Assessing characteristics of RNA amplification methods for single cell RNA sequencing.

Autor:	Dueck HR; Department of Genomics and Computational Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Ai R; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA., Camarena A; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Ding B; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA., Dominguez R; Department of Physiology & Biophysics, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA., Evgrafov OV; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Fan JB; Illumina, Inc., San Diego, CA, USA., Fisher SA; Department of Biology, University of Pennsylvania, 415 S. University Ave, Philadelphia, PA, 19104, USA., Herstein JS; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Kim TK; Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.; Present address: Allen Institute for Brain Science, Seattle, WA, USA., Kim JM; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Lin MY; Department of Physiology & Biophysics, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA., Liu R; Department of Bioengineering, University of California at San Diego, La Jolla, CA, USA., Mack WJ; Department of Neurological Surgery, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA., McGroty S; Department of Biology, University of Pennsylvania, 415 S. University Ave, Philadelphia, PA, 19104, USA., Nguyen JD; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Salathia N; Illumina, Inc., San Diego, CA, USA., Shallcross J; Department of Biology, University of Pennsylvania, 415 S. University Ave, Philadelphia, PA, 19104, USA., Souaiaia T; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Spaethling JM; Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Walker CP; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Wang J; Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Wang K; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Wang W; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA., Wildberg A; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA., Zheng L; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA, USA., Chow RH; Department of Physiology & Biophysics, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, USA., Eberwine J; Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Knowles JA; Department of Psychiatry & The Behavioral Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA., Zhang K; Department of Bioengineering, University of California at San Diego, La Jolla, CA, USA., Kim J; Department of Biology, University of Pennsylvania, 415 S. University Ave, Philadelphia, PA, 19104, USA. Junhyong@upenn.edu.
Jazyk:	angličtina
Zdroj:	BMC genomics [BMC Genomics] 2016 Nov 24; Vol. 17 (1), pp. 966. Date of Electronic Publication: 2016 Nov 24.
DOI:	10.1186/s12864-016-3300-3
Abstrakt:	Background: 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. Results: 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. Conclusions: Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.
Databáze:	MEDLINE
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