The Xenopus embryo has provided key insights into fate specification, the cell cycle, and other fundamental developmental and cellular processes, yet a comprehensive understanding of its ...transcriptome is lacking. Here, we used paired end RNA sequencing (RNA-seq) to explore the transcriptome of Xenopus tropicalis in 23 distinct developmental stages. We determined expression levels of all genes annotated in RefSeq and Ensembl and showed for the first time on a genome-wide scale that, despite a general state of transcriptional silence in the earliest stages of development, approximately 150 genes are transcribed prior to the midblastula transition. In addition, our splicing analysis uncovered more than 10,000 novel splice junctions at each stage and revealed that many known genes have additional unannotated isoforms. Furthermore, we used Cufflinks to reconstruct transcripts from our RNA-seq data and found that ∼13.5% of the final contigs are derived from novel transcribed regions, both within introns and in intergenic regions. We then developed a filtering pipeline to separate protein-coding transcripts from noncoding RNAs and identified a confident set of 6686 noncoding transcripts in 3859 genomic loci. Since the current reference genome, XenTro3, consists of hundreds of scaffolds instead of full chromosomes, we also performed de novo reconstruction of the transcriptome using Trinity and uncovered hundreds of transcripts that are missing from the genome. Collectively, our data will not only aid in completing the assembly of the Xenopus tropicalis genome but will also serve as a valuable resource for gene discovery and for unraveling the fundamental mechanisms of vertebrate embryogenesis.
Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed ...which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.
Vaccines in emergency use are efficacious against COVID-19, yet vaccine-induced prevention against nasal SARS-CoV-2 infection remains suboptimal.
Since mucosal immunity is critical for nasal ...prevention, we investigated the efficacy of an intramuscular PD1-based receptor-binding domain (RBD) DNA vaccine (PD1-RBD-DNA) and intranasal live attenuated influenza-based vaccines (LAIV-CA4-RBD and LAIV-HK68-RBD) against SARS-CoV-2.
Substantially higher systemic and mucosal immune responses, including bronchoalveolar lavage IgA/IgG and lung polyfunctional memory CD8 T cells, were induced by the heterologous PD1-RBD-DNA/LAIV-HK68-RBD as compared with other regimens. When vaccinated animals were challenged at the memory phase, prevention of robust SARS-CoV-2 infection in nasal turbinate was achieved primarily by the heterologous regimen besides consistent protection in lungs. The regimen-induced antibodies cross-neutralized variants of concerns. Furthermore, LAIV-CA4-RBD could boost the BioNTech vaccine for improved mucosal immunity.
Our results demonstrated that intranasal influenza-based boost vaccination induces mucosal and systemic immunity for effective SARS-CoV-2 prevention in both upper and lower respiratory systems.
This study was supported by the Research Grants Council Collaborative Research Fund, General Research Fund and Health and Medical Research Fund in Hong Kong; Outbreak Response to Novel Coronavirus (COVID-19) by the Coalition for Epidemic Preparedness Innovations; Shenzhen Science and Technology Program and matching fund from Shenzhen Immuno Cure BioTech Limited; the Health@InnoHK, Innovation and Technology Commission of Hong Kong; National Program on Key Research Project of China; donations from the Friends of Hope Education Fund; the Theme-Based Research Scheme.
Rapid advances in nanopore technologies for sequencing single long DNA and RNA molecules have led to substantial improvements in accuracy, read length and throughput. These breakthroughs have ...required extensive development of experimental and bioinformatics methods to fully exploit nanopore long reads for investigations of genomes, transcriptomes, epigenomes and epitranscriptomes. Nanopore sequencing is being applied in genome assembly, full-length transcript detection and base modification detection and in more specialized areas, such as rapid clinical diagnoses and outbreak surveillance. Many opportunities remain for improving data quality and analytical approaches through the development of new nanopores, base-calling methods and experimental protocols tailored to particular applications.
Landmark events occur in a coordinated manner during pre‐implantation development of the mammalian embryo, yet the regulatory network that orchestrates these events remains largely unknown. Here, we ...present the first systematic investigation of the network in pre‐implantation mouse embryos using morpholino‐mediated gene knockdowns of key embryonic stem cell (ESC) factors followed by detailed transcriptome analysis of pooled embryos, single embryos, and individual blastomeres. We delineated the regulons of Oct4, Sall4, and Nanog and identified a set of metabolism‐ and transport‐related genes that were controlled by these transcription factors in embryos but not in ESCs. Strikingly, the knockdown embryos arrested at a range of developmental stages. We provided evidence that the DNA methyltransferase Dnmt3b has a role in determining the extent to which a knockdown embryo can develop. We further showed that the feed‐forward loop comprising Dnmt3b, the pluripotency factors, and the miR‐290‐295 cluster exemplifies a network motif that buffers embryos against gene expression noise. Our findings indicate that Oct4, Sall4, and Nanog form a robust and integrated network to govern mammalian pre‐implantation development.
Coordination of many biological processes is necessary for mammalian pre‐implantation embryo development. The underlying regulatory network was mapped through mathematical modeling, gene‐specific knockdowns, and profiling of pooled embryos, single embryos, and single cells.
Synopsis
Coordination of many biological processes is necessary for mammalian pre‐implantation embryo development. The underlying regulatory network was mapped through mathematical modeling, gene‐specific knockdowns, and profiling of pooled embryos, single embryos, and single cells.
An integrated Oct4‐Sall4‐Nanog regulatory network of protein‐coding genes and microRNAs governs developmental progression in pre‐implantation mouse embryos.
