BACKGROUND: DNA demethylation occurs in many species and is involved in diverse biological processes. However, the occurrence and role of DNA demethylation in maize remain unknown. RESULTS: We analyze loss-of-function mutants of two major genes encoding DNA demethylases. No significant change in DNA methylation has been detected in these mutants. However, we detect increased DNA methylation levels in the mutants around genes and some transposons. The increase in DNA methylation is accompanied by alteration in gene expression, with a tendency to show downregulation, especially for the genes that are preferentially expressed in endosperm. Imprinted expression of both maternally and paternally expressed genes changes in F1 hybrid with the mutant as female and the wild-type as male parental line, but not in the reciprocal hybrid. This alteration in gene expression is accompanied by allele-specific DNA methylation differences, suggesting that removal of DNA methylation of the maternal allele is required for the proper expression of these imprinted genes. Finally, we demonstrate that hypermethylation in the double mutant is associated with reduced binding of transcription factor to its target, and altered gene expression. CONCLUSIONS: Our results suggest that active removal of DNA methylation is important for transcription factor binding and proper gene expression in maize endosperm.

A major advantage of single cell RNA-sequencing (scRNA-Seq) data is the ability to reconstruct continuous ordering and trajectories for cells. Here we present TraSig, a computational method for improving the inference of cell-cell interactions in scRNA-Seq studies that utilizes the dynamic information to identify significant ligand-receptor pairs with similar trajectories, which in turn are used to score interacting cell clusters. We applied TraSig to several scRNA-Seq datasets and obtained unique predictions that improve upon those identified by prior methods. Functional experiments validate the ability of TraSig to identify novel signaling interactions that impact vascular development in liver organoids.Software https://github.com/doraadong/TraSig .

Human-associated microbial communities comprise not only complex mixtures of bacterial species, but also mixtures of conspecific strains, the implications of which are mostly unknown since strain level dynamics are underexplored due to the difficulties of studying them. We introduce the Strain Genome Explorer (StrainGE) toolkit, which deconvolves strain mixtures and characterizes component strains at the nucleotide level from short-read metagenomic sequencing with higher sensitivity and resolution than other tools. StrainGE is able to identify strains at 0.1x coverage and detect variants for multiple conspecific strains within a sample from coverages as low as 0.5x.

BACKGROUND: Spinach (Spinacia oleracea L.) is a dioecious species with an XY sex chromosome system, but its Y chromosome has not been fully characterized. Our knowledge about the history of its domestication and improvement remains limited. RESULTS: A high-quality YY genome of spinach is assembled into 952 Mb in six pseudo-chromosomes. By a combination of genetic mapping, Genome-Wide Association Studies, and genomic analysis, we characterize a 17.42-Mb sex determination region (SDR) on chromosome 1. The sex chromosomes of spinach evolved when an insertion containing sex determination genes occurred, followed by a large genomic inversion about 1.98 Mya. A subsequent burst of SDR-specific repeats (0.1-0.15 Mya) explains the large size of this SDR. We identify a Y-specific gene, NRT1/PTR 6.4 which resides in this insertion, as a strong candidate for the sex determination or differentiation factor. Resequencing of 112 spinach genomes reveals a severe domestication bottleneck approximately 10.87 Kya, which dates the domestication of spinach 7000 years earlier than the archeological record. We demonstrate that a strong selection signal associated with internode elongation and leaf area expansion is associated with domestication of edibility traits in spinach. We find that several strong genomic introgressions from the wild species Spinacia turkestanica and Spinacia tetrandra harbor desirable alleles of genes related to downy mildew resistance, frost resistance, leaf morphology, and flowering-time shift, which likely contribute to spinach improvement. CONCLUSIONS: Analysis of the YY genome uncovers evolutionary forces shaping nascent sex chromosome evolution in spinach. Our findings provide novel insights about the domestication and improvement of spinach.

BACKGROUND: Host-microbe interactions are crucial for normal physiological and immune system development and are implicated in a variety of diseases, including inflammatory bowel disease (IBD), colorectal cancer (CRC), obesity, and type 2 diabetes (T2D). Despite large-scale case-control studies aimed at identifying microbial taxa or genes involved in pathogeneses, the mechanisms linking them to disease have thus far remained elusive. RESULTS: To identify potential pathways through which human-associated bacteria impact host health, we leverage publicly-available interspecies protein-protein interaction (PPI) data to find clusters of microbiome-derived proteins with high sequence identity to known human-protein interactors. We observe differential targeting of putative human-interacting bacterial genes in nine independent metagenomic studies, finding evidence that the microbiome broadly targets human proteins involved in immune, oncogenic, apoptotic, and endocrine signaling pathways in relation to IBD, CRC, obesity, and T2D diagnoses. CONCLUSIONS: This host-centric analysis provides a mechanistic hypothesis-generating platform and extensively adds human functional annotation to commensal bacterial proteins.

