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.
Highlights
Xenopus laevis and Xenopus tropicalis are important model organisms for the study of developmental biology and cell cycle control[1]
The quality and evenness of coverage of the genome or transcriptome is a function of the input material for library construction. In this current work we have shown that combining different mRNA capture strategies results in a wider coverage of transcribed sequences
This suggests that combining mRNA capture techniques will be a widely applicable method to generate more complete transcriptomes from a range of organisms
Summary
Xenopus laevis and Xenopus tropicalis are important model organisms for the study of developmental biology and cell cycle control[1]. In addition to discipline-specific tools it is necessary to have broad community resources that aid all researchers studying an organism. One of the most important resources for any model organism community is a high-quality, well-annotated genome sequence. In 2010 the genome sequence of Xenopus tropicalis was published[2], which provided a tremendous improvement in the genomic resources available to the community. For many experiments (cell cycle extracts and many developmental techniques) Xenopus laevis remains the preferred (or exclusive) frog of choice[1] and the genome sequence of X. tropicalis does not directly aid X. laevis research. Genome sequencing of X. laevis has proceeded at a slower pace (due partly to the fact that X. laevis has an allotetraploid genome compared to the diploid genome of X. tropicalis), but will eventually provide genomic resources to the frog community
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