| dc.description.abstract | Brusdalsvannet is a 7.52 km² drinking water reservoir located in Aalesund commune, surrounded by forest
with high canopy cover on one side, dense habitation and the main highway into Aalesund on the other
side. Consequently, there are a number of different sources of pollution of Brusdalsvannet, and amongst
these, wildlife and humans may be sources of waterborne pathogens. The presence of waterborne
Escherichia coli (E. coli) in drinking water indicates that other pathogens may be present and can therefore
be used as an indicator organism for water pollution. There have been frequent findings of E.coli in the
drinking water reservoir in Aalesund, which triggered a search for contaminating source and an interest to
better understand the epidemiology of E.coli. Several techniques are available to detect and genotype
E.coli. In this thesis, the aim is to establish an in-house workflow for detection and genotyping E.coli by
Next Generation Sequencing (NGS) on an Ion Torrent Platform. Overall purpose is to see if NGS can give
more details of the species of E.coli, to help tracking the source of the pollution and to establish a rapid
workflow for continuous detection and genotyping of E.coli. Workflow for NGS on Ion Torrent PGM contains
several steps and it would be too time-consuming and expensive to optimize every step within this study’s
limits. Some optimizing procedures are included in the study, not enough to give exact answers, but
enough to give some recommendations for further analysis. Focus has been on pure culture selection,
DNA isolation and library preparation, as the largest challenges have been in those steps of the workflow.
The separation and detection of colonies has been challenging, but the present work shows that optimizing
the growth temperature and adding quality assurance steps may help to separate E.coli from other
coliforms. DNA input in library preparation should be relatively high in order to achieve a representative
selection of fragments to sequence. It was shown that DNA isolation from bacteria suspension did not give
the wanted DNA yield. This study gives an indication that time of lysis of the cells plays a role in DNA
output. It was shown that DNA input to library preparation might influence on how long the enzyme in the
fragmentation step should work. Large amount of input DNA to library preparation, even within
recommended limits, lead to problems later in the sequencing and influenced on the result. Results from
this study shows that it is possible to detect and genotype E.coli in drinking water by NGS on Ion Torren
PGM within 5-6 days, but results have to be validated further | en |
