Whole Genome Shotgun Sequencing Detects Greater Lichen Fungal Diversity Than Amplicon-Based Methods in Environmental Samples

In this study we demonstrate the utility of whole genome shotgun (WGS) metagenomics in study organisms with small genomes to improve upon amplicon-based estimates of biodiversity and microbial diversity in environmental samples for the purpose of understanding ecological and evolutionary processes. We generated a database of full-length and near-full-length ribosomal DNA sequence complexes from 273 lichenized fungal species and used this database to facilitate fungal species identification in the southern Appalachian Mountains using low coverage WGS at higher resolution and without the biases of amplicon-based approaches. Using this new database and methods herein developed, we detected between 2.8 and 11 times as many species from lichen fungal propagules by aligning reads from WGS-sequenced environmental samples compared to a traditional amplicon-based approach. We then conducted complete taxonomic diversity inventories of the lichens in each one-hectare plot to assess overlap between standing taxonomic diversity and diversity detected based on propagules present in environmental samples (i.e., the &ldquo;potential&rdquo; of diversity). From the environmental samples, we detected 94 species not observed in organism-level sampling in these ecosystems with high confidence using both WGS and amplicon-based methods. This study highlights the utility of WGS sequence-based approaches in detecting hidden species diversity and demonstrates that amplicon-based methods likely miss important components of fungal diversity. We suggest that the adoption of this method will not only improve understanding of biotic constraints on the distributions of biodiversity but will also help to inform important environmental policy.</p

Symptom-based stratification of patients with primary Sjögren's syndrome: multi-dimensional characterisation of international observational cohorts and reanalyses of randomised clinical trials

Background Heterogeneity is a major obstacle to developing effective treatments for patients with primary Sjögren's syndrome. We aimed to develop a robust method for stratification, exploiting heterogeneity in patient-reported symptoms, and to relate these differences to pathobiology and therapeutic response. Methods We did hierarchical cluster analysis using five common symptoms associated with primary Sjögren's syndrome (pain, fatigue, dryness, anxiety, and depression), followed by multinomial logistic regression to identify subgroups in the UK Primary Sjögren's Syndrome Registry (UKPSSR). We assessed clinical and biological differences between these subgroups, including transcriptional differences in peripheral blood. Patients from two independent validation cohorts in Norway and France were used to confirm patient stratification. Data from two phase 3 clinical trials were similarly stratified to assess the differences between subgroups in treatment response to hydroxychloroquine and rituximab. Findings In the UKPSSR cohort (n=608), we identified four subgroups: Low symptom burden (LSB), high symptom burden (HSB), dryness dominant with fatigue (DDF), and pain dominant with fatigue (PDF). Significant differences in peripheral blood lymphocyte counts, anti-SSA and anti-SSB antibody positivity, as well as serum IgG, κ-free light chain, β2-microglobulin, and CXCL13 concentrations were observed between these subgroups, along with differentially expressed transcriptomic modules in peripheral blood. Similar findings were observed in the independent validation cohorts (n=396). Reanalysis of trial data stratifying patients into these subgroups suggested a treatment effect with hydroxychloroquine in the HSB subgroup and with rituximab in the DDF subgroup compared with placebo. Interpretation Stratification on the basis of patient-reported symptoms of patients with primary Sjögren's syndrome revealed distinct pathobiological endotypes with distinct responses to immunomodulatory treatments. Our data have important implications for clinical management, trial design, and therapeutic development. Similar stratification approaches might be useful for patients with other chronic immune-mediated diseases. Funding UK Medical Research Council, British Sjogren's Syndrome Association, French Ministry of Health, Arthritis Research UK, Foundation for Research in Rheumatology