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We compiled DNA and RNA isolation protocols for sediment bulk extraction and their yields from Guaymas Basin subsurface sediments recovered during International Ocean Discovery Program Expedition 385 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 downhole. Metagenomic library construction and sequencing was possible from as little as 0.2 to 0.5 ng DNA/cm3 sediment; polymerase chain reaction (PCR) amplification of 16S rRNA genes required in most cases approximately 1–2 ng DNA/cm3 sediment. At in situ temperatures of 50° to 60°C, decreasing DNA recovery leads to increasingly uncertain hit or miss outcomes and failures for metagenomic and amplicon analyses. DNA concentration profiles show that, even before these hot temperatures are reached, relatively moderate temperatures (near 40°C) have a major effect on microbial abundance and DNA yield. Comparison with cell count profiles shows that hydrothermal influence reduces downhole cell densities by multiple orders of magnitude compared to nonhydrothermal sediments. This effect is also visible at relatively moderate temperatures. RNA recovery is highly sensitive to downhole increasing temperatures and decreasing cell numbers, and was most efficient for microbial communities in cool, relatively shallow subsurface sediments.