Comparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling

Background The microbiota of the lower female genital tract plays an important role in women’s health. Microbial profiling using the chaperonin60 (cpn60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn60 UT. Methods DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. Results DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn60 based microbial profiling of cervicovaginal samples.

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Performance comparison of different microbial DNA extraction methods on bird feces

Avian Research ◽

10.1186/s40657-021-00254-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xian Hou ◽

Shengkai Pan ◽

Zhenzhen Lin ◽

Jiliang Xu ◽

Xiangjiang Zhan

Keyword(s):

Dna Extraction ◽

Relative Abundance ◽

Alpha Diversity ◽

Cell Lysis ◽

Extraction Methods ◽

Dna Integrity ◽

Microbial Composition ◽

Dna Yield ◽

Dna Extraction Methods ◽

Microbial Dna

Abstract Background As an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not been systematacially evaluated yet. Methods In this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild. Results Bead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversity and cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity. Conclusions DNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.

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Comparison of different conditions for DNA extraction in sputum - a pilot study

Multidisciplinary Respiratory Medicine ◽

10.4081/mrm.2019.4 ◽

2019 ◽

Vol 14 ◽

Author(s):

Martina Oriano ◽

Leonardo Terranova ◽

Antonio Teri ◽

Samantha Sottotetti ◽

Luca Ruggiero ◽

...

Keyword(s):

Real Time ◽

16S Rdna ◽

Dna Extraction ◽

Real Time Pcr ◽

Alpha Diversity ◽

Diversity Indices ◽

Extraction Yield ◽

Chronic Respiratory Diseases ◽

Dna Yield ◽

Selection Of

Background: The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis. Methods: Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and S. aureus and, then, microbiome was evaluated. Results: Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for S. aureus was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT. Conclusions: None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield.

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Optimized DNA extraction and purification method for characterization of bacterial and fungal communities in lung tissue samples

Scientific Reports ◽

10.1038/s41598-020-74137-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Vicente Pérez-Brocal ◽

Fabien Magne ◽

Susana Ruiz-Ruiz ◽

Carolina A. Ponce ◽

Rebeca Bustamante ◽

...

Keyword(s):

Microbial Community ◽

Dna Extraction ◽

Lung Tissue ◽

Dna Amount ◽

Purification Method ◽

Tissue Samples ◽

Dna Yield ◽

Microbial Profile ◽

Extraction And Purification ◽

Pre Treatment

Abstract Human lungs harbor a scarce microbial community, requiring to develop methods to enhance the recovery of nucleic acids from bacteria and fungi, leading to a more efficient analysis of the lung tissue microbiota. Here we describe five extraction protocols including pre-treatment, bead-beating and/or Phenol:Chloroform:Isoamyl alcohol steps, applied to lung tissue samples from autopsied individuals. The resulting total DNA yield and quality, bacterial and fungal DNA amount and the microbial community structure were analyzed by qPCR and Illumina sequencing of bacterial 16S rRNA and fungal ITS genes. Bioinformatic modeling revealed that a large part of microbiome from lung tissue is composed of microbial contaminants, although our controls clustered separately from biological samples. After removal of contaminant sequences, the effects of extraction protocols on the microbiota were assessed. The major differences among samples could be attributed to inter-individual variations rather than DNA extraction protocols. However, inclusion of the bead-beater and Phenol:Chloroform:Isoamyl alcohol steps resulted in changes in the relative abundance of some bacterial/fungal taxa. Furthermore, inclusion of a pre-treatment step increased microbial DNA concentration but not diversity and it may contribute to eliminate DNA fragments from dead microorganisms in lung tissue samples, making the microbial profile closer to the actual one.

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Comparison of DNA Extraction and Purification Methods from Different Soils for Metagenomic Sequencing

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.955-959.306 ◽

2014 ◽

Vol 955-959 ◽

pp. 306-309 ◽

Cited By ~ 1

Author(s):

Lin Hui Wu ◽

Jian Li Liu ◽

Jing Zeng ◽

Ji Zhao

Keyword(s):

