Benchmarking microbial DNA enrichment protocols from human intestinal biopsies.

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Shotgun metagenomic sequencing is vital for studying unculturable bacteria.
• Host DNA contamination significantly reduces microbial signal sensitivity in metagenomic data.
• Existing host DNA depletion methods require further evaluation for human intestinal samples.

Purpose of the Study:

• To assess the efficacy of wet-lab kits and software approaches for host DNA depletion in human intestinal microbiome samples.
• To compare the performance of commercial kits (NEBNext, Molzym, QIAamp, Zymo HostZERO) and adaptive sampling (ONT AS).
• To identify optimal strategies for enhancing microbial DNA detection in complex samples.

Main Methods:

• Evaluation of four commercial DNA enrichment kits: NEBNext Microbiome DNA Enrichment, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome, and Zymo HostZERO microbial DNA.
• Assessment of Oxford Nanopore Technologies (ONT) adaptive sampling (AS) for real-time host DNA sequencing abortion.
• Optimization of less effective protocols using detergents and bead-beating.

Main Results:

• NEBNext and QIAamp kits significantly reduced host DNA, increasing bacterial sequences to 24% and 28% respectively.
• ONT AS enhanced bacterial read numbers, leading to improved metagenomic assembly and contig completeness.
• ONT AS identified antimicrobial resistance markers and plasmids but also introduced shifts in bacterial abundance (e.g., increased E. coli).

Conclusions:

• NEBNext and QIAamp kits are effective for reducing host DNA in shotgun metagenomics of intestinal samples.
• ONT adaptive sampling offers advantages for microbial signal enrichment and targeted sequencing but requires careful interpretation due to abundance shifts.
• Optimizing existing protocols can improve host DNA depletion, enhancing the utility of metagenomic sequencing for complex human microbiome studies.