BACKGROUND: Neuroblastoma is a pediatric malignancy with a high frequency of metastatic disease at initial diagnosis. Neuroblastoma tumors have few recurrent protein-coding mutations but contain extensive somatic copy number alterations (SCNAs) suggesting that mutations that alter gene dosage are important drivers of tumorigenesis. Here, we analyze allele-specific expression in 96 high-risk neuroblastoma tumors to discover genes impacted by cis-acting mutations that alter dosage. RESULTS: We identify 1043 genes with recurrent, neuroblastoma-specific allele-specific expression. While most of these genes lie within common SCNA regions, many of them exhibit allele-specific expression in copy neutral samples and these samples are enriched for mutations that are predicted to cause nonsense-mediated decay. Thus, both SCNA and non-SCNA mutations frequently alter gene expression in neuroblastoma. We focus on genes with neuroblastoma-specific allele-specific expression in the absence of SCNAs and find 26 such genes that have reduced expression in stage 4 disease. At least two of these genes have evidence for tumor suppressor activity including the transcription factor TFAP2B and the protein tyrosine phosphatase PTPRH. CONCLUSIONS: In summary, our allele-specific expression analysis discovers genes that are recurrently dysregulated by both large SCNAs and other cis-acting mutations in high-risk neuroblastoma.

BACKGROUND: Vascular invasion with tumor thrombus frequently occurs in advanced renal cell carcinoma (RCC). Thrombectomy is one of the most challenging surgeries with high rate of perioperative morbidity and mortality. However, the mechanisms driving tumor thrombus formation are poorly understood which is required for designing effective therapy for eliminating tumor thrombus. RESULTS: We perform single-cell RNA sequencing analysis of 19 surgical tissue specimens from 8 clear cell renal cell carcinoma (ccRCC) patients with tumor thrombus. We observe tumor thrombus has increased tissue resident CD8+ T cells with a progenitor exhausted phenotype compared with the matched primary tumors. Remarkably, macrophages, malignant cells, endothelial cells and myofibroblasts from TTs exhibit enhanced remodeling of the extracellular matrix. The macrophages and malignant cells from primary tumors represent proinflammatory states, but also increase the expression of immunosuppressive markers compared to tumor thrombus. Finally, differential gene expression and interaction analyses reveal that tumor-stroma interplay reshapes the extracellular matrix in tumor thrombus associated with poor survival. CONCLUSIONS: Our comprehensive picture of the ecosystem of ccRCC with tumor thrombus provides deeper insights into the mechanisms of tumor thrombus formation, which may aid in the design of effective neoadjuvant therapy to promote downstaging of tumor thrombus and decrease the perioperative morbidity and mortality of thrombectomy.

Cost-efficient library generation by early barcoding has been central in propelling single-cell RNA sequencing. Here, we optimize and validate prime-seq, an early barcoding bulk RNA-seq method. We show that it performs equivalently to TruSeq, a standard bulk RNA-seq method, but is fourfold more cost-efficient due to almost 50-fold cheaper library costs. We also validate a direct RNA isolation step, show that intronic reads are derived from RNA, and compare cost-efficiencies of available protocols. We conclude that prime-seq is currently one of the best options to set up an early barcoding bulk RNA-seq protocol from which many labs would profit.

Comment in Nat Methods. 2022 Apr;19(4):401.

Erratum in Genome Biol. 2022 Sep 20;23(1):198.

BACKGROUND: Cohesin is a chromosome-associated SMC-kleisin complex that mediates sister chromatid cohesion, recombination, and most chromosomal processes during mitosis and meiosis. However, it remains unclear whether meiosis-specific cohesin complexes are functionally active in mitotic chromosomes. RESULTS: Through high-resolution 3D-structured illumination microscopy (3D-SIM) and functional analyses, we report multiple biological processes associated with the meiosis-specific cohesin components, α-kleisin REC8 and STAG3, and the distinct loss of function of meiotic cohesin during the cell cycle of embryonic stem cells (ESCs). First, we show that STAG3 is required for the efficient localization of REC8 to the nucleus by interacting with REC8. REC8-STAG3-containing cohesin regulates topological properties of chromosomes and maintains sister chromatid cohesion. Second, REC8-cohesin has additional sister chromatid cohesion roles in concert with mitotic RAD21-cohesin on ESC chromosomes. SIM imaging of REC8 and RAD21 co-staining revealed that the two types of α-kleisin subunits exhibited distinct loading patterns along ESC chromosomes. Third, knockdown of REC8 or RAD21-cohesin not only leads to higher rates of premature sister chromatid separation and delayed replication fork progression, which can cause proliferation and developmental defects, but also enhances chromosome compaction by hyperloading of retinoblastoma protein-condensin complexes from the prophase onward. CONCLUSIONS: Our findings indicate that the delicate balance between mitotic and meiotic cohesins may regulate ESC-specific chromosomal organization and the mitotic program.

