Quantifying Soil Microbiome Abundance by Metatranscriptomics and Complementary Molecular Techniques-Cross-Validation...

Area of Science:

• Soil microbiology
• Molecular ecology
• Biogeochemistry

Background:

• Linking soil microbial community structure and function to biogeochemical processes is crucial but methodologically challenging.
• Quantitative soil metatranscriptomics requires accurate estimation of microbial biomass.
• Internal standards can improve the reliability of nucleic acid extraction and quantification.

Purpose of the Study:

• To evaluate an internal RNA extraction standard (NAE_std) for quantitative microbial biomass estimation in soil metatranscriptomics.
• To compare biomass estimates derived from NAE_std, qRT-PCR, and a previous method (qMeTra) with soil microbial biomass carbon (MBC) and nitrogen (MBN).
• To assess the impact of laboratory protocols and soil properties on RNA extraction efficiency and biomass quantification.

Main Methods:

• Metatranscriptomic sequencing of soil samples using two library preparation protocols in triplicates.
• Addition of an internal RNA extraction standard (NAE_std) from *Saccharolobus solfataricus* to soil samples.
• Quantitative reverse transcription polymerase chain reaction (qRT-PCR) for 16S rRNA transcript abundance.
• Comparison of biomass estimates with microbial biomass carbon (MBC) and nitrogen (MBN) measurements.

Main Results:

• High accuracy (99.9%) in identifying NAE_std reads and good replication consistency between triplicates (Bray-Curtis dissimilarity 0.03).
• A clear bias was observed between library preparation protocols, although it did not affect the between-sample patterns.
• Biomass estimates derived from different methods (NAE_std, qRT-PCR, qMeTra) varied by orders of magnitude, with no correlation between NAE_std and MBC.
• Significant correlations were found between most biomass estimation methods, except for NAE_std and MBC.

Conclusions:

• The internal RNA standard (NAE_std) shows potential for assessing nucleic acid retention during soil extraction.
• Accurate microbial biomass estimation in soil metatranscriptomics remains challenging due to variations in community size and soil nucleic acid retention.
• Further research is needed to refine methods for robust quantitative biomass estimation in complex soil environments.