A discriminatory study of the total DNA pool

Most commonly, NGS-based microbial ecology studies are performed on the total DNA pool (totDNA), which comprises extracellular (exDNA) and intracellular (iDNA) DNA fractions. Although exDNA is quantitatively relevant in many habitats, a discrimination of both fractions is commonly not applied. The present study investigates two different batch anaerobic digester microbiomes over the course of 14 days by comparative discriminatory 16S rRNA sequencing of exDNA and iDNA vs. totDNA. Our results show that totDNA yields lower diversities and less unique reads as compared to iDNA and that exDNA-derived sequences are more similar to those deriving from totDNA than from iDNA. iDNA, however, reveals a more complex microbial community with high numbers of sequences being unique for this DNA fraction. Thus, we postulate that abundant sequences present within the exDNA fraction are masking the overall results of totDNA. We provide evidence that exDNA qualitatively biases microbiome studies and that iDNA might be the appropriate DNA fraction to gain genetic information of the target habitats. To the best of our knowledge, this is the first study of in depth discrimination and characterisation of extracellular- and intracellular fractions as compared to the total DNA pool by means of next generation sequencing.