Haptophyte mock community explored by 454-pyrosequencing.
A comparison of proportional species distribution and spurious diversity obtained by 454-pyrosequencing DNA and RNA/cDNA extracted from a mock community of 1 million cells of each of eleven species of haptophytes.
These data are from a study on a mock community of haptophytes, where we pooled 1 million cells of each of the species Chrysochromulina throndsenii (UIO048), Diacronema ennorea (UIO021), Emiliania huxleyi (UIO061), Haptolina fragaria (UIO029), Imantonia rotunda (UIO138), Isochrysis galbana (UIO102), Phaeocystis globosa (UIO062), Pleurochrysis pseudoroscoffensis (UIO094), Prymnesium kappa (UIO033), Prymnesium parvum (UIO054) and Prymnesium polylepis (UIO036). From this pool of cells we extracted DNA and RNA (reverse-transcribed into cDNA). In addition we created a sample of equal concentrations of DNA extracted separately from each of the species. For each sample we PCR amplified and pyrosequenced the V4 region of the 18S rDNA. After taxonomical assignation of the reads, we found that the proportional species distribution in the reads was significantly different from the species distribution by both cell numbers and biomass. Further, the species distribution in the reads obtained from cDNA was significantly different from DNA. We subsequently tested four bioinformatic filtering methods to remove errors from the read data; (1): culling of reads with >0 mismatches in barcode and primer, (2): (1) + trimming the reads when average quality score was <30 over a 50 bp moving window, (3): (1) + PyroNoise, (4): (1) + AmpliconNoise. Of these, we found AmpliconNoise to be the most efficient.
These data are from a study on a mock community of haptophytes, where we pooled 1 million cells of each of the species Chrysochromulina throndsenii (UIO048), Diacronema ennorea (UIO021), Emiliania huxleyi (UIO061), Haptolina fragaria (UIO029), Imantonia rotunda (UIO138), Isochrysis galbana (UIO102), Phaeocystis globosa (UIO062), Pleurochrysis pseudoroscoffensis (UIO094), Prymnesium kappa (UIO033), Prymnesium parvum (UIO054) and Prymnesium polylepis (UIO036). From this pool of cells we extracted DNA and RNA (reverse-transcribed into cDNA). In addition we created a sample of equal concentrations of DNA extracted separately from each of the species. For each sample we PCR amplified and pyrosequenced the V4 region of the 18S rDNA. After taxonomical assignation of the reads, we found that the proportional species distribution in the reads was significantly different from the species distribution by both cell numbers and biomass. Further, the species distribution in the reads obtained from cDNA was significantly different from DNA. We subsequently tested four bioinformatic filtering methods to remove errors from the read data; (1): culling of reads with >0 mismatches in barcode and primer, (2): (1) + trimming the reads when average quality score was <30 over a 50 bp moving window, (3): (1) + PyroNoise, (4): (1) + AmpliconNoise. Of these, we found AmpliconNoise to be the most efficient.