Refinement of 16S rRNA gene analysis for low biomass biospecimens

Villette, Remy; Autaa, Gaëlle; Hind, Sophie; Holm, Johanna B.; Moreno-Sabater, Alicia; Larsen, Martin

doi:10.1038/s41598-021-90226-2

Cited by 19 publications

(10 citation statements)

References 42 publications

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“…Variable feature counts also suggest differential abundance in sample biomass that can significantly affect alpha and beta-diversity analysis. 70 A proposed methodology to standardize the concentration of samples has been documented by Doan and colleagues. 18 A limitation to our methodology was not performing quantitative polymerase chain reaction (qPCR) to control for sample concentration.…”

Section: Discussionmentioning

confidence: 99%

Topical Glaucoma Therapy Is Associated With Alterations of the Ocular Surface Microbiome

Chang

Somohano

Zemsky

et al. 2022

Invest. Ophthalmol. Vis. Sci.

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Purpose To investigate the ocular surface microbiome of patients with unilateral or asymmetric glaucoma being treated with topical ophthalmic medications in one eye and to determine whether microbial community changes were related to measures of ocular surface disease. Methods V3-V4 16S rRNA sequencing was conducted on ocular surface swabs collected from both eyes of 17 subjects: 10 patients with asymmetric/unilateral glaucoma using topical glaucoma therapy on only one eye and seven age-matched, healthy controls with no history of ocular disease or eyedrop use. Samples were categorized into three groups: patients’ glaucomatous eye treated with eyedrops, patients’ contralateral eye without eyedrops, and healthy control eyes. Comparisons were made for microbial diversity and composition, with differences in composition tested for association with ocular surface disease measures including tear meniscus height, tear break-up time, and Dry Eye Questionnaire. Results Samples obtained from the patients’ treated and untreated eyes both had significantly greater alpha-diversity and relative abundance of gram-negative organisms compared to healthy controls. The microbial composition of patient eyes was associated with decreased tear meniscus height and tear break-up time, whereas metagenomic predictions, based on 16S rRNA data, suggested increased synthesis of lipopolysaccharide. Conclusions The ocular surface microbiome of patients taking unilateral preserved glaucoma drops is characterized by a highly diverse array of gram-negative bacteria that is significantly different from the predominantly gram-positive microbes detected on healthy control eyes. These compositional differences were associated with decreased tear film measures and distinct inferred protein synthesis pathways, suggesting a potential link between microbial alterations and ocular surface inflammation.

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Section: Discussionmentioning

confidence: 99%

Topical Glaucoma Therapy Is Associated With Alterations of the Ocular Surface Microbiome

Chang

Somohano

Zemsky

et al. 2022

Invest. Ophthalmol. Vis. Sci.

View full text Add to dashboard Cite

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“…Additionally, sample collection strategies may need to be optimized specifically for these low biomass samples; fortunately, recommendations exist [ 45 ] . Previous research has shown that larger volumes of low-biomass samples don’t necessarily lead to significantly increased DNA biomass [ 46 ], and therefore future efforts may yield more success by focusing on improved extraction methods [ 47 ].…”

Section: Discussionmentioning

confidence: 99%

The microbiome of common bedding materials before and after use on commercial dairy farms

et al. 2022

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Background Bovine mastitis is one of the most economically important diseases affecting dairy cows. The choice of bedding material has been identified as an important risk factor contributing to the development of mastitis. However, few reports examine both the culturable and nonculturable microbial composition of commonly used bedding materials, i.e., the microbiome. Given the prevalence of nonculturable microbes in most environments, this information could be an important step to understanding whether and how the bedding microbiome acts as a risk factor for mastitis. Therefore, our objective was to characterize the microbiome composition and diversity of bedding material microbiomes, before and after use. Methods We collected 88 bedding samples from 44 dairy farms in the U.S. Unused (from storage pile) and used (out of stalls) bedding materials were collected from four bedding types: new sand (NSA), recycled manure solids (RMS), organic non-manure (ON) and recycled sand (RSA). Samples were analyzed using 16S rRNA sequencing of the V3–V4 region. Results The overall composition as well as the counts of several microbial taxa differed between bedding types, with Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes dominating across all types. Used bedding contained a significantly different microbial composition than unused bedding, but the magnitude of this difference varied by bedding type, with RMS bedding exhibiting the smallest difference. In addition, positive correlations were observed between 16S rRNA sequence counts of potential mastitis pathogens (bacterial genera) and corresponding bedding bacterial culture data. Conclusion Our results strengthen the role of bedding as a potential source of mastitis pathogens. The consistent shift in the microbiome of all bedding types that occurred during use by dairy cows deserves further investigation to understand whether this shift promotes pathogen colonization and/or persistence, or whether it can differentially impact udder health outcomes. Future studies of bedding and udder health may be strengthened by including a microbiome component to the study design.

