Primer, Pipelines, Parameters: Issues in 16S rRNA Gene Sequencing

Abellan-Schneyder, Isabel; Matchado, Monica Steffi; Reitmeier, Sandra; Sommer, Alina; Sewald, Zeno; Baumbach, Jan; List, Markus; Neuhaus, Klaus

doi:10.1128/msphere.01202-20

Cited by 207 publications

(209 citation statements)

References 115 publications

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“…However, the failure of the detection of Collinsella and Eggerthella in the LoopSeq results could not be explained by using this particular forward primer. In contrast, it was shown in a previous study that the use of primer pair 1115F and 1492R led to an underrepresentation of those two genera [26], which might suggest that the reverse primer 1492R is responsible for this inferior result concerning the latter two genera. This highlights that studies must carefully test whether the primers used are suitable for the expected results.…”

Section: Full-length Ssu Rrna Gene Sequencing Improves Species-level Classification But Shows Primer Issuesmentioning

confidence: 61%

“…A variety of different factors can bias sequencing approaches targeting the 16S rRNA gene. The most well-known and studied are the sample collection, stabilization and transport to the laboratory, DNA extraction method, DNA concentration, primers targeting different V-regions, PCR condition and settings, laboratory practice, analysis pipelines, and reference databases [24,26,[36][37][38][39]. In our results, it became obvious that, e.g., primer pairs 27F and 338R underrepresent Akkermansia, whereas this is not the case for 338F and 785R.…”

Section: Full-length Ssu Rrna Gene Sequencing Improves Species-level Classification But Shows Primer Issuesmentioning

confidence: 71%

“…The details can be found in Reitmeier et al [3]. The ZIEL2 mock community was prepared and extracted as described in Abellan-Schneyder et al [26]. Briefly, 19 bacterial strains (18 different bacterial genera) of diverse taxonomy were cultured and harvested afterward by centrifugation.…”

Section: Dna Extraction Of the Mock Communitiesmentioning

confidence: 99%

“…For the amplification of the V1-V2 and V3-V4 regions of the 16S rRNA genes, 2-step PCRs of 20 and 10 cycles for the milk samples and 15 and 10 cycles for the mock communities were performed as described in Table S1 (first step PCR), Table S2 (second step PCR) and Abellan-Schneyder et al [26].…”

Section: Short Amplicon 16s Rrna Gene Library Preparationmentioning

confidence: 99%

“…Furthermore, the LoopGenomics pipeline conducts the pre-processing into full-length reads, which is easy to use. Next, the reads are identified using the SILVA database as a reference for both short and full-length sequences, which currently is one of the best databases available for 16S rRNA sequences [26]. In addition, as previously stated, not only bacteria but also eukaryotic and archaeal microorganisms are discovered due to the primer mix included.…”

Section: Using Full-length Sequencing Approaches For Microbial Monitoringmentioning

confidence: 99%

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Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk

et al. 2021

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Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing. Therefore, we anticipate that this approach might be useful for the detection of possible mastitis-causing species, as well as for the control of spoilage-associated microorganisms. In a proof of concept, we showed that we were able to identify several putative mastitis-causing or mastitis-associated species such as Streptococcusuberis, Streptococcusagalactiae, Streptococcusdysgalactiae, Escherichiacoli and Staphylococcusaureus, as well as several Candida species. Overall, the presented full-length approach for the sequencing of SSU rRNA is easy to conduct, able to be standardized, and allows the screening of microorganisms in labs with Illumina sequencing machines.

