Oxford Nanopore User Group Meeting, NIH

Long-read RNA sequencing has significantly improved our ability to resolve complex transcript structures often missed by short-read technologies. Yet, its broader adoption has been hindered by challenges in achieving sufficient sequencing depth and accurately assembling isoforms at scale. To overcome these limitations, we developed an integrated wet-lab and computational framework optimized for transcriptome profiling from postmortem human brain tissue. Our workflow combines Oxford Nanopore R10.4 flow cells, Dorado basecalling, and PyChopper-based processing to recover and orient full-length cDNA reads. Our library preparation protocol yields high sequencing depth comparable to short read sequencing per sample with Q-scores exceeding 20. We complement the experimental workflow with a reproducible Snakemake-based computational pipeline that performs isoform assembly using both StringTie and IsoQuant, followed by stringent counts-per-million (CPM) filtering via featureCounts to minimize false positives and improve annotation precision. Applied to initial brain samples, this framework uncovered thousands of high-confidence isoforms, including numerous novel transcripts not captured in existing reference annotations. We are now applying this approach to the full North American Brain Expression Consortium (NABEC) cohort (n=200). This will, generateing one of the most comprehensive long-read RNA resources from the human brain to date. This dataset will facilitate discovery of transcriptomic signatures associated with aging and serve as a valuable reference for the neuroscience and genomics communities.

Long-read RNA sequencing has significantly improved our ability to resolve complex transcript structures often missed by short-read technologies. Yet, its broader adoption has been hindered by challenges in achieving sufficient sequencing depth and accurately assembling isoforms at scale. To overcome these limitations, we developed an integrated wet-lab and computational framework optimized for transcriptome profiling from postmortem human brain tissue. Our workflow combines Oxford Nanopore R10.4 flow cells, Dorado basecalling, and PyChopper-based processing to recover and orient full-length cDNA reads. Our library preparation protocol yields high sequencing depth comparable to short read sequencing per sample with Q-scores exceeding 20. We complement the experimental workflow with a reproducible Snakemake-based computational pipeline that performs isoform assembly using both StringTie and IsoQuant, followed by stringent counts-per-million (CPM) filtering via featureCounts to minimize false positives and improve annotation precision. Applied to initial brain samples, this framework uncovered thousands of high-confidence isoforms, including numerous novel transcripts not captured in existing reference annotations. We are now applying this approach to the full North American Brain Expression Consortium (NABEC) cohort (n=200). This will, generateing one of the most comprehensive long-read RNA resources from the human brain to date. This dataset will facilitate discovery of transcriptomic signatures associated with aging and serve as a valuable reference for the neuroscience and genomics communities.

Cervical cancer (CC) causes ~660,000 new cases and 350,000 deaths annually, with the greatest burden in low- and middle-income countries. In Guatemala and Venezuela, CC incidence remains high, yet the molecular basis of aggressive disease subtypes remains poorly defined.

We analyzed 700 cervical tumors using Oxford Nanopore long-read sequencing and complementary genomic approaches. Among 52 tumors carrying one of 11 rare or probable hrHPVs (HPV26, 32, 35, 39, 51, 52, 53, 58, 59, 68, 69). The most frequent were HPV52 (19%) and HPV58 (17%). Out of two HPV patterns, integrated forms were slightly more common than episomal (54% vs 46%). Classification by HPV genera revealed representation of alpha 5, 6, 7, and 9, with alpha 7 and 9 predominating. Notably, one tumor contained a novel extrachromosomal DNA hybrid of human and HPV sequences, suggesting an alternative route of oncogene activation.

In parallel, YAP1 amplification was identified in 45 of 380 tumors and confirmed in 24 by barcode sequencing. Deep sequencing of these tumors showed frequent HPV integration (80%) and recurrent co-amplification of YAP1 with BIRC2/3, consistent with Breakage-Fusion-Bridge events. Clinically, YAP1 amplification defined a distinct aggressive subtype, with diagnosis a median of 12 years earlier, with significantly poorer survival and greater prevalence in minority populations.

Together, these findings highlight two major contributors to cervical carcinogenesis in underserved populations: rare hrHPV-driven tumors and YAP1-amplified aggressive subtypes. Integration of viral and host genomic landscapes provides new insights into CC disparities and points to potential biomarkers for early detection and therapeutic targeting.

Cervical cancer (CC) causes ~660,000 new cases and 350,000 deaths annually, with the greatest burden in low- and middle-income countries. In Guatemala and Venezuela, CC incidence remains high, yet the molecular basis of aggressive disease subtypes remains poorly defined.

We analyzed 700 cervical tumors using Oxford Nanopore long-read sequencing and complementary genomic approaches. Among 52 tumors carrying one of 11 rare or probable hrHPVs (HPV26, 32, 35, 39, 51, 52, 53, 58, 59, 68, 69). The most frequent were HPV52 (19%) and HPV58 (17%). Out of two HPV patterns, integrated forms were slightly more common than episomal (54% vs 46%). Classification by HPV genera revealed representation of alpha 5, 6, 7, and 9, with alpha 7 and 9 predominating. Notably, one tumor contained a novel extrachromosomal DNA hybrid of human and HPV sequences, suggesting an alternative route of oncogene activation.

In parallel, YAP1 amplification was identified in 45 of 380 tumors and confirmed in 24 by barcode sequencing. Deep sequencing of these tumors showed frequent HPV integration (80%) and recurrent co-amplification of YAP1 with BIRC2/3, consistent with Breakage-Fusion-Bridge events. Clinically, YAP1 amplification defined a distinct aggressive subtype, with diagnosis a median of 12 years earlier, with significantly poorer survival and greater prevalence in minority populations.

Together, these findings highlight two major contributors to cervical carcinogenesis in underserved populations: rare hrHPV-driven tumors and YAP1-amplified aggressive subtypes. Integration of viral and host genomic landscapes provides new insights into CC disparities and points to potential biomarkers for early detection and therapeutic targeting.

RNA modifications are critical regulators of gene expression and cellular processes; however, the epitranscriptome is less well studied than the epigenome. Here, we studied transcriptome-wide changes in RNA modifications and expression levels in two human pancreatic beta-cell lines, EndoC-BH1 and EndoC-BH3, after one hour of glucose stimulation. Using direct RNA nanopore sequencing (dRNA-seq), we measured N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, and pseudouridine concurrently across the transcriptome. We developed a differential RNA modification method and identified 1,697 differentially modified sites (DMSs) across all modifications. These DMSs were largely independent of changes in gene expression levels and enriched in transcripts for type 2 diabetes (T2D) genes. Our study demonstrates how dRNA-seq can be used to detect and quantify RNA modification changes in response to cellular stimuli at the single-nucleotide level and provides new insights into RNA-mediated mechanisms that may contribute to normal beta-cell response and potential dysfunction in T2D.