Part of the research team traveling on the Amazon River, one of the freshwater systems sampled in the studyM. LENKETwo sequencing methods often used in metagenomics analyses give strikingly different results when applied to relatively understudied microbial communities, according to research led by a team at the American Museum of Natural History (AMNH). The study, published July 31 in Scientific Reports, found that shotgun sequencing, generally thought to be the superior method for characterizing microbial communities, detected considerably less diversity than amplicon sequencing and frequently missed entire phyla when applied to samples of freshwater bacteria.
“It’s surprising that there was such a large difference,” says James Cole, a molecular microbial ecologist at Michigan State University, who was not involved in the work. “It sends out a caution to people using these types of tools to make sure that their tool is actually performing well on the types of samples that they’re working on.”
The amplicon approach matches samples to known bacterial taxa based on a single sequence: that of the highly conserved gene for bacterial 16S rRNA. Shotgun sequencing instead takes a genome-wide approach, targeting random sections of bacterial DNA and matching the resulting profile to a database using common sequences or clade-specific marker genes.
The Pantanal, the world's largest tropical wetland and one of the freshwater systems sampled in the studyM. LENKEComparisons of the two methods in well-studied systems such as the gut microbiome have generally found that shotgun sequencing identifies more microbial diversity—a result attributed to its reliance on more than one part of the genome, and the much larger volume of data it generates.
