High-resolution sequencing of DNA G-quadruplex secondary structures in the human genome

This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nbt.3295During active transcription and replication chromatin architecture is altered, allowing formation of DNA secondary structures. G-quadruplexes (G4s) have emerged as important regulatory DNA structures and have been associated with genomic instability, genetic diseases and cancer progression. Experimental evidence for G4 prevalence in the entire human genome is still lacking. We present a high-resolution sequencing-based method that detected 716,310 distinct G4s in the human genome, more than predicted by computational methods, including structural variants previously uncharacterised in a genomic context. We observed high G4-density in functional regions, such as 5’ UTRs and splicing sites, and in genes not predicted to have such structures (BRCA1 and BRCA2). We found a significant association of G4 formation with oncogenes and tumor suppressors, and with Somatic Copy-Number Alterations (SCNAs) that act as cancer drivers. Our results support that G4s are promising targets for cancer intervention and suggest novel candidates for further biological and mechanistic studies.We are grateful to the Biotechnology and Biological Sciences Research Council (BBSRC) and Illumina® for the studentship supporting V.C (BB/I015477/1). The S.B. research group is supported by programme funding from Cancer Research UK and from the European Research Council and project funding from BBSRC

Whole genome experimental maps of DNA G-quadruplexes in multiple species.

Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.The S.B. research group is supported by programme grant funding from Cancer Research UK (C9681/A18618), European Research Council Advanced Grant No. 339778, a Wellcome Trust Senior Investigator Award (grant 209441/z/17/z) and by core funding from Cancer Research UK (C14303/A17197). We are grateful to the Biotechnology and Biological Sciences Research Council (BBSRC) and Illumina for the CASE studentship supporting V.S.C. (BB/I015477/1)

Accurate whole human genome sequencing using reversible terminator chemistry

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications