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Title: Impact of gene annotation choice on the quantification of RNA-seq data.
Austin Authors: Chisanga, David;Liao, Yang;Shi, Wei
Affiliation: School of Computing and Information Systems, The University of Melbourne, Melbourne, Australia..
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia..
Department of Medical Biology, The University of Melbourne, Melbourne, Australia..
Olivia Newton-John Cancer Research Institute
School of Cancer Medicine, La Trobe University, Melbourne, Australia..
Issue Date: 30-Mar-2022 2022
Publication information: BMC bioinformatics 2022; 23(1): 107
Abstract: RNA sequencing is currently the method of choice for genome-wide profiling of gene expression. A popular approach to quantify expression levels of genes from RNA-seq data is to map reads to a reference genome and then count mapped reads to each gene. Gene annotation data, which include chromosomal coordinates of exons for tens of thousands of genes, are required for this quantification process. There are several major sources of gene annotations that can be used for quantification, such as Ensembl and RefSeq databases. However, there is very little understanding of the effect that the choice of annotation has on the accuracy of gene expression quantification in an RNA-seq analysis. In this paper, we present results from our comparison of Ensembl and RefSeq human annotations on their impact on gene expression quantification using a benchmark RNA-seq dataset generated by the SEQC consortium. We show that the use of RefSeq gene annotation models led to better quantification accuracy, based on the correlation with ground truths including expression data from >800 real-time PCR validated genes, known titration ratios of gene expression and microarray expression data. We also found that the recent expansion of the RefSeq annotation has led to a decrease in its annotation accuracy. Finally, we demonstrated that the RNA-seq quantification differences observed between different annotations were not affected by the use of different normalization methods. In conclusion, our study found that the use of the conservative RefSeq gene annotation yields better RNA-seq quantification results than the more comprehensive Ensembl annotation. We also found that, surprisingly, the recent expansion of the RefSeq database, which was primarily driven by the incorporation of sequencing data into the gene annotation process, resulted in a reduction in the accuracy of RNA-seq quantification.
DOI: 10.1186/s12859-022-04644-8
Journal: BMC bioinformatics
PubMed URL: 35354358
PubMed URL:
Type: Journal Article
Subjects: Gene annotation
Gene expression quantification
Appears in Collections:Journal articles

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