Soil metatranscriptomics: An improved RNA extraction method toward functional analysis using nanopore direct RNA sequencing

Soil microbes play an undeniable role in sustainable agriculture, plant health, and soil management. A deeper understanding of soil microbial composition and function has been gained through next-generation sequencing. While soil metagenomics has provided valuable information about microbial diversity, issues stemming from RNA extraction, low RNA abundance in some microbial populations (e.g., viruses), and mRNA enrichment have slowed the progress of soil metatranscriptomics. A variety of soil RNA extraction methods have been developed so far. Yet none of the available protocols can obtain RNA with high quality, purity, and yield for third-generation sequencing. This latter requires RNA with high quality and large quantities (with no or low contamination, such as humic acids). Also, use of commercial kits for in-batch soil RNA extraction is quite expensive, and these commercial kits lack buffer composition details, which prevents the optimization of protocols for different soil types. An improved and cost-effective method for extracting RNAs from mineral and organic soils is presented in this paper. An acidic sodium acetate buffer and phosphate buffer with modifications to bead-beating and nucleic acid precipitation lead to higher RNA yields and quality. Using this method, we obtained almost DNA-free RNA. By using nanopore’s direct RNA sequencing, the extracted contamination-free RNAs were successfully sequenced. Lastly, taxonomic groups such as bacteria, fungi, archaea, and viruses were classified and profiled as well as functional annotation of the datasets was carried out using an in-house customized bioinformatics workflow.