Quantitative Single-cell RNA-seq with Unique Molecular Identifiers

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2013 Dec 24

PMID 24363023

Citations 587

Authors

Saiful Islam

Amit Zeisel

Simon Joost

Gioele La Manno

Pawel Zajac

Maria Kasper

Peter Lonnerberg

Sten Linnarsson

Affiliations

Soon will be listed here.

Abstract

Single-cell RNA sequencing (RNA-seq) is a powerful tool to reveal cellular heterogeneity, discover new cell types and characterize tumor microevolution. However, losses in cDNA synthesis and bias in cDNA amplification lead to severe quantitative errors. We show that molecular labels--random sequences that label individual molecules--can nearly eliminate amplification noise, and that microfluidic sample preparation and optimized reagents produce a fivefold improvement in mRNA capture efficiency.

Citing Articles

Absolute Quantification of Nucleotide Variants in Cell-Free DNA via Quantitative NGS: Clinical Application in Non-Small Cell Lung Cancer Patients.

Herbreteau G, Marcq M, Sauzay C, Carpentier M, Pierre-Noel E, Pons-Tostivint E Cancers (Basel). 2025; 17(5).

PMID: 40075630 PMC: 11898635. DOI: 10.3390/cancers17050783.

Shaping early neural development by timed elevated tissue oxygen tension: Insights from multiomic analysis on human cerebral organoids.

Liu Y, Chung M, Lin H, Lee T, Cheng Y, Huang C Sci Adv. 2025; 11(11):eado1164.

PMID: 40073136 PMC: 11900884. DOI: 10.1126/sciadv.ado1164.

Identifying Essential Hub Genes and circRNA-Regulated ceRNA Networks in Hepatocellular Carcinoma.

Yu X, Xu H, Xing Y, Sun D, Li D, Shi J Int J Mol Sci. 2025; 26(4).

PMID: 40003874 PMC: 11855757. DOI: 10.3390/ijms26041408.

scCobra allows contrastive cell embedding learning with domain adaptation for single cell data integration and harmonization.

Zhao B, Song K, Wei D, Xiong Y, Ding J Commun Biol. 2025; 8(1):233.

PMID: 39948393 PMC: 11825689. DOI: 10.1038/s42003-025-07692-x.

Stoichiometric constraints for detection of EV-borne biomarkers in blood.

Zarovni N, Mladenovic D, Brambilla D, Panico F, Chiari M J Extracell Vesicles. 2025; 14(2):e70034.

PMID: 39901737 PMC: 11791308. DOI: 10.1002/jev2.70034.

References

Langmead B, Trapnell C, Pop M, Salzberg S . Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3):R25. PMC: 2690996. DOI: 10.1186/gb-2009-10-3-r25. View

Huang D, Sherman B, Lempicki R . Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4(1):44-57. DOI: 10.1038/nprot.2008.211. View

Picelli S, Bjorklund A, Faridani O, Sagasser S, Winberg G, Sandberg R . Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods. 2013; 10(11):1096-8. DOI: 10.1038/nmeth.2639. View

Galvin-Burgess K, Travis E, Pierson K, Vivian J . TGF-β-superfamily signaling regulates embryonic stem cell heterogeneity: self-renewal as a dynamic and regulated equilibrium. Stem Cells. 2012; 31(1):48-58. PMC: 3528825. DOI: 10.1002/stem.1252. View

Dar R, Razooky B, Singh A, Trimeloni T, McCollum J, Cox C . Transcriptional burst frequency and burst size are equally modulated across the human genome. Proc Natl Acad Sci U S A. 2012; 109(43):17454-9. PMC: 3491463. DOI: 10.1073/pnas.1213530109. View

Casbon J, Osborne R, Brenner S, Lichtenstein C . A method for counting PCR template molecules with application to next-generation sequencing. Nucleic Acids Res. 2011; 39(12):e81. PMC: 3130290. DOI: 10.1093/nar/gkr217. View

Kobayashi T, Mizuno H, Imayoshi I, Furusawa C, Shirahige K, Kageyama R . The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells. Genes Dev. 2009; 23(16):1870-5. PMC: 2725939. DOI: 10.1101/gad.1823109. View

Maurer J, Nelson B, Cecena G, Bajpai R, Mercola M, Terskikh A . Contrasting expression of keratins in mouse and human embryonic stem cells. PLoS One. 2008; 3(10):e3451. PMC: 2565505. DOI: 10.1371/journal.pone.0003451. View

Mortazavi A, Williams B, McCue K, Schaeffer L, Wold B . Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7):621-8. DOI: 10.1038/nmeth.1226. View

10.

Shalek A, Satija R, Adiconis X, Gertner R, Gaublomme J, Raychowdhury R . Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells. Nature. 2013; 498(7453):236-40. PMC: 3683364. DOI: 10.1038/nature12172. View

11.

Meyer L, Zweig A, Hinrichs A, Karolchik D, Kuhn R, Wong M . The UCSC Genome Browser database: extensions and updates 2013. Nucleic Acids Res. 2012; 41(Database issue):D64-9. PMC: 3531082. DOI: 10.1093/nar/gks1048. View

12.

Chambers I, Silva J, Colby D, Nichols J, Nijmeijer B, Robertson M . Nanog safeguards pluripotency and mediates germline development. Nature. 2007; 450(7173):1230-4. DOI: 10.1038/nature06403. View

13.

Raj A, Peskin C, Tranchina D, Vargas D, Tyagi S . Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 2006; 4(10):e309. PMC: 1563489. DOI: 10.1371/journal.pbio.0040309. View

14.

Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N . mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods. 2009; 6(5):377-82. DOI: 10.1038/nmeth.1315. View

15.

Kivioja T, Vaharautio A, Karlsson K, Bonke M, Enge M, Linnarsson S . Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2011; 9(1):72-4. DOI: 10.1038/nmeth.1778. View

16.

Shiroguchi K, Jia T, Sims P, Xie X . Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. Proc Natl Acad Sci U S A. 2012; 109(4):1347-52. PMC: 3268301. DOI: 10.1073/pnas.1118018109. View

17.

Islam S, Kjallquist U, Moliner A, Zajac P, Fan J, Lonnerberg P . Highly multiplexed and strand-specific single-cell RNA 5' end sequencing. Nat Protoc. 2012; 7(5):813-28. DOI: 10.1038/nprot.2012.022. View

18.

Carter M, Stagg C, Falco G, Yoshikawa T, Bassey U, Aiba K . An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells. Gene Expr Patterns. 2008; 8(3):181-98. PMC: 2238805. DOI: 10.1016/j.gep.2007.10.009. View

19.

Hashimshony T, Wagner F, Sher N, Yanai I . CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. Cell Rep. 2012; 2(3):666-73. DOI: 10.1016/j.celrep.2012.08.003. View

20.

Islam S, Kjallquist U, Moliner A, Zajac P, Fan J, Lonnerberg P . Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res. 2011; 21(7):1160-7. PMC: 3129258. DOI: 10.1101/gr.110882.110. View