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

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.

Liang Gong

Genome Technologies

Jackson Laboratory for Genomic Medicine

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Liang Gong

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Jackson Laboratory for Genomic Medicine

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Genome Technologies