IMPLEMENTING DNA AND RNA NANOPORE SEQUENCING TECHNOLOGY IN UNDERGRADUATE TEACHING

Considering the fast pace at which sequencing technologies progress and at which genomics data are generated, it is no longer possible to ignore the urgency of equipping undergraduate students in science programs with the required skills to manage the amount and type of sequencing data being generated. In particular, the third-generation technologies, such as nanopore sequencing, became highly relevant for genomic research and clinical diagnostics due to low capital cost, device portability and real-time data acquisition. One of the stage 3 undergraduate courses our School offers is Molecular Frontiers (BABS 3281). The course focuses on cutting-edge molecular biology techniques and their applications in biomedical research. We recently included nanopore DNA and direct RNA sequencing as a part of practical laboratory classes in the course and introduced lectures constructively aligned to the experimental protocol of the practical. The implementation of nanopore technology, where students could generate genome-wide RNA sequencing data and explore biological meaning of gene expression profiles using online bioinformatics pipeline, resulted in significant increase in students’ satisfaction with BABS 3281 and 3-fold increase in student enrolments in the following year. In this presentation, I will reflect on the students’ experience and discuss lessons learned as a course coordinator

Identification of Specific Circular RNA Expression Patterns and MicroRNA Interaction Networks in Mesial Temporal Lobe Epilepsy

Circular RNAs (circRNAs) regulate mRNA translation by binding to microRNAs (miRNAs), and their expression is altered in diverse disorders, including cancer, cardiovascular disease, and Parkinson’s disease. Here, we compare circRNA expression patterns in the temporal cortex and hippocampus of patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) and healthy controls. Nine circRNAs showed significant differential expression, including circRNA-HOMER1, which is expressed in synapses. Further, we identified miRNA binding sites within the sequences of differentially expressed (DE) circRNAs; expression levels of mRNAs correlated with changes in complementary miRNAs. Gene set enrichment analysis of mRNA targets revealed functions in heterocyclic compound binding, regulation of transcription, and signal transduction, which maintain the structure and function of hippocampal neurons. The circRNA–miRNA–mRNA interaction networks illuminate the molecular changes in MTLE, which may be pathogenic or an effect of the disease or treatments and suggests that DE circRNAs and associated miRNAs may be novel therapeutic target

Moving towards third-generation sequencing technologies

Within just the past few years, next-generation sequencing (NGS) platforms have reduced the cost of DNA sequencing by several orders of magnitude. With these lower costs, DNA sequencing is increasingly powerful as a single-data format onto which a broad range of biological processes can be projected for high-throughput molecular profiling. Although significant challenges remain, translating these powerful technologies into useful laboratory tests has great potential. This chapter provides a comparative analysis of currently available NGS techniques, including those of Illumina, 454/Roche, Applied Biosystems, and Helicos BioSciences, and addresses emerging techniques, such as Pacific Biosciences single-molecule sequencing and nanopore sequencing. Furthermore, very recent electronic sequencing from Ion Torrent is elaborated. The chapter discusses advantages and disadvantages of individual platforms, and those common to all techniques, including template preparation and genome enrichment

Transcriptome profiling in neurodegenerative disease

Changes in gene expression and splicing patterns (that occur prior to the onset and during the progression of complex diseases) have become a major focus of neurodegenerative disease research. These signature patterns of gene expression provide clues about the mechanisms involved in the molecular pathogenesis of neurodegenerative disease and may facilitate the discovery of novel therapeutic drugs. With the development of array technologies and the very recent RNA-seq technique, our understanding of the pathogenesis of neurodegenerative disease is expanding exponentially. Here, we review the technologies involved in gene expression and splicing analysis and the related literature on three common neurodegenerative diseases: Alzheimer's disease, Parkinson's disease and Huntington's disease

Sequencing of hippocampal and cerebellar transcriptomes provides new insights into the complexity of gene regulation in the human brain

The hippocampus and cerebellum represent anatomically and functionally distinct parts of the human brain. The RNA-Seq technique makes it possible to investigate the human transcriptome with unprecedented resolution, allowing identification of differential mRNA splicing and promoter usage on a genome-wide scale. We undertook whole-mRNA sequencing of samples from the human hippocampus and cerebellum. A bioinformatic analysis revealed distinct expression patterns of genes related to the molecular physiology of neurons and glial cells. Upregulated genes in hippocampal tissue included serpin peptidase inhibitor, clade A (SERPINA3), lymphocyte antigen 6 complex, locus H (LY6H) and transthyretin (TTR). In cerebellum, the cerebellin 3 precursor (CLBN3) and Zic family member 4 (ZIC4) genes were significantly upregulated. These changes were validated in independent donor samples by qRT-PCR. The hippocampus and the cerebellum showed striking differences in splicing patterns and promoter usage. A notable example of this was the gene for NGFI-A binding protein 2 (NAB2), which displayed tissue-specific isoforms which may affect its function as a transcriptional repressor

RNA-Seq analysis of the parietal cortex in Alzheimer's disease reveals alternatively spliced isoforms related to lipid metabolism

