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Generation of Centered Log-Ratio Normalized Antibody-Derived Tag Counts from Large Single-Cell Sequencing Datasets

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Single-Cell Protein Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2386))

Abstract

Recent developments in single-cell analysis has provided the ability to assay >50 surface-level proteins by combining oligo-conjugated antibodies with sequencing technology. These methods, such as CITE-seq and REAP-seq, have added another modality to single-cell analysis, enhancing insight across many biological subdisciplines. While packages like Seurat have greatly facilitated analysis of single-cell protein expression, the practical steps to carry out the analysis with increasingly larger datasets have been fragmented. In addition, using data visualizations, I will highlight some details about the centered log-ratio (CLR) normalization of antibody-derived tag (ADT) counts that may be overlooked. In this method chapter, I provide detailed steps to generate CLR-normalized CITE-seq data using cloud computing from a large CITE-seq dataset.

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References

  1. Peterson VM, Zhang KX, Kumar N, Wong J, Li L, Wilson DC, Moore R, McClanahan TK, Sadekova S, Klappenbach JA (2017) Multiplexed quantification of proteins and transcripts in single cells. Nat Biotechnol 35(10):936–939. https://doi.org/10.1038/nbt.3973

    Article  CAS  PubMed  Google Scholar 

  2. Stoeckius M, Hafemeister C, Stephenson W, Houck-Loomis B, Chattopadhyay PK, Swerdlow H, Satija R, Smibert P (2017) Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14(9):865–868. https://doi.org/10.1038/nmeth.4380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mulè MP, Martins AJ, Tsang JS (2020) Normalizing and denoising protein expression data from droplet-based single cell profiling. bioRxiv 2020.02.24.963603. https://doi.org/10.1101/2020.02.24.963603

  4. Navale V, Bourne PE (2018) Cloud computing applications for biomedical science: a perspective. PLoS Comput Biol 14(6):e1006144. https://doi.org/10.1371/journal.pcbi.1006144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM 3rd, Hao Y, Stoeckius M, Smibert P, Satija R (2019) Comprehensive integration of single-cell data. Cell 177(7):1888–1902.e21. https://doi.org/10.1016/j.cell.2019.05.031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Stoeckius M, Zheng S, Houck-Loomis B et al (2018) Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol 19:224. https://doi.org/10.1186/s13059-018-1603-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Becht E, McInnes L, Healy J et al (2019) Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol 37:38–44. https://doi.org/10.1038/nbt.4314

    Article  CAS  Google Scholar 

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Correspondence to Benjamin Lacar .

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Lacar, B. (2022). Generation of Centered Log-Ratio Normalized Antibody-Derived Tag Counts from Large Single-Cell Sequencing Datasets. In: Ooi, A.T. (eds) Single-Cell Protein Analysis. Methods in Molecular Biology, vol 2386. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1771-7_14

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  • DOI: https://doi.org/10.1007/978-1-0716-1771-7_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1770-0

  • Online ISBN: 978-1-0716-1771-7

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