Obtaining deeper insights into microbiome diversity using a simple method to block host and nontargets in amplicon...

Area of Science:

• Microbiome research
• Molecular biology
• Bioinformatics

Background:

• Microbiome profiling is crucial for understanding host-microbe interactions and ecological processes.
• Amplicon sequencing is a key tool, but often hampered by non-target DNA amplification, reducing sequencing depth.
• Host DNA and overabundant microbes frequently interfere with accurate microbiome analysis.

Purpose of the Study:

• To introduce a cost-effective method using blocking oligonucleotides (oligos) to prevent non-target DNA amplification during sequencing.
• To develop software for designing these blocking oligos for diverse applications.
• To demonstrate the efficacy of blocking oligos in plant-leaf microbiomes and other systems.

Main Methods:

• Designed and applied A. thaliana-specific blocking oligos targeting eight loci to reduce host DNA amplification.
• Developed universal 16S and 18S rRNA gene plant blocking oligos effective across multiple plant species.
• Tested blocking oligos against an oomycete pathogen and utilized them for a comprehensive A. thaliana leaf microbiome survey.

Main Results:

• A. thaliana-specific blocking oligos reduced host DNA amplification by up to 90% across eight target loci.
• Universal plant blocking oligos successfully inhibited amplification in five diverse plant species.
• Blocking oligos increased alpha diversity discovery without biasing beta diversity or microbial load data, and were effective against oomycete DNA.

Conclusions:

• Blocking oligos offer a flexible and low-cost solution to improve amplicon sequencing specificity and depth.
• This method enhances microbial discovery and facilitates deeper, systems-based insights in various study systems, including plant microbiomes.
• A publicly available R package supports the design of blocking oligos for broad applicability in microbiome research.