Biology subjects

Hwang, B.

Publications and source records attributed to Hwang, B..

2 recordsLinked to original sources

bioRxiv2018

TnClone: high-throughput clonal analysis usingTn5-mediated library construction and de novo assembly

A typical molecular cloning procedure requires Sanger sequencing for validation, which becomes cost-prohibitive and labour-intensive for large-scale clonal analysis of genotype-phenotype studies. Here we present a Tn5-mediated clonal analysis platform TnClone, which uses next-generation sequencing (NGS) to rapidly and cost-effectively analyze a large number of clones. We also developed a user-friendly graphical user interface and have provided general guidelines for conducting validation experiments. Using TnClone, we achieved more than 20-fold cost reduction compared with the cost incurred using conventional Sanger sequencing and detected low-frequency mutant clones (~10%) in mixed samples. We tested our programme and achieved 99.4% sensitivity. Our platform provides rapid turnaround with minimal hands-on time for secondary evaluation as NGS technology continues to evolve.

synthetic biology↗

bioRxiv2018

Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements

Determining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods have provided new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we developed a genome editing strategy using a cytidine deaminase fused with inactive Cas9 (dCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns served as a genetic barcode, which was induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.

bioengineering↗