Dataset Information

Influence of 16S rRNA target region on the outcome of microbiome studies in soil and saliva samples.

ABSTRACT: Next generation sequencing methods are widely used in evaluating the structure and functioning of microbial communities, especially those centered on 16S rRNA subunit. Since Illumina Miseq, the most used sequencing platform, does not allow the full sequencing of 16S rRNA gene, this study aims to evaluate whether the choice of different target regions might affect the outcome of microbiome studies regarding soil and saliva samples. V1V3, V3V4, V4V5 and V6V8 domains were studied, finding that while some regions showed differences in the detection of certain bacterial taxa and in the calculation of alpha diversity, especially in soil samples, the overall effect did not compromise the differentiation of any sample type in terms of taxonomic analysis at the genus level. 16S rRNA target regions did affect the detection of specific bacteria related to soil quality and development, and microbial genera used as health biomarkers in saliva. V1V3 region showed the closest similarity to internal sequencing control mock community B, suggesting it might be the most preferable choice regarding data reliability.

SUBMITTER: Soriano-Lerma A

PROVIDER: S-EPMC7423937 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Influence of 16S rRNA target region on the outcome of microbiome studies in soil and saliva samples.

Soriano-Lerma Ana A Pérez-Carrasco Virginia V Sánchez-Marañón Manuel M Ortiz-González Matilde M Sánchez-Martín Victoria V Gijón Juan J Navarro-Mari José María JM García-Salcedo José Antonio JA Soriano Miguel M

Scientific reports 20200812 1

Next generation sequencing methods are widely used in evaluating the structure and functioning of microbial communities, especially those centered on 16S rRNA subunit. Since Illumina Miseq, the most used sequencing platform, does not allow the full sequencing of 16S rRNA gene, this study aims to evaluate whether the choice of different target regions might affect the outcome of microbiome studies regarding soil and saliva samples. V1V3, V3V4, V4V5 and V6V8 domains were studied, finding that whil ...[more]

PMID: 32788589

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Project description:16S rRNA gene sequences are commonly analyzed for taxonomic and phylogenetic studies because they contain variable regions that can help distinguish different genera. However, intra-genus distinction using variable region homology is often impossible due to the high overall sequence identities among closely related species, even though some residues may be conserved within respective species. Using a computational method that included the allelic diversity within individual genomes, we discovered that certain Escherichia and Shigella species can be distinguished by a multi-allelic 16S rRNA variable region single nucleotide polymorphism (SNP). To evaluate the performance of 16S rRNAs with altered variable regions, we developed an in vivo system that measures the acceptance and distribution of variant 16S rRNAs into a large pool of natural versions supporting normal translation and growth. We found that 16S rRNAs containing evolutionarily disparate variable regions were underpopulated both in ribosomes and in active translation pools, even for an SNP. Overall, this study revealed that variable region sequences can substantially influence the performance of 16S rRNAs and that this biological constraint can be leveraged to justify refining taxonomic assignments of variable region sequence data. IMPORTANCE This study reevaluates the notion that 16S rRNA gene variable region sequences are uninformative for intra-genus classification and that single nucleotide variations within them have no consequence to strains that bear them. We demonstrated that the performance of 16S rRNAs in Escherichia coli can be negatively impacted by sequence changes in variable regions, even for single nucleotide changes that are native to closely related Escherichia and Shigella species; thus, biological performance is likely constraining the evolution of variable regions in bacteria. Further, the native nucleotide variations we tested occur in all strains of their respective species and across their multiple 16S rRNA gene copies, suggesting that these species evolved beyond what would be discerned from a consensus sequence comparison. Therefore, this work also reveals that the multiple 16S rRNA gene alleles found in most bacteria can provide more informative phylogenetic and taxonomic detail than a single reference allele.

