title: Assessment of the impact of using a reference transcriptome in mapping short RNA-Seq reads.
abstract: RNA-Seq has become increasingly popular in transcriptome profiling. The major challenge in RNA-Seq data analysis is the accurate mapping of junction reads to their genomic origins. To detect splicing sites in short reads, many RNA-Seq aligners use reference transcriptome to inform placement of junction reads. However, no systematic evaluation has been performed to assess or quantify the benefits of incorporating reference transcriptome in mapping RNA-Seq reads. In this paper, we have studied the impact of reference transcriptome on mapping RNA-Seq reads, especially on junction ones. The same dataset were analysed with and without RefGene transcriptome, respectively. Then a Perl script was developed to analyse and compare the mapping results. It was found that about 50-55% junction reads can be mapped to the same genomic regions regardless of the usage of RefGene model. More than one-third of reads fail to be mapped without the help of a reference transcriptome. For "Alternatively" mapped reads, i.e., those reads mapped differently with and without RefGene model, the mappings without RefGene model are usually worse than their corresponding alignments with RefGene model. For junction reads that span more than two exons, it is less likely to align them correctly without the assistance of reference transcriptome. As the sequencing technology evolves, the read length is becoming longer and longer. When reads become longer, they are more likely to span multiple exons, and thus the mapping of long junction reads is actually becoming more and more challenging without the assistance of reference transcriptome. Therefore, the advantages of using reference transcriptome in the mapping demonstrated in this study are becoming more evident for longer reads. In addition, the effect of the completeness of reference transcriptome on mapping of RNA-Seq reads is discussed.
authors.affiliation: Systems Pharmacology and Biomarkers, Janssen Research & Development, LLC, San Diego, California, United States of America.
authors.forename: Shanrong
authors.initials: S
authors.lastname: Zhao
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping; D020869:Gene Expression Profiling; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D017423:Sequence Analysis, RNA; D059467:Transcriptome; D014584:User-Computer Interface
chemical_list: D012313:RNA