Blood RNA alternative splicing events as diagnostic biomarkers for infectious disease.

Assays detecting blood transcriptome changes are studied for infectious disease diagnosis. Blood-based RNA alternative splicing (AS) events, which have not been well characterized in pathogen infection, have potential normalization and assay platform stability advantages over gene expression for diagnosis. Here, we present a computational framework for developing AS diagnostic biomarkers. Leveraging a large prospective cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and whole-blood RNA sequencing (RNA-seq) data, we identify a major functional AS program switch upon viral infection. Using an independent cohort, we demonstrate the improved accuracy of AS biomarkers for SARS-CoV-2 diagnosis compared with six reported transcriptome signatures. We then optimize a subset of AS-based biomarkers to develop microfluidic PCR diagnostic assays. This assay achieves nearly perfect test accuracy (61/62 = 98.4%) using a naive principal component classifier, significantly more accurate than a gene expression PCR assay in the same cohort. Therefore, our RNA splicing computational framework enables a promising avenue for host-response diagnosis of infection.

Pre-infection antiviral innate immunity contributes to sex differences in SARS-CoV-2 infection.

Male sex is a major risk factor for SARS-CoV-2 infection severity. To understand the basis for this sex difference, we studied SARS-CoV-2 infection in a young adult cohort of United States Marine recruits. Among 2,641 male and 244 female unvaccinated and seronegative recruits studied longitudinally, SARS-CoV-2 infections occurred in 1,033 males and 137 females. We identified sex differences in symptoms, viral load, blood transcriptome, RNA splicing, and proteomic signatures. Females had higher pre-infection expression of antiviral interferon-stimulated gene (ISG) programs. Causal mediation analysis implicated ISG differences in number of symptoms, levels of ISGs, and differential splicing of CD45 lymphocyte phosphatase during infection. Our results indicate that the antiviral innate immunity set point causally contributes to sex differences in response to SARS-CoV-2 infection. A record of this paper's transparent peer review process is included in the supplemental information.

Asymptomatic SARS-CoV-2 Infection Is Associated With Higher Levels of Serum IL-17C, Matrix Metalloproteinase 10 and Fibroblast Growth Factors Than Mild Symptomatic COVID-19.

Young adults infected with SARS-CoV-2 are frequently asymptomatic or develop only mild disease. Because capturing representative mild and asymptomatic cases require active surveillance, they are less characterized than moderate or severe cases of COVID-19. However, a better understanding of SARS-CoV-2 asymptomatic infections might shed light into the immune mechanisms associated with the control of symptoms and protection. To this aim, we have determined the temporal dynamics of the humoral immune response, as well as the serum inflammatory profile, of mild and asymptomatic SARS-CoV-2 infections in a cohort of 172 initially seronegative prospectively studied United States Marine recruits, 149 of whom were subsequently found to be SARS-CoV-2 infected. The participants had blood samples taken, symptoms surveyed and PCR tests for SARS-CoV-2 performed periodically for up to 105 days. We found similar dynamics in the profiles of viral load and in the generation of specific antibody responses in asymptomatic and mild symptomatic participants. A proteomic analysis using an inflammatory panel including 92 analytes revealed a pattern of three temporal waves of inflammatory and immunoregulatory mediators, and a return to baseline for most of the inflammatory markers by 35 days post-infection. We found that 23 analytes were significantly higher in those participants that reported symptoms at the time of the first positive SARS-CoV-2 PCR compared with asymptomatic participants, including mostly chemokines and cytokines associated with inflammatory response or immune activation (i.e., TNF-α, TNF-ß, CXCL10, IL-8). Notably, we detected 7 analytes (IL-17C, MMP-10, FGF-19, FGF-21, FGF-23, CXCL5 and CCL23) that were higher in asymptomatic participants than in participants with symptoms; these are known to be involved in tissue repair and may be related to the control of symptoms. Overall, we found a serum proteomic signature that differentiates asymptomatic and mild symptomatic infections in young adults, including potential targets for developing new therapies and prognostic tests.

dNEMO: a tool for quantification of mRNA and punctate structures in time-lapse images of single cells.

