Limitations of alignment-free tools in total RNA-seq quantification

Douglas C. Wu, Jun Yao, Kevin S. Ho, Alan M. Lambowitz, Claus O. Wilke
2018 BMC Genomics  
Alignment-free RNA quantification tools have significantly increased the speed of RNA-seq analysis. However, it is unclear whether these state-of-the-art RNA-seq analysis pipelines can quantify small RNAs as accurately as they do with long RNAs in the context of total RNA quantification. Result: We comprehensively tested and compared four RNA-seq pipelines for accuracy of gene quantification and fold-change estimation. We used a novel total RNA benchmarking dataset in which small non-coding
more » ... are highly represented along with other long RNAs. The four RNA-seq pipelines consisted of two commonly-used alignment-free pipelines and two variants of alignment-based pipelines. We found that all pipelines showed high accuracy for quantifying the expression of long and highly-abundant genes. However, alignment-free pipelines showed systematically poorer performance in quantifying lowly-abundant and small RNAs. Conclusion: We have shown that alignment-free and traditional alignment-based quantification methods perform similarly for common gene targets, such as protein-coding genes. However, we have identified a potential pitfall in analyzing and quantifying lowly-expressed genes and small RNAs with alignment-free pipelines, especially when these small RNAs contain biological variations.
doi:10.1186/s12864-018-4869-5 pmid:29969991 pmcid:PMC6042521 fatcat:k5bjud7pz5dybh7ntsiwn2ewfq