Nucleic Acids ◽

Dna Extraction ◽

Dna Isolation ◽

Metagenomic Sequencing ◽

Soil Dna ◽

Dna Yield ◽

Extraction And Purification ◽

Purification Methods ◽

The Impact ◽

Different Soils

There is an increased interest in the extraction of nucleic acids from various environmental samples, since only a minority of naturally occurring microbes can be cultured using standard techniques. Nucleic acids extraction and purification from soils are extremely challenging due to the low biomass, high organic contents and high variability of soil types. This has been regarded as one of the major difficulties that hamper the development of soil microbial ecology study. No commercial nucleic acids kits currently available are capable of preparing the DNAs without modifications. The cost can be very high for DNA extraction from extreme environmental soil samples, such as soils that have extreme high or low pHs. In this work, we developed and optimized soil DNA extraction and purification methods on different soils and compared the impact of three different DNA extraction protocols on DNA yield and purity. For the three different types of soil we used, direct extraction obtained the highest DNA recover rate, but required more cleanup steps. MoBio PowerSoil® DNA Isolation Kit yields less but do not require as many downstream cleaning steps. Both of the two methods obtained a more abundant microbial community than Meta-G-NomeTMDNA Isolation Kit.

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269 Influence of the maternal rumen microbiome on development of the calf meconium and rumen microbiome

Journal of Animal Science ◽

10.1093/jas/skaa278.359 ◽

2020 ◽

Vol 98 (Supplement_4) ◽

pp. 195-195

Author(s):

Kelly Woodruff ◽

Gwendolynn Hummel ◽

Kathleen Austin ◽

Travis Smith ◽

Hannah Cunningham

Keyword(s):

Beta Diversity ◽

Management Practices ◽

Sequence Data ◽

Rumen Fluid ◽

Alpha Diversity ◽

Illumina Miseq ◽

Sample Type ◽

Microbial Composition ◽

Alpha And Beta Diversity ◽

Rumen Microbiome

Abstract Optimization of host performance may be achieved through programming of the rumen microbiome. Thus, understanding maternal influences on the development of the calf rumen microbiome is critical. We hypothesized that the cow maternal rumen microbiome would influence colonization of the calf rumen microbiome. Our objective was to relate the microbiome of the cow rumen fluid prior to parturition (RFC) and at weaning (RFCw) to the calf’s meconium microbiome (M) and calf rumen fluid microbiome at birth (RFd1), d 2 (RFd2), d 28 (RFd28), and weaning (RFNw). Multiparous Angus crossbred cows (n = 10) from the University of Wyoming beef herd were used. Rumen fluid was collected from the cows prior to parturition and at weaning. Immediately following parturition, meconium and rumen fluid were collected from the calf. Rumen fluid was collected again at d 2, 28, and at weaning. Microbial DNA was isolated and 16S rRNA sequencing was completed on the Illumina MiSeq. Sequence data were analyzed with QIIME2 to determine both alpha and beta diversity by sample type and day. Alpha diversity metrics reported similarities in the early gut microbiome (M, RFd1, and RFD2; q ≥ 0.12) and between the cow and calf at weaning (q ≥ 0.06). Microbial composition as determined by beta diversity differed in the early rumen microbiome (RFd1, RFd2, and RFd28; q ≤ 0.04). There were similarities in composition between M, RFCw, and RFd1 (q ≥ 0.09). These data can be used to develop hypotheses for the pathway of colonization in the early gut and can provide insight into management practices affecting the microbiome, improving host performance.

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Microbiota Changes in Fathers Consuming a High Prebiotic Fiber Diet Have Minimal Effects on Male and Female Offspring in Rats

Nutrients ◽

10.3390/nu13030820 ◽

2021 ◽

Vol 13 (3) ◽

pp. 820

Author(s):

Faye Chleilat ◽

Alana Schick ◽

Raylene A. Reimer

Keyword(s):

