin-associated transcription factors and cofactors (TFCs) using HCD fragmentation and 669 O-GlcNAc sites using EThcD fragmentation. In addition, the distributions of O-GlcNAcylation across the genome and the dynamic interactions of OCAPs upon O-GlcNAc regulation were obtained.

Scarless Genomic Protein Labeling in Saccharomyces cerevisiae.

Labeling a protein of interest is widely used to examine its quantity, modification, localization, and dynamics in the budding yeast Saccharomyces cerevisiae. Fluorescent proteins and epitope tags are often used as protein fusion tags to study target proteins. One prevailing technique is to fuse these tags to a target gene at the precise chromosomal location via homologous recombination. Here we describe a protein labeling strategy based on the URA3 pop-in/pop-out and counterselection system to fuse a fluorescent protein or epitope tag scarlessly to a target protein at its native locus in S. cerevisiae.

Not All That Glitters Is Gold: The Paradox of CO-dependent Hydrogenogenesis in Parageobacillus thermoglucosidasius.

The thermophilic bacterium Parageobacillus thermoglucosidasius has recently gained interest due to its ability to catalyze the water gas shift reaction, where the oxidation of carbon monoxide (CO) is linked to the evolution of hydrogen (H2) gas. This phenotype is largely predictable based on the presence of a genomic region coding for a carbon monoxide dehydrogenase (CODH-Coo) and hydrogen evolving hydrogenase (Phc). In this work, seven previously uncharacterized strains were cultivated under 50% CO and 50% air atmosphere. Despite the presence of the coo-phc genes in all seven strains, only one strain, Kp1013, oxidizes CO and yields H2. The genomes of the H2 producing strains contain unique genomic regions that code for proteins involved in nickel transport and the detoxification of catechol, a by-product of a siderophore-mediated iron acquisition system. Combined, the presence of these genomic regions could potentially drive biological water gas shift (WGS) reaction in P. thermoglucosidasius.

A Universal Surrogate Reporter for Efficient Enrichment of CRISPR/Cas9-Mediated Homology-Directed Repair in Mammalian Cells.

CRISPR/Cas9-mediated homology-directed repair (HDR) can be leveraged to precisely engineer mammalian genomes. However, the inherently low efficiency of HDR often hampers to identify the desired modified cells. Here, we developed a novel universal surrogate reporter system that efficiently enriches for genetically modified cells arising from CRISPR/Cas9-induced HDR events (namely, the "HDR-USR" system). This episomally based reporter can be self-cleaved and self-repaired via HDR to create a functional puromycin selection cassette without compromising genome integrity. Co-transfection of the HDR-USR system into host cells and transient puromycin selection efficiently achieves enrichment of HDR-modified cells. We tested the system for precision point mutation at 16 loci in different human cell lines and one locus in two rodent cell lines. This system exhibited dramatic improvements in HDR efficiency at a single locus (up to 20.7-fold) and two loci at once (42% editing efficiency compared to zero in the control), as well as greatly improved knockin efficiency (8.9-fold) and biallelic deletion (35.9-fold) at test loci. Further increases were achieved by co-expression of yeast Rad52 and linear single-/double-stranded DNA donors. Taken together, our HDR-USR system provides a simple, robust and efficient surrogate reporter for the enrichment of CRISPR/Cas9-induced HDR-based precision genome editing across various targeting loci in different cell lines.

Visualization of Genomic Loci in Living Cells with BiFC-TALE.

Tracking the dynamics of genomic loci is essential for understanding a variety of cellular processes. However, earlier methods have all suffered from a low signal-to-background ratio (SBR), mainly caused by the background fluorescence from diffuse full-length fluorescent proteins in the nucleus. We have developed a novel method (BiFC-TALE) for labeling and tracking genomic loci in live mammalian cells, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies. Since only the sequences-targeted BiFC fragments can be pulled together by TALE modules to recombine intact fluorescent proteins, the background fluorescence in the living nucleus can be largely reduced, which significantly improves SBR. Using telomere and centromere labeling as examples, this unit describes in detail the design and implementation of BiFC-TALE system. © 2018 by John Wiley & Sons, Inc.

Genetics in Keratoconus - What is New?

BACKGROUND: Keratoconus is characterized as a bilateral, progressive, non-inflammatory thinning of the cornea resulting in blurred vision due to irregular astigmatism. Keratoconus has a multifactorial etiology, with multiple genetic and environmental components contributing to the disease pathophysiology. Several genomic loci and genes have been identified that highlight the complex molecular etiology of this disease. CONCLUSION: The review focuses on current knowledge of these genetic risk factors associated with keratoconus.

Identification of a novel phylogenetic lineage of Alternaria alternata causing citrus brown spot in China.

Alternaria alternata sensu lato, casual agent of citrus brown spot, first identified in Yunnan province in 2010 and subsequently found in Zhejiang, Hunan, Guangdong provinces, Chongqing municipality andGuangxi autonomous region in China. During 2010-2012, 86 isolates were collected from diseased citrus, of which 85 % isolates were pathogenic to Ponkan tangerine. Phylogenetic analyses of Chinese and worldwide isolates using partial sequences of an endopolygalacturonase gene (endoPG) and combined dataset ofendoPG and two anonymous loci (OPA1-3, OPA2-1) found that Chinese isolates fell into two of three previously described clades. One clade ('clade 3') contained isolates from Turkey and Israel, and the other clade ('clade 1') contained isolates from Florida, USA. None of the isolates from China fell into the last previously described clade ('clade 2'). However, 24 isolates from Hunan, Guangdong and Guangxi fell into a fourth clade ('clade 4') not previously reported to be associated with citrus brown spot. This clade included multilocus haplotypes known to infect Japanese pear and strawberry. The observation that Chinese brown spot isolates fell into only two of three known worldwide lineages suggests that this fungus may not have co-evolved with its host in China but elsewhere in Southeast Asia and introduced to China.