Project description:BackgroundSequencing variable regions of the 16S rRNA gene (≃300 bp) with Illumina technology is commonly used to study the composition of human microbiota. Unfortunately, short reads are unable to differentiate between highly similar species. Considering that species from the same genus can be associated with health or disease it is important to identify them at the lowest possible taxonomic rank. Third-generation sequencing platforms such as PacBio SMRT, increase read lengths allowing to sequence the whole gene with the maximum taxonomic resolution. Despite its potential, full length 16S rRNA gene sequencing is not widely used yet. The aim of the current study was to compare the sequencing output and taxonomic annotation performance of the two approaches (Illumina short read sequencing and PacBio long read sequencing of 16S rRNA gene) in different human microbiome samples. DNA from saliva, oral biofilms (subgingival plaque) and faeces of 9 volunteers was isolated. Regions V3-V4 and V1-V9 were amplified and sequenced by Illumina Miseq and by PacBio Sequel II sequencers, respectively.ResultsWith both platforms, a similar percentage of reads was assigned to the genus level (94.79% and 95.06% respectively) but with PacBio a higher proportion of reads were further assigned to the species level (55.23% vs 74.14%). Regarding overall bacterial composition, samples clustered by niche and not by sequencing platform. In addition, all genera with > 0.1% abundance were detected in both platforms for all types of samples. Although some genera such as Streptococcus tended to be observed at higher frequency in PacBio than in Illumina (20.14% vs 14.12% in saliva, 10.63% vs 6.59% in subgingival plaque biofilm samples) none of the differences were statistically significant when correcting for multiple testing.ConclusionsThe results presented in the current manuscript suggest that samples sequenced using Illumina and PacBio are mostly comparable. Considering that PacBio reads were assigned at the species level with higher accuracy than Illumina, our data support the use of PacBio technology for future microbiome studies, although a higher cost is currently required to obtain an equivalent number of reads per sample.

Project description:BACKGROUND: Sample storage conditions, extraction methods, PCR primers, and parameters are major factors that affect metagenomics analysis based on microbial 16S rRNA gene sequencing. Most published studies were limited to the comparison of only one or two types of these factors. Systematic multi-factor explorations are needed to evaluate the conditions that may impact validity of a microbiome analysis. This study was aimed to improve methodological options to facilitate the best technical approaches in the design of a microbiome study. Three readily available mock bacterial community materials and two commercial extraction techniques, Qiagen DNeasy and MO BIO PowerSoil DNA purification methods, were used to assess procedures for 16S ribosomal DNA amplification and pyrosequencing-based analysis. Primers were chosen for 16S rDNA quantitative PCR and amplification of region V3 to V1. Swabs spiked with mock bacterial community cells and clinical oropharyngeal swabs were incubated at respective temperatures of -80°C, -20°C, 4°C, and 37°C for 4 weeks, then extracted with the two methods, and subjected to pyrosequencing and taxonomic and statistical analyses to investigate microbiome profile stability. RESULTS: The bacterial compositions for the mock community DNA samples determined in this study were consistent with the projected levels and agreed with the literature. The quantitation accuracy of abundances for several genera was improved with changes made to the standard Human Microbiome Project (HMP) procedure. The data for the samples purified with DNeasy and PowerSoil methods were statistically distinct; however, both results were reproducible and in good agreement with each other. The temperature effect on storage stability was investigated by using mock community cells and showed that the microbial community profiles were altered with the increase in incubation temperature. However, this phenomenon was not detected when clinical oropharyngeal swabs were used in the experiment. CONCLUSIONS: Mock community materials originated from the HMP study are valuable controls in developing 16S metagenomics analysis procedures. Long-term exposure to a high temperature may introduce variation into analysis for oropharyngeal swabs, suggestive of storage at 4°C or lower. The observed variations due to sample storage temperature are in a similar range as the intrapersonal variability among different clinical oropharyngeal swab samples.

Dataset Information

Influence of 16S rRNA target region on the outcome of microbiome studies in soil and saliva samples.

Publications

Influence of 16S rRNA target region on the outcome of microbiome studies in soil and saliva samples.

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