Data related to the article "Evaluation of Oxford Nanopore MinION RNA-seq performance for human primary cells"

This record contains raw data related to the article “Evaluation of Oxford Nanopore MinION RNA-seq performance for human primary cells". Abstract: Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real-time via portable protein nanopore USB devices at a reasonable cost. This work aimed to provide the user with the number of reads that should be sequenced, through ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained increasing the sequencing depth, in particular for the non-coding genes. The reliability of expression levels was assayed by i) comparison with PCR quantifications of selected genes and ii) by the implementation of a user-friendly multiplexing method in a single run.

本数据集包含与论文《牛津纳米孔MinION RNA测序平台在人类原代细胞中的测序表现评估》相关的原始数据。
摘要：转录组测序是深入解析诊断与临床医学领域生物学过程的核心工具。长读长测序技术具备解析新型复杂真核生物转录组的潜力，可克服短读长RNA测序（RNA-seq）方法的部分局限。牛津纳米孔技术公司（Oxford Nanopore Technologies，ONT）推出的便携式蛋白纳米孔USB测序设备，能够以合理成本实时产出长读长测序数据。本研究旨在为使用者提供借助ONT MinION测序平台开展人类细胞RNA研究时，达到预期精度水平所需的测序读段（Reads）数量。我们对从人类原代成纤维细胞的聚腺苷酸RNA中制备的3个互补DNA（cDNA）文库进行了测序。由于各测序运行结果具有可比性，我们将其合并为包含4800万条读段的总数据集。基于该总数据集生成不同规模的合成数据集，并从已鉴定基因数目与基因表达水平两方面开展分析。正如预期，随着测序深度增加，检测灵敏度得以提升，尤其在非编码基因的检测中表现突出。我们通过两种方式验证了基因表达水平的可靠性：一是与选定基因的聚合酶链式反应（Polymerase Chain Reaction，PCR）定量结果进行比对；二是在单次测序运行中应用简便易用的多重测序方法。