PT  - JOURNAL ARTICLE
AU  - Lukas M. Weber
AU  - Ariel A. Hippen
AU  - Peter F. Hickey
AU  - Kristofer C. Berrett
AU  - Jason Gertz
AU  - Jennifer Anne Doherty
AU  - Casey S. Greene
AU  - Stephanie C. Hicks
TI  - Genetic demultiplexing of pooled single-cell RNA-sequencing samples in cancer facilitates effective experimental design
AID  - 10.1101/2020.11.06.371963
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2020.11.06.371963
4099  - http://biorxiv.org/content/early/2021/07/19/2020.11.06.371963.short
4100  - http://biorxiv.org/content/early/2021/07/19/2020.11.06.371963.full
AB  - Background Pooling cells from multiple biological samples prior to library preparation within the same single-cell RNA sequencing experiment provides several advantages, including lower library preparation costs and reduced unwanted technological variation, such as batch effects. Computational demultiplexing tools based on natural genetic variation between individuals provide a simple approach to demultiplex samples, which does not require complex additional experimental procedures. However, these tools have not been evaluated in cancer, where somatic variants, which could differ between cells from the same sample, may obscure the signal in natural genetic variation.Results Here, we performed in silico benchmark evaluations by combining raw sequencing reads from multiple single-cell samples in high-grade serous ovarian cancer, which has a high copy number burden, and lung adenocarcinoma, which has a high tumor mutational burden. Our results confirm that genetic demultiplexing tools can be effectively deployed on cancer tissue using a pooled experimental design, although high proportions of ambient RNA from cell debris reduce performance.Conclusions This strategy provides significant cost savings through pooled library preparation. To facilitate similar analyses at the experimental design phase, we provide freely accessible code and a reproducible Snakemake workflow built around the best-performing tools found in our in silico benchmark evaluations, available at https://github.com/lmweber/snp-dmx-cancer.Competing Interest StatementThe authors have declared no competing interest.