Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

Polona Rajar; Achal Dhariwal; Gabriela Salvadori; Roger Junges; Heidi Aarø Åmdal; Dag Berild; Drude Fugelseth; Ola Didrik Saugstad; Ulrik Lausten-Thomsen; Gorm Greisen; Kirsti Haaland; Fernanda Cristina Petersen

doi:10.3389/fmicb.2022.1038120

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies

Front Microbiol. 2022 Dec 21:13:1038120. doi: 10.3389/fmicb.2022.1038120. eCollection 2022.

Authors

Affiliations

¹ Department of Neonatal Intensive Care, Division of Paediatric and Adolescent Medicine, Oslo University Hospital Ullevål, Oslo, Norway.
² Institute of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
³ Department of Infectious Diseases, Oslo University Hospital, Oslo, Norway.
⁴ Institute of Clinical Medicine, Faculty of Medicine, Oslo University, Oslo, Norway.
⁵ Department of Paediatric Research, University of Oslo, Oslo, Norway.
⁶ Department of Neonatology, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark.

Abstract

Introduction: Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization.

Methods: Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples.

Results: The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness.

Discussion: Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

Keywords: antimicrobial resistance; host DNA depletion; low biomass; microbiome; premature infant; resistome; respiratory microbiome; whole metagenomic sequencing.