Is the rare biosphere real or a technical artifact? A case study involving a multi-year deep subsurface time series

Abstract:

The question of “who” is present in extreme environments is one of the major questions surrounding geobiological research. Techniques such as high-throughput sequencing and single cell genomics are illuminating previously unseen organisms that exist in low abundance. Commonly referred to as the “rare biosphere”, the contribution of these low abundance organisms and the effect that they have on traditional diversity estimates is poorly understood. Here, we present a study that critically examines the effect that sequencing depth has on the Chao1 alpha diversity estimator and the Sørensen similarity index. This is achieved through (1) a theoretical analysis of simulated community distributions and (2) a comparison between subsurface Sanger-sequenced clone libraries and high-throughput amplicon sequences generated from a 1.3 km deep fracture (Be326_Bh2). The screening of single cells, metagenomics, and metatranscriptomics are then used to assess the authenticity and contribution of rare members identified in the deeply sequenced amplicon datasets of Be326_Bh2. In particular, a “rare” (<1% relative abundance in 16S rDNA V6 amplicon libraries) member of Ignavibacteria that has been identified and sequenced through single cell genomics is molecularly tracked over the course of a 3-year sampling campaign in Be326_Bh2 while other “rare” members prove to be merely sequencing artifacts. Ultimately, these results help us better understand the limitations of methods used to calculate diversity in long-tail environments like the terrestrial deep subsurface and the role that the rare biosphere plays in Be326_Bh2.