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16s rRNA Sequencing with MR DNA

16S ribosomal  (rRNA) sequencing using next generation sequencing is a method used to identify and compare bacteria and archaea present within almost any type of sample. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study.

 

19. Am J Obstet Gynecol. 2015 May;212(5):653.e1-16. doi: 10.1016/j.ajog.2014.12.041.

Epub 2014 Dec 31.

 

The preterm placental microbiome varies in association with excess maternal

gestational weight gain.

 

Antony KM(1), Ma J(1), Mitchell KB(1), Racusin DA(1), Versalovic J(2), Aagaard

K(3).

 

Author information:

(1)Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology,

Baylor College of Medicine and Texas Children's Hospital, Houston, TX.

(2)Department of Virology and Microbiology, Baylor College of Medicine and Texas

Children's Hospital, Houston, TX; Department of Pathology and Immunology, Baylor

College of Medicine and Texas Children's Hospital, Houston, TX. (3)Division of

Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Baylor College

of Medicine and Texas Children's Hospital, Houston, TX. Electronic address:

aagaardt@bcm.edu.

 

OBJECTIVE: Although a higher maternal body mass index is associated with preterm

birth, it is unclear whether excess gestational weight gain (GWG) or obesity

drives increased risk. We and others have shown that the placenta harbors

microbiota, which is significantly different among preterm births. Our aim in

this study was to investigate whether the preterm placental microbiome varies by

virtue of obesity or alternately by excess GWG.

STUDY DESIGN: Placentas (n=320) were collected from term and preterm pregnancies.

Genomic DNA was extracted and subjected to metagenomic sequencing. Data were

analyzed by clinical covariates that included the 2009 Institute of Medicine's

GWG guideline and obesity.

RESULTS: Analysis of 16S recombinant RNA-based metagenomics revealed no

clustering of the microbiome by virtue of obesity (P=.161). Among women who

spontaneously delivered preterm, there was again no clustering by obesity

(P=.480), but there was significant clustering by excess GWG (P=.022). Moreover,

among preterm births, detailed analysis identified microbial genera (family and

genus level) and bacterial metabolic gene pathways that varied among pregnancies

with excess GWG. Notably, excess GWG was associated with decreased microbial

folate biosynthesis pathways and decreased butanoate metabolism (linear

discriminate analysis, >3.0-fold).

CONCLUSION: Although there were no significant alterations in the microbiome by

virtue of obesity per se, excess GWG was associated with an altered microbiome

and its metabolic profile among those women who experienced a preterm birth.

 

Copyright © 2015 Elsevier Inc. All rights reserved.

 

DOI: 10.1016/j.ajog.2014.12.041

PMCID: PMC4892181

PMID: 25557210  [PubMed - indexed for MEDLINE]

 

 

20. J Allergy Clin Immunol. 2016 Mar;137(3):852-60. doi: 10.1016/j.jaci.2015.08.021.

Epub 2015 Oct 1.

 

Faecalibacterium prausnitzii subspecies-level dysbiosis in the human gut

microbiome underlying atopic dermatitis.

 

Song H(1), Yoo Y(2), Hwang J(1), Na YC(3), Kim HS(4).

 

Author information:

(1)Department of Biomedical Sciences, Korea University, Seoul, Korea.

(2)Department of Pediatrics, Korea University, Seoul, Korea. (3)Western Seoul

Center, Korea Basic Science Institute, Seoul, Korea. (4)Department of Biomedical

Sciences, Korea University, Seoul, Korea. Electronic address:

hstanleykim@korea.ac.kr.

 

BACKGROUND: Atopic dermatitis (AD) is a serious global epidemic associated with a

modern lifestyle.

OBJECTIVE: Although aberrant interactions between gut microbes and the intestinal

immune system have been implicated in this skin disease, the nature of the

microbiome dysfunction underlying the disease remains unclear.

METHODS: The gut microbiome from 132 subjects, including 90 patients with AD, was

analyzed by using 16S rRNA gene and metagenome sequence analyses. Reference

genomes from the Human Microbiome Project and the KEGG Orthology database were

used for metagenome analyses. Short-chain fatty acids in fecal samples were

compared by using gas chromatographic-mass spectrometric analyses.

