Development of a protocol for environmental PCR of 18S rDNA V4 region and diversity survey of freshwater protists using 454 titanium pyrosequencing

Abstract

Abstract

The application of molecular methods to biodiversity studies of fresh water environments has revealed that there is a large amount of species that have so far gone undetected, and little has been known of the diversity of single celled eukaryotes (protists) in the freshwater. To investigate the eukaryotic diversity in freshwater sediments we developed primers targeting the V4 region of 18S rDNA that would amplify a broad diversity of eukaryotes, and a PCR protocol suitable for the 454 GS FLX Titanium pyrosequencing platform. A small pilot sequencing showed that we were able to retrieve sequences from all eukaryotic supergroups except excavata. This included novel groups that rarely have been described in the freshwater environments, such as relatives of the marine parasites Perkinsus and Parvilucifera, as well as the first truly freshwater sequence from the haptophyte group Pavlovophyceae.

A replicate of the proposed protocol and a threefold increase of the sequencing effort did not result in any significant increase in the revealed diversity among the common groups. The more rare groups however were not consistently retrieved in both rounds of sequencing. This indicates that several PCR amplifications and subsequent sequencing can reveal more of the uncommon groups than deeper sequencing of a single PCR amplification.

The most obvious difference between the two rounds of sequencing is the large difference of abundance in some of the retrieved groups. This difference is most likely the effect of bias in the PCR since all amplifications were performed on the same DNA isolate. Closer inspection of a single group, the cryptomonads, show that the differences are not as prominent when looking at the species detected at a genus level. This shows that quantitative measures from PCR based diversity studies should be used with caution.