Anatomy and Neuroscience - Research Publications
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ItemNanopore direct RNA sequencing detects differential expression between human cell populations( 2020)Accurately quantifying gene and isoform expression changes is essential to understanding cell functions, differentiation and disease. Therefore, a crucial requirement of RNA sequencing is identifying differential expression. The recent development of long-read direct RNA (dRNA) sequencing has the potential to overcome many limitations of short and long-read sequencing methods that require RNA fragmentation, cDNA synthesis or PCR. dRNA sequences native RNA and can encompass an entire RNA in a single read. However, its ability to identify differential gene and isoform expression in complex organisms is poorly characterised. Using a mixture of synthetic controls and human SH-SY5Y cell differentiation into neuron-like cells, we show that dRNA sequencing accurately quantifies RNA expression and identifies differential expression of genes and isoforms. We generated ∼4 million dRNA reads with a median length of 991 nt. On average, reads covered 74% of SH-SY5Y transcripts and 29% were full-length. Measurement of expression and fold changes between synthetic control RNAs confirmed accurate quantification of genes and isoforms. Differential expression of 231 genes, 291 isoforms, plus 27 isoform switches were detected between undifferentiated and differentiated SH-SY5Y cells and samples clustered by differentiation state at the gene and isoform level. Genes upregulated in neuron-like cells were associated with neurogenesis. We further identified >30,000 expressed transcripts including thousands of novel splice isoforms and transcriptional units. Our results establish the ability of dRNA sequencing to identify biologically relevant differences in gene and isoform expression and perform the key capabilities of expression profiling methodologies.
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ItemNo Preview AvailableTranscriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection.( 2020-12-22)SARS-CoV-2 uses subgenomic (sg)RNA to produce viral proteins for replication and immune evasion. We applied long-read RNA and cDNA sequencing to in vitro human and primate infection models to study transcriptional dynamics. Transcription-regulating sequence (TRS)-dependent sgRNA was upregulated earlier in infection than TRS-independent sgRNA. An abundant class of TRS-independent sgRNA consisting of a portion of ORF1ab containing nsp1 joined to ORF10 and 3’UTR was upregulated at 48 hours post infection in human cell lines. We identified double-junction sgRNA containing both TRS-dependent and independent junctions. We found multiple sites at which the SARS-CoV-2 genome is consistently more modified than sgRNA, and that sgRNA modifications are stable across transcript clusters, host cells and time since infection. Our work highlights the dynamic nature of the SARS-CoV-2 transcriptome during its replication cycle. Our results are available via an interactive web-app at http://coinlab.mdhs.unimelb.edu.au/ .
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ItemTargeted RNA sequencing enhances gene expression profiling of ultra-low input samples(TAYLOR & FRANCIS INC, 2020-12-01)RNA-seq is the standard method for profiling gene expression in many biological systems. Due to the wide dynamic range and complex nature of the transcriptome, RNA-seq provides an incomplete characterization, especially of lowly expressed genes and transcripts. Targeted RNA sequencing (RNA CaptureSeq) focuses sequencing on genes of interest, providing exquisite sensitivity for transcript detection and quantification. However, uses of CaptureSeq have focused on bulk samples and its performance on very small populations of cells is unknown. Here we show CaptureSeq greatly enhances transcriptomic profiling of target genes in ultra-low-input samples and provides equivalent performance to that on bulk samples. We validate the performance of CaptureSeq using multiple probe sets on samples of iPSC-derived cortical neurons. We demonstrate up to 275-fold enrichment for target genes, the detection of 10% additional genes and a greater than 5-fold increase in identified gene isoforms. Analysis of spike-in controls demonstrated CaptureSeq improved both detection sensitivity and expression quantification. Comparison to the CORTECON database of cerebral cortex development revealed CaptureSeq enhanced the identification of sample differentiation stage. CaptureSeq provides sensitive, reliable and quantitative expression measurements on hundreds-to-thousands of target genes from ultra-low-input samples and has the potential to greatly enhance transcriptomic profiling when samples are limiting.
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ItemLong-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain(SPRINGERNATURE, 2020-01)RNA splicing is a key mechanism linking genetic variation with psychiatric disorders. Splicing profiles are particularly diverse in brain and difficult to accurately identify and quantify. We developed a new approach to address this challenge, combining long-range PCR and nanopore sequencing with a novel bioinformatics pipeline. We identify the full-length coding transcripts of CACNA1C in human brain. CACNA1C is a psychiatric risk gene that encodes the voltage-gated calcium channel CaV1.2. We show that CACNA1C's transcript profile is substantially more complex than appreciated, identifying 38 novel exons and 241 novel transcripts. Importantly, many of the novel variants are abundant, and predicted to encode channels with altered function. The splicing profile varies between brain regions, especially in cerebellum. We demonstrate that human transcript diversity (and thereby protein isoform diversity) remains under-characterised, and provide a feasible and cost-effective methodology to address this. A detailed understanding of isoform diversity will be essential for the translation of psychiatric genomic findings into pathophysiological insights and novel psychopharmacological targets.