Strain-resolved microbiome sequencing reveals mobile elements that drive bacterial competition on a clinical timescale.

BACKGROUND: Populations of closely related microbial strains can be simultaneously present in bacterial communities such as the human gut microbiome. We recently developed a de novo genome assembly approach that uses read cloud sequencing to provide more complete microbial genome drafts, enabling precise differentiation and tracking of strain-level dynamics across metagenomic samples. In this case study, we present a proof-of-concept using read cloud sequencing to describe bacterial strain diversity in the gut microbiome of one hematopoietic cell transplantation patient over a 2-month time course and highlight temporal strain variation of gut microbes during therapy. The treatment was accompanied by diet changes and administration of multiple immunosuppressants and antimicrobials. METHODS: We conducted short-read and read cloud metagenomic sequencing of DNA extracted from four longitudinal stool samples collected during the course of treatment of one hematopoietic cell transplantation (HCT) patient. After applying read cloud metagenomic assembly to discover strain-level sequence variants in these complex microbiome samples, we performed metatranscriptomic analysis to investigate differential expression of antibiotic resistance genes. Finally, we validated predictions from the genomic and metatranscriptomic findings through in vitro antibiotic susceptibility testing and whole genome sequencing of isolates derived from the patient stool samples. RESULTS: During the 56-day longitudinal time course that was studied, the patient's microbiome was profoundly disrupted and eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allowed us to identify differences in substrain populations over time. Based on this, we predicted that particular mobile element integrations likely resulted in increased antibiotic resistance, which we further supported using in vitro antibiotic susceptibility testing. CONCLUSIONS: We find read cloud assembly to be useful in identifying key structural genomic strain variants within a metagenomic sample. These strains have fluctuating relative abundance over relatively short time periods in human microbiomes. We also find specific structural genomic variations that are associated with increased antibiotic resistance over the course of clinical treatment.

Single-cell transcriptome analysis identifies distinct cell types and niche signaling in a primary gastric organoid model.

The diverse cellular milieu of the gastric tissue microenvironment plays a critical role in normal tissue homeostasis and tumor development. However, few cell culture model can recapitulate the tissue microenvironment and intercellular signaling in vitro. We used a primary tissue culture system to generate a murine p53 null gastric tissue model containing both epithelium and mesenchymal stroma. To characterize the microenvironment and niche signaling, we used single cell RNA sequencing (scRNA-Seq) to determine the transcriptomes of 4,391 individual cells. Based on specific markers, we identified epithelial cells, fibroblasts and macrophages in initial tissue explants during organoid formation. The majority of macrophages were polarized towards wound healing and tumor promotion M2-type. During the course of time, the organoids maintained both epithelial and fibroblast lineages with the features of immature mouse gastric stomach. We detected a subset of cells in both lineages expressing Lgr5, one of the stem cell markers. We examined the lineage-specific Wnt signaling activation, and identified that Rspo3 was specifically expressed in the fibroblast lineage, providing an endogenous source of the R-spondin to activate Wnt signaling. Our studies demonstrate that this primary tissue culture system enables one to study gastric tissue niche signaling and immune response in vitro.

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA.

We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring.

Stress during Adolescence Alters Palatable Food Consumption in a Context-Dependent Manner.

Food consumption and preferences may be shaped by exposure to stressful environments during sensitive periods in development, and even small changes in consumption can have important effects on long term health. Adolescence is increasingly recognized as a sensitive period, in which adverse experiences can alter development, but the specific programming effects that may occur during adolescence remain incompletely understood. The current study seeks to explore the effects of stress during late adolescence on consumption of a palatable, high-fat, high-sugar food in adulthood-under basal conditions, as well following acute stress. Male Long-Evans rats were exposed to a regimen of variable stress for seven days in late adolescence (PND 45-51). During the stress regimen, stressed animals gained significantly less weight than control animals, but weight in adulthood was unaffected by adolescent stress. Palatable food consumption differed between experimental groups, and the direction of effect depended on context; stressed rats ate significantly more palatable food than controls upon first exposure, but ate less following an acute stressor. Leptin levels and exploratory behaviors did not differ between stressed and non-stressed groups, suggesting that other factors regulate preference for a palatable food. Altered food consumption following adolescent stress suggests that rats remain sensitive to stress during late adolescence, and that adult feeding behavior may be affected by previous adverse experiences. Such programming effects highlight adolescence as a period of plasticity, with the potential to shape long term food consumption patterns and preferences.