WYMM Tour: Los Angeles

High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy, killing more than 9,000 women each year in the United States alone. Nearly 80% of patients with HGSOC tumors will experience recurrence within 5 years, but little is known about the mechanisms that drive this process. Up to 60% of the genome in an HGSOC tumor is impacted by structural variant mutations and pronounced intra-tumoral heterogeneity. Intratumor heterogeneity is believed to be a key feature of recurrence and resistance within HGSOC tumors, with some evidence coming from relatively small studies indicating diverse and complex mechanisms of the seeding of metastatic sites, including metastasis reseeding the primary tumor. Few studies have investigated clonality and the role of structural variants due to low sample availability. In collaboration with CSMC we identified a cohort of 36 HGSOC patients for whom paired chemo-naive and chemoresistant tumors as well as germline DNA was available. Single nucleotide variants, indels, and structural variant calls were generated for each tumor. Somatic variant burdens did not significantly differ across recurrence but was driven by homologous recombination repair deficiency status. Clonal composition and dynamics were measured through variant allele frequency alterations as tumors progressed from primary, chemo-naïve, to recurrent, chemo-resistant, tumors. Surprisingly, few changes were observed in clonal abundance and complexity. Taking structural variants into account, homologous recombination repair proficient (HRP) tumors tend to be polyclonal while homologous recombination repair deficient (HRD) tumors tend to be monoclonal, accompanied by a longer progression free survival than the HRP patients. Oxford nanopore technologies ultra long read sequencing was used to confirm the structural variants observed in short read sequencing.

Faithful maintenance of DNA methylation is imperative for maintaining cellular identity and genome stability and the disruption of DNA methylation patterns is a characteristic of many diseases. Metabolic diseases, such as type 2 diabetes and obesity, are characterized by metabolic dysfunction resulting in high glucose levels (hyperglycemia) in blood serum and have been shown to result in increased cellular levels of UDP-GlcNAc and bulk levels of O-GlcNAcylation on proteins. We recently reported that hyperglycemia leads to O-GlcNAcylation of the maintenance methyltransferase DNMT1, impairing its maintenance methyltransferase function and resulting in loss of DNA methylation across the genome. This general loss of DNA methylation results in the creation of partially methylated domains (PMDs), resembling cancer specific methylomes. While there is a general loss of methylation at many repetitive elements in these conditions, some evolutionarily young subfamilies maintain methylation, suggesting continuous targeting for epigenetic repression. Our data provides novel evidence that the DNA methyltransferase function of DNMT1, the key methyltransferase needed for maintenance of DNA methylation, is regulated by nutrient availability through O-GlcNAcylation. Our results furthermore demonstrate that inhibition of DNMT1 results in the generation of partially methylated domains.

Lynch syndrome is a hereditary cancer predisposition condition caused by a heterozygous germline pathogenic variant within a DNA mismatch repair (MMR) gene that confers elevated risks for MMR-deficient colorectal, endometrial, and other cancers. An alternative cause for Lynch syndrome is constitutional MLH1 methylation, or "epimutation", found instead of a coding germline pathogenic variant in a subset of patients. Constitutional MLH1 epimutation is characterized by aberrant methylation of a single allele of the MLH1 CpG island promoter in germline DNA, which is accompanied by monoallelic transcriptional silencing in normal tissues. In some familial cases, the constitutional MLH1 methylation is inherited in a classic autosomal dominant pattern linked in cis to an underlying genetic variant that may be cryptic, non-coding, and unidentifiable by diagnostic NGS-based multi-gene panel tests, such as a promoter or structural variant. In other cases, the constitutional MLH1 epimutation arose de novo or was inherited in a non-Mendelian pattern, and so it remains unknown if there is any genetic basis to these cases. We applied ONT sequencing to 12 patients previously confirmed to carry constitutional MLH1 methylation via targeted assays performed on bisulfite-converted DNA. We aimed to determine if the monoallelic constitutional MLH1 methylation could be readily detected and resolved by ONT, and if so, to screen for any candidate genetic variants in cis that might underlie the epimutation. We found that ONT detected the constitutional MLH1 methylation in all 12 cases and assigned the methylation to the same genetic allele at common promoter SNP sites in patient heterozygous for these SNPs. Furthermore, ONT sequencing was able to extend the genetic haplotypes linked to constitutional MLH1 methylation at some distance from the MLH1 promoter region. Underlying cis genetic variants were identified in two familial cases. No evidence for a genetic basis in cis was found in 10 isolated probands.

Investigating the impact of genetic variation on epigenetics and epitranscriptomics provides valuable insights into the regulation of gene expression. As part of the IGVF consortium, we are generating a reference dataset of short-read as well as long-read functional genomics assays using the Oxford Nanopore Technologies platform. We are profiling the adult cerebral cortex of the mouse reference strain C57BL6/J and wild-derived CAST/EiJ with two biological replicates per sex to map the interactions between genetic variation and epigenetic mechanisms and to elucidate impact of variation on function on both the genomic and transcriptomic level. We measure chromatin accessibility with Fiber-seq, which is an assay that labels open chromatin regions using exogenous m6A methyltransferases. Sequencing of the resulting DNA identifies open chromatin regions using m6A that are orthogonal to endogenous 5mC and 5hmC methylation, thereby simultaneously measuring three distinct epigenetic signatures on the same DNA molecule. We also sequenced single-nuclei barcoded with the Parse Bio platform using the LR-Split-seq protocol to identify cell-type specific splicing and transcript isoform expression. We compare both the Fiber-seq open chromatin regions and the long-read single-nucleus results to short-read counterparts also generated in the consortium, such as Parse and 10x Multiome. Finally we sequenced RNA directly to profile m6A, inosine, pseudoUridine, and m5c in mRNA to characterize distinct patterns of modifications and to assess the extent of their conservation across both strains. The IGVF bridge sample dataset is designed to allow for the direct comparison of short-read and long-read assays as they relate to functional annotation of the genome.

Plasmidsaurus is on a mission to accelerate human and planetary health by unlocking a new level of productivity for bioengineers. Our global network of labs allows any scientist in the world to drop samples in a Plasmidsaurus collection box on the way out of the lab and have full-plasmid sequencing results in their inbox before they get to work in the morning. Sequencing the entire plasmid molecule in a continuous nanopore read eliminates the need for primers, simplifies analysis, and identifies backbone mutations, rearrangements, and duplications that are missed by Sanger sequencing. The elimination of sample verification wait times and lost months or years of experiments on faulty DNA constructs, integrated over our community of tens of thousands of innovative customers (including Nobel prize-winners, dynamic biotech startups, pharma companies, and DIY biohackers), is making a real difference in realizing the promise of biotechnology to solve the problems facing our planet.

To build an artificial kidney from pluripotent cells we must understand developmental programs and apply engineered synthetic biology approaches to deconstruct and reconstruct development. The Lindström lab uses in vivo inspired molecular mechanisms to control the production of clinically important kidney cells. Our work sheds light on the genetic circuitry that regulates development and disease and provides inspiration for how to generate functional kidney cells in vitro.