Simultaneous Profiling of Chromatin Accessibility and DNA Methylation in Complete Plant Genomes Using Long-Read Sequencing
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PROVIDER: PRJEB69301 | ENA |
REPOSITORIES: ENA
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Project description:Epigenetic regulations, including chromatin accessibility, nucleosome positioning and DNA methylation intricately shape genome function. However, current chromatin profiling techniques relying on short-read sequencing technologies fail to characterise highly repetitive genomic regions and cannot detect multiple chromatin features simultaneously. Here, we performed Simultaneous Accessibility and DNA Methylation Sequencing (SAM-seq) of purified plant nuclei. Thanks to the use of long-read nanopore sequencing, SAM-seq enables high-resolution profiling of m6A-tagged chromatin accessibility together with endogenous cytosine methylation in plants. Analysis of naked genomic DNA revealed significant sequence preference biases of m6A-MTases, controllable through a normalisation step. By applying SAM-seq to Arabidopsis and maize nuclei we obtained fine-grained accessibility and DNA methylation landscapes genome-wide. We uncovered crosstalk between chromatin accessibility and DNA methylation within nucleosomes of genes, TEs, and centromeric repeats. SAM-seq also detects DNA footprints over cis-regulatory regions. Furthermore, using the single-molecule information provided by SAM-seq we identified extensive cellular heterogeneity at chromatin domains with antagonistic chromatin marks, suggesting that bivalency reflects cell-specific regulations. SAM-seq is a powerful approach to simultaneously study multiple epigenetic features over unique and repetitive sequences, opening new opportunities for the investigation of epigenetic mechanisms.
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Project description:Understanding the genetic causes of trait variation is a primary goal of genetic research. One way that individuals can vary genetically is through variable pangenomic genes: genes that are only present in some individuals in a population. The presence or absence of entire genes could have large effects on trait variation. However, variable pangenomic genes can be missed in standard genotyping workflows, owing to reliance on aligning short-read sequencing to reference genomes. A popular method for studying the genetic basis of trait variation is linkage mapping, which identifies quantitative trait loci (QTLs), regions of the genome that harbor causative genetic variants. Large-scale linkage mapping in the budding yeast Saccharomyces cerevisiae has found thousands of QTLs affecting myriad yeast phenotypes. To enable the resolution of QTLs caused by variable pangenomic genes, we used long-read sequencing to generate highly complete de novo genome assemblies of 16 diverse yeast isolates. With these assemblies, we resolved QTLs for growth on maltose, sucrose, raffinose, and oxidative stress to specific genes that are absent from the reference genome but present in the broader yeast population at appreciable frequency. Copies of genes also duplicate onto chromosomes where they are absent in the reference genome, and we found that these copies generate additional QTLs whose resolution requires pangenome characterization. Our findings show the need for highly complete genome assemblies to identify the genetic basis of trait variation.
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Project description:BackgroundLong-read sequencing in metagenomics facilitates the assembly of complete genomes out of complex microbial communities. These genomes include essential biologic information such as the ribosomal genes or the mobile genetic elements, which are usually missed with short-reads. We applied long-read metagenomics with Nanopore sequencing to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a dog fecal sample.ResultsWe used nanopore long-read metagenomics and frameshift aware correction on a canine fecal sample and retrieved eight single-contig HQ MAGs, which were > 90% complete with < 5% contamination, and contained most ribosomal genes and tRNAs. At the technical level, we demonstrated that a high-molecular-weight DNA extraction improved the metagenomics assembly contiguity, the recovery of the rRNA operons, and the retrieval of longer and circular contigs that are potential HQ MAGs. These HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Catenibacterium, Blautia, and Enterococcus genera. Linking our results to previous gastrointestinal microbiome reports (metagenome or 16S rRNA-based), we found that some bacterial species on the gastrointestinal tract seem to be more canid-specific -Succinivibrio, Prevotellamassilia, Phascolarctobacterium, Blautia_A sp900541345-, whereas others are more broadly distributed among animal and human microbiomes -Sutterella, Catenibacterium, Enterococcus, and Blautia sp003287895. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Moreover, we show that long reads are essential to detect mobilome functions, usually missed in short-read MAGs.ConclusionsWe recovered eight single-contig HQ MAGs from canine feces of a healthy dog with nanopore long-reads. We also retrieved relevant biological insights from these specific bacterial species previously missed in public databases, such as complete ribosomal operons and mobilome functions. The high-molecular-weight DNA extraction improved the assembly's contiguity, whereas the high-accuracy basecalling, the raw read error correction, the assembly polishing, and the frameshift correction reduced the insertion and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.
