Why 16s rdna used

The significance and implications of the surprisingly large numbers of bands per OTU are discussed further. OTUs were chosen to illustrate the variety of banding patterns and counts observed from individual Alu I digestions of the 72 culturable isolates. Arrows illustrate 5-bp size differences between fragments. Surprisingly, the observed fingerprinting patterns indicated very little variation in number and position of bands Fig.

In other words, assuming complete digestion for all samples see Methods , the total number of representative fragments for the communities indicative of OTU richness did not change, or increased very little with higher numbers of isolates in the MBCs. For example, in the MBCs from type II, additional bands in the community appeared between 5 and 15 OTUs, but only positional changes in the profiles was further observed above 20 OTUs; disappearance and emergence of bands were noticed, which roughly kept the same number of detectable bands among MBCs Fig.

Following a regression analyses for the best-fit curve, the results expectedly indicated that the numbers of Alu I fragments per no. For both in silico and in vitro analyses, restriction fragments equal to, or less than 5-bp difference were considered as a single band and counted only once per MBC see Methods.

In silico analysis: the number of Alu I fragments was defined from 16S rDNA sequences from various endophytic bacteria from database, followed by computational processing and restriction analysis by free online software see Methods. These experiments were repeated at least twice for all isolates and their MBCs. The respective regressions, equations and coefficients of determination are indicated in the graphs.

For the rice and bean sequences, smaller regression coefficients 9. The variation in the number of Alu I fragments per no. In addition, for the former, the maximum number of restriction fragments increases along with higher no. In fact, based on the shapes of the in vitro curves, it appears that the delta between theoretical and actual data is likely to become even larger if more OTUs are added to MBCs. The use of HTS methods has drastically increased the outcomes in information, precision and reaches of research in microbial diversity.

Yet, low-throughput methods remain useful when simple variation on profiles of detectable operational taxonomic units OTUs is a sufficient response variable [ 12 , 13 , 17 , 59 , 60 , 61 , 62 , 63 , 64 , 65 ]. Independently of the analyses scale fingerprinting or HTS , though, the uncertainty about the maximum possible number of accessible OTUs has been an issue, as rarefaction curves mostly suggest the sampling efforts are not enough to cover the totality of diversity [ 11 , 12 , 22 , 30 , 38 , 54 , 66 , 67 ].

It is our contention that the possibility of reaching the totality of a given microdiversity is in fact hampered by informational losses or bias typical from the investigation routine. For 16S rDNA-based amplification methods, such a detectability issue is caused by a variety of factors related to the PCR [ 1 , 27 , 32 , 41 , 42 , 43 , 46 , 54 , 59 , 62 ].

Nevertheless, despite such a limitation, this set of 72 unique culturable bacterial isolates OTUs allowed a proper composition of MBCs for the experiments Figs.

Highly specific primers are essential to investigate bacterial diversity in environmental samples [ 42 , 65 ], mainly in cases involving endophytes. Primer-pairs that include the F, covering the V5—V9 hypervariable region of 16S rDNA, have been suggested to either exclude amplification of cpDNA, or properly separate bacterial from chloroplast amplicons [ 2 , 24 , 25 , 51 , 54 , 67 ].

Virola officinalis is an endemic tree from this region [ 72 ] that has not been previously studied with respect to its chloroplast features. Hence, cpDNA from V. A similar result of cpDNA amplification with F has been reported [ 22 ]. It has to be acknowledged that, under high cpDNA interference, changes in the number of bacterial OTUs in a sample may be undetectable Fig. Therefore, a very careful experimental planning is needed when addressing endophytic bacterial diversity; a relevant alternative is using extraction procedures able to isolate chloroplasts from the total extractable DNA [ 25 , 73 , 75 , 76 ].

In addition to cpDNA interference, two other technical factors appeared to be an issue. The amount of template DNA we used is not unusual in bacterial diversity experiments [ 22 , 77 ], so that the smear observed, especially above the expected-size amplicons, may be due to chimeric amplification [ 1 , 41 , 48 , 78 ].

Also, the number of PCRs can interfere in the final amplification output [ 23 , 41 , 48 ], likely by interacting with the cpDNA and primers specificity Fig. Taken together, these results suggest that research on microdiversity in tropical plants will require efforts to check for applicability and efficiency on any low- or high-throughput platform of 16S rDNA specific primers, adjusting experimental settings for more consistent, reproducible and broad amplification of associated bacteria [ 30 , 35 , 40 , 43 , 78 ].

To obtain a theoretical maximum of Alu I restriction fragments closer to a practical reality, the culturable OTUs from cacao were assessed individually examples illustrated on Fig.

In this regard, various aspects are worth discussing. Second, the isolate-specific restriction patterns observed indicated that our strategy of considering only unique OTUs for the MBCs assembly, and so for the generation of the maximum theoretical number of bands, was appropriate. The presence of such extra Alu I bands could be alternatively explained by intragenomic heterogeneity [ 44 , 45 , 46 , 79 ], i. Furthermore, the variable band intensities among isolates might be related to different copy numbers of the sequences [ 45 , 46 , 82 ].

This whole view is consistent with results from a survey in Bacillus cereus -group strains that showed an average number of 6. The distinct Alu I profiles for the isolates were consistently reproducible between replicates and experiments not shown. These results contrasted to what would be more logically expected based on Fig.

