WYMM Tour: Nashville

DNA methylation at CpG dinucleotides is a heritable epigenetic mark which carries information about gene function and regulation independent of DNA sequence. However, with the exception of parent-of-origin-specific transmission of methylation patterns through genomic imprinting, comparatively little is known about the intergenerational transmission of DNA methylation from parents to offspring and its relationship to DNA sequence. To investigate the nature of this epigenetic inheritance, we developed a computational pipeline designed for the analysis of phased DNA methylation data from ONT long-read sequencing of genetically diverse mouse crosses and have applied this pipeline to DNA from inbred samples and F1 crosses of two such strains. Our analysis revealed a diverse set of epigenetic inheritance patterns genome-wide, including an abundance of cis- and trans-acting meQTLs, several novel imprinted genes, and widespread sex-specific methylation. We further validated these epigenetic inheritance patterns utilizing targeted ONT sequencing by adaptive sampling of F2 crosses of the same strains, successfully differentiating inheritance patterns mediated by cis- and trans-acting genetic determinants, as well as those transmitted through genetically independent mechanisms. The abundance and diversity of the identified patterns of intergenerational epigenetic inheritance underscores the critical importance of their consideration in the study of heredity, and our work provides both the experimental and computational methods necessary to do so.

Alternative splicing (AS) contributes to the biological heterogeneity between species, sexes, tissues, and cell types. Many diseases are either caused by alterations in AS or by alterations to AS. Therefore, measuring AS accurately and efficiently is critical for assessing molecular phenotypes, including those associated with disease. Long-read sequencing enables more accurate quantification of differentially spliced isoform expression than short-read sequencing approaches, and third-generation platforms facilitate high-throughput experiments. To assess differences in AS across the cerebellum, cortex, hippocampus, and striatum by sex, we generated and analyzed Oxford Nanopore Technologies (ONT) long-read RNA sequencing (lrRNA-Seq) C57BL/6J mouse brain cDNA libraries. From >85 million reads that passed quality control metrics, we calculated differential gene expression (DGE), differential transcript expression (DTE), and differential transcript usage (DTU) across brain regions and by sex. We found significant DGE, DTE, and DTU across brain regions and that the cerebellum had the most differences compared to the other three regions. Additionally, we found region-specific differential splicing between sexes, with the most sex differences in DTU in the cortex and no DTU in the hippocampus. We also report on two distinct patterns of sex DTU we observed, sex-divergent and sex-specific, that could potentially help explain sex differences in the prevalence and prognosis of various neurological and psychiatric disorders in future studies. Finally, we built a Shiny web application for researchers to explore the data further. Our study provides a resource for the community it underscores the importance of AS in biological heterogeneity and the utility of long-read sequencing to better understand AS in the brain.

Effective treatment of acute lymphoblastic leukemia (ALL) is dependent on accurate genomic classification, typically derived from a combination of multiple time-consuming and costly techniques such as flow cytometry, fluorescence in situ hybridization (FISH), karyotype analysis, targeted PCR, and microarrays. We investigate the feasibility of a comprehensive single-assay diagnostic approach using long-read sequencing, with real-time genome target enrichment, to classify chromosomal abnormalities and structural variants characteristic of B-cell ALL. We performed whole genome long-read sequencing with adaptive sampling on DNA from diagnostic peripheral blood or bone marrow for thirty-five pediatric acute leukemia cases with a diversity of genomics subtypes. Cases include hyperdiploid, hypodiploid, ETV6::RUNX1, TCF3::PBX1, BCR::ABL1, KMT2A-, ZNF384-, DUX4-, MEF2D-, and CRLF2-rearranged, and intrachromosomal amplification of chromosome 21. Adaptive sampling enriched sequencing depth for fifty-nine genes commonly involved in structural variants in B-ALL. We demonstrated characterization of known, clinically relevant karyotype abnormalities and structural variants, concordant with standard-of-care clinical cytogenetics (G-banding karyotype analysis, FISH, copy-number microarray). Precise genomic alterations were identified in all cases following a maximum of forty-eight hours of sequencing. We performed real-time analysis – concurrent with sequencing – of the eleven cases, we identified the driving alteration in as little as fifteen minutes (for karyotype) or up to six hours (for complex structural variants). Whole genome nanopore sequencing with adaptive sampling has the potential to provide rapid classification of pediatric ALL specimens while reducing the cost and throughput requirements.