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“…Therefore we focused on 16S rRNA gene sequencing data. 16S rRNA gene sequencing is a well-characterized way of detecting and classifying bacterial communities within biological samples (158–160), and it is potentially sensitive enough to detect the typically low number of 16S rRNA bacterial gene copies hypothesized to be in the placenta (34, 161). 33 of the 41 studies which implemented 16S rRNA gene sequencing included at least one placental sample from an uncomplicated delivery at term (27-29, 36-45, 47, 49-65, 86, 142).…”

Section: Methodsmentioning

confidence: 99%

Is there a placental microbiota? A critical review and re-analysis of published placental microbiota datasets

Panzer

Romero

Greenberg

et al. 2022

Preprint

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The existence of a placental microbiota is under debate. The human placenta has historically been considered sterile and microbial colonization has been associated with adverse pregnancy outcomes. Yet, recent investigations using DNA sequencing reported a microbiota in human placentas from typical term pregnancies. However, this detected microbiota could represent background DNA contamination. Using fifteen publicly available 16S rRNA gene datasets, existing data were uniformly re-analyzed. 16S rRNA gene Amplicon Sequence Variants (ASVs) identified as Lactobacillus were highly abundant in eight of fifteen studies. However, the prevalence of Lactobacillus, a typical vaginal bacterium, was clearly driven by bacterial contamination from vaginal delivery and background DNA. After removal of likely DNA contaminants, Lactobacillus ASVs were highly abundant in only one of five studies for which data analysis could be restricted to placentas from term cesarean deliveries. A six study sub-analysis targeting the 16S rRNA gene V4 hypervariable region demonstrated that bacterial profiles of placental samples and technical controls share principal bacterial ASVs and that placental samples clustered primarily by study origin and mode of delivery. Across studies, placentas from typical term pregnancies did not share a consistent bacterial taxonomic signal. Contemporary DNA- based evidence does not support the existence of a placental microbiota.IMPORTANCEEarly-gestational microbial influences on human development are unclear. By applying DNA sequencing technologies to placental tissue, bacterial DNA signals were observed, leading some to conclude that a live bacterial placental microbiome exists in typical term pregnancy. However, the low-biomass nature of the proposed microbiome and high sensitivity of current DNA sequencing technologies indicate that the signal may alternatively derive from environmental or delivery-associated bacterial DNA contamination. Here we address these alternatives with a re- analysis of 16S rRNA gene sequencing data from 15 publicly available placental datasets. After identical DADA2 pipeline processing of the raw data, subanalyses were performed to control for mode of delivery and environmental DNA contamination. Both environment and mode of delivery profoundly influenced the bacterial DNA signal from term-delivered placentas. Aside from these contamination-associated signals, consistency was lacking across studies. Thus, placentas delivered at term are unlikely to be the original source of observed bacterial DNA signals.

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Refinement of 16S rRNA gene analysis for low biomass biospecimens

Cited by 19 publications

References 42 publications

Topical Glaucoma Therapy Is Associated With Alterations of the Ocular Surface Microbiome

Topical Glaucoma Therapy Is Associated With Alterations of the Ocular Surface Microbiome

The microbiome of common bedding materials before and after use on commercial dairy farms

Is there a placental microbiota? A critical review and re-analysis of published placental microbiota datasets

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