title: Assessing characteristics of RNA amplification methods for single cell RNA sequencing.
abstract: Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known.
  
  
  
  Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules.
  
  
  
  Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.
authors.affiliation: Department of Genomics and Computational Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Hannah R
authors.initials: HR
authors.lastname: Dueck
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Rizi
authors.initials: R
authors.lastname: Ai
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Adrian
authors.initials: A
authors.lastname: Camarena
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Ding
authors.affiliation: Department of Physiology & Biophysics, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA.
authors.forename: Reymundo
authors.initials: R
authors.lastname: Dominguez
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Evgrafov
authors.affiliation: Illumina, Inc., San Diego, CA, USA.
authors.forename: Jian-Bing
authors.initials: JB
authors.lastname: Fan
authors.affiliation: Department of Biology, University of Pennsylvania, 415 S. University Ave, Philadelphia, PA, 19104, USA.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Fisher
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Herstein
authors.affiliation: Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Present address: Allen Institute for Brain Science, Seattle, WA, USA.
authors.forename: Tae Kyung
authors.initials: TK
authors.lastname: Kim
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Jae Mun Hugo
authors.initials: JM
authors.lastname: Kim
authors.affiliation: Department of Physiology & Biophysics, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA.
authors.forename: Ming-Yi
authors.initials: MY
authors.lastname: Lin
authors.affiliation: Department of Bioengineering, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.affiliation: Department of Neurological Surgery, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Mack
authors.affiliation: Department of Biology, University of Pennsylvania, 415 S. University Ave, Philadelphia, PA, 19104, USA.
authors.forename: Sean
authors.initials: S
authors.lastname: McGroty
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Nguyen
authors.affiliation: Illumina, Inc., San Diego, CA, USA.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Salathia
authors.affiliation: Department of Biology, University of Pennsylvania, 415 S. University Ave, Philadelphia, PA, 19104, USA.
authors.forename: Jamie
authors.initials: J
authors.lastname: Shallcross
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Tade
authors.initials: T
authors.lastname: Souaiaia
authors.affiliation: Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Spaethling
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Walker
authors.affiliation: Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Jinhui
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Andre
authors.initials: A
authors.lastname: Wildberg
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Lina
authors.initials: L
authors.lastname: Zheng
authors.affiliation: Department of Physiology & Biophysics, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Chow
authors.affiliation: Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: James
authors.initials: J
authors.lastname: Eberwine
authors.affiliation: Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Knowles
authors.affiliation: Department of Bioengineering, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Biology, University of Pennsylvania, 415 S. University Ave, Philadelphia, PA, 19104, USA. Junhyong@upenn.edu.
authors.forename: Junhyong
authors.initials: J
authors.lastname: Kim
mesh_terms: D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D021141:Nucleic Acid Amplification Techniques; D012313:RNA / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis / Q000379:methods
chemical_list: D012313:RNA
keywords: Bioinformatics; Biotechnology; Genomics; Single-cell RNA-sequencing