The DNA event horizon in the Guaymas Basin subsurface biosphere: technical advances and re-defined limits in bulk extractions of nucleic acids from deep marine sediments

We compiled DNA and RNA isolation protocols for sediment bulk extraction and their yields from Guaymas Basin subsurface sediments, and evaluated their sensitivity for metagenomic and amplicon analyses of subsurface microbial communities. Guaymas Basin sediments present a challenge for DNA and RNA recovery due to high concentrations of hydrocarbons, steep thermal gradients and rapidly declining cell numbers downcore. Metagenomic library construction and sequencing was possible from as little as 0.2 to 0.5 ng DNA/cm3 sediment; PCR amplification of 16S rRNA genes required in most cases approx. 1-2 ng DNA/cm3 sediment. At in-situ temperatures of 50 to 60 degrees C, decreasing DNA recovery leads to increasingly uncertain "hit or miss" outcomes and to failures for metagenomic and amplicon analyses. DNA concentration profiles show that, even before these hot temperatures are reached, relatively moderate temperatures have a major effect on microbial abundance and DNA yield. Comparison with cell count profiles shows that hydrothermal influence is reducing downcore cell densities by multiple orders of magnitude faster compared to non-hydrothermal sediments; this effect is also visible at relatively moderate temperatures. To an even greater degree than DNA, RNA recovery is highly sensitive to downcore increasing temperatures and decreasing cell numbers, and worked best for microbial communities in cool, relatively shallow subsurface sediments.