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Project description:Simultaneous profiling of transcriptome and chromatin accessibility in single nucleus
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Project description:IntroductionGenome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time.MethodsIn this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%.Results and discussionFor a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.
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Project description:Current metagenome assembled human gut phage catalogs contained mostly fragmented genomes. Here, comprehensive gut virome detection procedure is developed involving virus-like particle (VLP) enrichment from ≈500 g feces and combined sequencing of short- and long-read. Applied to 135 samples, a Chinese Gut Virome Catalog (CHGV) is assembled consisting of 21,499 non-redundant viral operational taxonomic units (vOTUs) that are significantly longer than those obtained by short-read sequencing and contained ≈35% (7675) complete genomes, which is ≈nine times more than those in the Gut Virome Database (GVD, ≈4%, 1,443). Interestingly, the majority (≈60%, 13,356) of the CHGV vOTUs are obtained by either long-read or hybrid assemblies, with little overlap with those assembled from only the short-read data. With this dataset, vast diversity of the gut virome is elucidated, including the identification of 32% (6,962) novel vOTUs compare to public gut virome databases, dozens of phages that are more prevalent than the crAssphages and/or Gubaphages, and several viral clades that are more diverse than the two. Finally, the functional capacities are also characterized of the CHGV encoded proteins and constructed a viral-host interaction network to facilitate future research and applications.
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Project description:Long-read DNA sequencing has propelled medicinal plant genomics forward, with over 400 genomes from 203 plants sequenced by February 2025. However, many genomes still have assembly and annotation flaws, with only 11 gapless telomere-to-telomere assemblies. The core challenge remains identifying genes linked to secondary metabolite biosynthesis, regulation and evolution. High-quality complete genomes are essential for characterizing biosynthetic gene clusters and for enabling robust functional genomics and synthetic biology applications. We propose to focus on achieving more complete genome assemblies in diverse varieties on the basis of refining the currently available ones, leverage lessons from crop genomics research, and apply the cutting-edge genomics technologies in research of medicinal plant genomics.
| S-EPMC11876585 | biostudies-literature
Project description:BACKGROUND:Nontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques provide new opportunities to sequence and construct complete bacterial genomes faster and at a lower cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to its slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA with no additional clean-up steps. RESULTS:Our DNA extraction method was compared to 6 other methods with variations in timing of mechanical disruption and enzymatic digestion of the cell wall, quantity of matrix material, and reagents used in extraction and precipitation. We tested our optimized method on 38 clinical isolates from the M. avium and M. abscessus complexes, which yielded optimal quality and quantity measurements for Oxford Nanopore Technologies sequencing. We also present the efficient completion of circularized M. avium subspecies hominissuis genomes using our extraction technique and the long-read sequencing MinION platform, including the identification of a novel plasmid. CONCLUSIONS:Our optimized extraction protocol and assembly pipeline was both sufficient and efficient for genome closure. We expect that our finely-tuned extraction method will prove to be a valuable tool in long-read sequencing and completion of mycobacterial genomes going forward. Utilization of comprehensive, long-read based approaches will advance the understanding evolution and pathogenicity of NTM infections.
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Project description:We present scNanoATAC-seq (Single-cell Assay for Transposase Accessible Chromatin by Oxford Nanopore Technologies Sequencing), an effective method for simultaneous detection of chromatin accessibility and genetic variation. Long fragments (about 4-5Kb) of single-cell ATAC-seq library were enriched and sequenced by Oxford Nanopore Technologies platform. Ends of long ATAC-seq fragments are regarded as chromatin accessibility signal in downstream analysis.
2022-09-07 | GSE194023 | GEO
Project description:We present scNanoATAC-seq (Single-cell Assay for Transposase Accessible Chromatin by Oxford Nanopore Technologies Sequencing), an effective method for simultaneous detection of chromatin accessibility and genetic variation. Long fragments (about 4-5Kb) of single-cell ATAC-seq library were enriched and sequenced by Oxford Nanopore Technologies platform. Ends of long ATAC-seq fragments are regarded as chromatin accessibility signal in downstream analysis.
2022-09-07 | GSE194022 | GEO