Multiplex amplicon sequencing for microbe identification in community-based culture collections

Area of Science:

• Microbiology
• Plant Science
• Bioinformatics

Background:

• Metagenomic sequencing reveals extensive plant-associated microbial diversity.
• Understanding plant-microbe molecular interactions is crucial for agricultural technology development.
• Traditional microbial isolation and identification methods are slow and costly.

Purpose of the Study:

• To introduce a novel method for identifying microbes in community-based culture collections (CBCs).
• To accelerate the discovery of beneficial plant-microbe interactions for agricultural applications.
• To enable accurate microbial identification from cultures containing single or multiple microorganisms.

Main Methods:

• Development of community-based culture collections (CBCs) by picking colonies.
• Application of multiplexing 16S rRNA gene amplicon sequencing with tagged primers for plates, rows, and columns.
• Utilizing PacBio sequencing for near-full-length 16S rRNA gene recovery and accurate microbial identification.
• Cross-referencing CBC data with plant microbiome structure and abundance.

Main Results:

• The multiplexing sequencing method accurately identified microbial composition in wells with single or multiple microorganisms.
• Pooling amplicons into a single tube streamlined the process.
• Near-full-length 16S rRNA gene sequences enabled precise identification of microorganisms.
• Estimated diversity and abundance representation of microorganisms within the CBCs.

Conclusions:

• The described method offers a rapid and cost-effective approach for microbial identification in culture collections.
• This technique facilitates the construction and analysis of CBCs for agricultural microbiome research.
• Accurate characterization of microbial communities in culture collections aids in understanding plant-microbe interactions and developing microbiome-derived technologies.