Title: Challenges in single cells sequencing Microbial community and biofilm: A case of Oleidesulfovibrio alaskensis G20 NGS protocol

Biofilm, is a special-complex organization of bacterial cells with multiple layers, formed in certain environmental conditions. In the complex biofilm different bacterial (single) cells perform different functions and contribute to overall biofilm activity, where single bacterial cells are in different physiological states of growth, stress and signaling. Sulphate reducing bacteria (SRB) contributes to huge economic loss (${\$}$4B) causing microbial induced corrosion. To effectively combat the challenges posed by SRB, it is essential to understand their molecular mechanisms of biofilm formation and biocorrosion. Single Cell Genomics (single cell transcriptomics) is a technique which investigates the gene expression at the level of a single biological cell and could not be attainable in bulk analysis (RNASeq) could decipher the uniqueness of each cell in complex group. We are developing a NGS (oxford Nanopore)-workflow for single cell transcriptomics of biofilms using Oleidesulfovibrio alaskensis (earlier Desulfovibrio alaskensis) strain G20 (OA-G20) as a model. We encountered several challenges to design and develop the workflow such 1) absence of ploy(A) tail in mRNAs, 2) amount of total RNA, 3) half-lives of mRNAs, 4) perforation of cell membrane, 5) low copy number of mRNAs, 6) single cell suspension and cell sorting, 7) quantification of bacterial cells, 8) fixation of cells, 9) designing of probes, 10) hybridization of probes and 11) amplification of mRNAs. Here, we are presenting how we resolved some of these challenges to design and develop a single cell genomics (transcriptomics) workflow for SRB biofilms.