Instructions for use Title Construction and Characterization of Normalized cDNA Libraries by 454 Pyrosequencing and Estimation of DNAMethylation Levels in Three Distantly Related Termite Species

In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colonylevel productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST) libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti, Reticulitermes speratus and Nasutitermes takasagoensis. We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3) and methyl-CpG binding domain (mbd) were performed against the EST libraries. All four of these genes were found in the H. sjostedti library, while all except dnmt3 were found in R. speratus and N. takasagoensis. The ratio of the observed to the expected CpG content (CpG O/E), which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1), and the distributions of CpG O/E were bimodal, suggesting the presence of DNA methylation. Citation: Hayashi Y, Shigenobu S, Watanabe D, Toga K, Saiki R, et al. (2013) Construction and Characterization of Normalized cDNA Libraries by 454 Pyrosequencing and Estimation of DNA Methylation Levels in Three Distantly Related Termite Species. PLoS ONE 8(9): e76678. doi:10.1371/journal.pone. 0076678 Editor: Ken Mills, Queen's University Belfast, United Kingdom Received July 10, 2013; Accepted September 1, 2013; Published September 30, 2013 Copyright: © 2013 Hayashi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a Grant-in-Aid for Young Scientists (No. 21677001 to TM) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.mext.go.jp/english/), and by Grant-in-Aid for Young Scientists (No. 23770274 to MH), for JSPS Fellows (Nos. 2469, 12J03468 and 249520 to YH, DW and KT respectively) and for Scientific Research (C) (No. 24570022 to KM) from the Japan Society for the Promotion of Science (http://www.jsps.go.jp/english/). YH was also supported by Grant for Basic Science Research Projects from Sumitomo Foundation (http:// www.sumitomo.or.jp/e/). NL was supported by the Australian Research Council (http://www.arc.gov.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that they have no competing interests. * E-mail: miu@ees.hokudai. ac.jp