Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding the various biologic compartments contributing to the cfDNA pool. We sought to define the technical feasibility of a high-intensity sequencing assay of cfDNA and matched white-blood cell (WBC) DNA covering a large genomic region (508 genes, 2Mb, >60,000X raw-depth) in a prospective study of 124 metastatic cancer patients, with contemporaneous matched tumor tissue biopsies, and 47 non-cancer controls. The assay displayed a high sensitivity and specificity, allowing for de novo detection of tumor-derived mutations and inference of tumor mutational burden, microsatellite instability, mutational signatures and sources of somatic mutations identified in cfDNA. The vast majority of cfDNA mutations (81.6% in controls and 53.2% in cancer patients) had features consistent with clonal hematopoiesis (CH). This cfDNA sequencing approach revealed that CH constitutes a pervasive biological phenomenon emphasizing the importance of matched cfDNA-WBC sequencing for accurate variant interpretation.

IntroductionSleep deprivation is common in critically ill patients in the intensive care unit (ICU). Noise and light in the ICU and the reduction in plasma melatonin play the essential roles. The aim of this study was to determine the effect of simulated ICU noise and light on nocturnal sleep quality, and compare the effectiveness of melatonin and earplugs and eye masks on sleep quality in these conditions in healthy subjects.MethodsThis study was conducted in two parts. In part one, 40 healthy subjects slept under baseline night and simulated ICU noise and light (NL) by a cross-over design. In part two, 40 subjects were randomly assigned to four groups: NL, NL plus placebo (NLP), NL plus use of earplugs and eye masks (NLEE) and NL plus melatonin (NLM). 1 mg of oral melatonin or placebo was administered at 21:00 on four consecutive days in NLM and NLP. Earplugs and eye masks were made available in NLEE. The objective sleep quality was measured by polysomnography. Serum was analyzed for melatonin levels. Subjects rated their perceived sleep quality and anxiety levels.ResultsSubjects had shorter total sleep time (TST) and rapid eye movement (REM) sleep, longer sleep onset latency, more light sleep and awakening, poorer subjective sleep quality, higher anxiety level and lower serum melatonin level in NL night (P <0.05). NLEE had less awakenings and shorter sleep onset latency (P <0.05). NLM had longer TST and REM and shorter sleep onset latency (P <0.05). Compared with NLEE, NLM had fewer awakenings (P = 0.004). Both NLM and NLEE improved perceived sleep quality and anxiety level (P = 0.000), and NLM showed better than NLEE in perceived sleep quality (P = 0.01). Compared to baseline night, the serum melatonin levels were lower in NL night at every time point, and the average maximal serum melatonin concentration in NLM group was significantly greater than other groups (P <0.001).ConclusionsCompared with earplugs and eye masks, melatonin improves sleep quality and serum melatonin levels better in healthy subjects exposed to simulated ICU noise and light.Trial registrationChinese Clinical Trial Registry ChiCTR-IPR-14005458. Registered 10 November 2014.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (Oncorhynchus mykiss). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity withinF. psychrophilum. At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North AmericanF. psychrophilumisolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largestF. psychrophilumCC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came fromO. mykissexperiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification ofF. psychrophilumin North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species.IMPORTANCEFlavobacterium psychrophilumis the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundredF. psychrophilumisolates that were recovered from diseased fish across North America. Results highlighted multipleF. psychrophilumgenetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.

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