Improved placement of multi-mapping small RNAs

Nathan R. Johnson, Jonathan M. Yeoh, Ceyda Coruh, Michael J. Axtell

Research output: Contribution to journalArticlepeer-review

162 Scopus citations


High-throughput sequencing of small RNAs (sRNA-seq) is a popular method used to discover and annotate microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs). One of the key steps in sRNA-seq data analysis is alignment to a reference genome. sRNA-seq libraries often have a high proportion of reads that align to multiple genomic locations, which makes determining their true origins difficult. Commonly used sRNA-seq alignment methods result in either very low precision (choosing an alignment at random), or sensitivity (ignoring multi-mapping reads). Here, we describe and test an sRNA-seq alignment strategy that uses local genomic context to guide decisions on proper placements of multi-mapped sRNA-seq reads. Tests using simulated sRNA-seq data demonstrated that this local-weighting method outperforms other alignment strategies using three different plant genomes. Experimental analyses with real sRNA-seq data also indicate superior performance of localweighting methods for both plant miRNAs and heterochromatic siRNAs. The local-weighting methods we have developed are implemented as part of the sRNA-seq analysis program ShortStack, which is freely available under a general public license. Improved genome alignments of sRNA-seq data should increase the quality of downstream analyses and genome annotation efforts.

Original languageEnglish (US)
Pages (from-to)2103-2111
Number of pages9
JournalG3: Genes, Genomes, Genetics
Issue number7
StatePublished - Jul 1 2016

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics
  • Genetics(clinical)


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