Evaluation of an Adapted Semi-Automated DNA Extraction for Human Salivary Shotgun Metagenomics

Abstract

Recent attention has highlighted the importance of oral microbiota in human health and disease, e.g., in Parkinson's disease, notably using shotgun metagenomics. One key aspect for efficient shotgun metagenomic analysis relies on optimal microbial sampling and DNA extraction, generally implementing commercial solutions developed to improve sample collection and preservation, and provide high DNA quality and quantity for downstream analysis. As metagenomic studies are today performed on a large number of samples, the next evolution to increase study throughput is with DNA extraction automation. In this study, we proposed a semi-automated DNA extraction protocol for human salivary samples collected with a commercial kit, and compared the outcomes with the DNA extraction recommended by the manufacturer. While similar DNA yields were observed between the protocols, our semi-automated DNA protocol generated significantly higher DNA fragment sizes. Moreover, we showed that the oral microbiome composition was equivalent between DNA extraction methods, even at the species level. This study demonstrates that our semi-automated protocol is suitable for shotgun metagenomic analysis, while allowing for improved sample treatment logistics with reduced technical variability and without compromising the structure of the oral microbiome.

Keywords: DNA extraction; automation; oral microbiome; shotgun metagenomics.

Figure 1

General workflow of the study strategy. Before DNA extraction, samples were pre-treated (step 1) and prepared for DNA extraction procedures, by pooling randomly chosen samples when necessary (step 2). Two different DNA extraction procedures were used: Protocol P1, MGP SOP01 v1 adapted from the IHMS (International Human Microbiome Standards) SOP 07 V2 and Protocol P2 QIAGEN QIAamp PowerFecal Pro (step 3). Finally, samples were sequenced using the WGS Ion Proton sequencing technology before being analyzed using bioinformatical and biostatistical tools (step 4; Methods).

Figure 2

Detailed DNA extraction procedures. Diagrams describing each step for the DNA extraction protocols used in this study. Procotol P1 corresponds to the MGP SOP 01V1 procedure, which is fully available at https://mgps.eu/standard-operating-procedure (accessed on 2 October 2023). Protocol P2 corresponds to the Qiagen QIAamp PowerFecal Pro and is fully available at https://www.qiagen.com (accessed on 2 October 2023).

Figure 3

Effects of DNA extraction procedures on technical parameters. (A) DNA quantity (µg) obtained using Quant-it fluorescent-based quantification method; (B) Size of the genomic peak (bp), an indicator of the DNA quality, obtained using Fragment Analyzer; (C) Number of high-quality reads, after removing low-quality and host-related reads; and (D) Percentage of reads that mapped onto the 8.4 million oral genes catalog. p-values from unpaired Wilcoxon tests are reported.

Figure 4

Effects of DNA extraction procedures on the oral microbiome richness and structure. (A) Impact on alpha-diversity metrics measured using gene and species richness per protocol on the pooled samples only. p-values from unpaired Wilcoxon test are reported; (B) Heatmap for Spearman correlations between all pairs of samples using the gene count table. Colored strips referred to Protocols and IDs for raw and pooled samples (Pool sample), respectively; (C) Principal Component Analysis (PCA) for all samples using the MSP species count table and colored by protocol. Black circles indicate pooled samples.

Figure 5

Effects of DNA extraction procedures on the oral microbiome composition. Consistent microbial composition at phylum (A), family (B), and genus (C) taxonomic ranks between samples obtained from P1 and P2 protocols. Boxplots were ordered from the most to the least abundant taxa.