Nematode metagenetic/metagenomic control sample
Evaluating high-throughput sequencing as a method of metagenomic analysis of nematode diversity
It is well established that nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecosystem ecology is still an enigma.  Because nematode identification of all individuals at the species level using standard techniques is difficult, laborious, and extremely time consuming, the characterization of nematode communities continues to be resolved at coarser than the species level leaving ecological analysis partially ambiguous or superficial.  Novel cloning-independent pyrosequencing may offer a potentially rapid tool to inventory nematode fauna at previously unparalleled levels of resolution at faster speeds and lower cost. The main objective of our study was to assess the suitability of massively parallel sequencing using GS FLX technology for analysis of nematode diversity from metagenomic samples.   Two rapidly evolving ~ 400 bp sections of 18S and 28S rRNA loci flanked by “universal” primers were used to identify nematode taxa. To test this approach, we hand-picked 41 diverse reference nematodes (1 – 10 individuals per species) to set up 4 artificial metagenomic samples. These nematode samples were amplified individually (one species at a time) and collectively (all species together).  Two samples came from pooling PCR products amplified from single nematode species (18S sPCR and 28S sPCR).  Two additional metagenomic samples consisted of DNA amplified from DNA extracted from pooled nematode species (18S mPCR and 28S mPCR). All metagenomic samples were sequenced in the same direction on Genome Sequencer. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, ~81% were >199 bp in length. Within those reads ~86% matched referenced 41 species. While ~47% of these reads matched a reference perfectly, 38% differed from reference by 1-2 bp, and the rest of the reads varied by >3 bp. Overall, results strongly support the suitability of massively parallel sequencing technology for identification of nematode individuals from environmental samples. Although neither rRNA section recovered all nematode species, the 18S locus resulted in higher species recovery (~95%) than the 28S locus (~90%) and the use of both loci improved the detection level of nematode species (~97%). Of the reads >199 bp that did not match our reference database (~14%), 2% were identified as of nematode origin, 5% as chimeras, and most of the remaining reads as fungal. By contrast, the frequency of reads representing individual species did not strongly correlate with the number of individuals in the artificial metagenomic samples suggesting that inferring the relative abundances of species within a nematode community maybe premature.