Optimisation of the Split-Pool Barcoding Technique for Microbial Community Analysis

Abstract

Understanding microbial community assembly and the role of horizontal gene transfer (HGT) and metabolic cross-feeding in community dynamics is essential for advancing microbial ecology. Traditional methods, including microscopy, culture-dependent techniques, and some culture-independent sequencing approaches, provide valuable insights but fall short in revealing fine-scale interactions and gene flow within complex microbial communities. This study aims to develop an optimised split and pool barcoding protocol for high-throughput, single-cell analysis of microbial interactions, which offers an approach for studying gene dynamics and functional diversity in varying ecosystems.

The split and pool barcoding method combines the modular barcoding approach of SPLiT-seq protocol with polyacrylamide beads and semi-permeable capsules to tag and sequence individual microbial cells. Key optimisations included reducing PCR cycles to minimise signal crosstalk and refining ligation conditions to prevent unwanted hybridisations. Efforts to use semi-permeable capsules faced fabrication challenges and signal mixing, resulting in their exclusion from the final protocol.

Results demonstrated successful targeting of the 16S rRNA V4 gene region, within synthetic microbial communities, achieving reliable single cell barcoding with minimal signal mix-up. The protocol provides a cost-effective, high-throughput method for microbial community analysis, addressing limitations of existing methods and enabling more precise tracking of transfer of genes and other community interactions. This advancement lays a solid foundation for further applications, such as studying the transfer of other genes, for example antibiotic resistance genes, in various increasingly complex microbial ecosystems.