A Caltech Library Service

BUTTERFLY: addressing the pooled amplification paradox with unique molecular identifiers in single-cell RNA-seq

Gustafsson, Johan and Robinson, Jonathan and Nielsen, Jens and Pachter, Lior (2021) BUTTERFLY: addressing the pooled amplification paradox with unique molecular identifiers in single-cell RNA-seq. Genome Biology, 22 . Art. No. 174. ISSN 1474-760X. PMCID PMC8188791. doi:10.1186/s13059-021-02386-z.

[img] PDF - Published Version
Creative Commons Attribution.

[img] PDF (August 5, 2020) - Submitted Version
Creative Commons Attribution.

[img] PDF - Supplemental Material
Creative Commons Attribution.

[img] MS Excel (Table S1) - Supplemental Material
Creative Commons Attribution.

[img] MS Word (Review history) - Supplemental Material
Creative Commons Attribution.


Use this Persistent URL to link to this item:


The incorporation of unique molecular identifiers (UMIs) in single-cell RNA-seq assays makes possible the identification of duplicated molecules, thereby facilitating the counting of distinct molecules from sequenced reads. However, we show that the naïve removal of duplicates can lead to a bias due to a “pooled amplification paradox,” and we propose an improved quantification method based on unseen species modeling. Our correction called BUTTERFLY uses a zero truncated negative binomial estimator implemented in the kallisto bustools workflow. We demonstrate its efficacy across cell types and genes and show that in some cases it can invert the relative abundance of genes.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle ItemCode ItemCode Paper
Gustafsson, Johan0000-0001-5072-2659
Robinson, Jonathan0000-0001-8567-5960
Nielsen, Jens0000-0002-9955-6003
Pachter, Lior0000-0002-9164-6231
Alternate Title:Addressing the pooled amplification paradox with unique molecular identifiers in single-cell RNA-seq
Additional Information:© The Author(s). 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data. Received 27 August 2020; Accepted 21 May 2021; Published 08 June 2021. We thank Pall Melsted, Sina Booeshaghi, and Joseph Min for helpful suggestions on the project and on the integration of BUTTERFLY in bustools. Availability of data and materials: Means to access the datasets analyzed during the current study are listed in Additional file 1: Table S2. The source code as well as Jupyter notebooks for generating the figures is available in GitHub [36], as well as the source code for the branch of bustools used in this project [37]. Snapshots of the repositories are available in Zenodo [38]. Jupiter notebooks have not been produced for Fig. 5 and the data generation for Additional file 1: Fig. S6, S24, and S25, due to difficulties in setting up the right versions of R packages, but the code is directly available in the Github repository. All code is released under the BSD 2-clause license, except for the R file “modZTNB.R,” which is released under a GPL3 license to comply with the GPL3 license of PreseqR. Review history: The review history is available as Additional file 3. Peer review information: Barbara Cheifet was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. This work was supported by funding from the Knut and Alice Wallenberg foundation (J.N.), the National Cancer Institute of the National Institutes of Health under award number F32CA220848 (J.R.), and NIH U19MH114830 (L.P.). Author Contributions: Conceptualization, J.G., L.P.; Methodology, J.G, L.P.; software, J.G. ; writing—original draft, J.G., L.P.; writing—review and editing, J.G., L.P., J.R., J.N.; supervision, L.P., J.R., J.N.; funding acquisition, L.P., J.R., J.N. The authors read and approved the final manuscript. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. The authors declare that they have no competing interests.
Funding AgencyGrant Number
Knut and Alice Wallenberg FoundationUNSPECIFIED
NIH Postdoctoral FellowshipF32CA220848
Subject Keywords:Single-cell RNA-Seq; UMI; Droplet-based; PCR; Bias; Amplification; Batch correction; Correction
PubMed Central ID:PMC8188791
Record Number:CaltechAUTHORS:20200707-114817234
Persistent URL:
Official Citation:Gustafsson, J., Robinson, J., Nielsen, J. et al. BUTTERFLY: addressing the pooled amplification paradox with unique molecular identifiers in single-cell RNA-seq. Genome Biol 22, 174 (2021).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104254
Deposited By: Tony Diaz
Deposited On:07 Jul 2020 18:57
Last Modified:12 Jul 2021 17:58

Repository Staff Only: item control page