Hybrid-seq deciphers the complex transcriptional profile of the human <i>BRCA1</i> DNA repair associated gene

Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of <i>BRCA1</i> (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect <i>BRCA1</i> alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of <i>BRCA1</i> splice variants (<i>BRCA1</i> sv.7 – sv.52), thus deciphering the transcriptional landscape of the human <i>BRCA1</i> gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described <i>BRCA1</i> mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, <i>in silico</i> analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

乳腺癌基因1（BRCA1）是一类肿瘤抑制蛋白，可调控基因组稳定性、DNA损伤修复等多种生物学过程。尽管主流BRCA1蛋白的功能特性已得到充分阐释，但后续蛋白质组学研究已发现了更多分子量更低的BRCA1剪接异构体。然而，准确测定这些异构体对应的mRNA核苷酸序列仍是一大技术瓶颈，这主要源于BRCA1的mRNA长度较长（约5.5 kb），且现有测序技术存在固有局限。本研究设计并采用了一种基于纳米孔测序与半导体测序的多重混合测序技术（Hybrid-seq），旨在检测一组人类癌症与非癌细胞系中的BRCA1可变剪接转录本。该Hybrid-seq技术的应用成功产出了高精度长读长测序数据，使得我们能够鉴定出覆盖广泛范围的BRCA1剪接变体（BRCA1 sv.7至sv.52），从而完整解析了人类BRCA1基因的转录组图谱。此外，对测序数据进行解复用分析后，我们揭示了上述BRCA1 mRNA在乳腺癌、卵巢癌、前列腺癌、结直肠癌、肺癌、脑癌以及非癌人类细胞系中的表达谱与丰度。最后，计算机模拟（in silico）分析结果显示，本次检测到的多款mRNA均携带开放阅读框，极有可能编码带有保守结构域的潜在蛋白异构体，从而为解析BRCA1在基因组稳定性与DNA损伤修复中的复杂调控作用提供了全新的研究视角。