ABSTRACT
BACKGROUND: Some clinically important genetic variants are not easily evaluated with next-generation sequencing (NGS) methods due to technical challenges arising from high- similarity copies (e.g., PMS2, SMN1/SMN2, GBA1, HBA1/HBA2, CYP21A2), repetitive short sequences (e.g., ARX polyalanine repeats, FMR1 AGG interruptions in CGG repeats, CFTR poly-T/TG repeats), and other complexities (e.g., MSH2 Boland inversions). METHODS: We customized our NGS processes to detect the technically challenging variants mentioned above with adaptations including target enrichment and bioinformatic masking of similar sequences. Adaptations were validated with samples of known genotypes. RESULTS: Our adaptations provided high-sensitivity and high-specificity detection for most of the variants and provided a high-sensitivity primary assay to be followed with orthogonal disambiguation for the others. The sensitivity of the NGS adaptations was 100% for all of the technically challenging variants. Specificity was 100% for those in PMS2, GBA1, SMN1/SMN2, and HBA1/HBA2, and for the MSH2 Boland inversion; 97.8%-100% for CYP21A2 variants; and 85.7% for ARX polyalanine repeats. CONCLUSIONS: NGS assays can detect technically challenging variants when chemistries and bioinformatics are jointly refined. The adaptations described support a scalable, cost-effective path to identifying all clinically relevant variants within a single sample.
Subject(s)
Fragile X Mental Retardation Protein , High-Throughput Nucleotide Sequencing , Humans , Mismatch Repair Endonuclease PMS2 , Glycated Hemoglobin , MutS Homolog 2 Protein , High-Throughput Nucleotide Sequencing/methods , Genotype , Steroid 21-HydroxylaseABSTRACT
Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells.
Subject(s)
Cell-Free System/metabolism , Exosomes/metabolism , MicroRNAs/metabolism , Y-Box-Binding Protein 1/metabolism , Cell Movement , Cell-Free System/chemistry , Centrifugation, Density Gradient , Exosomes/chemistry , Gene Expression Profiling , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Luciferases/genetics , Luciferases/metabolism , MicroRNAs/classification , MicroRNAs/genetics , Tetraspanin 30/genetics , Tetraspanin 30/metabolism , Y-Box-Binding Protein 1/geneticsABSTRACT
BACKGROUND: Understanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults. RESULTS: Using a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages. CONCLUSIONS: We conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.
Subject(s)
Gene Expression Profiling/methods , Neurogenesis , Neurons/cytology , Zebrafish/embryology , Zebrafish/genetics , Animals , Cell Differentiation , Gene Expression Regulation , Neuropeptides/genetics , Sequence Analysis, RNA/methods , Signal Transduction , Transcription Factors/geneticsABSTRACT
Transcriptional profiling is a powerful approach for studying mouse development, physiology and disease models. Here we describe a protocol for mouse thiouracil tagging (TU tagging), a transcriptome analysis technology that includes in vivo covalent labeling, purification and analysis of cell type-specific RNA. TU tagging enables the isolation of RNA from a given cell population of a complex tissue, avoiding transcriptional changes induced by cell isolation trauma, as well as the identification of actively transcribed RNAs and not preexisting transcripts. Therefore, in contrast to other cell-specific transcriptional profiling methods based on the purification of tagged ribosomes or nuclei, TU tagging provides a direct examination of transcriptional regulation. We describe how to (i) deliver 4-thiouracil to transgenic mice to thio-label cell lineage-specific transcripts, (ii) purify TU-tagged RNA and prepare libraries for Illumina sequencing and (iii) follow a straightforward bioinformatics workflow to identify cell type-enriched or differentially expressed genes. Tissue containing TU-tagged RNA can be obtained in 1 d, RNA-seq libraries can be generated within 2 d and, after sequencing, an initial bioinformatics analysis can be completed in 1 additional day.
