title: Combining different mRNA capture methods to analyze the transcriptome: analysis of the Xenopus laevis transcriptome.
abstract: mRNA sequencing (mRNA-seq) is a commonly used technique to survey gene expression from organisms with fully sequenced genomes. Successful mRNA-seq requires purification of mRNA away from the much more abundant ribosomal RNA, which is typically accomplished by oligo-dT selection. However, mRNAs with short poly-A tails are captured poorly by oligo-dT based methods. We demonstrate that combining mRNA capture via oligo-dT with mRNA capture by the 5' 7-methyl guanosine cap provides a more complete view of the transcriptome and can be used to assay changes in mRNA poly-A tail length on a genome-wide scale. We also show that using mRNA-seq reads from both capture methods as input for de novo assemblers provides a more complete reconstruction of the transcriptome than either method used alone. We apply these methods of mRNA capture and de novo assembly to the transcriptome of Xenopus laevis, a well-studied frog that currently lacks a finished sequenced genome, to discover transcript sequences for thousands of mRNAs that are currently absent from public databases. The methods we describe here will be broadly applicable to many organisms and will provide insight into the transcriptomes of organisms with sequenced and unsequenced genomes.
authors.affiliation: Department of Molecular Biology Massachusetts General Hospital, Boston, Massachusetts, United States of America ; Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Blower
authors.forename: Ashwini
authors.initials: A
authors.lastname: Jambhekar
authors.forename: Dianne S
authors.initials: DS
authors.lastname: Schwarz
authors.forename: James A
authors.initials: JA
authors.lastname: Toombs
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011061:Poly A / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D017423:Sequence Analysis, RNA; D014982:Xenopus laevis / Q000235:genetics*
chemical_list: D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT); D011061:Poly A