Project description:To investigate immunoediting at the primary tumour, we used DNA barcoding combined with NGS. By stably integrating 4T1 murine cancer cell line with 250000 unique DNA barcodes (1 barcode per cell), we can trace how barcode (and therefore subclonal) diversity changes over time and after treatment with immunotherapy.
Project description:To investigate immunoediting at the primary tumour, we used DNA barcoding combined with NGS. By stably integrating 4T1 murine cancer cell line with 5000 unique DNA barcodes (1 barcode per cell), we can trace how barcode (and therefore subclonal) diversity changes over time and after treatment with immunotherapy.
Project description:To investigate immunoediting at the primary tumour, we used DNA barcoding combined with NGS. By stably integrating 4T1 murine cancer cell line with 5000 unique DNA barcodes (1 barcode per cell), we can trace how barcode (and therefore subclonal) diversity changes over time and after treatment with immunotherapy.
Project description:To investigate immunoediting at the primary tumour, we used DNA barcoding combined with NGS. By stably integrating 4T1 murine cancer cell line with 5000 unique DNA barcodes (1 barcode per cell), we can trace how barcode (and therefore subclonal) diversity changes over time and after treatment with immunotherapy.
Project description:To investigate immunoediting at the primary tumour, we used DNA barcoding combined with NGS. By stably integrating EMT6 murine cancer cell line with 5000 unique DNA barcodes (1 barcode per cell), we can trace how barcode (and therefore subclonal) diversity changes over time and after treatment with immunotherapy.
Project description:DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.
Project description:Droplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct both short-read and long-read sequencing, thereby allowing users to recover more reads per cell that permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and multiple myeloma cell lines to evaluate differential isoform usage and Ewing’s sarcoma cells to demonstrate Ig fusion transcript analysis.
Project description:Here we compare the performance of these three approaches (inDrop, Drop-seq and 10x) using the same kind of sample with a unified data processing pipeline. We generated 2-3 replicates for each method using lymphoblastoid cell line GM12891. The average sequencing depth was around 50-60k reads per cell barcode. We also developed a versatile and rapid data processing workflow and applied it for all datasets. Cell capture efficiency, effective read ratio, barcode detection error and transcript detection sensitivity were analyzed as well.
