BACKGROUND: High-throughput sequencing (HTS) has revolutionized the way in which epigenetic research is conducted. When coupled with fully-sequenced genomes, millions of small RNA (sRNA) reads are mapped to regions of interest and the results scrutinized for clues about epigenetic mechanisms. However, this approach requires careful consideration in regards to experimental design, especially when one investigates repetitive parts of genomes such as transposable elements (TEs), or when such genomes are large, as is often the case in plants. RESULTS: Here, in an attempt to shed light on complications of mapping sRNAs to TEs, we focus on the 2,300 Mb maize genome, 85% of which is derived from TEs, and scrutinize methodological strategies that are commonly employed in TE studies. These include choices for the reference dataset, the normalization of multiply mapping sRNAs, and the selection among sRNA metrics. We further examine how these choices influence the relationship between sRNAs and the critical feature of TE age, and contrast their effect on low copy genomic regions and other popular HTS data. CONCLUSIONS: Based on our analyses, we share a series of take-home messages that may help with the design, implementation, and interpretation of high-throughput TE epigenetic studies specifically, but our conclusions may also apply to any work that involves analysis of HTS data.

Central European Institute of Technology Masaryk University Brno 62500 Czech Republic

Department of Ecology and Evolutionary Biology UC Irvine Irvine CA 92697 USA

School of Life Sciences University of Sussex Brighton East Sussex BN1 9RH UK

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Castel SE, Martienssen RA. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nat Rev Genet. 2013;14(2):100–112. doi: 10.1038/nrg3355. PubMed DOI PMC

Axtell MJ. Classification and comparison of small RNAs from plants. Annu Rev Plant Biol. 2013;64:137–159. doi: 10.1146/annurev-arplant-050312-120043. PubMed DOI

Borges F, Martienssen RA. The expanding world of small RNAs in plants. Nat Rev Mol Cell Biol. 2015;16(12):727–741. doi: 10.1038/nrm4085. PubMed DOI PMC

Matzke MA, Mosher RA. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat Rev Genet. 2014;15(6):394–408. doi: 10.1038/nrg3683. PubMed DOI

An JY, Lai J, Lehman ML, Nelson CC. miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data. Nucleic Acids Res. 2013;41(2):727–737. doi: 10.1093/nar/gks1187. PubMed DOI PMC

Garmire LX, Subramaniam S. Evaluation of normalization methods in mammalian microRNA-Seq data. RNA. 2012;18(6):1279–1288. doi: 10.1261/rna.030916.111. PubMed DOI PMC

Hackenberg M, Rodriguez-Ezpeleta N, Aransay AM. miRanalyzer: an update on the detection and analysis of microRNAs in high-throughput sequencing experiments. Nucleic Acids Res. 2011;39:W132–W138. doi: 10.1093/nar/gkr247. PubMed DOI PMC

Li Y, Zhang Z, Liu F, Vongsangnak W, Jing Q, Shen B. Performance comparison and evaluation of software tools for microRNA deep-sequencing data analysis. Nucleic Acids Res. 2012;40(10):4298–4305. doi: 10.1093/nar/gks043. PubMed DOI PMC

Sablok G, Milev I, Minkov G, Minkov I, Varotto C, Baev V. isomiRex: Web-based identification of microRNAs, isomiR variations and differential expression using next-generation sequencing datasets. Febs Letters. 2013;587(16):2629–2634. doi: 10.1016/j.febslet.2013.06.047. PubMed DOI

Srivastava PK, Moturu TR, Pandey P, Baldwin IT, Pandey SP. A comparison of performance of plant miRNA target prediction tools and the characterization of features for genome-wide target prediction. Bmc Genomics. 2014;15. PubMed PMC

Zhu EL, Zhao FQ, Xu G, Hou HB, Zhou LL, Li XK, Sun ZS, Wu JY. mirTools: microRNA profiling and discovery based on high-throughput sequencing. Nucleic Acids Res. 2010;38:W392–W397. doi: 10.1093/nar/gkq393. PubMed DOI PMC

Axtell MJ. ShortStack: Comprehensive annotation and quantification of small RNA genes. RNA. 2013;19(6):740–751. doi: 10.1261/rna.035279.112. PubMed DOI PMC

Hardcastle TJ, Kelly KA, Baulcombe DC. Identifying small interfering RNA loci from high-throughput sequencing data. Bioinformatics. 2012;28(4):457–463. doi: 10.1093/bioinformatics/btr687. PubMed DOI

Luo G-Z, Yang W, Ma Y-K, Wang X-J. ISRNA: an integrative online toolkit for short reads from high-throughput sequencing data. Bioinformatics. 2014;30(3):434–436. doi: 10.1093/bioinformatics/btt678. PubMed DOI

McCormick KP, Willmann MR, Meyers BC. Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments. Silence. 2011;2(1):2–2. doi: 10.1186/1758-907X-2-2. PubMed DOI PMC

Rueda A, Barturen G, Lebron R, Gomez-Martin C, Alganza A, Oliver JL, Hackenberg M. sRNAtoolbox: an integrated collection of small RNA research tools. Nucleic Acids Res. 2015;43(W1):W467–W473. doi: 10.1093/nar/gkv555. PubMed DOI PMC

