Design and Testing of a Simple and Efficient 2-step Method for 16s Metabarcode Amplification and Illumina Library Preparation

Abstract

Metabarcoding is a DNA sequencing strategy that allows for high throughput sequencing and DNA-based identification of complex microbial communities. This method is particularly useful for identifying microorganisms based on taxonomically informative DNA sequences rather than morphological or metabolic characteristics. Our project seeks to apply DNA metabarcoding techniques to characterize microbial diversity present on the shells of freshwater turtle species in Oklahoma. Results will ultimately provide insight into aquatic microbial ecology and response to habitat perturbation. Our preliminary efforts involve designing a 2-step PCR protocol to simultaneously amplify the V3-V4 hypervariable regions of the prokaryotic 16s ribosomal RNA locus and incorporate sequence motifs necessary for sequencing on the Illumina MiSeq platform. Our technique relies on two sets of "fusion" primers for PCR amplification: 1) a first set with complementarity to conserved regions flanking the V3-V4 regions and a tail incorporating short, sample-specific index sequences and complementarity to Illumina sequencing primers; 2) a second set allowing amplification from the first PCR reaction and containing a tail allowing binding to the Illumina flowcell. I will present initial results of primer design and testing on several microbial communities isolated from aquatic substrates based on amplification strength and consistency.