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Nature Nov 2021Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different...
Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response.
Topics: Animals; Apoptosis; Cells, Cultured; Chromosome Mapping; DNA Fragmentation; DNA Ligase ATP; DNA, Circular; Endodeoxyribonucleases; Gene Expression Regulation; Genome; Immunity, Innate; Male; Membrane Proteins; Mice; Poly-ADP-Ribose Binding Proteins
PubMed: 34671165
DOI: 10.1038/s41586-021-04009-w -
Cell Dec 2020We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via...
We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-μm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.
Topics: Animals; Automation; Brain; Cluster Analysis; DNA Barcoding, Taxonomic; DNA, Complementary; Embryo, Mammalian; Eye; Female; Gene Expression Regulation, Developmental; Genomics; Human Umbilical Vein Endothelial Cells; Humans; Mice, Inbred C57BL; Microfluidics; Organ Specificity; RNA, Messenger; Reproducibility of Results; Single-Cell Analysis; Transcriptome
PubMed: 33188776
DOI: 10.1016/j.cell.2020.10.026 -
Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA.Genome Research Jul 2021Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased...
Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.
PubMed: 34140313
DOI: 10.1101/gr.266551.120 -
Nature Biotechnology Apr 2020Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current...
Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3 μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.
Topics: Animals; CRISPR-Associated Protein 9; Cells, Cultured; Chromosomes, Human; Genetic Loci; Genetic Variation; Genotype; High-Throughput Nucleotide Sequencing; Humans; Nanopore Sequencing; Sequence Analysis, DNA; RNA, Guide, CRISPR-Cas Systems
PubMed: 32042167
DOI: 10.1038/s41587-020-0407-5 -
Computational and Structural... 2022Transcriptome level expression data connected to the spatial organization of the cells and molecules would allow a comprehensive understanding of how gene expression is... (Review)
Review
Transcriptome level expression data connected to the spatial organization of the cells and molecules would allow a comprehensive understanding of how gene expression is connected to the structure and function in the biological systems. The spatial transcriptomics platforms may soon provide such information. However, the current platforms still lack spatial resolution, capture only a fraction of the transcriptome heterogeneity, or lack the throughput for large scale studies. The strengths and weaknesses in current ST platforms and computational solutions need to be taken into account when planning spatial transcriptomics studies. The basis of the computational ST analysis is the solutions developed for single-cell RNA-sequencing data, with advancements taking into account the spatial connectedness of the transcriptomes. The scRNA-seq tools are modified for spatial transcriptomics or new solutions like deep learning-based joint analysis of expression, spatial, and image data are developed to extract biological information in the spatially resolved transcriptomes. The computational ST analysis can reveal remarkable biological insights into spatial patterns of gene expression, cell signaling, and cell type variations in connection with cell type-specific signaling and organization in complex tissues. This review covers the topics that help choosing the platform and computational solutions for spatial transcriptomics research. We focus on the currently available ST methods and platforms and their strengths and limitations. Of the computational solutions, we provide an overview of the analysis steps and tools used in the ST data analysis. The compatibility with the data types and the tools provided by the current ST analysis frameworks are summarized.
PubMed: 36147664
DOI: 10.1016/j.csbj.2022.08.043 -
The Journal of Clinical Investigation Apr 2023Sepsis pathogenesis is complex and heterogeneous; hence, a precision-medicine strategy is needed. Acute kidney injury (AKI) following sepsis portends higher mortality....