Stocks MB, Moxon S, Mapleson D, Woolfenden HC, Mohorianu I, Folkes L, Schwach F, Dalmay T, Moulton V. The UEA sRNA workbench: a suite of tools for analysing and visualizing next generation sequencing microRNA and small RNA datasets. Bioinformatics. 2012;28(15):2059–2061. doi: 10.1093/bioinformatics/bts311. PubMed DOI PMC

Johnson NR, Yeoh JM, Coruh C, Axtell MJ. Improved Placement of Multi-mapping Small RNAs. G3. 2016;6(7):2103–2111. doi: 10.1534/g3.116.030452. PubMed DOI PMC

MacLean D, Moulton V, Studholme DJ. Finding sRNA generative locales from high-throughput sequencing data with NiBLS. BMC Bioinformatics. 2010;11. PubMed PMC

Moxon S, Schwach F, Dalmay T, MacLean D, Studholme DJ, Moulton V. A toolkit for analysing large-scale plant small RNA datasets. Bioinformatics. 2008;24(19):2252–2253. doi: 10.1093/bioinformatics/btn428. PubMed DOI

Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10(3). PubMed PMC

Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26(5):589–595. doi: 10.1093/bioinformatics/btp698. PubMed DOI PMC

Tenaillon MI, Hollister JD, Gaut BS. A triptych of the evolution of plant transposable elements. Trends Plant Sci. 2010;15(8):471–478. doi: 10.1016/j.tplants.2010.05.003. PubMed DOI

Hollister JD, Smith LM, Guo Y-L, Ott F, Weigel D, Gaut BS. Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata. Proc Natl Acad Sci U S A. 2011;108(6):2322–2327. doi: 10.1073/pnas.1018222108. PubMed DOI PMC

Bousios A, Diez CM, Takuno S, Bystry V, Darzentas N, Gaut BS. A role for palindromic structures in the cis-region of maize Sirevirus LTRs in transposable element evolution and host epigenetic response. Genome Res. 2016;26(2):226–37. doi: 10.1101/gr.193763.115. PubMed DOI PMC

Flutre T, Duprat E, Feuillet C, Quesneville H. Considering Transposable Element Diversification in De Novo Annotation Approaches. Plos One. 2011;6(1). PubMed PMC

Ragupathy R, You FM, Cloutier S. Arguments for standardizing transposable element annotation in plant genomes. Trends Plant Sci. 2013;18(7):367–376. doi: 10.1016/j.tplants.2013.03.005. PubMed DOI

Diez CM, Meca E, Tenaillon MI, Gaut BS. Three Groups of Transposable Elements with Contrasting Copy Number Dynamics and Host Responses in the Maize (Zea mays ssp mays) Genome. Plos Genet. 2014;10(4). PubMed PMC

Gent JI, Ellis NA, Guo L, Harkess AE, Yao Y, Zhang X, Dawe RK. CHH islands: de novo DNA methylation in near-gene chromatin regulation in maize. Genome Res. 2013;23(4):628–637. doi: 10.1101/gr.146985.112. PubMed DOI PMC

Regulski M, Lu Z, Kendall J, Donoghue MTA, Reinders J, Llaca V, Deschamps S, Smith A, Levy D, McCombie WR, et al. The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA. Genome Res. 2013;23(10):1651–1662. doi: 10.1101/gr.153510.112. PubMed DOI PMC

Baucom RS, Estill JC, Chaparro C, Upshaw N, Jogi A, Deragon JM, Westerman RP, SanMiguel PJ, Bennetzen JL. Exceptional Diversity, Non-Random Distribution, and Rapid Evolution of Retroelements in the B73 Maize Genome. Plos Genet. 2009;5(11). PubMed PMC

Bousios A, Kourmpetis YAI, Pavlidis P, Minga E, Tsaftaris A, Darzentas N. The turbulent life of Sirevirus retrotransposons and the evolution of the maize genome: more than ten thousand elements tell the story. Plant J. 2012;69(3):475–488. doi: 10.1111/j.1365-313X.2011.04806.x. PubMed DOI

Ellinghaus D, Kurtz S, Willhoeft U. LTRharvest, an efficient and flexible software for de novo detection of LTR retrotransposons. Bmc Bioinformatics. 2008;9. PubMed PMC

Katoh K, Standley DM. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol Biol Evol. 2013;30(4):772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Ma JX, Bennetzen JL. Rapid recent growth and divergence of rice nuclear genomes. Proc Natl Acad Sci U S A. 2004;101(34):12404–12410. doi: 10.1073/pnas.0403715101. PubMed DOI PMC

Bousios A, Gaut BS. Mechanistic and evolutionary questions about epigenetic conflicts between transposable elements and their plant hosts. Curr Opin Plant Biol. 2016;30:123–133. doi: 10.1016/j.pbi.2016.02.009. PubMed DOI

Fultz D, Choudury SG, Slotkin RK. Silencing of active transposable elements in plants. Curr Opin Plant Biol. 2015;27:67–76. doi: 10.1016/j.pbi.2015.05.027. PubMed DOI

