Impact of DNA Sequencing and Analysis Methods on 16S rRNA Gene Bacterial Community Analysis of Dairy Products

DNA sequencing and analysis methods were compared for 16S rRNA V4 PCR amplicon and genomic DNA (gDNA) mock communities encompassing nine bacterial species commonly found in milk and dairy products. The two communities comprised strain-specific DNA that was pooled before (gDNA) or after (PCR amplicon) the PCR step. The communities were sequenced on the Illumina MiSeq and Ion Torrent PGM platforms and then analyzed using the QIIME 1 (UCLUST) and Divisive Amplicon Denoising Algorithm 2 (DADA2) analysis pipelines with taxonomic comparisons to the Greengenes and Ribosomal Database Project (RDP) databases. Examination of the PCR amplicon mock community with these methods resulted in operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) that ranged from 13 to 118 and were dependent on the DNA sequencing method and read assembly steps. The additional 4 to 109 OTUs/ASVs (from 9 OTUs/ASVs) included assignments to spurious taxa and sequence variants of the 9 species included in the mock community. Comparisons between the gDNA and PCR amplicon mock communities showed that combining gDNAs from the different strains prior to PCR resulted in up to 8.9-fold greater numbers of spurious OTUs/ASVs. However, the DNA sequencing method and paired-end read assembly steps conferred the largest effects on predictions of bacterial diversity, with effect sizes of 0.88 (Bray-Curtis) and 0.32 (weighted Unifrac), independent of the mock community type. Overall, DNA sequencing performed with the Ion Torrent PGM and analyzed with DADA2 and the Greengenes database resulted in the most accurate predictions of the mock community phylogeny, taxonomy, and diversity. IMPORTANCE Validated methods are urgently needed to improve DNA sequence-based assessments of complex bacterial communities. In this study, we used 16S rRNA PCR amplicon and gDNA mock community standards, consisting of nine, dairy-associated bacterial species, to evaluate the most commonly applied 16S rRNA marker gene DNA sequencing and analysis platforms used in evaluating dairy and other bacterial habitats. Our results show that bacterial metataxonomic assessments are largely dependent on the DNA sequencing platform and read curation method used. DADA2 improved sequence annotation compared with QIIME 1, and when combined with the Ion Torrent PGM DNA sequencing platform and the Greengenes database for taxonomic assignment, the most accurate representation of the dairy mock community standards was reached. This approach will be useful for validating sample collection and DNA extraction methods and ultimately investigating bacterial population dynamics in milk- and dairy-associated environments.