Search bioRxiv⌕ Search

Biology subjects

Deslattes Mays, A.

Publications and source records attributed to Deslattes Mays, A..

5 recordsLinked to original sources

A trustworthy semantic knowledge network linking data-driven cell phenotypes to diseases and drugs

Genome-wide association and other genetic studies have identified genes involved in disease pathogenesis, and their encoded proteins have provided therapeutic targets. However, little is known about which cells are the functional home of these disease-associated genes and proteins. Single-cell technologies are enabling high-resolution profiling of cell phenotypes and revealing cellular contexts of disease, yet much of the resulting knowledge remains embedded in individual datasets and scientific literature. To address these gaps, the National Library of Medicine has developed Cell Knowledge Network (NLM-CKN, https://nlm-ckn.org). Reproducible computational workflows have generated cell phenotype knowledge for 2,585 cell sets and 5,139 selectively expressed genes from 88 datasets covering 10 human organs. Mapping of these cell sets to cell types grounds Cell Ontology terms in experimental evidence. This experimental knowledge is integrated with external knowledge of anatomical and cell-type hierarchies, gene-disease associations, and therapeutic interventions. A semantic schema defines node types and relations, enabling knowledge to be transformed into triple assertions and loaded into an ArangoDB database with provenance and quantitative evidence. The NLM-CKN interface supports graphical exploration of the cellular constituents of healthy human tissues. Finally, topological analyses generate insights into cellular contributions to disease pathogenesis, cellular contexts for therapeutic intervention, and drug-repurposing hypotheses.

bioinformatics↗

Full-length, single-cell RNA-sequencing of human bone marrow subpopulations reveals hidden complexity

Bone marrow progenitor cell differentiation has frequently been used as a model for studying cellular plasticity and cell-fate decisions. Recent analysis at the level of single-cells has expanded knowledge of the transcriptional landscape of human hematopoietic cell lineages. Using single-molecule real-time (SMRT) full-length RNA sequencing, we have previously shown that human bone marrow lineage-negative (Lin-neg) cell populations contain a surprisingly diverse set of mRNA isoforms. Here, we report from single cell, full-length RNA sequencing that this diversity is also reflected at the single-cell level. From fresh human bone marrow unselected and lineage-negative progenitor cells were isolated by droplet-based single-cell selection (10xGenomics). The single cell-derived mRNAs were analyzed by full-length SMRT and short-read sequencing. In both samples we detected an average of 8000 different genes using short-read sequencing. Differential expression analysis arranged the single-cells of the total bone marrow into only four clusters whereas the Lin-neg population was much more diverse with nine clusters. mRNA isoform analysis of the single-cell populations using full-length sequencing revealed that Lin-neg cells contain on average 24% more novel splice variants than the total bone marrow cells. Interestingly, among the most frequent genes expressing novel isoforms were members of the spliceosome, e.g. HNRNPs, DEAD box helicases and SRSFs. Mapping the isoforms from all genes to the cell type clusters revealed that total bone marrow cells express novel isoforms only in a small subset of clusters. On the other hand, lineage-negative progenitor cells expressing novel isoforms were present in nearly all subpopulations. In conclusion, on a single-cell level lineage-negative cells express a higher diversity of genes and more alternatively spliced novel isoforms suggesting that cells in this subpopulation are poised for different fates. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/454226v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1fa661aorg.highwire.dtl.DTLVardef@454456org.highwire.dtl.DTLVardef@1897badorg.highwire.dtl.DTLVardef@116d516_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Long-read isoform sequencing reveals survival-associated splicing in breast cancer

Tumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is not known. We developed a long-read RNA sequencing and analytical platform that identifies and annotates full-length isoforms, and infers tumor-specific splicing events. Application of this platform to breast cancer samples vastly expands the known isoform landscape of breast cancer, identifying thousands of previously unannotated isoforms of which ~30% impact protein coding exons and are predicted to alter protein localization and function, including of the breast cancer-associated genes ESR1 and ERBB2. We performed extensive cross-validation with -omics data sets to support transcription and translation of novel isoforms. We identified 3,059 breast tumor-specific splicing events, including 35 that are significantly associated with patient survival. Together, our results demonstrate the complexity, cancer subtype-specificity, and clinical relevance of novel isoforms in breast cancer that are only annotatable by LR-seq, and provide a rich resource of immuno-oncology therapeutic targets.

genomics↗

Single molecule real time (SMRT) full length RNA-sequencing reveals novel and distinct mRNA isoforms in human bone marrow cell subpopulations

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single molecule, real time (SMRT) full length RNA sequencing. This analysis revealed a [~]5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of mRNA isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was validated by mass spectrometry and validated previously unknown protein isoforms predicted e.g. for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g. CFD, GATA2, HLA-A, B & C) also distinguished cell subpopulations but were only detectable by full length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

molecular biology↗

The impact of sex on alternative splicing

Over 95% of human genes undergo alternative splicing (AS) in a developmental, tissue-specific, or signal transduction-dependent manner. Here, we present a large-scale survey of sex-biased differential alternative splicing (DAS) across 7027 samples of 39 tissues from 532 individuals (351 males and 181 females) from the Genotype-Tissue Expression project. We detected a total of 1278 statistically significant DAS events affecting 888 different genes and 4417 significant differential gene expression (DGE) events in 3221 genes. Only 267 (29.3%) of the differentially spliced genes were also differentially expressed. Genes that displayed sex-biased DGE or DAS across multiple tissues were enriched in functions related to signaling including histone demethylation. The probability of a gene showing significant differential AS varies by chromosome and is highest for the X chromosome, with differentially spliced X chromosomal genes additionally being more likely to escape X chromosomal inactivation. A small but significant association was found between sex-biased AS and transcripts that undergo physiological nonsense-mediated decay (NMD). We show a significant overlap of differential splicing and genes that display estrogen-induced alternative splicing, that are involved in estrogen response pathway. Further, we show overlap of the involved exons with estrogen-receptor bindings sites. Our results provide a comprehensive survey of sex-biased AS and its characteristics across a large collection of human tissues.

genomics↗