Pedobiologia 47, 447–451, 2003© Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/pedo

0031-4056/03/47/05–06–447 $15.00/0

*E-mail corresponding author: SturzenbaumSR@Cardiff.ac.uk

The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

The earthworm Expressed Sequence Tag project

Stephen R. Stürzenbaum1*, John Parkinson2, Mark Blaxter2, A. John Morgan1, Peter Kille1

and Oleg Georgiev3

1 School of Biosciences, University of Wales, P.O. Box 915, Cardiff CF10 3TL, Wales, UK2 Institute of Cell, Animal and Population Biology, University of Edinburgh, UK3 Institute of Molecular Biology, Zurich University, Switzerland

Submitted September 6, 2002 · Accepted May 13, 2003

Summary

This paper aims to provide a brief description of the earthworm Expressed Sequence Tag (EST) project. ESTs are short sin-gle read sequences randomly derived from cDNA libraries and provide the means to acquire large scale sequence infor-mation of coding DNA. The earthworm EST project is growing rapidly and the analysis of the first 577 sequences corre-sponded to ~ 400 different genes, with 79 represented by two or more ESTs. Significant sequence similarity to known pro-teins was observed in 76 % of cases and the remaining 24 % were classified as novel genes. Using a combination of bioin-formatic tools the sequence information was used to build a relational database, LumbriBASE, which can be queried viaan internet interface by sequence similarity and key word searches (see http://www.earthworms.org).

Key words: Expressed Sequence Tag, EST, sequencing project, earthworm

The genome of any given animal comprises 5 % to25 % coding DNA that is transcribed into mRNA. Invitro, these mRNAs can be reverse transcribed, result-ing in stable complementary DNAs (cDNAs) which inturn can be cloned into cDNA library vectors. Ex-pressed Sequence Tags (ESTs) are short single read se-quences derived from cDNA library clones selected atrandom (see Fig. 1 for schematic diagram). Assumingthat the number of ESTs is a reflection of actualmRNA levels, the EST strategy provides informationon mRNA expression and the metabolic activity, andhighlights important processes. Unfortunately, mostsequence data submitted to public databases show anextreme species bias, with the majority of sequencesderived from a few “model” species. EST analysis is arapid and effective way to redress this phylogenetic

bias by providing the tools to acquire genomic data forneglected but by no means unimportant species. Priorto the year 2000, the entire taxonomic group of Lumb-ricidae was represented by 60 sequence data deposi-tions with the majority originating from Lumbricusrubellus (22), L. terrestris (21) and Eisenia fetida (12).For this reason it was considered important to initiatean earthworm EST project. Here we provide a primarydescription of the earthworm EST project, the analysisof the first 577 single pass sequences and a brief intro-duction to LumbriBASE.

Adult earthworms were sampled from pastures nearDinas Powys, Wales and whole worm total RNA pre-pared using TRI reagent (Sigma) followed by mRNAisolation by oligo dT-cellulose chromatography (Phar-macia). A λ-ZAP Express cDNA library was con-

448 Stephen R. Stürzenbaum et al.

Pedobiologia (2003) 47, 447–451

ters of two or more reads. The remaining 320 se-quences (55 %) were single copy sequences and thusdesignated as ‘singletons’ (Fig. 2 C). These results re-flect other EST project outputs (e.g. Inaba et al. 2002)and thus are a typical representation of a eukaryoticcell. The most abundant transcripts identified were ofmitochondrial, ribosomal or housekeeping origin(Fig. 2 D). Exceptional, maybe, was the high incidenceof blood coagulating enzymes / fibrinolytic enzymeswith a proportional representation of nearly 6 %. Nosignificant similarity to known proteins was observedin 24 % of cases. The remaining clustered nicely into afunctional classification loosely adapted from Lee etal. (1999) (Fig. 2 E). The abundance of novel genes iscomparable with other EST projects for example in thehuman parasite Brugia malayi (Blaxter et al. 2002), incommon carp head kidney cells (Savan and Sakai2002) and Bos taurus mammary glands (Sonstegard etal. 2002).

The resultant information was used to build a rela-tional sequence database (LumbriBASE) that is freelyaccessible via the web (http://www.earthworms.org).LumbriBASE can be queried for sequence similarity(in house BLAST) and by annotation. Figure 3 showsa typical output of an annotation search. The input of akey word, in this case GAPDH (Fig. 3 A), provides in-formation on identified clusters (Fig. 3 B), a cartoon ofthe sequence alignments (Fig. 3 C) and the final con-sensus sequence (Fig. 2 D). The BLAST menu(Fig. 3 E) provides information on sequence identityand similarity on a protein or nucleotide level (Fig. 2 F)and is identical to an NCBI BLAST output (Altschul etal. 1997).

