title: Spliced synthetic genes as internal controls in RNA sequencing experiments.
abstract: RNA sequencing (RNA-seq) can be used to assemble spliced isoforms, quantify expressed genes and provide a global profile of the transcriptome. However, the size and diversity of the transcriptome, the wide dynamic range in gene expression and inherent technical biases confound RNA-seq analysis. We have developed a set of spike-in RNA standards, termed 'sequins' (sequencing spike-ins), that represent full-length spliced mRNA isoforms. Sequins have an entirely artificial sequence with no homology to natural reference genomes, but they align to gene loci encoded on an artificial in silico chromosome. The combination of multiple sequins across a range of concentrations emulates alternative splicing and differential gene expression, and it provides scaling factors for normalization between samples. We demonstrate the use of sequins in RNA-seq experiments to measure sample-specific biases and determine the limits of reliable transcript assembly and quantification in accompanying human RNA samples. In addition, we have designed a complementary set of sequins that represent fusion genes arising from rearrangements of the in silico chromosome to aid in cancer diagnosis. RNA sequins provide a qualitative and quantitative reference with which to navigate the complexity of the human transcriptome.
authors: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.|St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia.
authors: Simon A
authors: SA
authors: Hardwick
authors: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.|St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia.
authors: Wendy Y
authors: WY
authors: Chen
authors: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
authors: Ted
authors: T
authors: Wong
authors: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.|School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, New South Wales, Australia.
authors: Ira W
authors: IW
authors: Deveson
authors: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.|St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia.
authors: James
authors: J
authors: Blackburn
authors: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia.
authors: Stacey B
authors: SB
authors: Andersen
authors: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia.
authors: Lars K
authors: 0000-0001-8191-3511
authors: LK
authors: Nielsen
authors: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.|St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia.
authors: John S
authors: 0000-0002-7680-7527
authors: JS
authors: Mattick
authors: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.|St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia.
authors: Tim R
authors: TR
authors: Mercer
mesh_terms: D022201:Chromosomes, Artificial; D020869:Gene Expression Profiling / Q000592:standards*; D005813:Genes, Synthetic; D006801:Humans; D011786:Quality Control; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics*; D012015:Reference Standards; D017423:Sequence Analysis, RNA / Q000379:methods / Q000592:standards*
chemical_list: D012333:RNA, Messenger