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.

中文翻译：

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纳米孔直接RNA测序检测人类细胞群之间的差异表达

准确定量基因和同工型表达变化对于理解细胞功能，分化和疾病至关重要。因此，RNA测序的关键要求是鉴定差异表达。长读直接RNA（dRNA）测序的最新发展有可能克服短和长读测序方法的许多局限，这些方法需要RNA片段化，cDNA合成或PCR。dRNA可以对天然RNA进行测序，并且可以在一次读取中涵盖整个RNA。但是，其鉴定复杂生物中的差异基因和同工型表达的能力表征较差。使用合成控件和人类SH-SY5Y细胞分化为神经元样细胞的混合物，我们显示dRNA测序可准确定量RNA表达并鉴定基因和同工型的差异表达。我们产生了约400万条dRNA读数，中位长度为991 nt。平均而言，阅读覆盖了74％的SH-SY5Y转录本，其中29％为全长。合成对照RNA之间的表达和倍数变化的测量结果证实了基因和同工型的准确定量。在未分化和分化的SH-SY5Y细胞与通过基因和同工型水平的分化状态聚类的样品之间检测到231个基因，291个同工型和27个同工型开关的差异表达。神经元样细胞中上调的基因与神经发生有关。我们进一步确定了> 30,000个表达的转录本，包括成千上万的新型剪接亚型和转录单位。