Interpretations of Environmental Microbial Community Studies Are Biased by the Selected 16S rRNA (Gene) Amplicon Sequencing Pipeline

Straub, Daniel; Blackwell, Nia; Langarica-Fuentes, Adrián; Peltzer, Alexander; Nahnsen, Sven; Kleindienst, Sara

doi:10.3389/fmicb.2020.550420

Cited by 153 publications

(119 citation statements)

References 96 publications

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“…Statistical analysis (Wilcoxon signed-rank test) was performed at a depth of (A: qI) 29,998 and (B: qII) 30,000. Double asterisks indicate statistical significance (p < 0.01) DADA2 is used for ASV clustering and does not output unassigned ASVs, in contrast to OTU clustering with qI, and is reported to be superior in sequence clustering ability [32,36,37]. Our results indicated that the V34 results contain more filtered-out sequences that would be excluded when processed by DADA2, which clusters ASVs more accurately.…”

Section: Discussionmentioning

confidence: 80%

Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

et al. 2021

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Background 16S rRNA gene amplicon sequencing (16S analysis) is widely used to analyze microbiota with next-generation sequencing technologies. Here, we compared fecal 16S analysis data from 192 Japanese volunteers using the modified V1–V2 (V12) and the standard V3–V4 primer (V34) sets to optimize the gut microbiota analysis protocol. Results QIIME1 and QIIME2 analysis revealed a higher number of unclassified representative sequences in the V34 data than in the V12 data. The comparison of bacterial composition demonstrated that at the phylum level, Actinobacteria and Verrucomicrobia were detected at higher levels with V34 than with V12. Among these phyla, we observed higher relative compositions of Bifidobacterium and Akkermansia with V34. To estimate the actual abundance, we performed quantitative real-time polymerase chain reaction (qPCR) assays for Akkermansia and Bifidobacterium. We found that the abundance of Akkermansia as detected by qPCR was close to that in V12 data, but was markedly lower than that in V34 data. The abundance of Bifidobacterium detected by qPCR was higher than that in V12 and V34 data. Conclusions These results indicate that the bacterial composition derived from the V34 region might differ from the actual abundance for specific gut bacteria. We conclude that the use of the modified V12 primer set is more desirable in the 16S analysis of the Japanese gut microbiota.

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Section: Discussionmentioning

confidence: 80%

Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

et al. 2021

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“…Consequently, we reconstructed RF models with the same hyperparameters as the discovery RF models. Considering the limited resolution of the 16S rRNA gene and incomplete reference database 53 , not all ASVs could be assigned at the species level. Thus all ASVs with the same taxonomy assignments (at genus level), as well as patient metadata (only used ASVs for validation cohort2 for lack of the patient metadata), were used as the input features.…”

Section: Methodsmentioning

confidence: 99%

Identification of microbial markers across populations in early detection of colorectal cancer

et al. 2021

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Associations between gut microbiota and colorectal cancer (CRC) have been widely investigated. However, the replicable markers for early-stage adenoma diagnosis across multiple populations remain elusive. Here, we perform an integrated analysis on 1056 public fecal samples, to identify adenoma-associated microbial markers for early detection of CRC. After adjusting for potential confounders, Random Forest classifiers are constructed with 11 markers to discriminate adenoma from control (area under the ROC curve (AUC) = 0.80), and 26 markers to discriminate adenoma from CRC (AUC = 0.89), respectively. Moreover, we validate the classifiers in two independent cohorts achieving AUCs of 0.78 and 0.84, respectively. Functional analysis reveals that the altered microbiome is characterized with increased ADP-l-glycero-beta-d-manno-heptose biosynthesis in adenoma and elevated menaquinone-10 biosynthesis in CRC. These findings are validated in a newly-collected cohort of 43 samples using quantitative real-time PCR. This work proves the validity of adenoma-specific markers across multi-populations, which would contribute to the early diagnosis and treatment of CRC.

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“…Data with clean reads were analyzed by using the nf-core/ampliseqv1.2.0 pipeline, and the microbial diversity within our datasets were determined by setting the optional parameters “- -multiple Sequencing Runs,” “- -trunclenf 220,” and “- -trunclenr 180” (to resemble the truncation values of QIIME2 with DADA2) ( Straub et al, 2020 ). The 16s rRNA gene comparison database, SILVA v132 ( Quast et al, 2013 ), was used to perform clustering at 99% similarity.…”

Section: Methodsmentioning

confidence: 99%

Microbial Profiles of Retail Pacific Oysters (Crassostrea gigas) From Guangdong Province, China

Wang

2021

Front. Microbiol.

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Oysters are one of the main aquatic products sold in coastal areas worldwide and are popular among consumers because of their delicious taste and nutritional value. However, the microorganisms present in oysters may pose health risks to consumers. In this study, the microbial communities of Pacific oysters (Crassostrea gigas) collected from aquatic product markets in three cities (Guangzhou, Zhuhai, and Jiangmen) of Guangdong Province, China, where raw oysters are popular, were investigated. The plate counts of viable bacteria in oysters collected in the three cities were all approximately 2 log colony-forming units/g. High-throughput sequencing analysis of the V3–V4 region of the 16Sribosomal DNA gene showed a high level of microbial diversity in oysters, as evidenced by both alpha and beta diversity analysis. Proteobacteria, Bacteroidetes, and Firmicutes were the dominant phyla of the microorganisms present in these samples. A variety of pathogenic bacteria, including the fatal foodborne pathogen Vibrio vulnificus, were found, and Vibrio was the dominant genus. Additionally, the relationship between other microbial species and pathogenic microorganisms may be mostly symbiotic in oysters. These data provide insights into the microbial communities of retail oysters in the Guangdong region and indicate a considerable risk related to the consumption of raw oysters.

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Interpretations of Environmental Microbial Community Studies Are Biased by the Selected 16S rRNA (Gene) Amplicon Sequencing Pipeline

Cited by 153 publications

References 96 publications

Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

Identification of microbial markers across populations in early detection of colorectal cancer

Microbial Profiles of Retail Pacific Oysters (Crassostrea gigas) From Guangdong Province, China

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