While many target genes are common between embryos and embryonic stem cells (ESCs), pluripotency factors regulate the expression of many metabolism‐ and transport‐related genes only in embryos but not in stem cells.
The expression of some genes, including the DNA methyltransferase Dnmt3b, correlates strongly with the extent to which an embryo depleted of Oct4, Sall4, or Nanog can develop.
In wild‐type embryos and ESCs, a coherent feed‐forward loop buffers the expression of Dnmt3b against intrinsic fluctuations in the levels of the pluripotency factors.
We address the problem of observing personal diploid methylomes, CpG methylome pairs of homologous chromosomes that are distinguishable with respect to phased heterozygous variants (PHVs), which is ...challenging due to scarcity of PHVs in personal genomes. Single molecule real-time (SMRT) sequencing is promising as it outputs long reads with CpG methylation information, but a serious concern is whether reliable PHVs are available in erroneous SMRT reads with an error rate of ∼15%. To overcome the issue, we propose a statistical model that reduces the error rate of phasing CpG site to 1%, thereby calling CpG hypomethylation in each haplotype with >90% precision and sensitivity. Using our statistical model, we examined
complex locus known for a combination of maternally, paternally, or biallelically expressed isoforms, and observed allele-specific methylation pattern almost perfectly reflecting their respective allele-specific expression status, demonstrating the merit of elucidating comprehensive personal diploid methylomes and transcriptomes.
is an extremely valuable orchid used in traditional Chinese medicine, so sought after that it has a higher market value than gold. Although the expression profiles of some genes involved in the ...polysaccharide synthesis have previously been investigated, little research has been carried out on their alternatively spliced isoforms in
. In addition, information regarding the translocation of sugars from leaves to stems in
also remains limited. We analyzed the polysaccharide content of
leaves and stems, and completed in-depth transcriptome sequencing of these two diverse tissue types using second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing technology. The results of this study yielded a digital inventory of gene and mRNA isoform expressions. A comparative analysis of both transcriptomes uncovered a total of 1414 differentially expressed genes, including 844 that were up-regulated and 570 that were down-regulated in stems. Of these genes, one sugars will eventually be exported transporter (SWEET) and one sucrose transporter (SUT) are expressed to a greater extent in
stems than in leaves. Two glycosyltransferase (GT) and four cellulose synthase (Ces) genes undergo a distinct degree of alternative splicing. In the stems, the content of polysaccharides is twice as much as that in the leaves. The differentially expressed GT and transcription factor (TF) genes will be the focus of further study. The genes
and
are significantly expressed in the stem, and are likely to be involved in sugar loading in the phloem.
Chromosome instability and aneuploidies occur very frequently in human embryos, impairing proper embryogenesis and leading to cell cycle arrest, loss of cell viability, and developmental failures in ...50–80% of cleavage-stage embryos. This high frequency of cellular extinction events represents a significant experimental obstacle challenging analyses of individual cells isolated from human preimplantation embryos. We carried out single cell expression profiling of 241 individual cells recovered from 32 human embryos during the early and late stages of viable human blastocyst (VHB) differentiation. Classification of embryonic cells was performed solely based on expression patterns of human pluripotency-associated transcripts (HPAT), which represent a family of primate-specific transposable element-derived lincRNAs highly expressed in human embryonic stem cells and regulating nuclear reprogramming and pluripotency induction. We then validated our findings by analyzing transcriptomes of 1,708 individual cells recovered from more than 100 human embryos and 259 mouse cells from more than 40 mouse embryos at different stages of preimplantation embryogenesis. HPAT's expression-guided spatiotemporal reconstruction of human embryonic development inferred from single-cell expression analysis of VHB differentiation enabled identification of telomerase-positive embryonic cells co-expressing key pluripotency regulatory genes and genetic markers of three major lineages. Follow-up validation analyses confirmed the emergence in human embryos prior to lineage segregation of telomerase-positive cells co-expressing genetic markers of multiple lineages. Observations reported in this contribution support the hypothesis of a developmental pathway of creation embryonic lineages and extraembryonic tissues from telomerase-positive pre-lineage cells manifesting multi-lineage precursor phenotype.
Abstract
Motivation
Oxford Nanopore Technologies sequencing devices support adaptive sequencing, in which undesired reads can be ejected from a pore in real time. This feature allows targeted ...sequencing aided by computational methods for mapping partial reads, rather than complex library preparation protocols. However, existing mapping methods either require a computationally expensive base-calling procedure before using aligners to map partial reads or work well only on small genomes.
Results
In this work, we present a new streaming method that can map nanopore raw signals for real-time selective sequencing. Rather than converting read signals to bases, we propose to convert reference genomes to signals and fully operate in the signal space. Our method features a new way to index reference genomes using k-d trees, a novel seed selection strategy and a seed chaining algorithm tailored toward the current signal characteristics. We implemented the method as a tool Sigmap. Then we evaluated it on both simulated and real data and compared it to the state-of-the-art nanopore raw signal mapper Uncalled. Our results show that Sigmap yields comparable performance on mapping yeast simulated raw signals, and better mapping accuracy on mapping yeast real raw signals with a 4.4× speedup. Moreover, our method performed well on mapping raw signals to genomes of size >100 Mbp and correctly mapped 11.49% more real raw signals of green algae, which leads to a significantly higher F1-score (0.9354 versus 0.8660).
Availability and implementation
Sigmap code is accessible at https://github.com/haowenz/sigmap.
Supplementary information
Supplementary data are available at Bioinformatics online.
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