Abstract
Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000–20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing.
Highlights
Haptophytes are a major component in marine pico- and nanoplankton communities, occurring in all seas as important primary producers [1]
We explored the mock community further using 454 pyrosequencing, addressing the following questions: i) Do proportion of rDNA and rRNA pyrosequencing reads correlate to proportion of cell number or biomass of the haptophyte species present in the mock community? ii) Is there a difference between targeting the rRNA gene and the rRNA with respect to detection of species and estimation of relative abundance of the different species? iii) How can we process 454 pyrosequencing reads to keep as much of the taxonomic information as possible while minimising spurious phylotypes, in order to better infer species composition and diversity?
For a sample consisting of 11 species of haptophytes, with equal proportions in terms of cell number, rarefaction analysis suggested that 1400 reads were required to recover all of the species, with our primers and experimental setup
Summary
Haptophytes are a major component in marine pico- and nanoplankton communities, occurring in all seas as important primary producers [1]. Knowledge on the haptophyte diversity, distribution and dynamics at the species level is, limited because they are small and fragile, and species identification usually requires electron microscopy or molecular biological methods, except for a few recognisable bloom formers such as Emiliania huxleyi and Phaeocystis spp. During monitoring surveys by light microscopy haptophytes are usually identified only to genus level Because physiological and ecological functionalities, e.g. growth preferences and tolerances, growth rate, nutrition, swimming behaviour, toxicity, and life cycle differ among species [3,6], there is a strong need for more efficient and accurate methods to identify and quantify haptophytes at the species level in order to better understand their ecological and economical roles
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