scholarly journals Effects of sampling strategy and DNA extraction on human skin microbiome investigations

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Rie Dybboe Bjerre ◽  
Luisa Warchavchik Hugerth ◽  
Fredrik Boulund ◽  
Maike Seifert ◽  
Jeanne Duus Johansen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Dna Extraction ◽  
Human Skin ◽  
Alpha Diversity ◽  
Sampling Strategy ◽  
Sampling Technique ◽  
Rrna Gene ◽  
Skin Microbiome ◽  
Library Preparation ◽  
Reliable Technique

AbstractThe human skin is colonized by a wide array of microorganisms playing a role in skin disorders. Studying the skin microbiome provides unique obstacles such as low microbial biomass. The objective of this study was to establish methodology for skin microbiome analyses, focusing on sampling technique and DNA extraction. Skin swabs and scrapes were collected from 9 healthy adult subjects, and DNA extracted using 12 commercial kits. All 165 samples were sequenced using the 16S rRNA gene. Comparing the populations captured by eSwabs and scrapes, 99.3% of sequences overlapped. Using eSwabs yielded higher consistency. The success rate of library preparation applying different DNA extraction kits ranged from 39% to 100%. Some kits had higher Shannon alpha-diversity. Metagenomic shotgun analyses were performed on a subset of samples (N = 12). These data indicate that a reduction of human DNA from 90% to 57% is feasible without lowering the success of 16S rRNA library preparation and without introducing taxonomic bias. Using swabs is a reliable technique to investigate the skin microbiome. DNA extraction methodology is crucial for success of sequencing and adds a substantial amount of variation in microbiome analyses. Reduction of host DNA is recommended for interventional studies applying metagenomics.

scholarly journals Cutibacterium acnes (Propionibacterium acnes) 16S rRNA Genotyping of Microbial Samples from Possessions Contributes to Owner Identification

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Jiayue Yang ◽  
Tomoya Tsukimi ◽  
Mia Yoshikawa ◽  
Kenta Suzuki ◽  
Tomoki Takeda ◽  
...  
Keyword(s):  
16S Rrna ◽  
Human Skin ◽  
Rrna Gene ◽  
Skin Microbiome ◽  
Nucleotide Polymorphisms ◽  
Skin Microbiota ◽  
Content Type ◽  
Microbiome Profile ◽  
Cutibacterium Acnes ◽  
Genotype Composition

ABSTRACT The human skin surface harbors huge numbers of microbes. The skin microbiota interacts with its host and forms a skin microbiome profile that is specific for each individual. It has been reported that the skin microbiota that is left on an individual’s possessions can act as a sort of “fingerprint” and be used for owner identification. However, this approach needs to be improved to take into account any long-term instability of skin microbiota and contamination from nonspecific bacteria. Here, we took advantage of single-nucleotide polymorphisms (SNPs) in the 16S-encoding rRNA gene of Cutibacterium acnes, the most common and abundant bacterium on human skin, to perform owner identification. We first developed a high-throughput genotyping method based on next-generation sequencing to characterize the SNPs of the C. acnes 16S rRNA gene and found that the genotype composition of C. acnes 16S rRNA is individual specific. Owner identification accuracy of around 90% based on random forest machine learning was achieved by using a combination of C. acnes 16S rRNA genotype and skin microbiome profile data. Furthermore, our study showed that the C. acnes 16S rRNA genotype remained more stable over time than the skin microbiome profile. This characteristic of C. acnes was further confirmed by the analysis of publicly available human skin metagenome data. Our approach, with its high precision, good reproducibility, and low costs, thus provides new possibilities in the field of microbiome-based owner identification and forensics in general. IMPORTANCE Cutibacterium acnes is the most common and abundant bacterial species on human skin, and the gene that encodes its 16S rRNA has multiple single-nucleotide polymorphisms. In this study, we developed a method to efficiently determine the C. acnes 16S rRNA genotype composition from microbial samples taken from the hands of participants and from their possessions. Using the C. acnes 16S rRNA genotype composition, we could predict the owner of a possession with around 90% accuracy when the 16S rRNA gene-based microbiome profile was included. We also showed that the C. acnes 16S rRNA genotype composition was more stable over time than the skin microbiome profile and thus is more suitable for owner identification.

