Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hwasun Hospital, Hwasun, Republic of Korea.
Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hwasun Hospital, Hwasun, Republic of Korea; Brain Korea 21 Plus Project, Chonnam National University Medical School, Gwangju, Republic of Korea.
J Mol Diagn. 2021 Aug;23(8):1015-1029. doi: 10.1016/j.jmoldx.2021.05.008. Epub 2021 Jun 1.
Targeted RNA sequencing (RNA-seq) is a highly accurate method for sequencing transcripts of interest with a high resolution and throughput. However, RNA-seq has not been widely performed in clinical molecular laboratories because of the complexity of data processing and interpretation. We developed and validated a customized RNA-seq panel and data processing protocol for fusion detection using 4 analytical validation samples and 51 clinical samples, covering seven types of hematologic malignancies. Analytical validation showed that the results for target gene coverage and between- and within-run precision and linearity tests were reliable. Using clinical samples, RNA-seq based on filtering and prioritization strategies detected all 25 known fusions previously found by multiplex reverse transcriptase-PCR and fluorescence in situ hybridization. It also detected nine novel fusions. Known fusions detected by RNA-seq included two IGH rearrangements supported by expression analysis. Novel fusions included six that targeted just one partner gene. In addition, 18 disease- and drug resistance-associated transcript variants in ABL1, GATA2, IKZF1, JAK2, RUNX1, and WT1 were designated simultaneously. Expression analysis showed distinct clustering according to subtype and lineage. In conclusion, this study showed that our customized RNA-seq system had a reliable and stable performance for fusion detection, with enhanced diagnostic yield for hematologic malignancies in a clinical diagnostic setting.
靶向 RNA 测序（RNA-seq）是一种高度准确的方法，可以高分辨率和高通量地测序感兴趣的转录本。然而，由于数据处理和解释的复杂性，RNA-seq 尚未在临床分子实验室中广泛开展。我们开发并验证了一种定制的 RNA-seq 面板和数据处理方案，用于融合检测，使用 4 个分析验证样本和 51 个临床样本，涵盖七种血液系统恶性肿瘤。分析验证表明，目标基因覆盖度以及批内和批间精密度和线性测试的结果可靠。使用临床样本，基于过滤和优先级策略的 RNA-seq 检测到了以前通过多重逆转录-PCR 和荧光原位杂交发现的所有 25 种已知融合。它还检测到了 9 种新的融合。通过 RNA-seq 检测到的已知融合包括通过表达分析支持的两个 IGH 重排。新融合包括仅靶向一个伙伴基因的 6 个。此外，ABL1、GATA2、IKZF1、JAK2、RUNX1 和 WT1 中的 18 个与疾病和耐药性相关的转录变体也同时被指定。表达分析根据亚型和谱系显示出明显的聚类。总之，这项研究表明，我们的定制 RNA-seq 系统在融合检测方面具有可靠和稳定的性能，在临床诊断环境中提高了血液系统恶性肿瘤的诊断产量。
