Bourgeois, N. | bioRxiv Science

bioRxiv2023

Hidden rAAV Breakpoints Detected Using Single-Molecule, Modified Base Sequencing

The AAV genome is a single stranded DNA molecule packaged in an icosahedral protein capsid. Vector genomes of plus and minus polarities are packaged and complementary genomic strands hybridize when lysed in vitro. Standard sequencing library methods cause loss of information from individual genomes when mismatches and gaps are repaired. To retain original molecular information, modified bases are used during the repair step which allows pre-existing DNA to be distinguished from DNA added during library preparation. Modified bases introduced during repair are identified using the Sequel II system and used to detect HIDdEN DNA breakpoints (HIDEN-Seq). The most frequent breakpoints in an AAV vector subject to high strand breakage during packaging were linked to adjacent secondary structure, prompting changes in nearby sequences to reduce breakage. This use of modified bases for localizing DNA breaks enables better vector design, resulting in higher quality gene therapy vectors. The same approach can be used in other systems where knowledge of pre-existing sequence and structure is important.

genomics↗

bioRxiv2021

CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

bioinformatics↗