Question: How can we improve species assignment and multiplet rate estimation in barnyard samples using Loupe Browser?
Answer: In Barnyard samples, species assignment is executed for each genome. However, there can be instances where species assignment may lack robustness, leading to mis-assignment of species to a cell. This can affect the accuracy of multiplet rate estimates. To achieve more reliable species assignments, leveraging the .cloupe file is recommended. Specifically, the feature plot option can be employed to manually assign cells to species. Below is a step-by-step approach to manual species assignment using a public dataset from Loupe to illustrate this process.
- Initial Selection: Open the Loupe file using Loupe Browser and select the projection type as "feature plot". Select the GRCh38_Sum in the Y-axis and GRCm39_Sum in the X-axis from the Loupe Feature Plot.
Upon selection, the default view will display as shown in the screenshot below.
- Manual Assignment: Utilize the lasso tool to delineate barcodes for human, mouse and multiplet cells. This manual selection should give a better representation of species assignment. For example, select the cluster identified as mouse cells, assign them a cluster label "Mouse cells" and categorize them under a group named "ManualSpeciesAssignment". Repeat this process for and multiplet clusters (see screenshot below).
The reason for doing the above process, where we select cells along the axis is that, in some cases, you might encounter mis-classified multiplets in the web summary file. For example, illustrated below is a bi-plot from a web summary result from another dataset, where a small portion of the mouse cells was identified as multiplets (see red arrow).
- Final Review: After manual assignment, the final view will reflect the updated species distribution. As illustrated in the below screenshot, the revised assignment indicates a slightly higher number of mouse cells in this dataset. The barcodes corresponding to these clusters can then be utilized for further downstream analysis (see screenshot below).
Products: Single Cell Gene Expression
Last Updated: July 2024