Table 1.
Higher taxa composition and number of different species per taxa analyzed for the Sanger-sequence reference library used in the pooled DNA samples.
Table 2.
Species delineation results for the Sanger-generated reference library at a 98% similarity threshold.
For each cluster comprising more than one sequence a representative sequence* is presented.
Fig 1.
Cluster analysis conducted on modified reference libraries (characterized by 5-mer, 4-mer, 3-mer and 2-mer homopolymers, respectively) using different similarity thresholds (97–100%, at 1% intervals).
Table 3.
GS FLX sequences generated for each aliquot.
Fig 2.
Read coverage distribution of sample aliquots inferred from pyrosequencing after clustering against the reference library at 99% similarity.
(A) Dataset showing strong positive correlation between aliquot 1 (x-axis) and aliquot 2 (y-axis) of pooled ALLDNA sample (Spearman r = 0.98, P < 0.001). (B) Dataset showing strong positive correlation between aliquot 1 (x-axis) and aliquot 2 (y-axis) of pooled ZPDNA sample (Spearman r = 0.94, P < 0.001).
Fig 3.
A) Species composition of North Sea metazoa from four aliquots of pooled, known DNA samples (ALLDNA, ZPDNA) and two aliquots of one field-collected zooplankton sample (ZPHTS), as assigned by 454 sequencing of the 18S rDNA: V1-V2 region.
The tree, based on consensus sequences of the Sanger-generated reference library, was generated by the Neighbor-Joining algorithm / p-distance model. Clustering against the reference library was performed at 99% sequence similarity threshold using the program cd-hit-est-2d. Percentage of pyrosequencing reads for each species detected in each sample is shown by color-coded rectangles. Black asterisks denote species included in the artificially prepared ZPDNA sample. Red asterisks denote species not included, but detected in the ZPDNA sample. B) Figure 3A continued. C) Figure 3B continued.
Table 4.
Comparison of taxonomic composition of a morphologically analyzed (individuals/m3) and a pyrosequenced zooplankton sample, as aliquots ZPHTS1 and ZPHTS2 (no. of reads per species).
Identifications of the pyrosequenced sample determined by searching against a V1-V2 reference library (99% similarity) and the GenBank database (MEGABLAST search > 90% identity).
Fig 4.
Pie chart illustrating the relative proportion of each phylum detected by MEGABLAST analysis (score and sequence similarity > 100 and 90%, respectively).
(A) ZPHTS aliquot 1. (B) ZPHTS aliquot 2. MEGABLAST hits to ‘uncultured eukaryote clone’ represent unclassified taxa.