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Section: Full-length Ssu Rrna Gene Sequencing Improves Species-level Classification But Shows Primer Issuesmentioning

confidence: 61%

Section: Full-length Ssu Rrna Gene Sequencing Improves Species-level Classification But Shows Primer Issuesmentioning

confidence: 71%

Section: Dna Extraction Of the Mock Communitiesmentioning

confidence: 99%

Section: Short Amplicon 16s Rrna Gene Library Preparationmentioning

confidence: 99%

Section: Using Full-length Sequencing Approaches For Microbial Monitoringmentioning

confidence: 99%

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Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk

et al. 2021

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Characterization of the gastrointestinal microbiome of the Syrian hamster (Mesocricetus auratus) and comparison to data from mice

Böswald,

Popper,

Matzek

et al. 2024

FEBS Open Bio

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Syrian hamsters (Mesocricetus auratus) have been increasingly used as rodent models in recent years, especially for SARS‐CoV‐2 since the pandemic. However, the physiology of this animal model is not yet well‐understood, even less when considering the digestive tract. Generally, the gastrointestinal microbiome influences the immune system, drug metabolism, and vaccination efficacy. However, a detailed understanding of the gastrointestinal microbiome of hamsters is missing. Therefore, we analyzed 10 healthy 11‐week‐old RjHan:AURA hamsters fed a pelleted standard diet. Their gastrointestinal content was sampled (i.e., forestomach, glandular stomach, ileum, cecum, and colon) and analyzed using 16S rRNA gene amplicon sequencing. Results displayed a distinct difference in the bacterial community before and after the cecum, possibly due to the available nutrients and digestive functions. Next, we compared hamsters with the literature data of young‐adult C57BL/6J mice, another important animal model. We sampled the same gastrointestinal regions and analyzed the differences in the microbiome between both rodents. Surprisingly, we found strong differences in their specific gastrointestinal bacterial communities. For instance, Lactobacillaceae were more abundant in hamsters' forestomach and ileum, while Muribaculaceae dominated in the mouse forestomach and ileum. Similarly, in mouse cecum and colon, Muribaculaceae were dominant, while in hamsters, Lachnospiraceae and Erysipelotrichaceae dominated the bacterial community. Molecular strains of Muribaculaceae in both rodent species displayed some species specificity. This comparison allows a better understanding of the suitability of the Syrian hamster as an animal model, especially regarding its comparability to other rodent models. Thereby, this work contributes to the characterization of the hamster model and allows better experimental planning.

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16S rRNA gene sequencing analysis of gut microbiome in a mini‐pig diabetes model

Niu

Zhao

Xiang

et al. 2022

Anim Models and Exp Med

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Background: Currently, increasing attention is being paid to the important role of intestinal microbiome in diabetes. However, few studies have evaluated the characteristics of gut microbiome in diabetic miniature pigs, despite it being a good model animal for assessing diabetes. Methods:In this study, a mini-pig diabetes model (DM) was established by 9-month high-fat diet (HFD) combined with low-dose streptozotocin, while the animals fed standard chow diet constituted the control group. 16S ribosomal RNA (rRNA) gene sequencing was performed to assess the characteristics of the intestinal microbiome in diabetic mini-pigs. Results:The results showed that microbial structure in diabetic mini-pigs was altered, reflected by increases in levels of Coprococcus_3 and Clostridium_sensu_stricto_1, which were positively correlated with diabetes, and decreases in levels of the bacteria Rikenellaceae, Clostridiales_vadinBB60_group, and Bacteroidales_RF16_group, which were inversely correlated with blood glucose and insulin resistance. Moreover, PICRUSt-predicted pathways related to the glycolysis and Entner-Doudoroff superpathway, enterobactin biosynthesis, and the l-tryptophan biosynthesis were significantly elevated in the DM group. Conclusion:These results reveal the composition and predictive functions of the intestinal microbiome in the mini-pig diabetes model, further verifying the relationship between HFD, gut microbiome, and diabetes, and providing novel insights into the application of the mini-pig diabetes model in gut microbiome research.

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Primer, Pipelines, Parameters: Issues in 16S rRNA Gene Sequencing

Cited by 207 publications

References 115 publications

Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk

Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk

Characterization of the gastrointestinal microbiome of the Syrian hamster (Mesocricetus auratus) and comparison to data from mice

16S rRNA gene sequencing analysis of gut microbiome in a mini‐pig diabetes model

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