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Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota.

ABSTRACT: In this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish the experimental conditions. The results showed that 16S rRNA gene sequencing detects only part of the gut microbiota community revealed by shotgun sequencing. Specifically, when a sufficient number of reads is available, Shotgun sequencing has more power to identify less abundant taxa than 16S sequencing. Finally, we showed that the less abundant genera detected only by shotgun sequencing are biologically meaningful, being able to discriminate between the experimental conditions as much as the more abundant genera detected by both sequencing strategies.

SUBMITTER: Durazzi F

PROVIDER: S-EPMC7862389 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota.

Durazzi Francesco F Sala Claudia C Castellani Gastone G Manfreda Gerardo G Remondini Daniel D De Cesare Alessandra A

Scientific reports 20210204 1

In this paper we compared taxonomic results obtained by metataxonomics (16S rRNA gene sequencing) and metagenomics (whole shotgun metagenomic sequencing) to investigate their reliability for bacteria profiling, studying the chicken gut as a model system. The experimental conditions included two compartments of gastrointestinal tracts and two sampling times. We compared the relative abundance distributions obtained with the two sequencing strategies and then tested their capability to distinguish ...[more]

PMID: 33542369

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Project description:BackgroundObesity is a global epidemic in the industrialized and developing world, and many children suffer from obesity-related complications. Gut microbiota dysbiosis might have significant effect on the development of obesity. The microbiota continues to develop through childhood and thus childhood may be the prime time for microbiota interventions to realize health promotion or disease prevention. Therefore, it is crucial to understand the structure and function of pediatric gut microbiota.MethodsAccording to the inclusion criteria and exclusion criteria, twenty-three normal weight and twenty-eight obese children were recruited from Nanjing, China. Genomic DNA was extracted from fecal samples. The V4 region of the bacterial 16S rDNA was amplified by PCR, and sequencing was applied to analyze the gut microbiota diversity and composition using the Illumina HiSeq 2500 platform.ResultsThe number of operational taxonomic units (OTUs) showed a decrease in the diversity of gut microbiota with increasing body weight. The alpha diversity indices showed that the normal weight group had higher abundance and observed species than the obese group (Chao1: P < 0.001; observed species: P < 0.001; PD whole tree: P < 0.001; Shannon index: P = 0.008). Principal coordinate analysis (PCoA) and Nonmetric multidimensional scaling (NMDS) revealed significant differences in gut microbial community structure between the normal weight group and the obese group. The liner discriminant analysis (LDA) effect size (LEfSe) analysis showed that fifty-five species of bacteria were abundant in the fecal samples of the normal weight group and forty-five species of bacteria were abundant in the obese group. In regard to phyla, the gut microbiota in the obese group had lower proportions of Bacteroidetes (51.35%) compared to the normal weight group (55.48%) (P = 0.030). There was no statistical difference in Firmicutes between the two groups (P = 0.436), and the Firmicutes/Bacteroidetes between the two groups had no statistical difference (P = 0.983). At the genus level, Faecalibacterium, Phascolarctobacterium, Lachnospira, Megamonas, and Haemophilus were significantly more abundant in the obese group than in the normal weight group (P = 0.048, P = 0.018, P < 0.001, P = 0.040, and P = 0.003, respectively). The fecal microbiota of children in the obese group had lower proportions of Oscillospira and Dialister compared to the normal weight group (P = 0.002 and P = 0.002, respectively).ConclusionsOur results showed a decrease in gut microbiota abundance and diversity as the BMI increased. Variations in the bacterial community structure were associated with obesity. Gut microbiota dysbiosis might play a crucial part in the development of obesity in Chinese children.

Project description:Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies-ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies-ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies-ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable.

Project description:The ruminant gut microbial community has a strong impact on host health and can be altered during diarrhea disease. As an indigenous breed of the Tibetan Plateau, domestic yak displays a high diarrhea rate, but little research has been done to characterize the bacterial microbial structure in diarrheic yaks. In the present study, a total of 30 adult yaks, assigned to diarrhea (case, N = 15) and healthy (control, N = 15) groups, were subjected to gut microbiota profiling using the V3-V4 regions of the 16S rRNA gene. The results showed that the gut microbiome of the case group had a significant decrease in alpha diversity. Additionally, differences in beta diversity were consistently observed for the case and control groups, indicating that the microbial community structure was changed due to diarrhea. Bacterial taxonomic analysis indicated that the Bacteroidetes, Firmicutes, and Proteobacteria were the three most dominant phyla in both groups but different in relative abundance. Especially, the proportion of Proteobacteria in the case group was increased as compared with the control group, whereas Spirochaetota and Firmicutes were significantly decreased. At the genus level, the relative abundance of Escherichia-Shigella and Prevotellaceae_UCG-003 were dramatically increased, whereas that of Treponema, p-2534-18B5_gut_group, and Prevotellaceae_UCG-001 were observably decreased with the effect of diarrhea. Furthermore, based on our linear discriminant analysis (LDA) effect size (LEfSe) results, Alistipes, Solibacillus, Bacteroides, Prevotellaceae_UCG_003, and Bacillus were significantly enriched in the case group, while the other five genera, such as Alloprevotella, RF39, Muribaculaceae, Treponema, and Enterococcus, were the most preponderant in the control group. In conclusion, alterations in gut microbiota community composition were associated with yak diarrhea, differentially represented bacterial species enriched in case animals providing a theoretical basis for establishing a prevention and treatment system for yak diarrhea.

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Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota.

Publications

Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota.

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