Comparison of 16S rRNA gene based microbial profiling using five next-generation sequencers and various primers
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ABSTRACT:
PROVIDER: PRJEB45207 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR6001910.fastq.gz | Fastqsanger.gz | |||
| ERR6002390.fastq.gz | Fastqsanger.gz | |||
| ERR6002400.fastq.gz | Fastqsanger.gz | |||
| ERR6002410.fastq.gz | Fastqsanger.gz | |||
| ERR6002420.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:Microbial communities of the Arctic Ocean are poorly characterized in comparison to other aquatic environments as to their horizontal, vertical, and temporal turnover. Yet, recent studies showed that the Arctic marine ecosystem harbors unique microbial community members that are adapted to harsh environmental conditions, such as near-freezing temperatures and extreme seasonality. The gene for the small ribosomal subunit (16S rRNA) is commonly used to study the taxonomic composition of microbial communities in their natural environment. Several primer sets for this marker gene have been extensively tested across various sample sets, but these typically originated from low-latitude environments. An explicit evaluation of primer-set performances in representing the microbial communities of the Arctic Ocean is currently lacking. To select a suitable primer set for studying microbiomes of various Arctic marine habitats (sea ice, surface water, marine snow, deep ocean basin, and deep-sea sediment), we have conducted a performance comparison between two widely used primer sets, targeting different hypervariable regions of the 16S rRNA gene (V3-V4 and V4-V5). We observed that both primer sets were highly similar in representing the total microbial community composition down to genus rank, which was also confirmed independently by subgroup-specific catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) counts. Each primer set revealed higher internal diversity within certain bacterial taxonomic groups (e.g., the class Bacteroidia by V3-V4, and the phylum Planctomycetes by V4-V5). However, the V4-V5 primer set provides concurrent coverage of the archaeal domain, a relevant component comprising 10-20% of the community in Arctic deep waters and the sediment. Although both primer sets perform similarly, we suggest the use of the V4-V5 primer set for the integration of both bacterial and archaeal community dynamics in the Arctic marine environment.
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Project description:Tick-borne bacteria pose a significant threat to human and veterinary public health. Greece is a Mediterranean country with rich tick fauna and the most commonly detected tick-borne bacterial pathogens are members of the Rickettsia and Anaplasma species. The variable V2-V4 and V6-V9 regions of 16S rRNA gene of seven ticks belonging to four genera representative in Greece (Ixodes, Rhipicephalus, Dermacentor, Haemophyssalis) were analysed using multiple primer pairs by next generation sequencing (NGS). Nine bacterial phyla corresponding to 95 families, 116 genera and 172 species were identified. Proteobacteria was the predominant phylum in five of the seven ticks, followed by Actinobacteria, which predominated in two ticks. The tick-borne bacteria included Rickettsia and Anaplasma species, while "Candidatus Midichloria mitochondrii" were detected in high abundance in I. ricinus ticks and less in Rhipicephalus bursa; Coxiella-like endosymbionts were detected in Rh. sanguineus, H. parva, and less in Rh. bursa ticks. Co-infections with Rickettsia and Anaplasma were also observed. 16S rRNA NGS is a powerful tool to investigate the tick bacteriome and can improve the strategies for prevention and control of tick-borne diseases.
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Project description:Even though the nutritional and economic values of Solanum lycopersicum (tomato) are substantially impacted by microbial spoilage, the available data on its microbial community, particularly during spoilage, are limited and have primarily been characterized using conventional culture-dependent methods. This study employed a targeted high-throughput next-generation sequencing method to longitudinally characterize the microbial diversity of two South African tomato cultivars (jam and round) at varied storage intervals (1, 6, and 12 days). Throughout the storage period, the bacterial communities of the two cultivars were more diverse than the fungal communities. The microbial diversity of both bacteria and fungi was greater and comparable between the cultivars on day 1, but becomes distinct as the storage period increases, with round tomatoes being more diverse than jam tomato, though, on day 12, jam tomato develops greater diversity than round tomato. Overall, the most abundant phyla (though Proteobacteria was most dominant) were Proteobacteria, Firmicutes, and Bacteriodota in the bacterial communities, while Ascomycota and Basidiomycota formed most fungal communities with Ascomycota being dominant. At the genus level, Pantoea and Klebsiella (bacteria), Hanseniaspora, Stemphylium, and Alternaria (fungi) were prevalent. Taken together, this study casts light on a broad microbial diversity profile thus, confirms the cultivars' diversity and abundance differences.
