QUANTIFICATION OF HAPTOPHYTE SPECIES BY PYROSEQUENCING: IS READ ABUNDANCE PROPORTIONAL TO SPECIES BIOVOLUME?

Summary

Amplicon-sequencing of ribosomal RNA genes has become a standard for assessing microbial diversity in environmental samples. Abundance is of interest in addition to presence/absence of species to assess their ecological importance. The proportion of DNA sequences/reads from a particular species may, however, not reflect the proportion of the species in a sample in terms of cell number. We investigated the relationship between cell number and biovolume in an artificial community of haptophyte species, and the number of DNA sequences/reads appearing in libraries produced by cloning and 454-pyrosequencing nucleic acids extracted from this community. One million cells from each of 11 haptophyte species were pooled, and DNA and RNA extracted, and the V4 region of the small subunit ribosomal gene was amplified using haptophyte specific primers. The amplicons were sequenced by 454-pyrosequencing or Sanger sequencing. Pyrosequencing with 17000 reads per sample recovered all species in the culture mix, whereas clone libraries with 50-80 sequences per sample only recovered <50% of the species. Our results show that the proportion of reads from the different species in the 454-libraries were significantly different from the proportion of cells in the pool. There was a positive correlation between cell volume and read abundance, and thus cell size should be taken into account when interpreting 454-sequence data quantitatively.