Funds have been secured to start a follow-up projectthat will build on large scale amplification and se-quencing processes developed and refined primarilyfor the nematode EST project (Parkinson et al. 2001).In future the earthworm EST project will therefore ex-ploit fully automated high-throughput systems that userobotic facilities for plating and picking clones intohigh density bar coded micro-titre plates. Duplicateglycerol stocks will be generated and aliquots archivedat –80°C for future use in downstream post-genomicactivities, which will include amongst others micro-array technology. In short, LumbriBASE is set to grow!

References

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,Zhang, Z., Miller, W., Lipman, D. J. (1997) GappedBLAST and PSI-BLAST: a new generation of proteindatabase search programs. Nucleic Acids Research 25,3389–3402.

Fig. 1. Schematic diagram of the earthworm EST sequencing ap-proach

structed with 5 µg mRNA and rescued according to themanufacturer’s protocol (Stratagene). The cDNA li-brary approximated 0.5 × 106 primary recombinantswith an average size of 1400 bp (Fig. 2 A). DNA wasisolated by standard protocols and processed for fluo-rescent dye terminator sequencing. Single pass se-quences were determined from the 5’ end of each cloneusing an ABI Sequenator. DNAs and sequencing chro-matograms were collected and archived. ABI sequencereads were processed to derive high quality sequenceand clustered using customised software. The medianprocessed sequence length was 650 bp (Fig. 2 B) andthe “cap3” software was used to produce a single datafile from all overlapping /duplicate sequences. All se-quences were automatically submitted to the publicEST database, dbEST.

The entire dataset was analysed using the bioinfor-matic tools provided by the National Center forBiotechnology Information (NCBI). In detail, se-quences (consensi for the clusters, individual reads forthe singletons) were analysed using six frame trans-lated searches (BlastX against non-redundant data-base) and nucleotide searches (BlastN against non-re-dundant database and BlastN against dbEST). All se-quences were clustered using an algorithm based on‘BLAST’ and 257 reads could be grouped into 79 clus-

449The earthworm Expressed Sequence Tag project

Pedobiologia (2003) 47, 447–451

Fig. 2. Analysis of the ESTs, indicating digested vectors (V), cDNA inserts (I) and molecular weight markers (M) (Panel A), sequence length(Panel B), number of contig clusters (Panel C), list of abundant genes (Panel D) and representation within gene classes (Panel E)

450 Stephen R. Stürzenbaum et al.

Pedobiologia (2003) 47, 447–451

Fig. 3. A typical LumbriBASE search by annotation: after a key word input (Panel A) cluster match(es) will be displayed (Panel B) along withalignments (Panel C) and the resultant contig sequence (Panel D). Via the BLAST menu (Panel E) detailed information on sequence identityand similarity is provided (Panel F)

451The earthworm Expressed Sequence Tag project

Pedobiologia (2003) 47, 447–451

Blaxter, M., Daub, J., Guiliano, D., Parkinson, J., Whitton,C. (2002) The Brugia malayi genome project: expressedsequence tags and gene discovery. Transactions of theRoyal Society of Tropical Medicine and Hygiene 96,7–17.

Inaba, K., Padama, P., Satouh, Y., Shin-I, T., Kohara, Y.,Satoh, N., Satou, Y. (2002) EST analysis of gene expres-sion in testis of the ascidian Ciona intestinalis. MolecularReproduction and Development 62, 431–445.

Lee, Y. H., Huang, G. M., Cameron, R. A., Graham, G.,Davidson, E. H., Hood, L., Britten, R. J. (1999) ESTanalysis of gene expression in early cleavage-stage seaurchin embryos. Development 126, 3857–3867.

Parkinson, J., Whitton, C., Guiliano, D., Daub, J., Blaxter,M. (2001) 200 000 nematode expressed sequence tags onthe net. Trends in Parasitology 17, 394–396.

Savan, R., Sakai, M. (2002) Analysis of expressed sequencetags (ESTs) obtained from common carp, Cyprinus carpioL., head kidney cells after stimulation by two mitogens,lipopolysaccharide and concanavalin-A. ComparativeBiochemistry and Physiology B 131, 71–82.

Sonstegard, T. A., Capuco, A. V., White, J., Van Tassell, C. P.,Connor, E. E., Cho, J., Sultana, R., Shade, L., Wray, J. E.,Wells, K. D., Quackenbush, J. (2002) Analysis of bovinemammary gland EST and functional annotation of the Bostaurus gene index. Mammalian Genome 13, 373–379.