Determining the evolutionary relationships between genes is fundamental to comparative biological research. Here, we present SHOOT. SHOOT searches a user query sequence against a database of phylogenetic trees and returns a tree with the query sequence correctly placed within it. We show that SHOOT performs this analysis with comparable speed to a BLAST search. We demonstrate that SHOOT phylogenetic placements are as accurate as conventional tree inference, and it can identify orthologs with high accuracy. In summary, SHOOT is a fast and accurate tool for phylogenetic analyses of novel query sequences. It is available online at www.shoot.bio .

BACKGROUND: Brassica napus is an important vegetable oil source worldwide. Seed coat content is a complex quantitative trait that negatively correlates with the seed oil content in B. napus. RESULTS: Here we provide insights into the genetic basis of natural variation of seed coat content by transcriptome-wide association studies (TWAS) and genome-wide association studies (GWAS) using 382 B. napus accessions. By population transcriptomic analysis, we identify more than 700 genes and four gene modules that are significantly associated with seed coat content. We also characterize three reliable quantitative trait loci (QTLs) controlling seed coat content by GWAS. Combining TWAS and correlation networks of seed coat content-related gene modules, we find that BnaC07.CCR-LIKE (CCRL) and BnaTT8s play key roles in the determination of the trait by modulating lignin biosynthesis. By expression GWAS analysis, we identify a regulatory hotspot on chromosome A09, which is involved in controlling seed coat content through BnaC07.CCRL and BnaTT8s. We then predict the downstream genes regulated by BnaTT8s using multi-omics datasets. We further experimentally validate that BnaCCRL and BnaTT8 positively regulate seed coat content and lignin content. BnaCCRL represents a novel identified gene involved in seed coat development. Furthermore, we also predict the key genes regulating carbon allocation between phenylpropane compounds and oil during seed development in B. napus. CONCLUSIONS: This study helps us to better understand the complex machinery of seed coat development and provides a genetic resource for genetic improvement of seed coat content in B. napus breeding.

The recent advancement in spatial transcriptomics technology has enabled multiplexed profiling of cellular transcriptomes and spatial locations. As the capacity and efficiency of the experimental technologies continue to improve, there is an emerging need for the development of analytical approaches. Furthermore, with the continuous evolution of sequencing protocols, the underlying assumptions of current analytical methods need to be re-evaluated and adjusted to harness the increasing data complexity. To motivate and aid future model development, we herein review the recent development of statistical and machine learning methods in spatial transcriptomics, summarize useful resources, and highlight the challenges and opportunities ahead.

BACKGROUND: Most animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false-positive signals. RESULTS: We generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false-positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30-40 times using HiFi sequencing is required for phasing of the leaf rust genome, with 0.7% heterozygosity, and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. CONCLUSIONS: This first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.

Erratum in Genome Biol. 2022 Apr 21;23(1):104.

Cleavage Under Targets and Tagmentation (CUT&Tag) is an antibody-directed transposase tethering strategy for in situ chromatin profiling in small samples and single cells. We describe a modified CUT&Tag protocol using a mixture of an antibody to the initiation form of RNA polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution to produce high-resolution maps of both the active and repressive regulomes in single cells. The ability to seamlessly map active promoters, enhancers, and repressive regulatory elements using a single workflow provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.

When identifying differentially expressed genes between two conditions using human population RNA-seq samples, we found a phenomenon by permutation analysis: two popular bioinformatics methods, DESeq2 and edgeR, have unexpectedly high false discovery rates. Expanding the analysis to limma-voom, NOISeq, dearseq, and Wilcoxon rank-sum test, we found that FDR control is often failed except for the Wilcoxon rank-sum test. Particularly, the actual FDRs of DESeq2 and edgeR sometimes exceed 20% when the target FDR is 5%. Based on these results, for population-level RNA-seq studies with large sample sizes, we recommend the Wilcoxon rank-sum test.

Genomic prediction in crop breeding is hindered by modeling on limited phenotypic traits. We propose an integrative multi-trait breeding strategy via machine learning algorithm, target-oriented prioritization (TOP). Using a large hybrid maize population, we demonstrate that the accuracy for identifying a candidate that is phenotypically closest to an ideotype, or target variety, achieves up to 91%. The strength of TOP is enhanced when omics level traits are included. We show that TOP enables selection of inbreds or hybrids that outperform existing commercial varieties. It improves multiple traits and accurately identifies improved candidates for new varieties, which will greatly influence breeding.

