High-throughput dense reconstruction of cell lineages

The first meeting exclusively dedicated to the ‘High-throughput dense reconstruction of cell lineages' took place at Janelia Research Campus (Howard Hughes Medical Institute) from 14 to 18 April 2019. Organized by Tzumin Lee, Connie Cepko, Jorge Garcia-Marques and Isabel Espinosa-Medina, this meeting echoed the recent eruption of new tools that allow the reconstruction of lineages based on the phylogenetic analysis of DNA mutations induced during development. Combined with single-cell RNA sequencing, these tools promise to solve the lineage of complex model organisms at single-cell resolution. Here, we compile the conference consensus on the technological and computational challenges emerging from the use of the new strategies, as well as potential solutions.

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Single-Cell Signature Explorer for comprehensive visualization of single cell signatures across scRNA-seq datasets

Nucleic Acids Research ◽

10.1093/nar/gkz601 ◽

2019 ◽

Vol 47 (21) ◽

pp. e133-e133 ◽

Cited By ~ 23

Author(s):

Frédéric Pont ◽

Marie Tosolini ◽

Jean J Fournié

Keyword(s):

Lung Cancer ◽

Single Cell ◽

High Throughput ◽

Metabolic Pathways ◽

Single Cell Level ◽

Cell Level ◽

Human Pbmc ◽

Adult Testis ◽

Cell Lineages ◽

Gene Feature

Abstract The momentum of scRNA-seq datasets prompts for simple and powerful tools exploring their meaningful signatures. Here we present Single-Cell_Signature_Explorer (https://sites.google.com/site/fredsoftwares/products/single-cell-signature-explorer), the first method for qualitative and high-throughput scoring of any gene set-based signature at the single cell level and its visualization using t-SNE or UMAP. By scanning datasets for single or combined signatures, it rapidly maps any multi-gene feature, exemplified here with signatures of cell lineages, biological hallmarks and metabolic pathways in large scRNAseq datasets of human PBMC, melanoma, lung cancer and adult testis.

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Single cell based high-throughput Ig and TCR repertoire sequencing analysis in rhesus macaques

10.1101/2021.08.17.456682 ◽

2021 ◽

Author(s):

Evan S Walsh ◽

Tammy Tollison ◽

Hayden Brochu ◽

Brian Shaw ◽

Kayliegh Diveley ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

High Throughput ◽

Rhesus Macaques ◽

Expression Profiles ◽

Single Cells ◽

Model Organisms ◽

Sequencing Analysis ◽

Immune Repertoire ◽

Repertoire Sequencing

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired heavy- and light- chains of immunoglobulins (Ig) and VDJ- and VJ- chains of T cell receptors (TCR) from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. Here we employed custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single cell immune repertoire profiling. Using these rhesus specific assays, we sequenced Ig and TCR repertoires in over 60,000 cells from cryopreserved rhesus PBMC, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single cell level.

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Dense reconstruction of elongated cell lineages: overcoming suboptimum lineage encoding and sparse cell sampling

10.1101/2020.07.27.223321 ◽

2020 ◽

Author(s):

Ken Sugino ◽

Rosa L. Miyares ◽

Isabel Espinosa-Medina ◽

Hui-Min Chen ◽

Christopher J Potter ◽

...

Keyword(s):

Single Cell ◽

Cell Types ◽

Lineage Tracing ◽

Simple Method ◽

Cell Lineages ◽

Stem Cell Divisions ◽

Neuronal Lineage ◽

Dense Reconstruction ◽

Parameter Ranges ◽

Neuronal Diversification

AbstractAcquiring both lineage and cell-type information during brain development could elucidate transcriptional programs underling neuronal diversification. This is now feasible with single-cell RNA-seq combined with CRISPR-based lineage tracing, which generates genetic barcodes with cumulative CRISPR edits. This technique has not yet been optimized to deliver high-resolution lineage reconstruction of protracted lineages. Drosophila neuronal lineages are an ideal model to consider, as multiple lineages have been morphologically mapped at single-cell resolution. Here we find the parameter ranges required to encode a representative neuronal lineage emanating from 100 stem cell divisions. We derive the optimum editing rate to be inversely proportional to lineage depth, enabling encoding to persist across lineage progression. Further, we experimentally determine the editing rates of a Cas9-deaminase in cycling neural stem cells, finding near ideal rates to map elongated Drosophila neuronal lineages. Moreover, we propose and evaluate strategies to separate recurring cell-types for lineage reconstruction. Finally, we present a simple method to combine multiple experiments, which permits dense reconstruction of protracted cell lineages despite suboptimum lineage encoding and sparse cell sampling.

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