Comparison of 16S rRNA Gene Based Microbial Profiling Using Five Next-Generation Sequencers and Various Primers

Abstract

Microbial community analysis based on the 16S rRNA-gene is used to investigate both beneficial and harmful microorganisms in various fields and environments. Recently, the next-generation sequencing (NGS) technology has enabled rapid and accurate microbial community analysis. Despite these advantages of NGS based metagenomics study, sample transport, storage conditions, amplification, library preparation kits, sequencing, and bioinformatics procedures can bias microbial community analysis results. In this study, eight mock communities were pooled from genomic DNA of Lactobacillus acidophilus KCTC 3164T, Limosilactobacillus fermentum KCTC 3112T, Lactobacillus gasseri KCTC 3163T, Lacticaseibacillus paracasei subsp. paracasei KCTC 3510T, Limosilactobacillus reuteri KCTC 3594T, Lactococcus lactis subsp. lactis KCTC 3769T, Bifidobacterium animalis subsp. lactis KCTC 5854T, and Bifidobacterium breve KCTC 3220T. The genomic DNAs were quantified by droplet digital PCR (ddPCR) and were mixed as mock communities. The mock communities were amplified with various 16S rRNA gene universal primer pairs and sequenced by MiSeq, IonTorrent, MGIseq-2000, Sequel II, and MinION NGS platforms. In a comparison of primer-dependent bias, the microbial profiles of V1-V2 and V3 regions were similar to the original ratio of the mock communities, while the microbial profiles of the V1-V3 region were relatively biased. In a comparison of platform-dependent bias, the sequence read from short-read platforms (MiSeq, IonTorrent, and MGIseq-2000) showed lower bias than that of long-read platforms (Sequel II and MinION). Meanwhile, the sequences read from Sequel II and MinION platforms were relatively biased in some mock communities. In the data of all NGS platforms and regions, L. acidophilus was greatly underrepresented while Lactococcus lactis subsp. lactis was generally overrepresented. In all samples of this study, the bias index (BI) was calculated and PCA was performed for comparison. The samples with biased relative abundance showed high BI values and were separated in the PCA results. In particular, analysis of regions rich in AT and GC poses problems for genome assembly, which can lead to sequencing bias. According to this comparative analysis, the development of reference material (RM) material has been proposed to calibrate the bias in microbiome analysis.

First Bacteremia Due to Corynebacterium gottingense in an Immunocompromised Child: A Case Report, 16S rDNA-Based Phylogenetic Analyses and Review of the Literature

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A Novel Single-Tube Eicosaplex/Octaplex PCR System for the Detection of Extended-Spectrum β-Lactamases, Plasmid-Mediated AmpC β-Lactamases, and Integrons in Gram-Negative Bacteria

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The role of IL-10 in regulating inflammation and gut microbiome in mice consuming milk kefir and orally challenged with S. Typhimurium

“…Fecal DNA extraction was performed according to a protocol adapted from Zhang et al 18 The quality and quantification of the DNA were analyzed by Nanodrop with the calculation of the ratio 260/230 and 260/230, absence of degradation smear in the 0.8% agarose gel electrophoresis and positive amplification by Polymerase Chain Reaction (PCR) using 337F and 518R16s rRNA primers for the V3 hypervariable region. 19 Subsequently, the samples were sent to the company responsible for the sequencing, on an Illumina HiSeq 2500 platform. The V3-V4 hypervariable region of the 16S rRNA gene was amplified and the qualities of the sequences obtained were evaluated using the FastQC package (version 1.44.3).…”