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Small RNA-seq data of peripheral blood obtained from treatment-näive IBD patients and control individuals (small RNA-Seq I)

ABSTRACT: Comperhensive analysis of blood transcriptomes obtained from treatment-näive IBD patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE169569 | GEO | 2022/08/16

REPOSITORIES: GEO

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Project description:BackgroundThe molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples.ResultsWe compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding.ConclusionsGlobin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic range of detection and provides an accurate estimation of RNA concentration in peripheral whole blood. High-throughput sequencing is thus a promising technology for whole blood transcriptomics and biomarker discovery.

Project description:BackgroundSingle-cell transcriptomics provides means to study cell populations at the level of individual cells. In leukocyte biology this approach could potentially aid the identification of subpopulations and functions without the need to develop species-specific reagents. The present study aimed to evaluate single-cell RNA-seq as a tool for identification of chicken peripheral blood leukocytes. For this purpose, purified and thrombocyte depleted leukocytes from 4 clinically healthy hens were subjected to single-cell 3' RNA-seq. Bioinformatic analysis of data comprised unsupervised clustering of the cells, and annotation of clusters based on expression profiles. Immunofluorescence phenotyping of the cell preparations used was also performed.ResultsComputational analysis identified 31 initial cell clusters and based on expression of defined marker genes 28 cluster were identified as comprising mainly B-cells, T-cells, monocytes, thrombocytes and red blood cells. Of the remaining clusters, two were putatively identified as basophils and eosinophils, and one as proliferating cells of mixed origin. In depth analysis on gene expression profiles within and between the initial cell clusters allowed further identification of cell identity and possible functions for some of them. For example, analysis of the group of monocyte clusters revealed subclusters comprising heterophils, as well as putative monocyte subtypes. Also, novel aspects of TCRγ/δ + T-cell subpopulations could be inferred such as evidence of at least two subtypes based on e.g., different expression of transcription factors MAF, SOX13 and GATA3. Moreover, a novel subpopulation of chicken peripheral B-cells with high SOX5 expression was identified. An overall good correlation between mRNA and cell surface phenotypic cell identification was shown.ConclusionsTaken together, we were able to identify and infer functional aspects of both previously well known as well as novel chicken leukocyte populations although some cell types. e.g., T-cell subtypes, proved more challenging to decipher. Although this methodology to some extent is limited by incomplete annotation of the chicken genome, it definitively has benefits in chicken immunology by expanding the options to distinguish identity and functions of immune cells also without access to species specific reagents.

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Small RNA-seq data of peripheral blood obtained from treatment-näive IBD patients and control individuals (small RNA-Seq I)

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