title: Improved Placement of Multi-mapping Small RNAs.
abstract: 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 local-weighting 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.
authors.affiliation: Huck Institutes of the Life Sciences, Penn State University, Philadelphia 16802 Department of Biology, Knox College, Galesburg, Illinois, 61401.
authors.forename: Nathan R
authors.identifier: 0000-0002-5279-9964
authors.initials: NR
authors.lastname: Johnson
authors.affiliation: Department of Biology, Knox College, Galesburg, Illinois, 61401.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Yeoh
authors.affiliation: Huck Institutes of the Life Sciences, Penn State University, Philadelphia 16802 Department of Biology, Penn State University, Philadelphia 16802.
authors.forename: Ceyda
authors.initials: C
authors.lastname: Coruh
authors.affiliation: Huck Institutes of the Life Sciences, Penn State University, Philadelphia 16802 Department of Biology, Penn State University, Philadelphia 16802 mja18@psu.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Axtell
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D018745:Genome, Plant; D059014:High-Throughput Nucleotide Sequencing; D035683:MicroRNAs / Q000235:genetics*; D012275:Oryza / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D017423:Sequence Analysis, RNA; D012984:Software; D003313:Zea mays / Q000235:genetics
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering
keywords: alignment; annotation; bioinformatics; miRNA; sRNA-seq; siRNA