April 16, 2023
Recent advances in new molecular biology methods and next generation sequencing (NGS) technologies have revolutionized metabarcoding-based research to study complex microbial communities in diverse environments. The inevitable first step in sample preparation is the isolation of DNA, which introduces a number of biases and inconsistencies.In this study, we evaluated the effects of five DNA isolation methods (B1: standard phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitation, -both B1 modifications, K1: DNeasy PowerWater Kit (QIAGEN) , K2: a modified DNeasy PowerWater Kit (QIAGEN)) and a direct PCR approach (P), which completely bypasses the isolation step, were evaluated for DNA composition and yield of bacterial “mock-ups” and marine communities from the Adriatic Sea. Methods B1-B3 generally resulted in higher DNA yields and more similar microbial communities, but with greater interindividual variability, with each method showing significant differences in specific community structure in which rare taxa play a key role. None of the methods proved superior or came closest to the theoretically expected composition of the mock community; all showed skewed ratios of bacterial species but in similar ways, which could be attributed to other factors such as primer bias or taxon-specific 16S rRNA gene copy number. Direct PCR, which has been little used in environmental microbiology, is an interesting approach when large numbers of samples need to be processed. We emphasize the importance of making a careful decision about the choice of DNA isolation method or direct PCR approach, but even more important is its consistent application throughout the research. Read more here.