Nematode gene sequences: update for december 2003.

The molecular characterization of parasitic nematodes and development of novel control strategies can benefit from genomic approaches. The highthroughput generation of expressed sequence tags (ESTs) from numerous nematode cDNA libraries is now providing thousands of new gene sequences, and their availability in public databases will facilitate broad characterization of their function. A project at Washington University’s Genome Sequencing Center is nearing the completion of 235,000 5 ESTs from 28 nematode species (227,272 ESTs as of October 2003). Another 32,078 ESTs from seven species have been contributed by a sister project at the Sanger Institute and Edinburgh University. Sequences are immediately submitted to the database of expressed sequence tags (dbEST) at GenBank and are also available from specialized Web sites (Table 1A). Nematode ESTs have diverse applications (e.g., Blaxter et al., 2002; Jasmer et al., 2001; Murray et al., 2001; Pleasance et al., 2003; Scholl et al., 2003; Srinivasan et al., 2002), and strategies for their use have been reviewed (Blaxter et al., 1999; Grant and Viney, 2001; Marra et al., 1998; McCarter et al., 2000; Parkinson et al., 2001; Parkinson et al., 2003). Here we provide an update on the availability of nematode ESTs and genomes. Since our June 2002 report (McCarter et al., 2002) 153,750 new nematode ESTs have been submitted to dbEST including 129,818 from parasites. A review by Parkinson et al. (2003), focusing on NEMBASE and NemaGene clustering also provides EST totals from November 2002. Caenorhabditis elegans has long been a focus of genome studies (The C. elegans Sequencing Consortium, 1998), and 42% of all nematode ESTs are from C. elegans (Kohara, 1996; McCombie et al., 1992; Waterston et al., 1992). The 300,497 ESTs from 34 nematode species beyond Caenorhabditis include sequences from 22 mammalian parasites, 10 plant parasites, and two free-living bacteriovores (Table 2). For most species, ESTs dominate the conventionally submitted nucleotide and protein sequences in GenBank. ESTs are redundant, with common mRNAs being highly represented. Extrapolating from initial clustering of ESTs in 12 species, the 300,497 ESTs likely represent no more than 100,000 genes. For example, clustering of 12,269 ESTs from Onchocerca volvulus formed 4,208 groups (Williams et al., 2002), and 5,713 ESTs from Meloidogyne incognita formed 1,625 clusters (McCarter et al., 2003). New gene discovery can be maximized by sampling multiple cDNA libraries produced from different stages and tissues. In Strongyloides stercoralis, for example, several thousand ESTs were generated from both first (L1) and third (L3) larva stages. Despite redundancy within each library, only 12% of clusters contained ESTs from both stages (Mitreva et al., 2003). Generating two distinct libraries per source can also substantially increase the pool of identified transcripts. For example, Ancylostoma caninum L3 libraries produced by an SL1 splice leader PCR protocol vs. a modified SMARTTM protocol and sequenced to a depth of ∼800 and ∼2,800, respectively, found only 3.5% overlap (Mitreva, unpubl.). Ancylostoma caninum clusters, along with those from seven other species, are searchable on www.nematode.net, a Web-accessible resource for investigating nematode gene sequences (Fig. 1). Among its features, the site provides NemaGene EST cluster consensus sequences, enhanced online BLAST search tools, functional classifications of cluster sequences, and instructions for clone requests (Wylie et al., 2003). Hundreds of cDNA clones have been provided to 37 nematologists in 12 countries. Plant-parasitic nematode ESTs have been generated from root-knot nematodes (five species, 54,483 ESTs) (Dautova et al., 2001; McCarter et al., 2003), cyst nematodes (four species, 30,698 ESTs) (Popeijus et al., 2000), and one migratory endoparasite, Pratylenchus penetrans (1,928 ESTs, in preparation). Representation of multiple life-stages is improving with 20,114 ESTs now available from five life stages for Heterodera glycines (egg, J2, J3, J4, virgin female) and sequencing from five life stages of Meloidogyne species in progress. However, plant-parasitic nematode genomics is still a nascent field. Within clade IVB (Blaxter et al., 1998), sampling has yet to occur from most migratory Tylenchida species. Virtually no sequence information is available from the distant clade II nematodes including the Dorylaimida and Triplonchida parasites. Perhaps the most visible gap hindering the field is the lack of a draft or complete plant-parasitic nematode genome. The recent announced opening of the National Science Foundation/ Received for publication 14 October 2003. 1 Genome Sequencing Center, Department of Genetics, Box 8501, Washington University School of Medicine, St. Louis, MO 63108. 2 Plant Nematode Genetics Group, Box 7253, North Carolina State University, Raleigh, NC 27695. 3 Divergence Inc., 892 North Warson Road, St. Louis, MO 63141. 4 Department of Genome Sciences, Box 357730, University of Washington, Seattle, WA 98195. Nematode EST sequencing at Washington University was supported in part by NIH grant AI 46593 to R.H.W., NSF grant 0077503 to D.M.B. and S.W.C., and a Helen Hay Whitney/Merck Fellowship to J.P.M. The authors thank members of our EST lab, especially Deana Pape, John Martin, Todd Wylie, Mike Dante, Brandi Chiapelli, and Claire Murphy, and the many collaborators who have generously provided materials (www.nematode.net/Collaborators/), especially Thomas Baum for supplying staged libraries of Heterodera glycines. JPM and DMB are equity holders of Divergence Inc., and JPM is a Divergence employee; this research was not company funded. E-mail: [email protected] This paper was edited by J. L. Starr. Journal of Nematology 35(4):465–469. 2003. © The Society of Nematologists 2003.