Combining long-read sequencing with single cell assays enables the unambiguous identification of alternative splicing at single cell resolution. Traditional single cell assays have relied on short-read sequencing, which loses valuable information about transcript isoforms relevant to health, development, and disease. Long-read sequencing libraries were prepared from the 10x Genomics' Chromium Single Cell and Visium Spatial Gene Expression assays, and sequenced on an Oxford Nanopore Technologies’ PromethION sequencer. Shortread libraries were prepared in parallel and sequenced on an Illumina platform. We found cell calling and clustering from long-read data was comparable to short-read data and, upon further analysis of long-read data, identified isoforms differentially expressed between cell types.

A whirlwind tour of the bioinformatics behind the most common Nanopore long read applications, focusing on the simultanously user-friendly and ultra-flexible EPI2ME workflows. Highlights to be hit include characterization of human germline and somatic genetic and epigenetic variation, de novo genome assembly including T2T genome assembly, and metagenomic de novo assembly and taxonomic and functional profiling.

Reconstruction of complex genomic regions containing recent segmental duplications, repeat expansions or complex structural variants has been challenging using short read sequencing methods. These regions, however, potentially harbor a substantial fraction of disease-causing variants missed in unsolved rare disease cases (still around 50% of all cases). The introduction of Nanopore long-read genome sequencing (LR-GS) into clinical genetic testing promises to reduce this gap by resolving complex genomic regions, facilitating haplotype-phasing and revealing complex SVs that often consist of closely located inversions, tandem-duplications and deletions.

Therefore, a study group consisting of four German university hospitals was launched aiming to evaluate the added diagnostic value of Nanopore LR-GS and to demonstrate its sustainability in clinical practice. The study group relies on established collaborative structures with multidisciplinary teams, data analysis task forces (DATFs), and data interpretation task forces (DITFs). We analyzed a cohort of solved cases featuring causal repeat expansions, variants in duplicate genes, complex rearrangements, and aberrant DNA methylation. We included multiple GiaB samples to benchmark variant detection methods for accreditation of LR-GS. The experience gain from the initial pilot serves as a blueprint for the analysis of a larger clinical cohort (>1000 samples).

In this talk I will focus on our experience with the bioinformatics analysis of Nanopore LR-GS data for rare disease diagnostics, including development and/or application of tools and pipelines for SNV, indel and SV detection, haplotype phasing, length estimation for repeat expansions, genotyping in duplicate genes and haplotype-specific DNA methylation analysis. I will discuss our quality criteria and benchmarking efforts and demonstrate the need for generating large Nanopore LR-GS background dataset to facilitate systemic filtering and efficient clinical interpretation of SVs in clinical diagnostics.

Reconstruction of complex genomic regions containing recent segmental duplications, repeat expansions or complex structural variants has been challenging using short read sequencing methods. These regions, however, potentially harbor a substantial fraction of disease-causing variants missed in unsolved rare disease cases (still around 50% of all cases). The introduction of Nanopore long-read genome sequencing (LR-GS) into clinical genetic testing promises to reduce this gap by resolving complex genomic regions, facilitating haplotype-phasing and revealing complex SVs that often consist of closely located inversions, tandem-duplications and deletions.

Therefore, a study group consisting of four German university hospitals was launched aiming to evaluate the added diagnostic value of Nanopore LR-GS and to demonstrate its sustainability in clinical practice. The study group relies on established collaborative structures with multidisciplinary teams, data analysis task forces (DATFs), and data interpretation task forces (DITFs). We analyzed a cohort of solved cases featuring causal repeat expansions, variants in duplicate genes, complex rearrangements, and aberrant DNA methylation. We included multiple GiaB samples to benchmark variant detection methods for accreditation of LR-GS. The experience gain from the initial pilot serves as a blueprint for the analysis of a larger clinical cohort (>1000 samples).

In this talk I will focus on our experience with the bioinformatics analysis of Nanopore LR-GS data for rare disease diagnostics, including development and/or application of tools and pipelines for SNV, indel and SV detection, haplotype phasing, length estimation for repeat expansions, genotyping in duplicate genes and haplotype-specific DNA methylation analysis. I will discuss our quality criteria and benchmarking efforts and demonstrate the need for generating large Nanopore LR-GS background dataset to facilitate systemic filtering and efficient clinical interpretation of SVs in clinical diagnostics.