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

1992 ◽  
Vol 100 (1-3) ◽  
pp. 59-65 ◽  
Author(s):  
Paul A. Rochelle ◽  
John C. Fry ◽  
R. John Parkes ◽  
Andrew J. Weightman
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Gene Analysis ◽  
Rrna Gene ◽  
16S Rrna Gene Analysis ◽  
Deep Sediment

scholarly journals Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor

2017 ◽  
Vol 28 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Anniina Rintala ◽  
Sami Pietilä ◽  
Eveliina Munukka ◽  
Erkki Eerola ◽  
Juha-Pekka Pursiheimo ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Rrna Gene ◽  
Gene Target ◽  
Target Region ◽  
Microbiota Analysis ◽  
The Impact ◽  
The 16S Rrna Gene

scholarly journals The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Ingo C. Starke ◽  
Wilfried Vahjen ◽  
Robert Pieper ◽  
Jürgen Zentek
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Dna Extraction ◽  
16S Rrna Genes ◽  
Read Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Variable Regions ◽  
Extraction Procedures ◽  
Primer Sets

In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

scholarly journals A contribution to the larval amphibian microbiome: characterization of bacterial microbiome of Ichthyophis bannanicus (Order: Gymnophiona) and comparison with the other two amphibian orders

2021 ◽  
Author(s):  
Amrapali Rajput ◽  
Shipeng Zhou ◽  
Madhava Meegaskumbura
Keyword(s):  
Gut Microbiome ◽  
Alpha Diversity ◽  
The Other ◽  
Rrna Gene ◽  
Scaling Analysis ◽  
Skin Microbiome ◽  
Sequencing Platform ◽  
Bacterial Phyla ◽  
Faecal Samples ◽  
Skin Samples

It is known that animal-associated microbiomes form indispensable relationships with hosts and are responsible for many functions important for host-survival. Next-gen driven approaches documenting the remarkable diversity of microbiomes have burgeoned, with amphibians too, benefiting from such treatments. The microbiome of Gymnophiona (caecilians), one of the three amphibian orders, constituting of 3% of amphibians, however, remains almost unknown. The present study aims to address this knowledge gap through analysis of the microbiome of Ichthyophis bannanicus. As these caecilian larvae are aquatic and hence exposed to a greater propensity for bacterial microbiomic interchange, we hypothesize that bacterial phyla would overlap between gut and skin. Further, from the host-specificity patterns observed in other vertebrate taxa, we hypothesize that Gymnophiona have different dominant gut bacterial microbiomes at a higher taxonomic level when compared to the larvae of the other two amphibian orders (Anura and Caudata). We used 16S rRNA gene amplicon sequencing based on Illumina Nova sequencing platform to characterize and compare the gut (represented by faecal samples) and skin microbiome of I. bannanicus larvae (N = 13), a species distributed across South-East-Asia and the only caecilian species occurring in China. We compared our gut microbiome results with published anuran and caudate larval microbiomes. For I. bannanicus, a total of 4,053 operational taxonomic units (OTU) across 13 samples were detected. Alpha-diversity indices were significant between gut and skin samples. Non-metric multidimensional scaling analysis suggest that gut and skin samples each contained a distinct microbiome at OTU level. We record significant differences between the bacterial phyla of gut and skin samples in larvae of I. bannanicus. The study provides an overview of gut and skin bacterial microbiomes of a caecilian, while highlighting the major differences between larval microbiomes of the three amphibian orders. We find a partial overlap of gut bacterial microbiomes at phylum level for the three orders; however, the relative abundance of the dominant phyla is distinct. The skin and gut microbiomes are distinct with little overlap of species, highlighting that gut-skin axis is weak. This in turn suggests that many of the microbial species on skin and gut are functionally specialized to those locations. We also show that the skin microbiome is more diverse than the gut microbiome at species level; however, a reason for this could be a portion of the gut microbiome not being represented in faecal samples. These first microbiome information from a caecilian lay the foundation for comparative studies of the three amphibian orders.

scholarly journals Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiome profiling of olive xylem sap

2020 ◽  
Author(s):  
CARMEN HARO ◽  
MANUEL ANGUITA-MAESO ◽  
Madis Metsis ◽  
JUAN A NAVAS-CORTES ◽  
BLANCA LANDA
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Bacterial Communities ◽  
Methodological Approach ◽  
Xylem Sap ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Community Assessment ◽  
Woody Crop

Next Generation Sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a nonbiased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from 'Picual' and 'Arbequina' olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UNIFRAC distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, and 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignation, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction Kit, the combination of 799F/1193R primers amplifying the hypervariable V5-V7 region and the Silva 132 database for taxonomic assignation. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter, Granulicatella, Prevotella and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium. The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting within the xylem sap of olives or other woody crop species.

