Biology subjects

John D Blischak

Publications and source records attributed to John D Blischak.

3 recordsLinked to original sources

bioRxiv2016

Batch effects and the effective design of single-cell gene expression studies

Single cell RNA sequencing (scRNA-seq) can be used to characterize variation in gene expression levels at high resolution. However, the sources of experimental noise in scRNA-seq are not yet well understood. We investigated the technical variation associated with sample processing using the single cell Fluidigm C1 platform. To do so, we processed three C1 replicates from three human induced pluripotent stem cell (iPSC) lines. We added unique molecular identifiers (UMIs) to all samples, to account for amplification bias. We found that the major source of variation in the gene expression data was driven by genotype, but we also observed substantial variation between the technical replicates. We observed that the conversion of reads to molecules using the UMIs was impacted by both biological and technical variation, indicating that UMI counts are not an unbiased estimator of gene expression levels. Based on our results, we suggest a framework for effective scRNA-seq studies.

Genomics↗

bioRxiv2015

Patterns of transcriptional response to 1,25-dihydroxyvitamin D3 and bacterial lipopolysaccharide in primary human monocytes

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), plays an important immunomodulatory role, regulating transcription of genes in the innate and adaptive immune system. The present study examines patterns of transcriptome-wide response to 1,25D and the bacterial lipopolysaccharide (LPS) in primary human monocytes, to elucidate pathways underlying the effects of 1,25D on the immune system. Monocytes obtained from healthy individuals of African-African and European-American ancestry were treated with 1,25D alone or in the presence of LPS, which induced significant up-regulation of genes in the antimicrobial and autophagy pathways, while pro-inflammatory response genes were significantly down-regulated. A joint Bayesian analysis enabled clustering of genes into patterns of shared transcriptional response across treatments. The biological pathways enriched within these expression patterns highlighted several mechanisms through which 1,25D could exert its immunomodulatory role. Pathways such as mTOR signaling, EIF2 signaling, IL-8 signaling and Tec Kinase signaling were enriched among genes with opposite transcriptional responses to 1,25D and LPS, respectively, highlighting the important roles of these pathways in mediating the immunomodulatory activity of 1,25D. Furthermore, a subset of genes with evidence of inter-ethnic differences in transcriptional response was also identified, suggesting that in addition to the well-established inter-ethnic variation in circulating levels of vitamin D, the intensity of transcriptional response to 1,25D and LPS also varies between ethnic groups. We propose that dysregulation of the pathways identified in this study could contribute to immune-mediated disease risk.

Genetics↗

bioRxiv2015

Mycobacterial infection induces a specific human innate immune response

The innate immune system provides the first response to pathogen infection and orchestrates the activation of the adaptive immune system. Though a large component of the innate immune response is common to all infections, pathogen-specific responses have been documented as well. The innate immune response is thought to be especially critical for fighting infection with Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB). While TB can be deadly, only 5-10% of individuals infected with MTB develop active disease. The risk for disease susceptibility is, at least partly, heritable. Studies of inter-individual variation in the innate immune response to MTB infection may therefore shed light on the genetic basis for variation in susceptibility to TB. Yet, to date, we still do not know which properties of the innate immune response are specific to MTB infection and which represent a general response to pathogen infection. To begin addressing this gap, we infected macrophages with eight different bacteria, including different MTB strains and related mycobacteria, and studied the transcriptional response to infection. Although the ensued gene regulatory responses were largely consistent across the bacterial infection treatments, we were able to identify a novel subset of genes whose regulation was affected specifically by infection with mycobacteria. Genetic variants that are associated with regulatory differences in these genes should be considered candidate loci for explaining inter-individual susceptibility TB.\n\nAuthor SummaryTuberculosis (TB) is a deadly disease responsible for millions of deaths annually. It is caused by infection with Mycobacterium tuberculosis (MTB), an ancient human pathogen. Approximately a third of the worlds population is infected with MTB, yet only an estimated 5-10% of individuals will develop an active form of the disease. While this variation in TB susceptibility has been demonstrated to be heritable, we still know little about its underlying genetic basis. The genetic variation that affects TB susceptibility likely involves the innate immune system, which is our first line of defense against invading pathogens, because infection with MTB does not prevent future infections. However, we do not fully understand how the innate immune system differs in its response to MTB versus other bacteria. To investigate this further, we infected macrophages with MTB, related mycobacteria, and other bacteria, and measured how their gene expression levels changed in response. We identified a subset of genes that respond preferentially to infection with mycobacterial species. These genes provide insight into the interactions between MTB and the innate immune system and are candidate loci for explaining inter-individual susceptibility to TB.

Genomics↗