WYMM Tour: NYC

The effects of treatments for depression and related disorders involve adaptation of cellular processes in the brain; however, underlying molecular mechanisms are ill defined. Notably, the acute mechanisms of ketamine and electroconvulsive therapy for treatment-resistant depression are poorly understood. We exposed adult mice to a single treatment of ketamine or electroconvulsive therapy and sacrificed 24 hours following treatment. Prefrontal cortex tissue punches were used for long-read, single-nucleus RNA sequencing using the 10x Genomics Chromium and Oxford Nanopore PromethION platforms.

Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully-phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells resulted in whole transcriptome characterization of each cell, as well as highly-accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrated binding to measles antigens and neutralization of measles live virus.

Germline mutations in homologous recombination repair (HRR) pathway genes, such as BRCA1/2, PALB2 and RAD51C, can lead to oncogenic transformation and cancer. PARP inhibitor therapies have been developed to selectively target HRR-deficient cancer cells in multiple cancer types, and therefore it is critical to identify germline HRR mutations in patients to inform the most effective treatment options.

Current diagnostic techniques for HRR mutation detection utilize short-read next generation approaches, like the MSK-IMPACT capture-based sequencing assay, which can be time-consuming and technically complex to run and analyse. We investigated the feasibility of using nanopore-based sequencing with adaptive sampling to detect the full spectrum of germline mutations relevant to HRR deficiency.

Blood collected from solid tumor patients previously profiled using MSK-IMPACT and known to harbor germline HRR mutations were used to benchmark adaptive sampling against capture-based short read data, in terms of accuracy and turnaround time. We demonstrate the concordance of HRR variant calls for pathogenic mutations between the two approaches, meaning that adaptive sampling has the potential to detect clinically relevant alterations in a time-effective and highly customizable way.

Further refinement of this workflow is needed to determine multiplexing capacity to reduce cost, and to establish the minimum coverage required for robust variant detection to reduce sequencing time. Also, processing more samples will further validate the accuracy and sensitivity of this methodology, enabling its adoption as a rapid low-cost diagnostic tool in clinical oncology.

Aneuploidy is a leading cause of pregnancy loss, fetal anomalies, and developmental delays. The identification of genetic abnormalities is a critical aspect of prenatal and fertility care. Current methods for aneuploidy testing in reproductive care, including rapid targeted approaches and comprehensive whole-genome approaches, are either limited to certain chromosomes or require lengthy processing times in specialized laboratories, making timely and comprehensive screening challenging.

This study introduces short-read transpore rapid karyotyping (STORK). STORK is a novel nanopore-based sequencing method designed for rapid aneuploidy screening across 24 chromosomes in reproductive care. It was validated with high accuracy compared with standard clinical methods. STORK delivers faster results and is more cost-effective, reducing testing time to as little as 10 minutes to 2 hours, and costs to under $50 when multiplexing samples. Although it cannot detect certain genetic variations, its speed, accessibility, and affordability make it a promising alternative for comprehensive genetic screening.

Recent development involving long-read single-cell transcriptome sequencing (lr-scRNA-Seq) represents a significant leap forward in single-cell genomics. With the recent introduction of R10 flowcells by Oxford Nanopore, we propose that previous computational methods designed to handle high sequencing error rates are no longer relevant, and that the prevailing approach using short reads to compile "barcode space" (candidate barcode list) to de-multiplex long reads are no longer necessary. Instead, computational methods should now shift focus on harnessing the unique benefits of long reads to analyze transcriptome complexity. In this context, we introduce a comprehensive suite of computational methods named Single-Cell Omics for Transcriptome CHaracterization (SCOTCH). Our method is compatible with the single-cell library preparation platform from both 10X Genomics and Parse Biosciences, facilitating the analysis of special cell populations, such as neurons, hepatocytes and developing cardiomyocytes. We specifically re-formulated the transcript mapping problem with a compatibility matrix and addressed the multiple-mapping issue using probabilistic inference, which allows the discovery of novel isoforms as well as the detection of differential isoform usage between cell populations. We evaluated SCOTCH through analysis of real data across different combinations of single-cell libraries and sequencing technologies (10X + Illumina, Parse + Illumina, 10X + Nanopore_R9, 10X + Nanopore_R10, Parse + Nanopore_R10), and showed its ability to infer novel biological insights on cell type-specific isoform expression. These datasets enhance the availability of publicly available data for continued development of computational approaches. In summary, SCOTCH allows extraction of more biological insights from the new advancements in single-cell library construction and sequencing technologies, facilitating the examination of transcriptome complexity at the single-cell level.

Multiway interactions between more than two genomic loci are common in human chromatin and are hypothesized to play a role in transcriptional regulation. Recent studies have implicated BRD4-mediated spatial colocalization of multiple extrachromosomal DNA (ecDNA), or hubbing, as a mechanism leading to oncogene overexpression in cancer. However, existing chromatin conformation capture techniques limited to pairwise interactions are unable to observe the multiway contacts induced by ecDNA hubbing and cannot differentiate between hubbed ecDNAs and integrated amplicons. Here we demonstrate that the multiway contacts observable with chromatin conformation capture technique Pore-C enable the identification of ecDNA hubs. We previously developed Chromunity to analyze multiway contacts in Pore-C data, however this method is computationally limited to small sliding genomic windows or loci at predefined regulatory elements and does not account for the dependence of contact counts on copy number, limiting its insight into ecDNA hubbing. To better understand the higher order structure of ecDNA hubs we developed Chromunity v2, which identifies significant genome-wide higher order interactions by directly modeling the dependence of higher order contact counts on distance, pairwise contacts, and copy number. We show Chromunity v2 successfully nominates synergistic interactions with dramatically increased precision and recall compared to Chromunity and with greatly improved computational efficiency. We note that Chromunity v2 may be able to more finely resolve higher order ecDNA structure by leveraging adaptive sampling to increase higher order contact counts in amplicon regions. We apply Chromunity v2 to Pore-C data collected from colorectal cancer cell lines in a control condition and exposed to BRD4 inhibitor JQ1, revealing that JQ1 reduces significant higher order amplicon contacts exclusively in cells containing hubbed ecDNAs and that this difference is not observable in pairwise contacts. We correlate the loss of higher order contacts with the dispersal of ecDNA hubs observed with fluorescence in situ hybridization (FISH), demonstrating the ability of Chromunity v2 to distinguish between hubbed ecDNAs and integrated amplicons in Pore-C data.

Chromatin architecture, histone modification, DNA methylation, epigenetic modifications, and TF activity all work in concert to orchestrate coordinated gene expression programs and respond to cellular stress and extracellular signals. These questions are at the very crux of cellular fate decision. The insulation factor CTCF and the cohesin complex are essential in maintaining the integrity of both short-range DNA-DNA interactions as well as providing regional structured boundaries between genomic neighborhoods. While large genomic structure remains relatively static, short-range DNA loop structures that facilitate cell-type specific promoter-enhancer contacts are highly dynamic and enable transcriptional programs critical for hematopoietic differentiation. Our preliminary findings identify that during hematopoietic differentiation, changes in DNA methylation at CTCF sites toggle CTCF and can alter chromatin architecture and key genes controlled by lineage-specific hematopoietic transcription factors such as CEBPA and PU.1. Using novel low input genomic approaches, we have been able to map the dynamic changes in chromatin architecture, DNA methylation, and CTCF binding during hematopoietic stem cell commitment towards a myeloid fate. Here we demonstrate that CTCF binding by ChIPseq correlates negatively with cytosine methylation of the CTCF motif in both gained and lost CTCF loci throughout the genome. Nanopore single-molecule dual 5-methylcytosine/5-hydroxymethylcytosine calling validates these interactions with the capability to identify novel 5-hydroxymethylcytosine as specific potential targets for APOBEC and Tet-dioxygenase enzymatic activity driving hematopoietic differentiation.