@article {10019,
	title = {Limitations of alignment-free tools in total RNA-seq quantification.},
	journal = {BMC Genomics},
	volume = {19},
	number = {1},
	year = {2018},
	pages = {510},
	abstract = {
	BACKGROUND:



	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 RNAs 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.

},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/29969991},
	author = {D.C. Wu and J. Yao and K.S. Ho and A.M. Lambowitz and C.O. Wilke}
}