The parietal cortex of the human brain plays a unique role in the coordination of movement and in the integration of signals from the other cortices. Because of its extensive connections and involvement in many higher-order cognitive functions, neurodegenerative changes in the parietal lobe are believed to be crucial in the early symptoms of Alzheimer's disease (AD). Little is known about the transcriptome of this part of the human brain or how it is perturbed by the neurodegenerative process. To that end, we performed mRNA sequencing using the Illumina RNA-Seq technique on samples derived from normal and AD parietal lobes. Gene expression analysis evaluating alternatively spliced isoform expression and promoter usage revealed surprisingly elevated transcriptome activity in the AD condition. This phenomenon was particularly apparent in the alternative usage of transcriptional start sites. A Gene Ontology analysis of the differentially expressed genes revealed enrichment in the functional pathways related to lipid metabolism, thus highlighting the importance of astrocyte activity in the neurodegenerative process. We also identified an upregulation of the diazepam-binding inhibitor (DBI) gene in AD, as the result of a splicing switch toward shorter, intron-retaining isoforms driven by alternative promoters and was coupled with a simultaneous decrease in the abundance of protein-coding transcripts. These two DBI isoforms have not been described previously

Investigation of Transcriptome Patterns in Endometrial Cancers from Obese and Lean Women

Endometrial cancer is the most common gynaecological malignancy in developed countries. One of the largest risk factors for endometrial cancer is obesity. The aim of this study was to determine whether there are differences in the transcriptome of endometrial cancers from obese vs. lean women. Here we investigate the transcriptome of endometrial cancer between obese and lean postmenopausal women using rRNA-depleted RNA-Seq data from endometrial cancer tissues and matched adjacent non-cancerous endometrial tissues. Differential expression analysis identified 12,484 genes (6370 up-regulated and 6114 down-regulated) in endometrial cancer tissues from obese women, and 6219 genes (3196 up-regulated and 3023 down-regulated) in endometrial cancer tissues from lean women (adjusted p-value < 0.1). A gene ontology enrichment analysis revealed that the top 1000 up-regulated genes (by adjusted p-value) were enriched for growth and proliferation pathways while the top 1000 down-regulated genes were enriched for cytoskeleton restructure networks in both obese and lean endometrial cancer tissues. In this study, we also show perturbations in the expression of protein coding genes (HIST1H2BL, HIST1H3F, HIST1H2BH, HIST1H1B, TTK, PTCHD1, ASPN, PRELP, and CDH13) and the lncRNA MBNL1-AS1 in endometrial cancer tissues. Overall, this study has identified gene expression changes that are similar and also unique to endometrial cancers from obese vs. lean women. Furthermore, some of these genes may serve as prognostic biomarkers or, possibly, therapeutic targets for endometrial cancer

Characterization of circular RNAs landscape in multiple system atrophy brain

Circular RNAs (circRNAs) have been recently identified as a naturally occurring family of widespread and diverse endogenous non-coding RNAs that may regulate gene expression in mammals. They are unusually stable RNA molecules with cell type- or developmental stage-specific expression patterns. However, the role of circRNAs in pathology of complex disease is entirely unknown. Here, we report the specific circular transcriptome in the multiple system atrophy (MSA) brain as determined by RNA sequencing. Five circRNAs, namely IQCK, MAP4K3, EFCAB11, DTNA, and MCTP1, were identified and validated as specifically over-expressed in MSA frontal cortex. The expression levels of linear transcripts were not significantly altered and thus did not follow the pattern of their circular counterparts. Further analysis of expression of five MSA-specific circRNAs revealed their over-expression in the white matter of the MSA cortical tissue. Together, this is the first report describing perturbation of circular transcriptome in α-synucleinopathie

LINC00507 Is Specifically Expressed in the Primate Cortex and Has Age-Dependent Expression Patterns

Over the past decade, there has been an increase in the appreciation of the role of non-coding RNA in the development of organism phenotype. It is possible to divide the non-coding elements of the transcriptome into three categories: short non-coding RNAs, circular RNAs and long non-coding RNAs. Long non-coding RNAs are those transcripts that are greater than 200 nts in length and lack any significant open reading frames that produce proteins greater then 100 amino acids. Long intervening non-coding RNAs (lincRNAs) are a subclass of long non-coding RNAs. In contrast to protein coding RNAs, lincRNAs are expressed in a more tissue- and species-specific manner. In particular, many lincRNAs are only conserved amongst higher primates. This coupled with the propensity of many lincRNAs to be expressed in the brain, suggests that they are in fact one of the major drivers of organism complexity. We analysed 39 lincRNAs that are expressed in the frontal cortex and identified LINC00507 as being expressed in a cortex-specific manner in non-human primates and humans. The expression patterns of LINC00507 appear to be age-dependent, suggesting it may be involved in brain development of higher primates. Moreover, the analysis of LINC00507 potential to bind ribosomes revealed that this previously identified non-coding transcript may harbour a micropeptid