Project description:Cellular barcoding using heritable synthetic barcodes coupled to high throughput sequencing is a powerful technique for the accurate tracing of clonal lineages in a wide variety of biological contexts. Recent studies have integrated cellular barcoding with a single-cell transcriptomics readout, extending the capabilities of these lineage tracing methods to the single-cell level. However there remains a lack of scalable and standardised open-source tools to pre-process and visualise both bulk and single-cell level cellular barcoding datasets. Here, we describe bartools, an open-source R-based toolkit that streamlines the pre-processing, analysis and visualisation of synthetic cellular barcoding datasets. In addition, we developed BARtab, a portable and scalable Nextflow pipeline that automates upstream barcode extraction, quality control, filtering and enumeration from high throughput sequencing data. In addition to population-level cellular barcoding datasets, BARtab and bartools contain methods for the extraction, annotation, and visualisation of transcribed barcodes from single-cell RNA-seq and spatial transcriptomics experiments, thus extending the analytical toolbox to also support novel expressed cellular barcoding methodologies. We showcase the integrated BARtab and bartools workflow through the analysis of bulk, single-cell, and spatial transcriptomics cellular barcoding datasets.
Project description:Detecting strain-specific barcodes with mass spectrometry can facilitate the screening of genetically engineered bacterial libraries. Here, we introduce intact protein barcoding, a method to measure protein-based library barcodes and metabolites using flow-injection mass spectrometry (FI-MS). Protein barcodes are based on ubiquitin with N-terminal tags of six amino acids. We demonstrate that FI-MS detects intact ubiquitin proteins and identifies the mass of N-terminal barcodes. In the same analysis, we measured relative concentrations of primary metabolites. We constructed 6 ubiquitin-barcoded CRISPRi strains targeting metabolic enzymes, and analyzed their metabolic profiles and ubiquitin barcodes. FI-MS detected barcodes and distinct metabolome changes in CRISPRi-targeted pathways. We demonstrate the scalability of intact protein barcoding by measuring 132 ubiquitin barcodes in microtiter plates. These results show that intact protein barcoding enables fast and simultaneous detection of library barcodes and intracellular metabolites, opening up new possibilities for mass spectrometry-based barcoding.
Project description:5000 target cells per sample where sequenced from 2 gastric tumor samples and 1 sample of adjacent gastric mucosa Twist2Cre;Lkb1fl/+;RosaRTdtomato+/- mice. Drop-seq 10X Genomics (10X v3 library kit) was used for single cell barcoding and library preparation. Samples were sequenced with Novaseq6000.
Project description:We present spatially resolved chromatin accessibility profiling of tissue sections via next-generation sequencing by combining in situ ATAC-seq chemistry and microfluidic deterministic barcoding. Mouse and human tissues were profiled by spatial-ATAC-seq.
Project description:we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultrahigh-throughput single-cell joint profiling of multiple epigenetic proteins. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets.
Project description:Mutation analysis in plasma samples with low ctDNA levels using a molecular barcoding technology, i.e. the single target approach SiMSen-seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing).
Project description:It is elusive whether clonal selection of tumor cells in response to ionizing radiation (IR) is a deterministic or stochastic process. With high resolution clonal barcoding and tracking of over 400.000 HNSCC patient-derived tumor cells the clonal dynamics of tumor cells in response to IR was analysed. Fractionated IR induced a strong selective pressure for clonal reduction. This significantly exceeded uniform clonal survival probabilities indicative for a strong clone-to clone difference within tumor cells. Survival to IR is driven by a deterministic clonal selection of a smaller population which commonly survives radiation, while increased clonogenic capacity is a result of clonal competition of cells which have been selected stochastically. The ratio of these parameters is amenable to radiation sensitivity which correlates to prognostic biomarkers of HNSCC. Evidence for the existence of a rare subpopulation with an intrinsically radiation resistant phenotype was found at a frequency of 0.6-3.3%. With cellular barcoding we introduce a novel functional heterogeneity associated qualitative readout for evaluating the contribution of stochastic and deterministic clonal selection processes in response to IR.
