Abstract
Protists are key players in microbial communities, yet our understanding of their role in ecosystem functioning is seriously impeded by difficulties in identification of protistan species and their quantification. Current microscopy-based methods used for determining the abundance of protists are tedious and often show a low taxonomic resolution. Recent development of next-generation sequencing technologies offered a very powerful tool for studying the richness of protistan communities. Still, the relationship between abundance of species and number of sequences remains subjected to various technical and biological biases. Here, we test the impact of some of these biological biases on sequence abundance of SSU rRNA gene in foraminifera. First, we quantified the rDNA copy number and rRNA expression level of three species of foraminifera by qPCR. Then, we prepared five mock communities with these species, two in equal proportions and three with one species ten times more abundant. The libraries of rDNA and cDNA of the mock communities were constructed, Sanger sequenced and the sequence abundance was calculated. The initial species proportions were compared to the raw sequence proportions as well as to the sequence abundance normalized by rDNA copy number and rRNA expression level per species. Our results showed that without normalization, all sequence data differed significantly from the initial proportions. After normalization, the congruence between the number of sequences and number of specimens was much better. We conclude that without normalization, species abundance determination based on sequence data was not possible because of the effect of biological biases. Nevertheless, by taking into account the variation of rDNA copy number and rRNA expression level we were able to infer species abundance, suggesting that our approach can be successful in controlled conditions.
Highlights
Determining the abundance of protists is essential for understanding ecosystem functioning, seasonal communities’ turnover and finding key-species in ecosystems
small subunit (SSU) rDNA copy number in Foraminifera Before the present study, nothing was known about the number of SSU rDNA copies in Foraminifera
It is widely accepted that SSU rDNA copy number varies considerably among eukaryotic species [28] but there are few systematic studies that examine the range of these variations in unicellular eukaryotes
Summary
Determining the abundance of protists is essential for understanding ecosystem functioning, seasonal communities’ turnover and finding key-species in ecosystems. Determination of protist abundance relied mainly on direct microscopic counting, quantitative PCR [4] and flow cytometry (FCM) [5,6]. It is useful to determine the broad functional groups that are present in an environmental sample, but its taxonomic resolution is low. This method is only suitable for quantification of phototrophic organisms in water samples. Quantitative PCR can accurately estimate the copy number of a targeted gene present in a sample [7], but it is difficult to link qPCR results with abundance of protists if the gene copy number per species is unknown. Scientists have to make compromises to choose between analysis speed and taxonomic resolution when studying protist abundance
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