Division of Translational Medical Oncology, National Center for Tumor Diseases (NCT) Heidelberg, German Cancer Research Center (DKFZ), Heidelberg, Germany.
German Cancer Consortium (DKTK), Heidelberg, Germany.
Sci Rep. 2023 Feb 9;13(1):2378. doi: 10.1038/s41598-023-29550-8.
Transcriptome profiling is a mainstay of translational cancer research and is increasingly finding its way into precision oncology. While bulk RNA sequencing (RNA-seq) is widely available, high investment costs and long data return time are limiting factors for clinical applications. We investigated a portable nanopore long-read sequencing device (MinION, Oxford Nanopore Technologies) for transcriptome profiling of tumors. In particular, we investigated the impact of lower coverage than that of larger sequencing devices by comparing shallow nanopore RNA-seq data with short-read RNA-seq data generated using reversible dye terminator technology (Illumina) for ten samples representing four cancer types. Coupled with ShaNTi (Shallow Nanopore sequencing for Transcriptomics), a newly developed data processing pipeline, a turnaround time of five days was achieved. The correlation of normalized gene-level counts between nanopore and Illumina RNA-seq was high for MinION but not for very low-throughput Flongle flow cells (r = 0.89 and r = 0.24, respectively). A cost-saving approach based on multiplexing of four samples per MinION flow cell maintained a high correlation with Illumina data (r = 0.56-0.86). In addition, we compared the utility of nanopore and Illumina RNA-seq data for analysis tools commonly applied in translational oncology: (1) Shallow nanopore and Illumina RNA-seq were equally useful for inferring signaling pathway activities with PROGENy. (2) Highly expressed genes encoding kinases targeted by clinically approved small-molecule inhibitors were reliably identified by shallow nanopore RNA-seq. (3) In tumor microenvironment composition analysis, quanTIseq performed better than CIBERSORT, likely due to higher average expression of the gene set used for deconvolution. (4) Shallow nanopore RNA-seq was successfully applied to detect fusion genes using the JAFFAL pipeline. These findings suggest that shallow nanopore RNA-seq enables rapid and biologically meaningful transcriptome profiling of tumors, and warrants further exploration in precision cancer medicine studies.
转录组谱分析是转化癌症研究的主要支柱,并且越来越多地应用于精准肿瘤学。尽管批量 RNA 测序(RNA-seq)广泛可用,但高投资成本和长数据回报时间是其临床应用的限制因素。我们研究了一种便携式纳米孔长读测序设备(MinION,Oxford Nanopore Technologies),用于肿瘤的转录组谱分析。特别是,我们通过比较使用可逆染料终止技术(Illumina)生成的短读 RNA-seq 数据和浅层纳米孔 RNA-seq 数据,研究了比更大的测序设备更低的覆盖度对转录组谱分析的影响,这十种样本代表四种癌症类型。结合新开发的数据处理管道 ShaNTi(用于转录组学的浅层纳米孔测序),实现了五天的周转时间。MinION 上的归一化基因水平计数与 Illumina RNA-seq 的相关性很高,但通量非常低的 Flongle 流池则不然(分别为 r=0.89 和 r=0.24)。基于每个 MinION 流池对四个样本进行多重化的节省成本方法,与 Illumina 数据保持了高度相关性(r=0.56-0.86)。此外,我们比较了纳米孔和 Illumina RNA-seq 数据在转化肿瘤学中常用分析工具的应用:(1)使用 PROGENy,浅层纳米孔和 Illumina RNA-seq 对于推断信号通路活性同样有用。(2)通过浅层纳米孔 RNA-seq 可以可靠地鉴定编码经临床批准的小分子抑制剂靶向的激酶的高表达基因。(3)在肿瘤微环境组成分析中,quanTIseq 的性能优于 CIBERSORT,可能是由于用于反卷积的基因集的平均表达更高。(4)使用 JAFFAL 管道,成功应用浅层纳米孔 RNA-seq 检测融合基因。这些发现表明,浅层纳米孔 RNA-seq 能够快速且具有生物学意义地进行肿瘤转录组谱分析,值得在精准癌症医学研究中进一步探索。