Linking meta-omics and biogeochemistry approaches in soils has remained challenging. This study evaluates the use of an internal RNA extraction standard and its potential for making quantitative estimates of a given microbial community size (biomass) in soil metatranscriptomics. We evaluate commonly used laboratory protocols for RNA processing, metatranscriptomic sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Metatranscriptomic profiles from soil samples were generated using two library preparation protocols and prepared in triplicates. RNA extracted from pure cultures of Saccharolobus solfataricus was added to the samples as an internal nucleic acid extraction standard (NAEstd). RNA reads originating from NAEstd were identified with a 99.9% accuracy. A remarkable replication consistency between triplicates was seen (average Bray-Curtis dissimilarity 0.03 ± 0.02), in addition to a clear library preparation bias. Nevertheless, the between-sample pattern was not affected by library type. Estimates of 16S rRNA transcript abundance derived from qRT-PCR experiments, NAEstd and a previously published quantification method of metatranscriptomics (hereafter qMeTra) were compared with microbial biomass carbon (MBC) and nitrogen (MBN) extracts. The derived biomass estimates differed by orders of magnitude. While most estimates were significantly correlated with each other, no correlation was observed between NAEstd and MBC extracts. We discuss how simultaneous changes in community size and the soils nucleic acid retention strength might hamper accurate biomass estimation. Adding NAEstd has the potential to shed important light on nucleic acid retention in the substance matrix (e.g., soil) during extraction.

Agricultural University of Iceland Hvanneyri Iceland

Archaea Biology and Ecogenomics Unit Department of Functional and Evolutionary Ecology University of Vienna Vienna Austria

Department of Arctic and Marine Biology UiT The Arctic University of Norway Tromsø Norway

Department of Bacterial Physiology University Greifswald Greifswald Germany

Department of Microbiology and Ecosystem Science University Vienna Vienna Austria

Institute of Bioinformatics University Medicine Greifswald Greifswald Germany

Institute of Mathematics and Computer Science University Greifswald Greifswald Germany

Institute of Soil Biology and Biogeochemistry Biology Centre CAS České Budějovice Czech Republic

Interfaculty Institute of Genetics and Functional Genomics University Greifswald Greifswald Germany

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