Biology subjects

Emiliani, F. E.

Publications and source records attributed to Emiliani, F. E..

2 recordsLinked to original sources

bioRxiv2024

Hierarchal single-cell lineage tracing reveals differential fate commitment of CD8 T-cell clones in response to acute infection.

Generating balanced populations of CD8 effector and memory T cells is necessary for immediate and durable immunity to infections and cancer. Yet, a definitive understanding of CD8 differentiation remains unclear. We used CARLIN, a processive lineage recording mouse model with single-cell RNA-seq and TCR-seq to track endogenous antigen-specific CD8 T cells during acute viral infection. We identified a diverse repertoire of expanded T-cell clones represented by seven transcriptional states. TCR enrichment analysis revealed differential memory- or effector-fate biases within clonal populations. Shared Vb segments and amino acid motifs were found within biased categories despite high TCR diversity. Using single-cell CARLIN barcode-seq we tracked multi-generational clones and found that unlike unbiased or memory-biased clones, which stably retain their fate profiles, effector-biased clones could adopt memory- or effector-bias within subclones. Collectively, our study demonstrates that a heterogenous T-cell repertoire specific for a shared antigen is composed of clones with distinct TCR-intrinsic fate-biases.

immunology↗

bioRxiv2022

Circuit-seq: Circular reconstruction of cut in vitro transposed plasmids using Nanopore sequencing

Recombinant DNA is a fundamental tool in biotechnology and medicine. Validation of the resulting plasmid sequence is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence for approximately $1.50 per plasmid. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating accurate and contiguous plasmid maps. We then leverage our long read-data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, with low startup costs make it practical for individual laboratories.

molecular biology↗