Shatalov P A, Raygorodskaya M P, Shinkarkina A P, Traspov A A, Murzaeva A V, Doroshenko Y A, Leuhina I A, Mileyko V A, Ivanov M V, Grigoreva T V, Lebedeva A A, Veselovsky E M, Belyaeva L D, Belova E V, Kaprin A D, Shegai P V
National Medical Research Radiological Centre of the Ministry of Health of the Russian Federation, Obninsk, Russia.
OncoAtlas LLC, Moscow, Russia.
PLoS One. 2025 Jun 4;20(6):e0323685. doi: 10.1371/journal.pone.0323685. eCollection 2025.
MGI platforms hold promise to become a widespread instrument for various clinical next-generation sequencing applications, from whole genome sequencing to COVID-19 genotyping. However, in the clinical oncology setting it is still restricted to large panel sequencing limiting capacity for routine biomarker screening. In this article, we describe our experience of tailoring amplicon-based library construction for the MGI platform. Illumina compatible reagents served as a prototype in order to introduce platform specific adapters. Elaborated reagent kits were used for BRCA1/2 or 34 oncogenes testing both with whole blood and FFPE-derived DNA. Our data show that amplicon-based DNBSEQ-tailored library preparation demonstrates sufficient analytical efficiency in terms of coverage uniformity (average MAPD 1.08 and 1.19 for ABC plus and Atlas plus panels) and amplicon drop-out rate (ranging from 0.3% to 2.5%). Additionally, it shows efficiency in terms of single sample sensitivity, maintaining 99% sensitivity compared to 99% for the Illumina prototype. We show that it also outreaches expected diagnostic parameters of MGI exome sequencing (99% vs 95% for WES). Per-amplicon coverage of sticky-end libraries sequenced on Illumina and MGI were highly correlated demonstrating that the platform itself does not introduce any bias to amplicon coverage. Across three tested variations of library preparation protocol, discordances were related to ligation mix component composition and resulted in underrepresentation of GC-low and GC-high amplicons and low uniformity as a result. Overall, we outline the successful adaptation of PCR-based library preparation for MGI signifying the importance of tailoring component composition of reagent kit for uniform coverage.
MGI平台有望成为各种临床下一代测序应用的广泛工具，从全基因组测序到新冠病毒基因分型。然而，在临床肿瘤学环境中，它仍仅限于大panel测序，限制了常规生物标志物筛查的能力。在本文中，我们描述了为MGI平台定制基于扩增子的文库构建的经验。以Illumina兼容试剂作为原型，以便引入平台特异性接头。精心制作的试剂盒用于BRCA1/2或34个癌基因检测，使用全血和FFPE来源的DNA。我们的数据表明，基于扩增子的DNBSEQ定制文库制备在覆盖均匀性（ABC plus和Atlas plus panel的平均MAPD分别为1.08和1.19）和扩增子缺失率（范围为0.3%至2.5%）方面表现出足够的分析效率。此外，它在单样本灵敏度方面也表现出效率，与Illumina原型的99%灵敏度相比，保持了99%的灵敏度。我们表明，它还超过了MGI外显子组测序的预期诊断参数（WES为99%对95%）。在Illumina和MGI上测序的粘性末端文库的每个扩增子覆盖度高度相关，表明该平台本身不会对扩增子覆盖度引入任何偏差。在文库制备方案的三个测试变体中，不一致与连接混合组分组成有关，导致GC含量低和GC含量高的扩增子代表性不足，结果均匀性较低。总体而言，我们概述了基于PCR的文库制备对MGI的成功适应性，这表明为实现均匀覆盖而定制试剂盒的组分组成非常重要。
