Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

View ORCID ProfileTerkild Brink Buus, View ORCID ProfileAlberto Herrera, View ORCID ProfileEllie Ivanova, View ORCID ProfileEleni Mimitou, View ORCID ProfileAnthony Cheng, View ORCID ProfileRamin Sedaghat Herati, View ORCID ProfileThales Papagiannakopoulos, View ORCID ProfilePeter Smibert, View ORCID ProfileNiels Ødum, View ORCID ProfileSergei B. Koralov
doi: https://doi.org/10.1101/2020.06.15.153080
Terkild Brink Buus
1Department of Pathology, New York University School of Medicine, New York, NY, USA
2LEO Foundation Skin Immunology Research Center, Department of Immunology & Microbiology, University of Copenhagen, Copenhagen, Denmark
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Terkild Brink Buus
  • For correspondence: Terkild.Buus@sund.ku.dk Sergei.Koralov@nyulangone.org
Alberto Herrera
1Department of Pathology, New York University School of Medicine, New York, NY, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Alberto Herrera
Ellie Ivanova
1Department of Pathology, New York University School of Medicine, New York, NY, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Ellie Ivanova
Eleni Mimitou
3Technology Innovation Lab, New York Genome Center, New York, NY, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Eleni Mimitou
Anthony Cheng
4Department of Genetic and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT, USA
5Department of Biostatistics and Epidemiology, School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Anthony Cheng
Ramin Sedaghat Herati
6NYU Langone Vaccine Center, Department of Medicine, New York University School of Medicine, New York, NY
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Ramin Sedaghat Herati
Thales Papagiannakopoulos
1Department of Pathology, New York University School of Medicine, New York, NY, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Thales Papagiannakopoulos
Peter Smibert
3Technology Innovation Lab, New York Genome Center, New York, NY, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Peter Smibert
Niels Ødum
2LEO Foundation Skin Immunology Research Center, Department of Immunology & Microbiology, University of Copenhagen, Copenhagen, Denmark
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Niels Ødum
Sergei B. Koralov
1Department of Pathology, New York University School of Medicine, New York, NY, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Sergei B. Koralov
  • For correspondence: Terkild.Buus@sund.ku.dk Sergei.Koralov@nyulangone.org
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Data/Code
  • Preview PDF
Loading

Abstract

Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. In this study, we investigated the response of oligo-conjugated antibodies towards four variables: Concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations cause unnecessarily high background and that concentrations can be drastically reduced without loss of biological information. Reducing volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background and reduces costs. Background signal can account for a major fraction of the total sequencing and is primarily derived from antibodies used at high concentrations. Together, this study provides new insight into the titration response and background of oligo-conjugated antibodies and offers concrete guidelines on how such panels can be improved.

Impact statement Oligo-conjugated antibodies are a powerful tool but require thorough optimization to reduce background signal, increase sensitivity, and reduce sequencing costs.

Competing Interest Statement

PS is co-inventor of a patent related to the single cell technology utilized in this study (US provisional patent application 62/515.180).

Footnotes

  • In the revised manuscript, we have done our best to address all the points raised by the preprint peer-reviewers. We have conducted an additional CITE-seq experiment comparing the signal and background from pre-titration concentrations with our suggested concentrations for all 52 antibodies as well as using our recommended reduced staining volume and cell numbers. This experiment shows marked improvements in signal and sequencing balance, as well as reduced background across the panel. In addition to the improved signal-to-noise, we estimate that the antibody costs of our suggested approach is ~34 fold cheaper than using vendor recommendations and requires less sequencing depth to acquire same signal. Prompted by the suggestions from our reviewers we have also updated the figures to improve clarity, show cell type annotations and included a guide to the high-information-content titration plots presented in the manuscript.

  • https://doi.org/10.6084/m9.figshare.c.5018987

  • https://github.com/Terkild/CITE-seq_optimization

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted March 13, 2021.
Download PDF
Data/Code
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Improving oligo-conjugated antibody signal in multimodal single-cell analysis
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Improving oligo-conjugated antibody signal in multimodal single-cell analysis
Terkild Brink Buus, Alberto Herrera, Ellie Ivanova, Eleni Mimitou, Anthony Cheng, Ramin Sedaghat Herati, Thales Papagiannakopoulos, Peter Smibert, Niels Ødum, Sergei B. Koralov
bioRxiv 2020.06.15.153080; doi: https://doi.org/10.1101/2020.06.15.153080
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
Improving oligo-conjugated antibody signal in multimodal single-cell analysis
Terkild Brink Buus, Alberto Herrera, Ellie Ivanova, Eleni Mimitou, Anthony Cheng, Ramin Sedaghat Herati, Thales Papagiannakopoulos, Peter Smibert, Niels Ødum, Sergei B. Koralov
bioRxiv 2020.06.15.153080; doi: https://doi.org/10.1101/2020.06.15.153080

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Genomics
Subject Areas
All Articles
  • Animal Behavior and Cognition (3482)
  • Biochemistry (7329)
  • Bioengineering (5301)
  • Bioinformatics (20212)
  • Biophysics (9985)
  • Cancer Biology (7706)
  • Cell Biology (11273)
  • Clinical Trials (138)
  • Developmental Biology (6425)
  • Ecology (9923)
  • Epidemiology (2065)
  • Evolutionary Biology (13292)
  • Genetics (9353)
  • Genomics (12559)
  • Immunology (7681)
  • Microbiology (18964)
  • Molecular Biology (7421)
  • Neuroscience (40915)
  • Paleontology (298)
  • Pathology (1226)
  • Pharmacology and Toxicology (2130)
  • Physiology (3145)
  • Plant Biology (6842)
  • Scientific Communication and Education (1271)
  • Synthetic Biology (1893)
  • Systems Biology (5299)
  • Zoology (1086)