A strategy for 454-pyrosequencing of ribosomal SSU DNA and RNA to more accurately assess protist diversity and relative abundance

Summary

Next generation sequencing of ribosomal DNA is increasingly used to assess the biodiversity and structure in microbial communities. Our aim was to develop a method to depict the diversity and community structure of marine planktonic haptophytes to species level. We used a mock community consisting of equal number of cells of 11 species representing all known haptophyte orders to investigate the ability to detect and assess the number of species present, and assess the relative abundance in terms of cell numbers and biomass by 454-pyrosequencing. We compared DNA to RNA/cDNA, 454-pyrosequencing to Sanger sequencing of clone libraries, and two different V4-SSU rDNA haptophyte biased primer pairs. Further, we tested 5 different strategies to clean the sequence data from errors. Up to 6000 unique tags per sample (with avg. 16500 reads) were obtained before sequence cleaning and clustering, and higher diversity was obtained from cDNA than DNA. The distribution of reads assigned to a species was significantly different from the initial proportions of cells or biomass in the culture mix, but cell numbers were better reflected by proportion of cDNA than DNA reads. Here we propose a strategy to more accurately depict the haptophyte diversity and assess relative abundance using 454-pyrosequencing.