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Series GSE77072 Query DataSets for GSE77072
Status Public on Jul 21, 2016
Title Spliced synthetic genes as internal controls in RNA sequencing experiments.
Organisms Homo sapiens; synthetic construct
Experiment type Expression profiling by high throughput sequencing
Summary 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 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 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.
Overall design Detailed transcriptomic analysis of two human cell lines with synthetic RNA spike-ins ('sequins'). Sequins were initially combined at equimolar concentrations (a "flat" mix) and sequenced neat (i.e. without any natural RNA added). We then prepared two staggered mixtures (Mix A & B) and sequenced them neat. Mix A was then spiked into total RNA extracted from K562 cells, while Mix B was spiked into total RNA extracted from GM12878 cells. Finally, we prepared a staggered mixture of fusion sequins and sequenced it neat.
Contributor(s) Hardwick SA, Mercer TR
Citation(s) 27502218
Submission date Jan 21, 2016
Last update date May 15, 2019
Contact name Simon Andrew Hardwick
Phone +61401264672
Organization name Garvan Institute of Medical Research
Lab Transcriptomic Research
Street address 384 Victoria Street
City Darlinghurst
State/province NSW
ZIP/Postal code 2010
Country Australia
Platforms (2)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
GPL19604 Illumina HiSeq 2500 (synthetic construct)
Samples (10)
GSM2225088 K562 spiked with Sequins Mix A rep1
GSM2225089 K562 spiked with Sequins Mix A rep2
GSM2225090 K562 spiked with Sequins Mix A rep3
BioProject PRJNA309391
SRA SRP068708

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Supplementary file Size Download File type/resource
GSE77072_RAW.tar 59.9 Mb (http)(custom) TAR (of GTF)
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Raw data are available in SRA
Processed data provided as supplementary file

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