Lister R, O’Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, Ecker JR. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell. 2008;133(3):523–536. doi: 10.1016/j.cell.2008.03.029. PubMed DOI PMC

Law JA, Du JM, Hale CJ, Feng SH, Krajewski K, Palanca AMS, Strahl BD, Patel DJ, Jacobsen SE. Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature. 2013;498(7454):385. doi: 10.1038/nature12178. PubMed DOI PMC

Panda K, Ji LX, Neumann DA, Daron J, Schmitz RJ, Slotkin RK. Full-length autonomous transposable elements are preferentially targeted by expression-dependent forms of RNA-directed DNA methylation. Genome Biol. 2016;17. PubMed PMC

Zhai JX, Bischof S, Wang HF, Feng SH, Lee TF, Teng C, Chen XY, Park SY, Liu LS, Gallego-Bartolome J, et al. A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis. Cell. 2015;163(2):445–455. doi: 10.1016/j.cell.2015.09.032. PubMed DOI PMC

Bousios A, Darzentas N, Tsaftaris A, Pearce SR. Highly conserved motifs in non-coding regions of Sirevirus retrotransposons: the key for their pattern of distribution within and across plants? BMC Genomics. 2010;11. PubMed PMC

Bennetzen JL, Schmutz J, Wang H, Percifield R, Hawkins J, Pontaroli AC, Estep M, Feng L, Vaughn JN, Grimwood J, et al. Reference genome sequence of the model plant Setaria. Nat Biotechnol. 2012;30(6):555. doi: 10.1038/nbt.2196. PubMed DOI

Shulaev V, Sargent DJ, Crowhurst RN, Mockler TC, Folkerts O, Delcher AL, Jaiswal P, Mockaitis K, Liston A, Mane SP, et al. The genome of woodland strawberry (Fragaria vesca) Nat Genet. 2011;43(2):109–116. doi: 10.1038/ng.740. PubMed DOI PMC

Diez CM, Vitte C, Ross-Ibarra J, Gaut BS, Tenaillon MI. Using Nextgen Sequencing to Investigate Genome Size Variation and Transposable Element Content. In: Grandbastien MA, Casacuberta JM, editors. Plant Transposable Elements Topics in Current Genetics. 2012. pp. 41–58.

Tenaillon MI, Hufford MB, Gaut BS, Ross-Ibarra J. Genome Size and Transposable Element Content as Determined by High-Throughput Sequencing in Maize and Zea luxurians. Genome Biol Evol. 2011;3:219–229. doi: 10.1093/gbe/evr008. PubMed DOI PMC

Bousios A, Minga E, Kalitsou N, Pantermali M, Tsaballa A, Darzentas N. MASiVEdb: the Sirevirus Plant Retrotransposon Database. BMC Genomics. 2012;13. PubMed PMC

Darzentas N, Bousios A, Apostolidou V, Tsaftaris AS. MASiVE: Mapping and Analysis of SireVirus Elements in plant genome sequences. Bioinformatics. 2010;26(19):2452–2454. doi: 10.1093/bioinformatics/btq454. PubMed DOI

He G, Chen B, Wang X, Li X, Li J, He H, Yang M, Lu L, Qi Y, Wang X, et al. Conservation and divergence of transcriptomic and epigenomic variation in maize hybrids. Genome Biology. 2013;14(6). PubMed PMC

McCue AD, Nuthikattu S, Slotkin RK. Genome-wide identification of genes regulated in trans by transposable element small interfering RNAs. RNA Biol. 2013;10(8):1379–1395. doi: 10.4161/rna.25555. PubMed DOI PMC

Wang X, Elling AA, Li X, Li N, Peng Z, He G, Sun H, Qi Y, Liu XS, Deng XW. Genome-Wide and Organ-Specific Landscapes of Epigenetic Modifications and Their Relationships to mRNA and Small RNA Transcriptomes in Maize. Plant Cell. 2009;21(4):1053–1069. doi: 10.1105/tpc.109.065714. PubMed DOI PMC

Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008;5(7):621–628. doi: 10.1038/nmeth.1226. PubMed DOI

Gong L, Masonbrink RE, Grover CE, Renny-Byfield S, Wendel JF. A Cluster of Recently Inserted Transposable Elements Associated with siRNAs in Gossypium raimondii. Plant Genome. 2015;8(2). PubMed

Law JA, Jacobsen SE. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet. 2010;11(3):204–220. doi: 10.1038/nrg2719. PubMed DOI PMC

Maumus F, Quesneville H. Ancestral repeats have shaped epigenome and genome composition for millions of years in Arabidopsis thaliana. Nat Commun. 2014;5. PubMed PMC

Ye RQ, Wang W, Iki T, Liu C, Wu Y, Ishikawa M, Zhou XP, Qi YJ. Cytoplasmic Assembly and Selective Nuclear Import of Arabidopsis ARGONAUTE4/siRNA Complexes. Mol Cell. 2012;46(6):859–870. doi: 10.1016/j.molcel.2012.04.013. PubMed DOI

Treangen TJ, Salzberg SL. Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet. 2012;13(1):36–46. PubMed PMC