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

Bernd Wollnik, Göttingen


The objective of the central project is providing access to state-of-the-art sequencing technology and methods as well as novel next-generation sequencing (NGS)-based approaches to all partners and individual projects within the consortium. Advances in NGS and NGS-based approaches such as whole-genome sequencing (WGS) have tremendously improved the detection of structural variants/copy number variations (CNVs) and single somatic variants and mutations as measures of genome integrity. With strand-sensitive sequencing in single cells, another novel NGS-based approach has recently emerged that allows to detect copy-number-neutral chromosomal rearrangements. All projects proposed for the research group “Chromosome Instability: Cross-talk of DNA replication stress and mitotic dysfunction” will require modern NGS-based analyses for their research, including low-coverage WGS for determination of whole chromosome instability (W-CIN) and structural chromosome instability (S-CIN) across the genome and high-coverage WGS to determine specific mutations, mutation signatures across genes or the overall load of low-frequency mutations as a measure of genome stability. In addition, specific ChIP-Sequencing is required for individual projects. Accurate and profound data analysis, however, is a particular challenge for research projects using NGS-based approaches. This central service project will be based on the extensive experience of the Wollnik group in various NGS-based analyses of the exome, genome, genome dynamics and genome instability that was demonstrated in the identification of a substantial number of novel disease-associated genes and appropriately established bioinformatics pipelines and on the broad spectrum of NGS-based methods and applications established at the Institute of Human Genetics Göttingen. In the sub-project Z, we aim to provide NGS-based data generations as well as a bioinformatics pipeline for the detection of CNVs from low-coverage genome sequencing data to all individual project partners. We will use our pipeline also for the detection of somatic mutations by generating 10x-coverage WGS datasets for all experiments, drawing from our previous proof-of-principle study, in which we were able to show that genomic instability syndromes and accelerated-aging phenotypes can give rise to an increased load of somatic mutations and that with adequate bioinformatics filtering, the load of somatic mutations can be analyzed directly in NGS data sets. Additionally, we will establish a method for single-cell-template-strand sequencing (Strand-seq) and a bioinformatics pipeline for processing and analyzing sequence data obtained from Strand-seq experiments, which will revolutionize the analysis of S-CIN for all projects. In addition, our highly specific aims will be complemented by general NGS-based analysis support, data handling, and bioinformatics support provided to all members of the research group.

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