## 3 Results
### High Throughput Nucleic Acid Sequencing  
The relative abundance of Operational Taxonomic Units (OTUs) was determined by High Throughput Sequencing (HTS), using both ends of DNA fragments in both reverse and forward orientation. No reference was made to microscopic data from shared samples in determining taxonomic grouping using HTS. Employing NGS Core Tools for longer reads and increased quality, a total of 26.5 million reads were reduced to 10.9 million usable sequences. Three poor quality samples (9, 12 and 21) were discarded after trimming and merging. The number of sequences examined per sample indicated that numbers of OTUs resolved stabilized at approximately 100,000 sequences per sample (Figure 1), where the accumulative differentiation of OTUs is expressed as rarefaction curves. Data indicated that additional sequencing beyond 100,000 will not provide greater coverage of the microbial assemblage. Sequencing data was edited for relevance to 18S. freshwater sample analysis. For example, major taxonomic groups identified were the algae Archaeplastida, Hartophyte, Cryptophyceaeae Archaeplastida, Hartophyte, Cryptophyceaeae and a super family of Stramenoplies. However, each sample contained protests such as _Centrohelida_, _Excavata_ and Amoebozoa, and slime molds such as _Protstiales_, which were irrelevant to 18S freshwater analysis and were not included in the analytical process. At 'Class' and 'Phylum' levels Stramenopiles, Archaeplastida and Chioroplasida were dominant groups, with Chlorophyllta and Chlorophyllvae abundant at the 'Family' level, but with  
Figure 1: Rarefaction curves showing the relationship between sequences per sample and Operational Taxonomic Unit (OTU) resolution. In the lower graph, samples are categorized by lake. few major differences between samples at this level. The data were too 'noisy' to interpret at the 'Genus' level. Using the QUIIIM script summarized_taxa_through_plots.py the summarized taxonomy was categorized according to three factors; sample, location and temperature. Detailed representations of alpha diversity determined through HTS and related to location and temperature are shown in Supplementary Materials B. Supplementary Materials B Figure S1 shows alpha diversity for all samples at the 'Phylum' level, and in Supplementary Materials B, Figures S2 and S3 alpha diversity at the 'phylum' level has been categorized according to temperature and lake respectively. Alpha diversity is extended to 'Class' level in Supplementary Materials B Figure S4 and categorized according to temperature and lake in Supplementary Materials B, Figures S5 and S6 respectively. For analytical purposes temperatures were divided into three groups: 4-10 \({}^{\circ}\)C, low; 10-20 \({}^{\circ}\)C, medium and 20-26.8 \({}^{\circ}\)C, high. At the 'Phylum' level, the low temperature samples contained relatively higher percentage of Archaeplastida. Interestingly, the abundance of Cryptophyceaeae and Hartophyta was higher in the low temperature samples. A similar phenomenon was also evident at the 'Class' level, where there was a higher abundance of Archaeplastida and Chloroplastica in samples at medium and high temperatures. Higher proportions of Cryptophyceaeae_ Cryptomonadales and Hartophyta_Prymnesiophyceaeae were found in low temperature samples. The same trend was seen at the 'Order' level where the percentage of Archaeplastida_Chloroplyta_Chloroplyta was higher in the medium and high temperature samples. Chlorophyta_Sphaeropheles and Chlorophyta_Chloroplyceae were in relatively low abundance in the low temperature samples. At the 'Genus' level, almost all of the OTUs were not assigned, suggesting that 'Genus' level data were inappropriate for this analysis. Overall, the low temperature environment exhibited different features compared to higher temperature environments, indicating a critical role in determining the differential abundance of phytoplankton taxa. Summarized taxonomy were also categorized according to the harbor where the samples were collected.