title: Digital gene expression for non-model organisms.
abstract: Next-generation sequencing technologies offer new approaches for global measurements of gene expression but are mostly limited to organisms for which a high-quality assembled reference genome sequence is available. We present a method for gene expression profiling called EDGE, or EcoP15I-tagged Digital Gene Expression, based on ultra-high-throughput sequencing of 27-bp cDNA fragments that uniquely tag the corresponding gene, thereby allowing direct quantification of transcript abundance. We show that EDGE is capable of assaying for expression in >99% of genes in the genome and achieves saturation after 6-8 million reads. EDGE exhibits very little technical noise, reveals a large (10(6)) dynamic range of gene expression, and is particularly suited for quantification of transcript abundance in non-model organisms where a high-quality annotated genome is not available. In a direct comparison with RNA-seq, both methods provide similar assessments of relative transcript abundance, but EDGE does better at detecting gene expression differences for poorly expressed genes and does not exhibit transcript length bias. Applying EDGE to laboratory mice, we show that a loss-of-function mutation in the melanocortin 1 receptor (Mc1r), recognized as a Mendelian determinant of yellow hair color in many different mammals, also causes reduced expression of genes involved in the interferon response. To illustrate the application of EDGE to a non-model organism, we examine skin biopsy samples from a cheetah (Acinonyx jubatus) and identify genes likely to control differences in the color of spotted versus non-spotted regions.
authors.affiliation: Department of Genetics, Stanford University, Stanford, California 94305, USA.
authors.forename: Lewis Z
authors.initials: LZ
authors.lastname: Hong
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Anne
authors.initials: A
authors.lastname: Schmidt-Küntzel
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Warren
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Barsh
mesh_terms: D002612:Acinonyx / Q000235:genetics; D000818:Animals; D019295:Computational Biology; D020224:Expressed Sequence Tags; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D007372:Interferons / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010858:Pigmentation / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D043562:Receptors, G-Protein-Coupled; D007372:Interferons