However, the scale and complexity of the data produced by these methods are rapidly becoming a bottleneck for the accurate inference of phylogenies. Finally, these trees (corresponding to the maximum parsimony solutions for each group) are returned and merged into a complete phylogeny Next, when these groups reach a predefined threshold, we infer Steiner trees, finding the tree of minimum weight connecting all observed cell states across all possible evolutionary histories in a “potential graph,” using integer linear programming (ILP). First, the greedy phase identifies mutations that likely occurred early in the lineage and splits cells recursively into groups based on the presence or absence of these mutations. Cassiopeia-Hybrid merges two novel algorithms: the “greedy” (Cassiopeia-Greedy) and “Steiner tree/integer linear programming” (Cassiopeia-ILP) approaches. Cassiopeia takes as input a “character matrix,” summarizing the mutations seen at heritable target sites across cells. c The Cassiopeia reconstruction framework. These clones are then passed to Cassiopeia’s reconstruction module for phylogenetic inference.
CASSIOPEIA COUNTERS SOFTWARE
The Cassiopeia software includes modules for the processing of target-site sequencing data: first, identical reads are collapsed together and similar reads are error corrected second, these reads are locally aligned to a reference sequence and indels are called from this alignment third, unique molecules are aggregated per cell and intra-doublets are called from this information finally, the cell population is segmented into clones (or lineage groups) and inter-doublets are called. Finally, the character matrix is used to infer phylogenies by one of various methods. Next, the indels are read off from single cells (e.g., by scRNA-seq) and summarized in a “character matrix,” where rows represent cells, columns represent individual target sites (or “characters”), and values represent the observed indel (or “character state”). First, cells are engineered with lineage tracing machinery, namely Cas9 that cuts a genomic target site the target site accrues heritable, Cas9-induced indels (“character states”). a The workflow of a lineage tracing experiment. Cassiopeia and its benchmarking resources are publicly available at A generalized approach to lineage tracing and lineage reconstruction. Together, these should broadly enable large-scale mammalian lineage tracing efforts. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Finally, we generate the most complex experimental lineage tracing dataset to date, 34,557 human cells continuously traced over 15 generations, and use it for benchmarking phylogenetic inference approaches. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. First, we introduce Cassiopeia-a suite of scalable maximum parsimony approaches for tree reconstruction. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. Contact us today for a tour and start investing in your future.The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing.
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