title: Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization.
abstract: There is an increasing interest in complementing RNA-seq experiments with small-RNA (sRNA) expression data to obtain a comprehensive view of a transcriptome. Currently, two main experimental challenges concerning sRNA-seq exist: how to check the size distribution of isolated sRNAs, given the sensitive size-selection steps in the protocol; and how to normalize data between samples, given the low complexity of sRNA types. We here present two separate sets of synthetic RNA spike-ins for monitoring size-selection and for performing data normalization in sRNA-seq. The size-range quality control (SRQC) spike-in set, consisting of 11 oligoribonucleotides (10-70 nucleotides), was tested by intentionally altering the size-selection protocol and verified via several comparative experiments. We demonstrate that the SRQC set is useful to reproducibly track down biases in the size-selection in sRNA-seq. The external reference for data-normalization (ERDN) spike-in set, consisting of 19 oligoribonucleotides, was developed for sample-to-sample normalization in differential-expression analysis of sRNA-seq data. Testing and applying the ERDN set showed that it can reproducibly detect differential expression over a dynamic range of 2(18). Hence, biological variation in sRNA composition and content between samples is preserved while technical variation is effectively minimized. Together, both spike-in sets can significantly improve the technical reproducibility of sRNA-seq.
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands.
authors: Mauro D
authors: MD
authors: Locati
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands.
authors: Inez
authors: I
authors: Terpstra
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands Netherlands eScience Center, Amsterdam 1098 XG, The Netherlands.
authors: Wim C
authors: WC
authors: de Leeuw
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands Netherlands eScience Center, Amsterdam 1098 XG, The Netherlands.
authors: Mateusz
authors: M
authors: Kuzak
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands Netherlands eScience Center, Amsterdam 1098 XG, The Netherlands.
authors: Han
authors: H
authors: Rauwerda
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands.
authors: Wim A
authors: WA
authors: Ensink
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands.
authors: Selina
authors: S
authors: van Leeuwen
authors: Department of Molecular Cell Biology, Institute of Biology, Leiden University, Gorlaeus Laboratories - Cell Observatorium, Leiden 2333 CE, The Netherlands.
authors: Ulrike
authors: U
authors: Nehrdich
authors: Department of Molecular Cell Biology, Institute of Biology, Leiden University, Gorlaeus Laboratories - Cell Observatorium, Leiden 2333 CE, The Netherlands.
authors: Herman P
authors: HP
authors: Spaink
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands.
authors: Martijs J
authors: MJ
authors: Jonker
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands t.m.breit@uva.nl.
authors: Timo M
authors: TM
authors: Breit
authors: RNA Biology & Applied Bioinformatics research group, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam 1090 GE, The Netherlands.
authors: Rob J
authors: RJ
authors: Dekker
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000592:standards*; D011786:Quality Control; D058727:RNA, Small Untranslated / Q000737:chemistry / Q000378:metabolism*; D012015:Reference Standards; D017423:Sequence Analysis, RNA / Q000592:standards*; D015027:Zebrafish / Q000235:genetics
chemical_list: D058727:RNA, Small Untranslated