Ivancic, D. | Search bioRxiv

bioRxiv2025

Parallel multiOMIC analysis reveals glutamine deprivation enhances directed differentiation of renal organoids

Metabolic pathways play a critical role in driving differentiation but remain poorly understood in the development of kidney organoids. In this study, parallel metabolite and transcriptome profiling of differentiating human pluripotent stem cells (hPSCs) to multicellular renal organoids revealed key metabolic drivers of the differentiation process. In the early stage, transitioning from hPSCs to nephron progenitor cells (NPCs), both the glutamine and the alanine-aspartate-glutamate pathways changed significantly, as detected by enrichment and pathway impact analyses. Intriguingly, hPSCs maintained their ability to generate NPCs, even when deprived of both glutamine and glutamate. Surprisingly, single cell RNA-Seq analysis detected enhanced maturation and enrichment for podocytes under glutamine-deprived conditions. Together, these findings illustrate a novel role of glutamine metabolism in regulating podocyte development.

bioengineering↗

bioRxiv2022

INSERT-seq enables high resolution mapping of genomically integrated DNA using nanopore sequencing

Comprehensive characterization of genome engineering with viral vectors, transposons, CRISPR/Cas mediated DNA integration and other DNA editors remains relevant for their development and safe use in human gene therapy. Currently, described methods for measuring DNA integration in edited cells rely on short read based technologies. Due to the repetitive nature of the human genome, short read based methods can potentially overlook insertion events in repetitive regions. We modelled the impact of read length in resolving insertion sites, which suggested a significant drop in insertion site detection with shorter read length. Based on that, we developed a method that combines targeted amplification of integrated DNA, UMI-based correction of PCR bias and Oxford Nanopore long-read sequencing for robust analysis of DNA integration in a genome. This method, called INSERT-seq, is capable of detecting events occurring at a frequency of up to 0.1%. INSERT-seq presents a complete handling of all insertions independently of repeat size. The experimental pipeline improves the number mappable insertions at repetitive regions by 7.3% and repeats larger than the long read sequencing size are processed computationally to perform a peak calling in a repeat database. INSERT-seq is a simple, cheap and robust method to quantitatively characterise DNA integration in diverse ex-vivo and in-vivo samples.

genomics↗