title: Fourth Generation of Next-Generation Sequencing Technologies: Promise and Consequences.
abstract: In this review, we discuss the emergence of the fourth-generation sequencing technologies that preserve the spatial coordinates of RNA and DNA sequences with up to subcellular resolution, thus enabling back mapping of sequencing reads to the original histological context. This information is used, for example, in two current large-scale projects that aim to unravel the function of the brain. Also in cancer research, fourth-generation sequencing has the potential to revolutionize the field. Cancer Research UK has named "Mapping the molecular and cellular tumor microenvironment in order to define new targets for therapy and prognosis" one of the grand challenges in tumor biology. We discuss the advantages of sequencing nucleic acids directly in fixed cells over traditional next-generation sequencing (NGS) methods, the limitations and challenges that these new methods have to face to become broadly applicable, and the impact that the information generated by the combination of in situ sequencing and NGS methods will have in research and diagnostics.
authors.affiliation: School of Biomedical Sciences, Huaqiao University, Quanzhou, Fujian, 362021, China.
authors.forename: Rongqin
authors.initials: R
authors.lastname: Ke
authors.affiliation: Department of Information Technology, Centre for Image Analysis, Science for Life Laboratory, Uppsala University, Uppsala, SE-75105, Sweden.|Department of Bioengineering, Stanford University, Stanford, California, 75105.
authors.forename: Marco
authors.initials: M
authors.lastname: Mignardi
authors.affiliation: Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, SE-171 21, Sweden.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hauling
authors.affiliation: Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, SE-171 21, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Nilsson
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods; D020022:Genetic Predisposition to Disease; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods
keywords: in situ sequencing; multiplex in situ RNA detection; next-generation sequencing; single cell sequencing; spatial gene expression