title: Using normalization to resolve RNA-Seq biases caused by amplification from minimal input.
abstract: RNA-Seq has become a widely used method to study transcriptomes, and it is now possible to perform RNA-Seq on almost any sample. Nevertheless, samples obtained from small cell populations are particularly challenging, as biases associated with low amounts of input RNA can have strong and detrimental effects on downstream analyses. Here we compare different methods to normalize RNA-Seq data obtained from minimal input material. Using RNA from isolated medaka pituitary cells, we have amplified material from six samples before sequencing. Both synthetic and real data are used to evaluate different normalization methods to obtain a robust and reliable pipeline for analysis of RNA-Seq data from samples with very limited input material. The analysis outlined here shows that quantile normalization outperforms other more commonly used normalization procedures when using amplified RNA as input and will benefit researchers employing low amounts of RNA in similar experiments.
authors.affiliation: Weltzien Laboratory, Department of Basic Sciences and Aquatic Medicine, Norwegian University of Life Sciences, Oslo, Norway;
authors.forename: Eirill
authors.initials: E
authors.lastname: Ager-Wick
authors.affiliation: Weltzien Laboratory, Department of Basic Sciences and Aquatic Medicine, Norwegian University of Life Sciences, Oslo, Norway; Institute of Biology, Leiden University, Leiden, The Netherlands; and.
authors.forename: Christiaan V
authors.initials: CV
authors.lastname: Henkel
authors.affiliation: Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Trude M
authors.initials: TM
authors.lastname: Haug
authors.affiliation: Weltzien Laboratory, Department of Basic Sciences and Aquatic Medicine, Norwegian University of Life Sciences, Oslo, Norway; Department of Biosciences, University of Oslo, Oslo, Norway finn-arne.weltzien@nmbu.no.
authors.forename: Finn-Arne
authors.initials: FA
authors.lastname: Weltzien
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D009990:Oryzias / Q000235:genetics*; D010902:Pituitary Gland / Q000166:cytology; D012313:RNA / Q000302:isolation & purification; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins; D012313:RNA
keywords: RNA-Seq; low RNA input; medaka; normalization; pituitary