These pieces of evidence suggest a strong bias in the PCRs, in which the primers likely had a binding preference for specific sequences OTUs within the MBCs [ 40 , 84 , 85 ]. Since the amount of template in the MBCs was always the same, made of equimolar amounts of DNA from participating isolates, our results also suggest that most abundant OTUs in a sample will not necessarily be amplified preferentially, as it has been long- and logically-assumed.

This certainly has a significant impact on estimates of diversity indexes in natural communities [ 11 , 62 , 79 , 85 , 86 , 87 ], independently of the analytical platform used. It is possible that working with more than 30 OTUs in the MBCs could have yielded more bands, although this trend was not observed Figs. Further experiments are warranted for an in-depth assessment of such a scale issue, as well as to test whether very low levels of template concentration such as 0.

It is important to highlight that only one restriction enzyme was used in this study to simplify the restriction profiles. The simultaneous use of other restriction enzyme s as in usual PCR-RFLP-type studies [ 9 , 18 , 88 ] would have increased the complexity of banding profiles, likely turning the data analysis into a cumbersome process; the fact that a higher number of Alu I fragments was found for many isolates individually Fig.

In view of the various interfering factors here discussed, a direct experimental access to all possible microbes in a sample through PCR will likely be unfeasible, even for high-throughput techniques. Therefore, for the vast majority of studies, the current explanation given for rarefaction curves that tend to, but not reach a plateau, might need to be reconsidered: this likely happens not because the sampling effort is insufficient, but rather because the totality of a microdiversity simply cannot be reached by PCR-based methods.

The direct access to microbial communities without relying on PCR, such as using a true metagenomics approach [ 31 ], may possibly be a feasible alternative to solve this whole issue. Our results indicated that, independently of the scale of the analysis, environmental samples of microorganisms subjected to universal-priming PCR can show a severely biased and misestimated number of OTUs.

If dealing with endophytic communities, further interfering effects on primer-binding can be caused by cpDNA. These confounding aspects must not be overlooked in studies on microbial diversity, as they can alter the outputs of richness, abundance and composition of OTUs [ 1 , 11 , 24 , 31 ]. It seems clear that true sources of variation among environmental microbial communities are not only the natural differences between samples, but also the intrinsic interfering effects of the research methodology.

Despite the analytical power, depth and reach of high-throughput sequencing approaches, there are circumstances where simple observation of changes in robustly detectable OTUs will suffice for the research objectives, such as in multi-samples assessments of treatments effects on structure and dynamics of microbial communities [ 15 , 21 , 59 ]. For research designs relying upon PCR-based methods for microdiversity studies [ 46 , 88 ], we hope this study has contributed to a greater awareness for the need of not only a comprehensive knowledge on the biological systems under study, but also a maximum control of intrinsic factors of variation, mainly those related to universal-primed PCR on 16S rDNA.

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Price, M. FastTree 2 — approximately maximum-likelihood trees for large alignments. Paradis, E. Linking genomics and population genetics with R. Bland, M. Statistics notes: measurement Error. Camacho, C. BMC Bioinformatics 10 , Download references. Jethro S. Johnson, Daniel J. Leopold, Blake M. Hanson, Hanako O. You can also search for this author in PubMed Google Scholar. Correspondence to Jethro S. Peer Review Information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work.

Peer reviewer reports are available. Reprints and Permissions. Johnson, J. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nat Commun 10, Download citation. Received : 09 January Accepted : 16 October Published : 06 November Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.

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If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Subjects Bacterial genes Bacterial techniques and applications Microbiome. Introduction Since the advent of high-throughput sequencing, PCR-amplified 16S sequences have typically been clustered based on similarity to generate operational taxonomic units OTUs and representative OTU sequences compared with reference databases to infer likely taxonomy.

Full size image. Discussion Here, we have presented the results of four experiments that collectively demonstrate the taxonomic resolution achievable in the current 16S gene-based microbiome studies. Methods In-silico comparison of full vs. Construction of a bacterial mock community Based on data available from the Human Microbiome Project and Human Oral Microbiome database, 36 bacterial strains were selected to represent microbes prevalent in the human body sites including the airways, gut, oral cavity, skin, and vaginal tract Supplementary Table 3.

Illumina library preparation shotgun sequencing and assembly WGS sequencing was performed for 19 members of the mock community that did not have WGS sequence data publicly available.

Analysis of the bacterial mock community Reference 16S rRNA gene sequences matching strains in the mock community were initially downloaded from the RDP database Sampling and sequencing of the human stool microbiome Stool samples were collected from four healthy, competitive cyclists enrolled in the study described by Petersen et al. Isolation and sequencing of bacteria from human stool Stool samples were again contributed by competitive cyclists enrolled in the study described by Petersen et al.

Computational analysis of individual isolates Sequence data for each isolate were quality filtered and adapters removed as described above. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. References 1. Google Scholar 4. Article Google Scholar CAS Google Scholar Google Scholar Author information Author notes These authors contributed equally: Jethro S. Johnson View author publications. View author publications.

Ethics declarations Competing interests The authors declare no conflict of interest. Additional information Peer Review Information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Supplementary information. Supplementary Information.

Peer Review File. Description of Additional Supplementary Files. Supplementary Data 1. Supplementary Data 2. Reporting Summary. Source data Source Data. About this article. Cite this article Johnson, J. Copy to clipboard. Further reading Methods for exploring the faecal microbiome of premature infants: a review Jacob A. Westaway , Roger Huerlimann , Catherine M. Chrisman , Kelley M. Wall BMC Bioinformatics Temporal changes in the gut microbiota in farmed Atlantic cod Gadus morhua outweigh the response to diet supplementation with macroalgae C.

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