Sepsis pathogenesis is complex and heterogeneous; hence, a precision-medicine strategy is needed. Acute kidney injury (AKI) following sepsis portends higher mortality. Overproduction of mitochondrial ROS (mtROS) is a potential mediator of sepsis and sepsis-induced AKI. BAM15, a chemical uncoupler, dissipates mitochondrial proton gradients without generating mtROS. We injected BAM15 into mice at 0, 6, or 12 hours after cecal ligation and puncture (CLP), and these mice were treated with fluids and antibiotics. BAM15 reduced mortality, even after 12 hours, when mice were ill, and BAM15 reduced kidney damage and splenic apoptosis. Serial plasma and urinary mitochondrial DNA (mtDNA) levels increased after CLP and decreased after BAM15 administration (at 0 or 6 hours). In vitro septic serum proportionately increased mtROS overproduction and mtDNA release from kidney tubule cells, which BAM15 prevented. BAM15 decreased neutrophil apoptosis and mtDNA release; neutrophil depletion counteracted BAM15 benefits. Further, mtDNA injection in vivo replicated inflammation and kidney injury, which was prevented by BAM15. A large dose of exogenous mtDNA reversed protection by BAM15. We conclude that BAM15 is an effective preventive and therapeutic candidate in experimental sepsis and that BAM15 and mtDNA, a potential drug-companion diagnostic/drug-efficacy pair for clinical sepsis, are mechanistically linked via mtROS.
Topics: Mice; Animals; DNA, Mitochondrial; Neutrophils; Kidney; Acute Kidney Injury; Sepsis; Mice, Inbred C57BL
PubMed: 36757801
DOI: 10.1172/JCI152401 -
Autophagy Jan 2024AKI: acute kidney injury; ATP: adenosine triphosphate; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; eGFR: estimated glomerular filtration rate; H&E:...
AKI: acute kidney injury; ATP: adenosine triphosphate; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; eGFR: estimated glomerular filtration rate; H&E: hematoxylin and eosin staining; LCN2/NGAL: lipocalin 2; LPS: lipopolysaccharide; LTL: lotus tetragonolobus lectin; mKeima: mitochondria-targeted Keima; mtDNA: mitochondrial DNA; PAS: periodic acid - Schiff staining; RTECs: renal tubular epithelial cells; SAKI: sepsis-induced acute kidney injury; Scr: serum creatinine; SIRT3: sirtuin 3; TFAM: transcription factor A, mitochondrial; TMRE: tetramethylrhodamine.
Topics: Humans; Sirtuin 3; Melatonin; Mitophagy; Autophagy; Acute Kidney Injury; Lipopolysaccharides; DNA, Mitochondrial; Sepsis; Kidney; DNA-Binding Proteins; Transcription Factors; Mitochondrial Proteins
PubMed: 37651673
DOI: 10.1080/15548627.2023.2252265 -
Nature Cell Biology Apr 2021Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library...
Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs-mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)-that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.
Topics: DNA, Complementary; HeLa Cells; Humans; MicroRNAs; RNA Processing, Post-Transcriptional; RNA, Ribosomal; RNA, Small Untranslated; RNA-Seq; Transcriptome
PubMed: 33820973
DOI: 10.1038/s41556-021-00652-7 -
Genome Research Mar 2023Tn5 transposon tagments double-stranded DNA and RNA/DNA hybrids to generate nucleic acids that are ready to be amplified for high-throughput sequencing. The nucleic acid...
Tn5 transposon tagments double-stranded DNA and RNA/DNA hybrids to generate nucleic acids that are ready to be amplified for high-throughput sequencing. The nucleic acid substrates for the Tn5 transposon must be explored to increase the applications of Tn5. Here, we found that the Tn5 transposon can transpose oligos into the 5' end of single-stranded DNA longer than 140 nucleotides. Based on this property of Tn5, we developed a tagmentation-based and ligation-enabled single-stranded DNA sequencing method called TABLE-seq. Through a series of reaction temperature, time, and enzyme concentration tests, we applied TABLE-seq to strand-specific RNA sequencing, starting with as little as 30 pg of total RNA. Moreover, compared with traditional dUTP-based strand-specific RNA sequencing, this method detects more genes, has a higher strand specificity, and shows more evenly distributed reads across genes. Together, our results provide insights into the properties of Tn5 transposons and expand the applications of Tn5 in cutting-edge sequencing techniques.
Topics: DNA, Single-Stranded; DNA; Base Sequence; RNA; Sequence Analysis, RNA; DNA Transposable Elements
PubMed: 36958795
DOI: 10.1101/gr.277213.122 -
Cell Nov 2022Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution....
Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.
Topics: Mice; Animals; Transcriptome; Chronic Pain; DNA; RNA; Gels
PubMed: 36368323
DOI: 10.1016/j.cell.2022.10.021