Beta Diversity ◽

Control Diet ◽

Gastrointestinal Hormones ◽

Alpha Diversity ◽

Female Offspring ◽

Sprague Dawley ◽

Microbial Composition ◽

Gut Hormone ◽

Satiety Hormone ◽

Prebiotic Fiber

Background: Consuming a diet high in prebiotic fiber has been associated with improved metabolic and gut microbial parameters intergenerationally, although studies have been limited to maternal intake with no studies examining this effect in a paternal model. Method: Male Sprague Dawley rats were allocated to either (1) control or (2) oligofructose-supplemented diet for nine weeks and then mated. Offspring consumed control diet until 16 weeks of age. Bodyweight, body composition, glycemia, hepatic triglycerides, gastrointestinal hormones, and gut microbiota composition were measured in fathers and offspring. Results: Paternal energy intake was reduced, while satiety inducing peptide tyrosine tyrosine (PYY) gut hormone was increased in prebiotic versus control fathers. Increased serum PYY persisted in female prebiotic adult offspring. Hepatic triglycerides were decreased in prebiotic fathers with a similar trend (p = 0.07) seen in female offspring. Gut microbial composition showed significantly reduced alpha diversity in prebiotic fathers at 9 and 12 weeks of age (p < 0.001), as well as concurrent differences in beta diversity (p < 0.001), characterized by differences in Bifidobacteriaceae, Lactobacillaceae and Erysipelotrichaceae, and particularly Bifidobacterium animalis. Female prebiotic offspring had higher alpha diversity at 3 and 9 weeks of age (p < 0.002) and differences in beta diversity at 15 weeks of age (p = 0.04). Increases in Bacteroidetes in female offspring and Christensenellaceae in male offspring were seen at nine weeks of age. Conclusions: Although paternal prebiotic intake before conception improves metabolic and microbiota outcomes in fathers, effects on offspring were limited with increased serum satiety hormone levels and changes to only select gut bacteria.

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Is Intestinal Bacterial Diversity Enhanced by Trans-Species Spread in the Mixed-Species Flock of Hooded Crane (Grus monacha) and Bean Goose (Anser fabalis) Wintering in the Lower and Middle Yangtze River Floodplain?

Animals ◽

10.3390/ani11010233 ◽

2021 ◽

Vol 11 (1) ◽

pp. 233

Author(s):

Zhuqing Yang ◽

Lizhi Zhou

Keyword(s):

Contact Time ◽

Alpha Diversity ◽

Gut Bacteria ◽

Species Flock ◽

Mixed Species ◽

Microbial Composition ◽

Indirect Contact ◽

Hooded Crane ◽

Bean Goose ◽

Intestinal Pathogens

Diversity of gut microbes is influenced by many aspects, including the host internal factors and even direct or indirect contact with other birds, which is particularly important for mixed-species wintering waterbird flocks. In this study, Illumina high-throughput sequencing was used to analyze the intestinal bacteria of the hooded crane and bean goose whose niches overlap at Shengjin Lake. We tested whether contact time enhances the trans-species spread of gut bacteria. Results indicate alpha-diversity and microbial composition displayed significant separation between the two hosts in every wintering period, although the number of bacteria types shared increased with increasing contact time. For the same species, with the lengthening of contact time, alpha-diversity and the number of operational taxonomic units (OTUs) in the host intestine augmented, and the common OTUs and structural similarity of microflora in the middle and late periods were more than in the early and middle periods. In addition, we found a very high proportion of shared pathogens. Our results indicate that, although intestinal microflora of different species were separated, direct or indirect contact in the mixed-species flock caused the spread of gut bacteria trans-species, indicating that more attention should be paid to intestinal pathogens in wild birds.

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Experimental infection with the hookworm, Necator americanus, is associated with stable gut microbial diversity in human volunteers with relapsing multiple sclerosis

BMC Biology ◽

10.1186/s12915-021-01003-6 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Timothy P. Jenkins ◽

David I. Pritchard ◽

Radu Tanasescu ◽

Gary Telford ◽

Marina Papaiakovou ◽

...

Keyword(s):

Multiple Sclerosis ◽

Experimental Infection ◽

High Throughput Sequencing ◽

Alpha Diversity ◽

Placebo Treatment ◽

Sequencing Data ◽

Faecal Microbiota ◽

Microbial Composition ◽

Necator Americanus ◽

Human Volunteers

Abstract Background Helminth-associated changes in gut microbiota composition have been hypothesised to contribute to the immune-suppressive properties of parasitic worms. Multiple sclerosis is an immune-mediated autoimmune disease of the central nervous system whose pathophysiology has been linked to imbalances in gut microbial communities. Results In the present study, we investigated, for the first time, qualitative and quantitative changes in the faecal bacterial composition of human volunteers with remitting multiple sclerosis (RMS) prior to and following experimental infection with the human hookworm, Necator americanus (N+), and following anthelmintic treatment, and compared the findings with data obtained from a cohort of RMS patients subjected to placebo treatment (PBO). Bacterial 16S rRNA high-throughput sequencing data revealed significantly decreased alpha diversity in the faecal microbiota of PBO compared to N+ subjects over the course of the trial; additionally, we observed significant differences in the abundances of several bacterial taxa with putative immune-modulatory functions between study cohorts. Parabacteroides were significantly expanded in the faecal microbiota of N+ individuals for which no clinical and/or radiological relapses were recorded at the end of the trial. Conclusions Overall, our data lend support to the hypothesis of a contributory role of parasite-associated alterations in gut microbial composition to the immune-modulatory properties of hookworm parasites.

