Background: Although single-cell RNA sequencing of xenograft samples has been widely used, no comprehensive bioinformatics pipeline is available for human and mouse mixed single-cell analyses. Considering the numerous homologous genes across the human and mouse genomes, misalignment errors should be evaluated, and a new algorithm is required. We assessed the extents and effects of misalignment errors and exonic multi-mapping events when using human and mouse combined reference data and developed a new bioinformatics pipeline with expression-based species deconvolution to minimize errors. We also evaluated false-positive signals presumed to originate from ambient RNA of the other species and address the importance to computationally remove them. Result: Error when using combined reference account for an average of 0.78% of total reads, but such reads were concentrated to few genes that were greatly affected. Human and mouse mixed single-cell data, analyzed using our pipeline, clustered well with unmixed data and showed higher k-nearest-neighbor batch effect test and Local Inverse Simpson’s Index scores than those derived from Cell Ranger (10 × Genomics). We also applied our pipeline to multispecies multisample single-cell library containing breast cancer xenograft tissue and successfully identified all samples using genomic array and expression. Moreover, diverse cell types in the tumor microenvironment were well captured. Conclusion: We present our bioinformatics pipeline for mixed human and mouse single-cell data, which can also be applied to pooled libraries to obtain cost-effective single-cell data. We also address misalignment, multi-mapping error, and ambient RNA as a major consideration points when analyzing multispecies single-cell data.

Original languageEnglish
Article number157
JournalBMC Bioinformatics
Issue number1
StatePublished - Dec 2022


  • Bioinformatics pipeline
  • Patient-derived xenograft
  • Single-cell sequencing


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