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Open Access Research article

Transcriptome assembly and microarray construction for Enchytraeus crypticus, a model oligochaete to assess stress response mechanisms derived from soil conditions

Marta P Castro-Ferreira12, Tjalf E de Boer1, John K Colbourne34, Riet Vooijs1, Cornelis AM van Gestel1, Nico M van Straalen1, Amadeu MVM Soares2, Mónica JB Amorim2 and Dick Roelofs1*

Author Affiliations

1 Department of Ecological Science, Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

2 Department of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal

3 The Center for Genomics and Bioinformatics, Indiana University, 1001 E. 3rd St., 47405-7005 Bloomington, IN, USA

4 Current Address: Environmental Genomics Group, School of Biosciences, University of Birmingham, Edgbaston, B15 2TT Birmingham, UK

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BMC Genomics 2014, 15:302  doi:10.1186/1471-2164-15-302

Published: 23 April 2014

Abstract

Background

The soil worm Enchytraeus crypticus (Oligochaeta) is an ecotoxicology model species that, until now, was without genome or transcriptome sequence information. The present research aims at studying the transcriptome of Enchytraeus crypticus, sampled from multiple test conditions, and the construction of a high-density microarray for functional genomic studies.

Results

Over 1.5 million cDNA sequence reads were obtained representing 645 million nucleotides. After assembly, 27,296 contigs and 87,686 singletons were obtained, from which 44% and 25% are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes. Concerning assembly quality, 84% of the contig sequences contain an open reading frame with a start codon while E. crypticus homologs were identified for 92% of the core eukaryotic genes. Moreover, 65% and 77% of the singletons and contigs without known homologs, respectively, were shown to be transcribed in an independent microarray experiment. An Agilent 180 K microarray platform was designed and validated by hybridizing cDNA from 4 day zinc- exposed E. crypticus to the concentration corresponding to 50% reduction in reproduction after three weeks (EC50). Overall, 70% of all probes signaled expression above background levels (mean signal + 1x standard deviation). More specifically, the probes derived from contigs showed a wider range of average intensities when compared to probes derived from singletons. In total, 522 significantly differentially regulated transcripts were identified upon zinc exposure. Several significantly regulated genes exerted predicted functions (e.g. zinc efflux, zinc transport) associated with zinc stress. Unexpectedly, the microarray data suggest that zinc exposure alters retro transposon activity in the E. crypticus genome.

Conclusion

An initial investigation of the E. crypticus transcriptome including an associated microarray platform for future studies proves to be a valuable resource to investigate functional genomics mechanisms of toxicity in soil environments and to annotate a potentially large number of lineage specific genes that are responsive to environmental stress conditions.

Keywords:
Ecotoxicogenomics; Next-generation pyrosequencing; Invertebrate; Zinc; Annelid; 454 sequencing