Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization.

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Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization.

Type: Article / Letter to editor
Title: Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization.
Author: Locati, M.D.Terpstra, I.Leeuw, W.C. deKuzak, M.Rauwerda, H.Ensink, W.A.Leeuwen, S. vanNehrdich, U.Spaink, H.P.Jonker, M.J.Breit, T.M.Dekker, R.J.
Journal Title: Nucleic acids research
Issue: 14
Volume: 43
Issue Date: 2015
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.
Handle: http://hdl.handle.net/1887/44373
 

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