ultra-long-read-sequencing-for-whole-genomic-dna-analysis

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

Jackson Laboratory for Genomic Medicine

In multicellular organisms, transcription regulation is one of the central mechanisms modelling lineage differentiation and cell-fate determination. Transcription requires dynamic chromatin configurations between promoters and their corresponding distal regulatory elements. It is believed that their communication occurs within large discrete foci of aggregated RNA polymerases termed transcription factories in three-dimensional nuclear space. However, the dynamic nature of chromatin connectivity has not been characterized at the genome-wide level. Here, through a chromatin interaction analysis with paired-end tagging approach using an antibody that primarily recognizes the pre-initiation complexes of RNA polymerase II, we explore the transcriptional interactomes of three mouse cells of progressive lineage commitment, including pluripotent embryonic stem cells, neural stem cells and neurosphere stem/progenitor cells. Our global chromatin connectivity maps reveal approximately 40,000 long-range interactions, suggest precise enhancer-promoter associations and delineate cell-type-specific chromatin structures. Analysis of the complex regulatory repertoire shows that there are extensive colocalizations among promoters and distal-acting enhancers. Most of the enhancers associate with promoters located beyond their nearest active genes, indicating that the linear juxtaposition is not the only guiding principle driving enhancer target selection. Although promoter-enhancer interactions exhibit high cell-type specificity, promoters involved in interactions are found to be generally common and mostly active among different cells. Chromatin connectivity networks reveal that the pivotal genes of reprogramming functions are transcribed within physical proximity to each other in embryonic stem cells, linking chromatin architecture to coordinated gene expression. Our study sets the stage for the full-scale dissection of spatial and temporal genome structures and their roles in orchestrating development. 

Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations.

The identification of microbial species has depended predominantly upon culture-based techniques. However, the difficulty with which types of organisms are cultured implies that the grown species may be overrepresented by both cultivation and plate counts. In recent years, culture-independent analysis using high-throughput sequencing has been advocated for use as a point-of-care diagnostic tool. Although it offers a rapid and unbiased survey to characterize the pathogens in clinical specimens, its accuracy is reduced by the high level of contamination of human DNA. In this paper, we propose using a culture-independent analysis for a Klebsiella pneumoniae clinical strain within a liver abscess using nanopore sequencing. Owing to the highly-contaminated cell population within a liver abscess, we managed to reduce the confounding effects of human DNA through the use of DNase and differential centrifugation. Genomic DNA was sequenced through the use of Nanopore MinION sequencer and analyzed using a suite of bioinformatics approaches. K. pneumoniae was successfully identified along with antibiotic-resistant genes. Our results indicate that, by integrating real-time nanopore sequencing and bioinformatics software, real-time pathogen identification in a liver abscess can be achieved.

Culture-independent analysis of liver abscess using nanopore sequencing.

Acquired genomic structural variants (SVs) are major hallmarks of cancer genomes, but they are challenging to reconstruct from short-read sequencing data. Here we exploited the long reads of the nanopore platform using our customized pipeline, Picky ( https://github.com/TheJacksonLaboratory/Picky ), to reveal SVs of diverse architecture in a breast cancer model. We identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses, and uncovered repetitive DNA as the major source of variation. Examination of genome-wide breakpoints at nucleotide resolution uncovered micro-insertions as the common structural features associated with SVs. Breakpoint density across the genome is associated with the propensity for interchromosomal connectivity and was found to be enriched in promoters and transcribed regions of the genome. Furthermore, we observed an over-representation of reciprocal translocations from chromosomal double-crossovers through phased SVs. We demonstrate that Picky analysis is an effective tool for comprehensive detection of SVs in cancer genomes from long-read data.

Picky comprehensively detects high-resolution structural variants in nanopore long reads.

We hypothesize that genomic variants including deletions, insertions, inversions, and tandem duplications beyond the changes in tumor suppressor NF2 gene affect gene expression of tumor-specific pathways in vestibular schwannomas (VS) patients with Neurofibromatosis type 2 (NF2), thus contributing to their clinical behavior.

Linked-read Sequencing Analysis Reveals Tumor-specific Genome Variation Landscapes in Neurofibromatosis Type 2 (NF2) Patients.

The genomes of multicellular organisms are extensively folded into 3D chromosome territories within the nucleus. Advanced 3D genome-mapping methods that combine proximity ligation and high-throughput sequencing (such as chromosome conformation capture, Hi-C), and chromatin immunoprecipitation techniques (such as chromatin interaction analysis by paired-end tag sequencing, ChIA-PET), have revealed topologically associating domains with frequent chromatin contacts, and have identified chromatin loops mediated by specific protein factors for insulation and regulation of transcription. However, these methods rely on pairwise proximity ligation and reflect population-level views, and thus cannot reveal the detailed nature of chromatin interactions. Although single-cell Hi-C potentially overcomes this issue, this method may be limited by the sparsity of data that is inherent to current single-cell assays. Recent advances in microfluidics have opened opportunities for droplet-based genomic analysis but this approach has not yet been adapted for chromatin interaction analysis. Here we describe a strategy for multiplex chromatin-interaction analysis via droplet-based and barcode-linked sequencing, which we name ChIA-Drop. We demonstrate the robustness of ChIA-Drop in capturing complex chromatin interactions with single-molecule precision, which has not been possible using methods based on population-level pairwise contacts. By applying ChIA-Drop to Drosophila cells, we show that chromatin topological structures predominantly consist of multiplex chromatin interactions with high heterogeneity; ChIA-Drop also reveals promoter-centred multivalent interactions, which provide topological insights into transcription.

Multiplex chromatin interactions with single-molecule precision.

The SOX2 transcription factor is critical for neural stem cell (NSC) maintenance and brain development. Through chromatin immunoprecipitation (ChIP) and chromatin interaction analysis (ChIA-PET), we determined genome-wide SOX2-bound regions and Pol II-mediated long-range chromatin interactions in brain-derived NSCs. SOX2-bound DNA was highly enriched in distal chromatin regions interacting with promoters and carrying epigenetic enhancer marks. Sox2 deletion caused widespread reduction of Pol II-mediated long-range interactions and decreased gene expression. Genes showing reduced expression in Sox2-deleted cells were significantly enriched in interactions between promoters and SOX2-bound distal enhancers. Expression of one such gene, Suppressor of Cytokine Signaling 3 (Socs3), rescued the self-renewal defect of Sox2-ablated NSCs. Our work identifies SOX2 as a major regulator of gene expression through connections to the enhancer network in NSCs. Through the definition of such a connectivity network, our study shows the way to the identification of genes and enhancers involved in NSC maintenance and neurodevelopmental disorders.

Article	Year
Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations. Nature\| PubMed ID: 24213634	2013
Culture-independent analysis of liver abscess using nanopore sequencing. PloS one\| PubMed ID: 29315344	2018
Picky comprehensively detects high-resolution structural variants in nanopore long reads. Nature methods\| PubMed ID: 29713081	2018
Linked-read Sequencing Analysis Reveals Tumor-specific Genome Variation Landscapes in Neurofibromatosis Type 2 (NF2) Patients. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology\| PubMed ID: 30624408	2019
Multiplex chromatin interactions with single-molecule precision. Nature\| PubMed ID: 30778195	2019
Mapping the Global Chromatin Connectivity Network for Sox2 Function in Neural Stem Cell Maintenance. Cell stem cell\| PubMed ID: 30849367	2019

Chee-Hong Wong

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