Dynamic changes in the human methylome during differentiation.

DNA methylation is a critical epigenetic regulator in mammalian development. Here, we present a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic differentiated derivative of the hESCs, and neonatal fibroblasts. As a reference, we compared our maps with a methylome map of a fully differentiated adult cell type, mature peripheral blood mononuclear cells (monocytes). We observed many notable common and cell-type-specific features among all cell types. Promoter hypomethylation (both CG and CA) and higher levels of gene body methylation were positively correlated with transcription in all cell types. Exons were more highly methylated than introns, and sharp transitions of methylation occurred at exon-intron boundaries, suggesting a role for differential methylation in transcript splicing. Developmental stage was reflected in both the level of global methylation and extent of non-CpG methylation, with hESC highest, fibroblasts intermediate, and monocytes lowest. Differentiation-associated differential methylation profiles were observed for developmentally regulated genes, including the HOX clusters, other homeobox transcription factors, and pluripotence-associated genes such as POU5F1, TCF3, and KLF4. Our results highlight the value of high-resolution methylation maps, in conjunction with other systems-level analyses, for investigation of previously undetectable developmental regulatory mechanisms.

Cartography of opportunistic pathogens and antibiotic resistance genes in a tertiary hospital environment.

Although disinfection is key to infection control, the colonization patterns and resistomes of hospital-environment microbes remain underexplored. We report the first extensive genomic characterization of microbiomes, pathogens and antibiotic resistance cassettes in a tertiary-care hospital, from repeated sampling (up to 1.5 years apart) of 179 sites associated with 45 beds. Deep shotgun metagenomics unveiled distinct ecological niches of microbes and antibiotic resistance genes characterized by biofilm-forming and human-microbiome-influenced environments with corresponding patterns of spatiotemporal divergence. Quasi-metagenomics with nanopore sequencing provided thousands of high-contiguity genomes, phage and plasmid sequences (>60% novel), enabling characterization of resistome and mobilome diversity and dynamic architectures in hospital environments. Phylogenetics identified multidrug-resistant strains as being widely distributed and stably colonizing across sites. Comparisons with clinical isolates indicated that such microbes can persist in hospitals for extended periods (>8 years), to opportunistically infect patients. These findings highlight the importance of characterizing antibiotic resistance reservoirs in hospitals and establish the feasibility of systematic surveys to target resources for preventing infections.

A Novel System Control for Quality Control of Diagnostic Tests Based on Next-Generation Sequencing.

BACKGROUND: We describe a novel system control (SC) implemented in an automated AmpliSeq™-based next-generation sequencing (NGS)2 run that simultaneously acts as (a) an external positive/sensitivity control, (b) a spike-in QC for DNA extraction, and (c) a nontemplate control to detect exogenous DNA contamination. METHODS: Plasmids carrying wild-type tobacco mosaic virus sequence and a sequence with three designed mutations were synthesized and mixed, such that the mutations are present at 5% variant frequency in the mixture designated as SC. SC was used as a stand-alone sample and spiked into each sample in each run. A cell line-derived reference material, in both a formalin-fixed paraffin-embedded (FFPE) sample and genomic DNA (gDNA), was sequenced in the same runs. RESULTS: By interpolation, 100 fg SC spiked in FFPE sample produced sequencing coverage equivalent to approximately 3 fg in the gDNA. In the SC-only sample, all three designed mutations were recovered around 5% as expected, while no significant reads of human genome were present. In samples with a common PCR inhibitor, coverage for both SC and target amplicons were eliminated. An inverse relationship between the coverage of SC and DNA input was observed. In clinical samples, the ratio of SC to the median coverage of sample can be used to indicate insufficient DNA input. CONCLUSIONS: The SC is an elegant and comprehensive QC concept for NGS-based diagnostic tests.