Theoretical framework for the difference of two negative binomial distributions and its application in comparative analysis of sequencing data.

High-throughput sequencing (HTS) technologies have been instrumental in investigating biological questions at the bulk and single-cell levels. Comparative analysis of two HTS data sets often relies on testing the statistical significance for the difference of two negative binomial distributions (DOTNB). Although negative binomial distributions are well studied, the theoretical results for DOTNB remain largely unexplored.

Detecting m6A RNA modification from nanopore sequencing using a semisupervised learning framework.

Direct nanopore-based RNA sequencing can be used to detect posttranscriptional base modifications, such as N6-methyladenosine (m6A) methylation, based on the electric current signals produced by the distinct chemical structures of modified bases. A key challenge is the scarcity of adequate training data with known methylation modifications. We present Xron, a hybrid encoder-decoder framework that delivers a direct methylation-distinguishing basecaller by training on synthetic RNA data and immunoprecipitation (IP)-based experimental data in two steps.

Genomic epidemiology of carbapenem-resistant Enterobacterales at a New York City hospital over a 10-year period reveals complex plasmid-clone dynamics and evidence for frequent horizontal transfer of .

Transmission of carbapenem-resistant Enterobacterales (CRE) in hospitals has been shown to occur through complex, multifarious networks driven by both clonal spread and horizontal transfer mediated by plasmids and other mobile genetic elements. We performed nanopore long-read sequencing on CRE isolates from a large urban hospital system to determine the overall contribution of plasmids to CRE transmission and identify specific plasmids implicated in the spread of (the carbapenemase [KPC] gene). Six hundred and five CRE isolates collected between 2009 and 2018 first underwent Illumina sequencing for genome-wide genotyping; 435 -positive isolates were then successfully nanopore sequenced to generate hybrid assemblies including circularized -harboring plasmids.

Nanopore strand-specific mismatch enables de novo detection of bacterial DNA modifications.

DNA modifications in bacteria present diverse types and distributions, playing crucial functional roles. Current methods for detecting bacterial DNA modifications via nanopore sequencing typically involve comparing raw current signals to a methylation-free control. In this study, we found that bacterial DNA modification induces errors in nanopore reads.

Genetics-driven risk predictions leveraging the Mendelian randomization framework.

Accurate predictive models of future disease onset are crucial for effective preventive healthcare, yet longitudinal data sets linking early risk factors to subsequent health outcomes are limited. To overcome this challenge, we introduce a novel framework, redictive sk modeling using ndelian andomization (PRiMeR), which utilizes genetic effects as supervisory signals to learn disease risk predictors without relying on longitudinal data. To do so, PRiMeR leverages risk factors and genetic data from a healthy cohort, along with results from genome-wide association studies of diseases of interest.

Rapid SARS-CoV-2 surveillance using clinical, pooled, or wastewater sequence as a sensor for population change.

The COVID-19 pandemic has highlighted the critical role of genomic surveillance for guiding policy and control. Timeliness is key, but sequence alignment and phylogeny slow most surveillance techniques. Millions of SARS-CoV-2 genomes have been assembled.

Designing realistic regulatory DNA with autoregressive language models.

-regulatory elements (CREs), such as promoters and enhancers, are DNA sequences that regulate the expression of genes. The activity of a CRE is influenced by the order, composition, and spacing of sequence motifs that are bound by proteins called transcription factors (TFs). Synthetic CREs with specific properties are needed for biomanufacturing as well as for many therapeutic applications including cell and gene therapy.

Mutational scanning of classifies clinical variants and reveals biochemical properties of the transcriptional effector domain.

The transcription factor (TF) cone-rod homeobox (CRX) is essential for the differentiation and maintenance of photoreceptor cell identity. Several human variants cause degenerative retinopathies, but most are variants of uncertain significance. We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitutions in CRX using a cell-based transcriptional reporter assay, curating a high-confidence list of nearly 2000 variants with altered transcriptional activity.

Contrasting and combining transcriptome complexity captured by short and long RNA sequencing reads.

Mapping transcriptomic variations using either short- or long-read RNA sequencing is a staple of genomic research. Long reads are able to capture entire isoforms and overcome repetitive regions, whereas short reads still provide improved coverage and error rates. Yet, open questions remain, such as how to quantitatively compare the technologies, can we combine them, and what is the benefit of such a combined view? We tackle these questions by first creating a pipeline to assess matched long- and short-read data using a variety of transcriptome statistics.

duplicons associate with structural diversity at Chromosome 10q11.22.

