Design and Testing of a Simple and Efficient 2-step Method for 18s Metabarcode Amplification and Illumina Library Preparation

Abstract

Metabarcoding methods allow rapid characterization of complex microbial communities through the sequencing of shared, taxonomically diagnostic genetic loci. These methods are particularly useful for identification of diverse microbiota lacking clear morphological traits. Our project seeks to apply DNA metabarcoding to characterize microbial communities 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 objective is to design a 2-step PCR amplification strategy to simultaneously amplify the V4 and V8-9 hypervariable regions of the eukaryotic 18s ribosomal DNA locus and prepare amplification products for sequencing on the Illumina MiSeq platform. We will use two sets of "fusion" primers: 1) a first set with complementarity to conserved flanking regions of target loci 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 describe my progress toward designing and testing of these fusion primers on several microbial communities isolated from aquatic substrates, based on amplification strength and consistency.