MOTIVATION: Many biological processes are regulated by single molecules and molecular assemblies within cells that are visible by microscopy as punctate features, often diffraction limited. Here, we present detecting-NEMO (dNEMO), a computational tool optimized for accurate and rapid measurement of fluorescent puncta in fixed-cell and time-lapse images. RESULTS: The spot detection algorithm uses the à trous wavelet transform, a computationally inexpensive method that is robust to imaging noise. By combining automated with manual spot curation in the user interface, fluorescent puncta can be carefully selected and measured against their local background to extract high-quality single-cell data. Integrated into the workflow are segmentation and spot-inspection tools that enable almost real-time interaction with images without time consuming pre-processing steps. Although the software is agnostic to the type of puncta imaged, we demonstrate dNEMO using smFISH to measure transcript numbers in single cells in addition to the transient formation of IKK/NEMO puncta from time-lapse images of cells exposed to inflammatory stimuli. We conclude that dNEMO is an ideal user interface for rapid and accurate measurement of fluorescent molecular assemblies in biological imaging data. AVAILABILITY AND IMPLEMENTATION: The data and software are freely available online at https://github.com/recleelab. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Analysis of the structural variability of topologically associated domains as revealed by Hi-C.

Three-dimensional chromosome structure plays an integral role in gene expression and regulation, replication timing, and other cellular processes. Topologically associated domains (TADs), building blocks of chromosome structure, are genomic regions with higher contact frequencies within the region than outside the region. A central question is the degree to which TADs are conserved or vary between conditions. We analyze 137 Hi-C samples from 9 studies under 3 measures to quantify the effects of various sources of biological and experimental variation. We observe significant variation in TAD sets between both non-replicate and replicate samples, and provide initial evidence that this variability does not come from genetic sequence differences. The effects of experimental protocol differences are also measured, demonstrating that samples can have protocol-specific structural changes, but that TADs are generally robust to lab-specific differences. This study represents a systematic quantification of key factors influencing comparisons of chromosome structure, suggesting significant variability and the potential for cell-type-specific structural features, which has previously not been systematically explored. The lack of observed influence of heredity and genetic differences on chromosome structure suggests that factors other than the genetic sequence are driving this structure, which plays an important role in human disease and cellular functioning.

Quantifying the similarity of topological domains across normal and cancer human cell types.

Motivation: Three-dimensional chromosome structure has been increasingly shown to influence various levels of cellular and genomic functions. Through Hi-C data, which maps contact frequency on chromosomes, it has been found that structural elements termed topologically associating domains (TADs) are involved in many regulatory mechanisms. However, we have little understanding of the level of similarity or variability of chromosome structure across cell types and disease states. In this study, we present a method to quantify resemblance and identify structurally similar regions between any two sets of TADs. Results: We present an analysis of 23 human Hi-C samples representing various tissue types in normal and cancer cell lines. We quantify global and chromosome-level structural similarity, and compare the relative similarity between cancer and non-cancer cells. We find that cancer cells show higher structural variability around commonly mutated pan-cancer genes than normal cells at these same locations. Availability and implementation: Software for the methods and analysis can be found at https://github.com/Kingsford-Group/localtadsim.

Chromosomal dynamics predicted by an elastic network model explains genome-wide accessibility and long-range couplings.

Understanding the three-dimensional (3D) architecture of chromatin and its relation to gene expression and regulation is fundamental to understanding how the genome functions. Advances in Hi-C technology now permit us to study 3D genome organization, but we still lack an understanding of the structural dynamics of chromosomes. The dynamic couplings between regions separated by large genomic distances (>50 Mb) have yet to be characterized. We adapted a well-established protein-modeling framework, the Gaussian Network Model (GNM), to model chromatin dynamics using Hi-C data. We show that the GNM can identify spatial couplings at multiple scales: it can quantify the correlated fluctuations in the positions of gene loci, find large genomic compartments and smaller topologically-associating domains (TADs) that undergo en bloc movements, and identify dynamically coupled distal regions along the chromosomes. We show that the predictions of the GNM correlate well with genome-wide experimental measurements. We use the GNM to identify novel cross-correlated distal domains (CCDDs) representing pairs of regions distinguished by their long-range dynamic coupling and show that CCDDs are associated with increased gene co-expression. Together, these results show that GNM provides a mathematically well-founded unified framework for modeling chromatin dynamics and assessing the structural basis of genome-wide observations.