Summary
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.
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