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Droplet scRNA-seq is not zero-inflated

Nature Biotechnology volume 38, pages 147–150 (2020)Cite this article

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To the Editor — Potential users of single-cell RNA-sequencing (scRNA-seq)1 often encounter a choice between high-throughput droplet-based methods and high-sensitivity plate-based methods. There is a widespread belief that scRNA-seq will often fail to generate measurements for some genes from some cells owing to technical molecular inefficiencies. It is believed that this causes data to have an overabundance of zero values compared to what is expected from random sampling and that this effect is particularly pronounced in droplet-based methods. Here I present an investigation of published data for technical controls in droplet-based scRNA-seq experiments that demonstrates that the number of zero values in the data is consistent with common distributional models of molecule sampling counts. Thus, any additional zero values in biological data likely result from biological variation or may reflect variation in gene abundance among cell types or cell states.

As scRNA-seq started to gain popularity, users expressed concern about the number of zero values among gene expression measures. That is, for any given gene, several cells showed no expression, even if expression was relatively high in other cells2,3,4. These ‘technical zeros’ are considered distinct from ‘biological zeros’, which are zero because the gene is not expressed5.

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Fig. 1: Comparing observed with expected zeros in scRNA-seq data.

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Acknowledgements

I thank E. da Veiga Beltrame for feedback on the manuscript, G. Eraslan for making fast code for fitting negative binomial models available, and the scientific community on Twitter for suggesting writing up this analysis as a manuscript. V.S. was funded in part by the EMBL International PhD Programme and NIH U19MH114830.

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  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Valentine Svensson

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  1. Valentine Svensson
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Correspondence to Valentine Svensson.

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Svensson, V. Droplet scRNA-seq is not zero-inflated. Nat Biotechnol 38, 147–150 (2020). https://doi.org/10.1038/s41587-019-0379-5

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