Not many of the abundant freshwater bacterial groups have a representative cultured isolate. In this master thesis project, some abundant bacterioplankton from two lakes (Lake Ekoln and Lake Erken) could be isolated by a dilution-to-extinction approach. Sterilized lake water which was obtained through an ultrafiltration system was used resembling a natural medium. Specific fragments of 16s rRNA of the isolates were amplified by universal bacterial primers (27f and 1492r, 341f and 805r.) for genotyping against a freshwater sequence database and RDP training set (Version 7). A total of 33 isolates from the two lakes were taxonomically classified and revealed the isolation of typical and abundant freshwater bacteria. Original bacterial community of Lake Ekoln was also analyzed by 16S rRNA clone library construction for diversity study. Phylogenetic trees were built through neighbor-joining method by Mega (Version 5) to reveal the evolutionary relationships among database entries, obtained isolates and clones.

The main goal of this thesis was to design and implement a tool that would be able to classify the sequences of the 16S rRNA gene into taxonomic categories using the properties of the 16S rRNA gene. The created tool analyzes all input sequences simultaneously, which differs from common classification approaches, which classify input sequences individually. This tool relies on the fact that bacteria contain several copies of the 16S rRNA gene, which may differ in sequence. The main contribution of this work is design, implementation and evaluation of the capabilities of this tool. Experiments have shown that the proposed tool is able to identify the corresponding bacteria for smaller datasets and determine the correct ratios of their abundances. However, with larger datasets, the state space becomes very large and fragmented, which requires further improvements in order for it to search the state space in an efficient way.

Polymerase chain reaction (PCR) is a method used in many research protocols to amplify a small amount of a short segment of DNA to millions of copies. PCR is used for many taxonomic studies, as well as for some medical diagnostic procedures. Through PCR, short DNA primers bind to the template DNA to allow the thermostable DNA polymerase to copy the DNA. Often, researchers create universal primers to target a conserved region of DNA in multiple species, for example, the 16S rRNA gene in bacteria. The problem with these universal primers is that they do not always perfectly match the target DNA. The mismatch primers can still bind to the template, but could affect the efficiency of the PCR amplification. The effect of mismatch primers on the efficiency of the amplification in PCR is the focus of this thesis. Four forward primers with various mismatch overhangs were generated and incorporated into a DNA template through an initial PCR. These primers contained the binding region complementary to the V3/V4 region of the 16S rRNA bacterial gene. Further quantitative PCR (qPCR) reactions were run on these newly-made templates using two sets of primers complementary to the 16S rRNA gene region – one with ambiguous base pairs, one with unambiguous base pairs. The qPCR amplification curves, the Cq values, and the initial concentrations of DNA products (seed values) were analyzed and compared. The results showed differences in the Cq values and seed values between the reactions containing mismatches and those not containing mismatches. Other variables including annealing temperature, addition of Illumina sequencing tails to the primers, and initial primer concentration were also tested to determine if these variables had an effect on the amplification. The results from these reactions using different variables were inconclusive.