The 10q11.22 chromosomal region is a duplication-rich interval of the human genome and one of the last to be fully assembled. It carries copy number-variable genes associated with intellectual disability, bipolar disorder, and obesity.

Chromosome-level subgenome-aware de novo assembly provides insight into genome divergence after hybridization.

Interspecies hybridization is prevalent in various eukaryotic lineages and plays important roles in phenotypic diversification, adaptation, and speciation. To better understand the changes that occurred in the different subgenomes of a hybrid species and how they facilitate adaptation, we have completed chromosome-level de novo assemblies of all chromosomes for a recently formed hybrid yeast, strain CBS380, using Oxford Nanopore Technologies' MinION long-read sequencing. We characterize the genome and compare it with its parent species, and , and other genomes to better understand genome evolution after a relatively recent hybridization event.

Full-length RNA transcript sequencing traces brain isoform diversity in house mouse natural populations.

The ability to generate multiple RNA transcript isoforms from the same gene is a general phenomenon in eukaryotes. However, the complexity and diversity of alternative isoforms in natural populations remain largely unexplored. Using a newly developed full-length transcript enrichment protocol with 5' CAP selection, we sequenced full-length RNA transcripts of 48 individuals from outbred populations and subspecies of , and from the closely related sister species and as outgroups.

Long-read RNA sequencing of archival tissues reveals novel genes and transcripts associated with clear cell renal cell carcinoma recurrence and immune evasion.

The use of long-read direct RNA sequencing (DRS) and PCR cDNA sequencing (PCS) in clinical oncology remains limited, with no direct comparison between the two methods. We used DRS and PCS to study clear cell renal cell carcinoma (ccRCC), focusing on new transcript and gene discovery. Twelve primary ccRCC archival tumors, six from patients who went on to relapse, were analyzed.

Measuring X-Chromosome inactivation skew for X-linked diseases with adaptive nanopore sequencing.

X-linked genetic disorders typically affect females less severely than males owing to the presence of a second X Chromosome not carrying the deleterious variant. However, the phenotypic expression in females is highly variable, which may be explained by an allelic skew in X-Chromosome inactivation. Accurate measurement of X inactivation skew is crucial to understand and predict disease phenotype in carrier females, with prediction especially relevant for degenerative conditions.

Long-read DNA and cDNA sequencing identify cancer-predisposing deep intronic variation in tumor-suppressor genes.

The vast majority of deeply intronic genomic variants are benign, but some extremely rare or private deep intronic variants lead to exonification of intronic sequence with abnormal transcriptional consequences. Damaging variants of this class are likely underreported as causes of disease for several reasons: Most clinical DNA and RNA testing does not include full intronic sequences; many of these variants lie in complex repetitive regions that cannot be aligned from short-read whole-genome sequence; and, until recently, consequences of deep intronic variants were not accurately predicted by in silico tools. We evaluated the frequency and consequences of rare deep intronic variants for families severely affected with breast, ovarian, pancreatic, and/or metastatic prostate cancer, but with no causal variant identified by any previous genomic or cDNA-based approach.

A novel approach for in vivo DNA footprinting using short double-stranded cell-free DNA from plasma.

Here, we present a method for enrichment of double-stranded cfDNA with an average length of ∼40 bp from cfDNA for high-throughput DNA sequencing. This class of cfDNA is enriched at gene promoters and binding sites of transcription factors or structural DNA-binding proteins, so that a genome-wide DNA footprint is directly captured from liquid biopsies. In short double-stranded cfDNA from healthy individuals, we find significant enrichment of 203 transcription factor motifs.

Comprehensive identification of genomic and environmental determinants of phenotypic plasticity in maize.

Maize phenotypes are plastic, determined by the complex interplay of genetics and environmental variables. Uncovering the genes responsible and understanding how their effects change across a large geographic region are challenging. In this study, we conducted systematic analysis to identify environmental indices that strongly influence 19 traits (including flowering time, plant architecture, and yield component traits) measured in the maize nested association mapping (NAM) population grown in 11 environments.

Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of mutations.

Mutations in splicing factor 3B subunit 1 () frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDSs). These mutations have different effects on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on mutation effects on RNA splicing and its contribution to patient survival and treatment options.

Evidence for compensatory evolution within pleiotropic regulatory elements.

Pleiotropy, measured as expression breadth across tissues, is one of the best predictors for protein sequence and expression conservation. In this study, we investigated its effect on the evolution of regulatory elements (CREs). To this end, we carefully reanalyzed the Epigenomics Roadmap data for nine fetal tissues, assigning a measure of pleiotropic degree to nearly half a million CREs.