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Project description:BackgroundSequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper analyses these primers using two databases containing 16S rRNA sequences from bacteria and archaea found in the human mouth and describes some of the best primers for each domain.ResultsA total of 369 distinct individual primers were identified from sequencing studies of the oral microbiome and other ecosystems. These were evaluated against a database reported in the literature of 16S rRNA sequences obtained from oral bacteria, which was modified by our group, and a self-created oral archaea database. Both databases contained the genomic variants detected for each included species. Primers were evaluated at the variant and species levels, and those with a species coverage (SC) ≥75.00% were selected for the pair analyses. All possible combinations of the forward and reverse primers were identified, with the resulting 4638 primer pairs also evaluated using the two databases. The best bacteria-specific pairs targeted the 3-4, 4-7, and 3-7 16S rRNA gene regions, with SC levels of 98.83-97.14%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-6, and 3-6, with SC estimates of 95.88%. Finally, the best pairs for detecting both domains targeted regions 4-5, 3-5, and 5-9, and produced SC values of 95.71-94.54% and 99.48-96.91% for bacteria and archaea, respectively.ConclusionsGiven the three amplicon length categories (100-300, 301-600, and >600 base pairs), the primer pairs with the best coverage values for detecting oral bacteria were as follows: KP_F048-OP_R043 (region 3-4; primer pair position for Escherichia coli J01859.1: 342-529), KP_F051-OP_R030 (4-7; 514-1079), and KP_F048-OP_R030 (3-7; 342-1079). For detecting oral archaea, these were as follows: OP_F066-KP_R013 (5-6; 784-undefined), KP_F020-KP_R013 (3-6; 518-undefined), and OP_F114-KP_R013 (3-6; 340-undefined). Lastly, for detecting both domains jointly they were KP_F020-KP_R032 (4-5; 518-801), OP_F114-KP_R031 (3-5; 340-801), and OP_F066-OP_R121 (5-9; 784-1405). The primer pairs with the best coverage identified herein are not among those described most widely in the oral microbiome literature. Video Abstract.
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Project description:IntroductionAs tick-borne diseases rise to become the second most prevalent arthropod-transmitted disease globally, the increasing investigations focus on ticks correspondingly. Factors contributed to this increase include anthropogenic influences, changes in vertebrate faunal composition, social-recreational shifts, and climatic variation. Employing the 16S gene sequence method in next-generation sequencing (NGS) allows comprehensive pathogen identification in samples, facilitating the development of refined approaches to tick research omnidirectionally.MethodsIn our survey, we compared the microbial richness and biological diversity of ticks in Wuwei City, Gansu province, differentiating between questing ticks found in grass and parasitic ticks collected from sheep based on 16S NGS method.ResultsThe results show Rickettsia, Coxiella, and Francisella were detected in all 50 Dermacentor nuttalli samples, suggesting that the co-infection may be linked to specific symbiotic bacteria in ticks. Our findings reveal significant differences in the composition and diversity of microorganisms, with the Friedmanniella and Bordetella genera existing more prevalent in parasitic ticks than in questing ticks (p < 0.05). Additionally, the network analysis demonstrates that the interactions among bacterial genera can be either promotive or inhibitive in ticks exhibiting different lifestyles with the correlation index |r| > 0.6. For instance, Francisella restrains the development of 10 other bacteria in parasitic ticks, whereas Phyllobacterium and Arthrobacter enhance colonization across all tick species.DiscussionBy leveraging NGS techniques, our study reveals a high degree of species and phylogenetic diversity within the tick microbiome. It further highlights the potential to investigate the interplay between bacterial genera in both parasitic and questing ticks residing in identical habitat environments.
| S-EPMC10773790 | biostudies-literature