Genome Sequence of the Microsporidian Species Nematocida sp1 Strain ERTm6 (ATCC PRA-372)

Microsporidia comprise a phylum of obligate intracellular pathogens related to fungi. Microsporidia Nematocida sp1 strain ERTm6 was isolated from wild-caught Caenorhabditis briggsae and causes a lethal intestinal infection in Caenorhabditis nematodes. We report the genome sequence of N. sp1 ERTm6, which will facilitate study of the Nematocida genus and other Microsporidia.

ment and infect virtually all animal phyla, including humans. Phylogenomic analysis revealed that microsporidia are in the earliest branching group of sequenced fungi and that Nematocida is in the earliest branching group of sequenced microsporidia (1)(2)(3). Outside of their hosts, microsporidia persist as environmentally resistant spores. They infect by deploying an infection apparatus (polar tube), through which spore contents are delivered into host cells. Microsporidia replicate intracellularly as meronts, which differentiate into spores and are shed from the host into the environment. The discovery of Nematocida parisii, a microsporidian species that infects the model organism Caenorhabditis elegans in the wild (4), opened up new avenues for investigating microsporidia pathogenesis (2,5). Several N. parisii and N. sp1 strains have been isolated from wild-caught Caenorhabditis nematodes throughout the world (6). Sequence analysis of two N. parisii (ERTm1 and ERTm3) and one N. sp1 genome (ERTm2) provided the first molecular evidence that microsporidia are likely to be diploid, with extensive heterozygote regions (2). N. sp1 was particularly polymorphic in these regions (1 single nucleotide polymorphism [SNP] every 82 bp). Thus, we sought to investigate the genome of a second N. sp1 strain for comparison.
N. sp1 ERTm6 was found infecting wild-caught Caenorhabditis briggsae JU1638, collected from soil next to a yam/taro plant in Cape Verde Islands (17.13768 N, 25.06689 W). It was subsequently isolated and transferred into the C. elegans wild-type N2 strain for propagation and harvest (4). Total DNA was extracted from Nematocida-infected C. elegans using standard nematode lysis, followed by phenol/chloroform extraction methods (2). The purity and concentration of DNA was checked using NanoDrop ND-8000 UV-Vis (Thermo Fisher Scientific). For genome sequencing, we constructed two genome shotgun libraries with average insert sizes of 178 bases and 2.6 kb and sequenced both using Illumina technology (7,8). Reads were assembled with ALLPATHS-LG version R41828 (9), generating a consensus sequence with a read depth of roughly 118ϫ.
The genome size was estimated to be 4.28 Mb, with a GC content of 38.30%. The assembly was organized in 57 contigs, linked by paired-end reads into 24 scaffolds. As found for other sequenced Nematocida (2), this is a compact genome and 67.48% of the genome sequence is predicted to be coding, with a mean distance between coding sequences of 578.63 bp. The average base is found in a scaffold with an N 50 of 797.7 kb and a contig with an N 50 of 219.3 kb. A total of 2,433 protein-coding genes, 51 tRNA genes, and 9 rRNA genes were predicted as previously described (2).
We found that N. sp1 ERTm6 is likely diploid, although the heterozygous regions are not as polymorphic as ERTm2. The ERTm6 strain has 1 SNP every 989 bp, identified from BWA-MEM (10) alignments of the Illumina reads using the GATK Unified Genotyper version 2.7 (11). This genome, together with the ERTm1, ERTm2, and ERTm3 genomes, provide an excellent resource for investigating microsporidia diversity and coevolution of parasites and hosts.
Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number AKIJ00000000. The version described in this paper is version AKIJ01000000.

ACKNOWLEDGMENTS
We thank the Broad Institute Genomics Platform for generating all of the DNA sequences described here. We also thank Marie-Anne Félix for collecting and generously providing infected nematodes, and Aurore Dubuffet and Hinrich Schulenburg for generously providing the ERTm6 rRNA sequence. This project has been funded in whole or in part with Federal funds from the National Institute of Allergy