SP-Z: NGS-based approaches for systematic analysis of genomic and chromosome instability

下一代测序(NGS)已被证明是一种强大的工具，可以提供对基因组完整性潜在机制的新见解，基于NGS的高端方法现在可以用来彻底研究细胞中的基因组不稳定性。在拟议的FOR2800(SP-Z)中央服务项目的范围内，我们打算提供基于NGS的成熟和创新的方法，用于在不同基因组水平上分析基因组不稳定性。使用一种新的单细胞(Sc)转录管道，我们将为所有单独的项目提供scRNA测序，以确定基因组不稳定对基因活性和转录图谱的影响。DNA修复机制的保真度降低或受损DNA的复制会导致体细胞突变的积累。此外，结构染色体不稳定(S-CIN)和整体染色体不稳定(W-CIN)可能促进体细胞变异的发生，从而导致基因组不稳定。因此，发现体细胞变异对于研究复制胁迫或有丝分裂改变后SNV积累的新机制至关重要。这就需要超高的NGS数据覆盖率。我们开发了一种新的流水线，用于分析超高覆盖率(10.000x)的多基因面板测序数据，以检测这些低频变异。例如，这种方法将被用来确定BLM和其他BTRR复杂成分中变异的积累，但也提供了对适应基因组不稳定和非整倍体的机制的见解。SP-Z的另一个重要目标是通过使用EDU-Seq来识别在复制胁迫下启动休眠起源激发的基因组位置。此外，我们将继续提供低覆盖率的批量或单细胞DNA测序，用于S-CIN和W-CIN的检测和定量，这对于各种项目确定基因组不稳定的程度是重要的。为此，我们可以依靠我们在FOR2800的第一个资助期建立的生物信息学管道来分析从批量DNA产生的数据以及单细胞数据集。为此，我们使用了软件工具AneuFinder，并在全自动管道中执行数据的预处理和结果的可视化。因此，SP-Z将提供集中的基于NGS的战略和方法(例如，单细胞WGS、超高覆盖率多基因小组、RNA-SEQ、单链RNA-SEQ和EDU-SEQ)，对FOR2800的子项目以及随后的数据生物信息学分析至关重要。

Next-generation sequencing (NGS) has proven to be a powerful tool to deliver new insights into mechanisms underlying genome integrity, and high-end NGS-based approaches can now be employed to thoroughly investigate genomic instability in cells. Within the scope of the proposed central service project of the FOR2800 (SP-Z), we intend to provide well-established as well as innovative NGS-based methods for analyzing genomic instability on different genomic levels. Using a novel single-cell (sc) transcriptomic pipeline, we will offer scRNA sequencing to all individual projects to determine the consequences of genome instability on gene activity and transcriptional profiles. Decreased fidelity of DNA repair mechanisms or replication of damaged DNA can lead to an accumulation of somatic mutations. Furthermore, structural chromosome instability (S-CIN) and whole chromosome instability (W-CIN) might promote the occurrence of somatic variants, which can additionally cause genomic instability. Uncovering somatic variants is therefore crucial to investigate novel mechanisms of SNV accumulation after replication stress or mitotic alterations. This requires an ultra-high coverage of NGS data. We have developed a novel pipeline for the analysis of ultra-high coverage (10.000x) multi-gene panel sequencing data to detect these low-frequency variants. For instance, this approach will be used to determine accumulation of variants in BLM and other BTRR complex components, but also to provide insights into the mechanisms of adaption to genome instability and aneuploidy. Another important aim of SP-Z is to identify genomic sites where dormant origin firing is initiated upon replication stress by employing EdU-Seq. In addition, we will continue to offer low-coverage bulk or single-cell DNA sequencing for the detection and quantification of S-CIN and W-CIN, which is important for various projects to determine the extent of genomic instability. For this purpose, we can rely on our bioinformatics pipeline established in the first funding period of the FOR2800 for analyzing both, data generated from bulk DNA as well as single cell data sets. For this, we employ the software tool AneuFinder and perform the pre-processing of the data as well as visualization of the results in a fully automated pipeline. SP-Z will thus offer centralized NGS-based strategies and approaches (e.g. single-cell WGS, ultra-high coverage multi-gene panel, RNA-seq, scRNA-seq, and EdU-seq) essential to the sub-projects of the FOR2800 as well as the subsequent bioinformatics analysis of data.