Nanopore native RNA sequencing of a human poly(A) transcriptome

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes

Effects of partial sleep restriction on biological markers of cardiovascular risk: Evidence of differential vulnerability within a healthy population

Accumulating epidemiological evidence suggests a relationship between short sleep duration and cardiovascular disease. The biological mechanisms underlying this relationship, however, are not yet fully understood. The three studies contained in this dissertation will contribute to our overall understanding of this relationship. In the first study, the effects of partial restriction on plasma leptin were examined. Contrary to previous research, it was observed that five nights of partial sleep restriction significantly increased plasma leptin levels. Increases were significantly greater among women, a population that has been inadequately represented in prior studies. Additionally, it was observed that recovery sleep, compared to further sleep restriction, decreased plasma leptin levels. The second study focused on the effects of sleep restriction on adiponectin. This study is the first to document changes in adiponectin levels in response to sleep restriction. Notably, a strong sex by ethnicity interaction was observed, in which female participants were observed to have significant changes in adiponectin: levels in Caucasian women decreased significantly while levels in African American women increased significantly. These changes could not be explained through differences in body mass index. The third study examined the effects of sleep restriction on levels of C-reactive protein (CRP), IL-6, and cortisol. Sleep restriction did not significantly change CRP levels in the total sample; however, levels of IL-6 and cortisol increased and differential vulnerability were observed for both IL-6 and cortisol. Further, a subset of the healthy participants was found to be in the \u27high risk for future cardiovascular events\u27 category, based on baseline CRP levels; this population responded differently to the effects of sleep restriction compared to those participants with \u27normal\u27 baseline CRP levels. Results from these studies provide preliminary evidence for a differential vulnerability to the effects of sleep restriction on biological markers of cardiovascular risk. In particular, women appear to have a more reactive adipocyte-derived response to sleep restriction, although the directionality of effects with respect to subsequent effects on health may be related to race/ethnicity in some domains. These findings suggest a more complex relationship between sleep duration and cardiovascular risk than has been previously proposed

Sleep Deprivation and Stressors: Evidence for Elevated Negative Affect in Response to Mild Stressors When Sleep Deprived

Stress often co-occurs with inadequate sleep duration, and both are believed to impact mood and emotion. It is not yet known whether inadequate sleep simply increases the intensity of subsequent stress responses or interacts with stressors in more complicated ways. To address this issue, we investigated the effects of one night of total sleep deprivation on subjective stress and mood in response to low-stress and high-stress cognitive testing conditions in healthy adult volunteers in two separate experiments (total N ϭ 53). Sleep was manipulated in a controlled, laboratory setting and stressor intensity was manipulated by changing difficulty of cognitive tasks, time pressure, and feedback about performance. Sleep-deprived participants reported greater subjective stress, anxiety, and anger than rested controls following exposure to the low-stressor condition, but not in response to the high-stressor condition, which elevated negative mood and stress about equally for both sleep conditions. These results suggest that sleep deprivation lowers the psychological threshold for the perception of stress from cognitive demands but does not selectively increase the magnitude of negative affect in response to high-stress performance demands

Nanopore native RNA sequencing of a human poly(A) transcriptome.

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes