Benchmarking Transcriptome Quantification Methods for Duplicated Genes in Xenopus laevis

Abstract

Xenopus is an important model organism for the study of genome duplication in vertebrates. With the full genome sequence of diploid Xenopus tropicalis available, and that of allotetraploid X. laevis close to being finished, we will be able to expand our understanding of how duplicated genes have evolved. One of the key features in the study of the functional consequence of gene duplication is how their expression patterns vary across different conditions, and RNA-seq seems to have enough resolution to discriminate the expression of highly similar duplicated genes. However, most of the current RNA-seq analysis methods were not designed to study samples with duplicate genes such as in X. laevis. Here, various computational methods to quantify gene expression in RNA-seq data were evaluated, using 2 independent X. laevis egg RNA-seq datasets and 2 reference databases for duplicated genes. The fact that RNA-seq can measure expression levels of similar duplicated genes was confirmed, but long paired-end reads are more informative than short single-end reads to discriminate duplicated genes. Also, it was found that bowtie, one of the most popular mappers in RNA-seq analysis, reports significantly smaller numbers of unique hits according to a mapping quality score compared to other mappers tested (BWA, GSNAP, STAR). Calculated from unique hits based on a mapping quality score, both expression levels and the expression ratio of duplicated genes can be estimated consistently among biological replicates, demonstrating that this method can successfully discriminate the expression of each copy of a duplicated gene pair. This comprehensive evaluation will be a useful guideline for studying gene expression of organisms with genome duplication using RNA-seq in the future.