Systematic evaluation of spliced alignment programs for RNA-seq data

Pär G. Engström, Tamara Steijger, Botond Sipos, Gregory R. Grant, André Kahles, Gunnar Rätsch, Nick Goldman, Tim J. Hubbard, Jennifer Harrow, Roderic Guigó, Paul Bertone, Tyler Alioto, Jonas Behr, Regina Bohnert, Davide Campagna, Carrie A. Davis, Alexander Dobin, Thomas R. Gingeras, Géraldine Jean, Peter KosarevSheng Li, Jinze Liu, Christopher E. Mason, Vladimir Molodtsov, Zemin Ning, Hannes Ponstingl, Jan F. Prins, Paolo Ribeca, Igor Seledtsov, Victor Solovyev, Giorgio Valle, Nicola Vitulo, Kai Wang, Thomas D. Wu, Georg Zeller

Research output: Contribution to journalArticlepeer-review

357 Scopus citations


High-throughput RNA sequencing is an increasingly accessible method for studying gene structure and activity on a genome-wide scale. A critical step in RNA-seq data analysis is the alignment of partial transcript reads to a reference genome sequence. To assess the performance of current mapping software, we invited developers of RNA-seq aligners to process four large human and mouse RNA-seq data sets. In total, we compared 26 mapping protocols based on 11 programs and pipelines and found major performance differences between methods on numerous benchmarks, including alignment yield, basewise accuracy, mismatch and gap placement, exon junction discovery and suitability of alignments for transcript reconstruction. We observed concordant results on real and simulated RNA-seq data, confirming the relevance of the metrics employed. Future developments in RNA-seq alignment methods would benefit from improved placement of multimapped reads, balanced utilization of existing gene annotation and a reduced false discovery rate for splice junctions.

Original languageEnglish
Pages (from-to)1185-1191
Number of pages7
JournalNature Methods
Issue number12
StatePublished - Dec 2013

Bibliographical note

Funding Information:
This work was supported by the European Molecular Biology Laboratory, US National Institutes of Health/NHGRI grants U54HG004555 and U54HG004557, Wellcome Trust grant WT09805, and grants BIO2011-26205 and CSD2007-00050 from the Ministerio de Educación y Ciencia.

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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