A More Accurate, Cheaper, Faster Microbiome Preparation Method: Application to Study the Microbiome in Cocaine Addiction

Background

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. All microbiome sequencing methods depend on recovering the DNA from a sample as a first step. Gentle DNA preparation methods preserve DNA quality but can bias the results by failing to open hard to lyse bacteria. Harsher methods like bead beating can also bias results by degrading the DNA of the more easily lysed microbes. A novel DNA extraction (K) method designed to maintain representation across the population of difficult and easily lysed bacteria was developed.

Methods

DNA extraction bias was evaluated for the novel K method and compared to 6 commercially and publicly available protocols including a method used in Human Microbiome Project (HMP) using a mock community. Additional testing on 20 human fecal samples compared microbes recovered by the novel K method with a HMP protocol. Mouse fecal samples from 227 mice with 29 tracked behavior tests were evaluated using the novel K method via 16S rRNA gene based sequencing before and after susceptibility to cocaine addiction test.

Results

Evaluation of the mock of community demonstrated that the novel protocol yielded optimal population representation, although amplicon-dependent variation was observed for all methods. Comparison of novel K and HMP methods using human fecal samples revealed that the novel protocol consistently recovered bacterial community structures with significantly higher levels of Firmicutes species containing thicker cell walls and are harder to lyse. Characterization of cocaine addiction mouse microbiome via novel K method showed Firmicutes species including Flavonifractor sp., Eisenbergiella sp., Lactobacillus sp., and Oscillibacter sp. that may play a significant role in cocaine addiction behavior.

Conclusions

Our findings suggest that the novel K method yields improved representation of hard to lyse microbes while reducing sample preparation hands-on time 20-fold for manual 96 sample methods. The novel K method reduces or eliminates microbial population bias due to DNA lysis and preparation, a vital first step for NGS assays, presenting opportunities for improved profiling of individual known and unknown microbes within microbial communities that are associated with healthy and dysbiotic conditions.