A comparison of next-generation sequencing protocols for microbial profiling : a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Genetics, Massey University, Palmerston North, New Zealand

Abstract

The introduction of massive parallel sequencing has revolutionized analyses of microbial communities. Illumina and other Whole Genome Shotgun Sequencing (WGS) sequencing protocols have promised improved opportunities for investigation of microbial communities. In the present work, we compared and contrasted the findings from different NGS library preparation protocols (Illumina Nextera, Nextera-XT, NEXTFlex PCR-free and Ion-Xpress-400bp) and two sequencing platforms (MiSeq and Ion-Torrent). Short reads were analysed using the rapid database matching software PAUDA and visualization software MEGAN5, which provides a conservative approach for taxonomic identification and functional analyses. In analyses of a Tamaki River water sample, biological inferences were made and compared across platforms and protocols. For even a relatively small number of reads generated on the MiSeq sequencing platform important pathogens were identified in the water sample. Far greater phylogenetic resolution was obtained with WGS sequencing protocols than has been reported in similar studies that have used 16S rDNA Illumina sequencing protocols. TruSeq and Nextera-XT sequencing protocols produced similar results. The latter protocol offered cheaper, and faster results from less DNA starting material. Proteobacteria (alpha, beta and gamma), Actinobacteria and Bacteroidetes were identified as major microbial elements in the Tamaki River sample. Our findings support the emerging view that short read sequence data and enzymatic library prep protocols provide a cost effective tool for evaluating, cataloguing and monitoring microbial species and communities. This is an approach that complements, and provides additional insight to microbial culture “water testing” protocols routinely used for analysing aquatic environments.