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Gut Microbiome in Psoriasis: An Updated Review

Pathogens ◽

10.3390/pathogens9060463 ◽

2020 ◽

Vol 9 (6) ◽

pp. 463

Author(s):

Mariusz Sikora ◽

Albert Stec ◽

Magdalena Chrabaszcz ◽

Aleksandra Knot ◽

Anna Waskiel-Burnat ◽

...

Keyword(s):

Gut Microbiome ◽

Beta Diversity ◽

Large Scale ◽

Skin Diseases ◽

Intestinal Barrier ◽

Alpha Diversity ◽

Current Data ◽

Intestinal Microbiome ◽

Microbial Composition ◽

Alpha And Beta Diversity

(1) Background: A growing body of evidence highlights that intestinal dysbiosis is associated with the development of psoriasis. The gut–skin axis is the novel concept of the interaction between skin diseases and microbiome through inflammatory mediators, metabolites and the intestinal barrier. The objective of this study was to synthesize current data on the gut microbial composition in psoriasis. (2) Methods: We conducted a systematic review of studies investigating intestinal microbiome in psoriasis, using the PRISMA checklist. We searched MEDLINE, EMBASE, and Web of Science databases for relevant published articles (2000–2020). (3) Results: All of the 10 retrieved studies reported alterations in the gut microbiome in patients with psoriasis. Eight studies assessed alpha- and beta-diversity. Four of them reported a lack of change in alpha-diversity, but all confirmed significant changes in beta-diversity. At the phylum-level, at least two or more studies reported a lower relative abundance of Bacteroidetes, and higher Firmicutes in psoriasis patients versus healthy controls. (4) Conclusions: There is a significant association between alterations in gut microbial composition and psoriasis; however, there is high heterogeneity between studies. More unified methodological standards in large-scale studies are needed to understand microbiota’s contribution to psoriasis pathogenesis and its modulation as a potential therapeutic strategy.

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The Effects of Genetic Relatedness on the Preterm Infant Gut Microbiota

Microorganisms ◽

10.3390/microorganisms9020278 ◽

2021 ◽

Vol 9 (2) ◽

pp. 278

Author(s):

Shen Jean Lim ◽

Miriam Aguilar-Lopez ◽

Christine Wetzel ◽

Samia V. O. Dutra ◽

Vanessa Bray ◽

...

Keyword(s):

Gut Microbiota ◽

Preterm Infant ◽

Neonatal Intensive Care ◽

Genetic Relatedness ◽

Bovine Milk ◽

Alpha Diversity ◽

Neonatal Intensive Care Units ◽

Rrna Gene ◽

Microbial Composition ◽

Operational Taxonomic Units

The preterm infant gut microbiota is influenced by environmental, endogenous, maternal, and genetic factors. Although siblings share similar gut microbial composition, it is not known how genetic relatedness affects alpha diversity and specific taxa abundances in preterm infants. We analyzed the 16S rRNA gene content of stool samples, ≤ and >3 weeks postnatal age, and clinical data from preterm multiplets and singletons at two Neonatal Intensive Care Units (NICUs), Tampa General Hospital (TGH; FL, USA) and Carle Hospital (IL, USA). Weeks on bovine milk-based fortifier (BMF) and weight gain velocity were significant predictors of alpha diversity. Alpha diversity between siblings were significantly correlated, particularly at ≤3 weeks postnatal age and in the TGH NICU, after controlling for clinical factors. Siblings shared higher gut microbial composition similarity compared to unrelated individuals. After residualizing against clinical covariates, 30 common operational taxonomic units were correlated between siblings across time points. These belonged to the bacterial classes Actinobacteria, Bacilli, Bacteroidia, Clostridia, Erysipelotrichia, and Negativicutes. Besides the influence of BMF and weight variables on the gut microbial diversity, our study identified gut microbial similarities between siblings that suggest genetic or shared maternal and environmental effects on the preterm infant gut microbiota.

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