BACKGROUND: Paralogs that arise from gene duplications during genome evolution enable genetic redundancy and phenotypic robustness. Variation in the coding or regulatory sequence of paralogous transcriptional regulators diversifies their functions and relationships, which provides developmental robustness against genetic or environmental perturbation. The fate transition of plant shoot stem cells for flowering and reproductive success requires a robust transcriptional control. However, how paralogs function and interact to achieve such robustness is unknown. RESULTS: Here, we explore the genetic relationship and protein behavior of ALOG family transcriptional factors with diverse transcriptional abundance in shoot meristems. A mutant spectrum covers single and higher-order mutant combinations of five ALOG paralogs and creates a continuum of flowering transition defects, showing gradually enhanced precocious flowering, along with inflorescence simplification from wild-type-like to progressively fewer flowers until solitary flower with sterile floral organs. Therefore, these paralogs play unequal roles and act together to achieve a robust genetic canalization. All five proteins contain prion-like intrinsically disordered regions (IDRs) and undergo phase separation. Accumulated mutations following gene duplications lead to IDR variations among ALOG paralogs, resulting in divergent phase separation and transcriptional regulation capabilities. Remarkably, they retain the ancestral abilities to assemble into a heterotypic condensate that prevents precocious activation of the floral identity gene ANANTHA. CONCLUSIONS: Our study reveals a novel genetic canalization mechanism enabled by heterotypic transcriptional condensates formed by paralogous protein interactions and phase separation, uncovering the molecular link between gene duplication caused IDR variation and robust transcriptional control of stem cell fate transition.

BACKGROUND: Nucleomorphs are remnants of secondary endosymbiotic events between two eukaryote cells wherein the endosymbiont has retained its eukaryotic nucleus. Nucleomorphs have evolved at least twice independently, in chlorarachniophytes and cryptophytes, yet they have converged on a remarkably similar genomic architecture, characterized by the most extreme compression and miniaturization among all known eukaryotic genomes. Previous computational studies have suggested that nucleomorph chromatin likely exhibits a number of divergent features. RESULTS: In this work, we provide the first maps of open chromatin, active transcription, and three-dimensional organization for the nucleomorph genome of the chlorarachniophyte Bigelowiella natans. We find that the B. natans nucleomorph genome exists in a highly accessible state, akin to that of ribosomal DNA in some other eukaryotes, and that it is highly transcribed over its entire length, with few signs of polymerase pausing at transcription start sites (TSSs). At the same time, most nucleomorph TSSs show very strong nucleosome positioning. Chromosome conformation (Hi-C) maps reveal that nucleomorph chromosomes interact with one other at their telomeric regions and show the relative contact frequencies between the multiple genomic compartments of distinct origin that B. natans cells contain. CONCLUSIONS: We provide the first study of a nucleomorph genome using modern functional genomic tools, and derive numerous novel insights into the physical and functional organization of these unique genomes.

Few methods have been developed to investigate copy number variants (CNVs) based on their predicted pathogenicity. We introduce TADA, a method to prioritise pathogenic CNVs through assisted manual filtering and automated classification, based on an extensive catalogue of functional annotation supported by rigourous enrichment analysis. We demonstrate that our classifiers are able to accurately predict pathogenic CNVs, outperforming current alternative methods, and produce a well-calibrated pathogenicity score. Our results suggest that functional annotation-based prioritisation of pathogenic CNVs is a promising approach to support clinical diagnostics and to further the understanding of mechanisms controlling the disease impact of larger genomic alterations.

BACKGROUND: Vaginal bacterial communities dominated by Lactobacillus species are associated with a reduced risk of various adverse health outcomes. However, somewhat unexpectedly, many healthy women have microbiota that are not dominated by lactobacilli. To determine the factors that drive vaginal community composition we characterized the genetic composition and transcriptional activities of vaginal microbiota in healthy women. RESULTS: We demonstrate that the abundance of a species is not always indicative of its transcriptional activity and that impending changes in community composition can be predicted from metatranscriptomic data. Functional comparisons highlight differences in the metabolic activities of these communities, notably in their degradation of host produced mucin but not glycogen. Degradation of mucin by communities not dominated by Lactobacillus may play a role in their association with adverse health outcomes. Finally, we show that the transcriptional activities of L. crispatus, L. iners, and Gardnerella vaginalis vary with the taxonomic composition of the communities in which they reside. Notably, L. iners and G. vaginalis both demonstrate lower expression of their cholesterol-dependent cytolysins when co-resident with Lactobacillus spp. and higher expression when co-resident with other facultative and obligate anaerobes. The pathogenic potential of these species may depend on the communities in which they reside and thus could be modulated by interventional strategies. CONCLUSIONS: Our results provide insight to the functional ecology of the vaginal microbiota, demonstrate the diagnostic potential of metatranscriptomic data, and reveal strategies for the management of these ecosystems.