BMC Genomics 2008, 9:200 http://www.biomedcentral.com/1471-2164/9/200 DNA was extracted from 2 × 107germinated Brachiola alge- rae spores (a strain originally isolated the mosquito Anopheles stephensi). Spores were cultivated in vitro in RK13 rabbit kidney cells at 30C in 5% CO2, purified and germinated by incubation in 0.3% H2O2 for 16 hours at 25°C. The germinated spores were concentrated by cen- trifugation suspended in 100 μg/ml Proteinase K-PBS and incubated for 15 min at 65°C. DNA was extracted using phenol-chloroform, followed by ethanol precipitation. DNA was then dissolved in TE buffer and stored at -70°C until use. A sample of purified Brachiola algerae DNA was amplified using Genomphi (Amersham) to produce 10 μg of DNA and another sample of 1.7 μg of purified DNA was processed directly to make two separate libraries. DNA was sheared, blunt-ended and cloned as described for E. aedis above. A respective 64,433 and 140,051 bases from the Genomiphi and non-Genomiphi treated librar- ies were sequenced with ABI Big Dye 3.1. This gave a total of 203,748 non-overlapping bases of sequence in 181 contigs with an average length of 1,125 bp. Areas of six representative B. algerae clones from the Genomiphi-amplified DNA library were reamplified by PCR from genomic DNA to confirm that the Genomiphi process had not amplified chimeric sequences. Primers were designed to areas of 6 clones. These fragments were between 525 and 1000 base pairs and included non-cod- ing areas, putative transposases, transposons, protein- coding genes, and an SSU gene area. Contigs were analysed by BlastX and BlastN to sequences in GenBank. Open reading frames were considered signif- icantly similar if E values were less than 0.00001. Contigs were further searched for stretches of nucleotides coding for sequences of at least 100 amino acids, and these were considered ORFs. New sequences were deposited in GenBank under acces- sion numbers ET437577–ET437812 and ET437979– ET437981 (E. aedis) and ET223031–ET223211 (B. alge- rae). Authors' contributions BAPW and RCHL constructed libraries, sequenced clones, analysed data and drafted the paper, JJB and LMW con- tributed microsporidian material and to the writing of the paper. NMF and PJK conceived of the study, analysed data and drafted the paper. Acknowledgements This work was supported by a grant from the Canadian Institutes for Health Research to PJK (MOP-42517) and a grant from the Natural Sciences and Engineering Research Council of Canada to NMF. PJK is a Fellow of the Canadian Institute for Advanced Research and senior investigator awards from the Michael Smith Foundation for Health Research. References 1. Didier ES: Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Trop 2005, 94:61-76. 2. Vossbrinck CR, Maddox JV, Friedman S, Debrunner-Vossbrinck BA, Woese CR: Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature 1987, 326:411-414. 3. Vossbrinck CR, Woese CR: Eukaryotic ribosomes that lack a 5.8S RNA. Nature 1986, 320:287-288. 4. Hirt RP, Logsdon JM Jr., Healy B, Dorey MW, Doolittle WF, Embley TM: Microsporidia are related to Fungi: evidence from the largest subunit of RNA polymerase II and other proteins. Proc Natl Acad Sci USA 1999, 96:580-585. 5. Edlind TD, Li J, Visversvara GS, Vodkin MH, McLaughlin GL, Katiyar SK: Phylogenetic analysis of the b-tubulin sequences from amitochondriate protozoa. Mol Phylogenet Evol 1996, 5:359-367. 6. Keeling PJ, Luker MA, Palmer JD: Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi. Mol Biol Evol 2000, 17:23-31. 7. Van de Peer Y, Ben Ali A, Meyer A: Microsporidia: accumulating molecular evidence that a group of amitochondriate and sus- pectedly primitive eukaryotes are just curious fungi. Gene 2000, 246:1-8. 8. Gill EE, Fast NM: Assessing the microsporidia-fungi relation- ship: Combined phylogenetic analysis of eight genes. Gene 2006, 375:103-109. 9. Peyretaillade E, Biderre C, Peyret P, Duffieux F, Méténier G, Gouy M, Michot B, Vivarès CP: Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dis- persion of ribosomal genes and a LSU rRNA reduced to the universal core. Nucleic Acids Res 1998, 26:3513-3520. 10. Katinka MD, Duprat S, Cornillot E, Méténier G, Thomarat F, Prensier G, Barbe V, Peyretaillade E, Brottier P, Wincker P, Delbac F, El Alaoui H, Peyret P, Saurin W, Gouy M, Weissenbach J, Vivarès CP: Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature 2001, 414:450-453. 11. [http://gmod.mbl.edu/perl/site/antonospora01?page=intro]. 12. Slamovits CH, Fast NM, Law JS, Keeling PJ: Genome compaction and stability in microsporidian intracellular parasites. Curr Biol 2004, 14:891-896. 13. Zhang J: Protein-length distributions for the three domains of life. Trends Genet 2000, 16:107-109. 14. Polonais V, Prensier G, Méténier G, Vivarès CP, Delbac F: Micro- sporidian polar tube proteins: highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evo- lutionarily distant Antonospora and Encephalitozoon groups. Fung Genet Biol 2005, 42:791-803. 15. Slamovits CH, Burri L, Keeling PJ: Characterization of a diver- gent Sec61beta gene in microsporidia. J Mol Biol 2006, 359:1196-1202. 16. Corradi N, Akiyoshi DE, Morrison HG, Feng X, Weiss LM, Tzipori S, Keeling PJ: Patterns of genome evolution among the micro- sporidian parasites Encephalitozoon cuniculi, Antonospora locustae and Enterocytozoon bieneusi. PLoS ONE 2007, 2:e1277. 17. Williams BA, Slamovits CH, Patron NJ, Fast NM, Keeling PJ: A high frequency of overlapping gene expression in compacted eukaryotic genomes. Proc Natl Acad Sci USA 2005, 102:10936-10941. 18. Corradi N, Gangaeva A, Keeling PJ: Comparative profiling of overlapping transcription in the compacted genomes of microsporidia Antonospora locustae and Encephalitozoon cuniculi. Genomics 2008 in press. 19. Bennetzen JL: Mechanisms and rates of genome expansion and contraction in flowering plants. Genetica 2002, 115:29-36. 20. Hinkle G, Morrison HG, Sogin ML: Genes coding for reverse transcriptase, DNA-directed RNA polymerase, and chitin synthase from the microsporidian Spraguea lophii. Biol Bull 1997, 193:250-251. 21. Mittleider D, Green LC, Mann VH, Michael SF, Didier ES, Brindley PJ: Sequence survey of the genome of the opportunistic micro- sporidian pathogen, Vittaforma corneae. J Eukaryot Microbiol 2002, 49:393-401. 22. Xu J, Pan G, Fang L, Li J, Tian X, Li T, Zhou Z, Xiang Z: The varying microsporidian genome: existence of long-terminal repeat retrotransposon in domesticated silkworm parasite Nosema bombycis. Int J Parasitol 2006, 36:1049-1056. Page 8 of 9 (page number not for citation purposes)