scholarly journals Surveying the sweetpotato rhizosphere, endophyte, and surrounding soil microbiomes at two North Carolina farms reveals underpinnings of sweetpotato microbiome community assembly

2019 ◽  
Author(s):  
C Pepe-Ranney ◽  
C Keyser ◽  
J Trimble ◽  
B Bissinger
Keyword(s):  
North Carolina ◽  
Community Assembly ◽  
Alpha Diversity ◽  
Rrna Gene ◽  
Sequence Variant ◽  
Skin Microbiome ◽  
Microbiome Composition ◽  
Pests And Diseases ◽  
Sub Saharan ◽  
Surrounding Soil

AbstractFarmers grow sweetpotatoes worldwide and some sub-Saharan African and Asian diets include sweetpotato as a staple, yet the sweetpotato microbiome is conspicuously less studied relative to crops such as maize, soybean, and wheat. Studying sweetpotato microbiome ecology may reveal paths to engineer the microbiome to improve sweetpotato yield, and/or combat sweetpotato pests and diseases. We sampled sweetpotatoes and surrounding soil from two North Carolina farms. We took samples from sweetpotato fields under two different land management regimes, conventional and organic, and collected two sweetpotato cultivars, ‘Beauregard’ and ‘Covington’. By comparing SSU rRNA gene amplicon sequence profiles from sweetpotato storage root skin, rhizosphere, and surrounding soil we found the skin microbiome possessed the least composition heterogeneity among samples and lowest alpha-diversity and was significantly nested by the rhizosphere in amplicon sequence variant (ASV) membership. Many ASVs were specific to a single field and/or only found in either the skin, rhizosphere, or surrounding soil. Notably, sweetpotato skin enriched for Planctomycetaceae in relative abundance at both farms. This study elucidates underpinnings of sweetpotato microbiome community assembly, quantifies microbiome composition variance within a single farm, and reveals microorganisms associated with sweetpotato skin that belong to common but uncultured soil phylotypes.

scholarly journals 2576. The Microbiome of Recurrent Bacterial Vaginosis Compared with Asymptomatic Controls

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S895-S895
Author(s):  
Elizabeth O Shay ◽  
Oluwatosin Goje ◽  
Roshan Padmanabhan ◽  
Charis Eng
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Vaginosis ◽  
Alpha Diversity ◽  
Dna Probe ◽  
Gardnerella Vaginalis ◽  
Rrna Gene ◽  
Vaginal Microbiome ◽  
Β Diversity ◽  
Probe Test

Abstract Background Bacterial vaginosis (BV) affects nearly 1 in 3 women in the United States and is poorly understood. The study of the vaginal microbiome, using 16S rRNA-gene amplicon sequencing, has increased our knowledge of BV. We aimed to characterize the vaginal microbiome of women with recurrent BV firstly in comparison to controls, and secondly in comparison to a sub-population of our asymptomatic controls, positive for Gardnerella vaginalis via a vaginal pathogens DNA direct probe test (DNA probe). Methods Women aged 18–40 years, with recurrent BV, and asymptomatic controls were prospectively enrolled. Vaginal samples were collected from each participant. DNA was extracted, amplified using primers targeting the V3-V4 variable region of the 16S rRNA-gene, and then sequenced and processed through a hybrid Qiime MICCA bioinformatics pipeline. We also tested for G. vaginalis using the DNA probe. Results Seventeen recurrent BV patients and 46 controls were enrolled. Β diversity (P = 0.045), but not alpha diversity (P = 0.076) differed between groups. The genera Gardnerella and Prevotella were relatively more abundant, while Lactobacillus was relatively less abundant in recurrent BV vs. control groups. Of the patients for whom results of the DNA probe for Gardnerella vaginalis were available, 11 (69%) recurrent BV patients and 14 (35%) controls were positive. Control patients, negative by the DNA probe test, showed decreased alpha diversity (P = 0.0001) and significantly different β diversity (P = 0.001) compared with recurrent BV patients. Neither alpha (P = 0.31) nor β (P = 0.096) diversity differed between recurrent BV patients and controls that were G. vaginalis positive. Conclusion The microbiome of recurrent BV patients is distinct from that of asymptomatic controls; recurrent BV patients exhibit different β diversity, less Lactobacillus and more Gardnerella and Prevotella. Asymptomatic Gardnerella vaginalis-colonized controls demonstrate similar microbiome profiles to those of recurrent BV patients. These findings suggest that individual factors may influence whether or not a patient with a BV microbiomic profile experiences symptoms. Further investigation into these mechanisms could yield insights into the treatment of recurrent BV. Disclosures All authors: No reported disclosures.

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