RESULTS: We show that enrichment of a subspecies of the major gut species

Faecalibacterium prausnitzii is strongly associated with AD. In addition, the AD

microbiome was enriched in genes encoding the use of various nutrients that could

be released from damaged gut epithelium, reflecting a bloom of auxotrophic

bacteria. Fecal samples from patients with AD showed decreased levels of butyrate

and propionate, which have anti-inflammatory effects. This is likely a

consequence of an intraspecies compositional change in F prausnitzii that reduces

the number of high butyrate and propionate producers, including those related to

the strain A2-165, a lack of which has been implicated in patients with Crohn

disease.

CONCLUSIONS: The data suggest that feedback interactions between dysbiosis in F

prausnitzii and dysregulation of gut epithelial inflammation might underlie the

chronic progression of AD by resulting in impairment of the gut epithelial

barrier, which ultimately leads to aberrant TH2-type immune responses to

allergens in the skin.

 

Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by

Elsevier Inc. All rights reserved.

 

DOI: 10.1016/j.jaci.2015.08.021

PMID: 26431583  [PubMed - indexed for MEDLINE]

 

 

 

16s sequencing illumina or PGM low cost prices with MR DNA

MR DNA is a next generation sequencing provider with low cost 16s sequencing services.

 

110. Inflamm Bowel Dis. 2015 Mar;21(3):556-63. doi: 10.1097/MIB.0000000000000307.

 

Fecal microbial transplant effect on clinical outcomes and fecal microbiome in

active Crohn's disease.

 

Suskind DL(1), Brittnacher MJ, Wahbeh G, Shaffer ML, Hayden HS, Qin X, Singh N,

Damman CJ, Hager KR, Nielson H, Miller SI.

 

Author information:

(1)*Department of Pediatrics, Division of Gastroenterology, Seattle Children's

Hospital, University of Washington, Seattle, Washington; Departments of

†Microbiology, and ‡Laboratory Medicine, University of Washington, Seattle,

Washington; §Department of Pediatrics, Division of Gastroenterology, Cedars-Sinai

Medical Center, Los Angeles, California; and Departments of ‖Medicine,

¶Immunology, and **Genome Sciences, University of Washington, Seattle,

Washington.

 

Comment in

    Inflamm Bowel Dis. 2015 Jun;21(6):E8.

    Inflamm Bowel Dis. 2015 Jun;21(6):E8.

 

BACKGROUND: Crohn's disease (CD) is a chronic idiopathic inflammatory intestinal

disorder associated with fecal dysbiosis. Fecal microbial transplant (FMT) is a

potential therapeutic option for individuals with CD based on the hypothesis that

changing the fecal dysbiosis could promote less intestinal inflammation.

METHODS: Nine patients, aged 12 to 19 years, with mild-to-moderate symptoms

defined by Pediatric Crohn's Disease Activity Index (PCDAI of 10-29) were

enrolled into a prospective open-label study of FMT in CD (FDA IND 14942).

Patients received FMT by nasogastric tube with follow-up evaluations at 2, 6, and

12 weeks. PCDAI, C-reactive protein, and fecal calprotectin were evaluated at

each study visit.

RESULTS: All reported adverse events were graded as mild except for 1 individual

who reported moderate abdominal pain after FMT. All adverse events were

self-limiting. Metagenomic evaluation of stool microbiome indicated evidence of

FMT engraftment in 7 of 9 patients. The mean PCDAI score improved with patients

having a baseline of 19.7 ± 7.2, with improvement at 2 weeks to 6.4 ± 6.6 and at

6 weeks to 8.6 ± 4.9. Based on PCDAI, 7 of 9 patients were in remission at 2

weeks and 5 of 9 patients who did not receive additional medical therapy were in

remission at 6 and 12 weeks. No or modest improvement was seen in patients who

did not engraft or whose microbiome was most similar to their donor.

CONCLUSIONS: This is the first study to demonstrate that FMT for CD may be a

possible therapeutic option for CD. Further prospective studies are required to

fully assess the safety and efficacy of the FMT in patients with CD.

 

DOI: 10.1097/MIB.0000000000000307

PMCID: PMC4329080

PMID: 25647155  [PubMed - indexed for MEDLINE]

 

 

111. J Pediatr Gastroenterol Nutr. 2016 Feb;62(2):193-4. doi:

10.1097/MPG.0000000000001068.

 

Intestinal Microbiota Studies in Preterm Infants.

 

Neu J(1).

 

Author information:

(1)University of Florida, Gainesville.

 

Comment on

    J Pediatr Gastroenterol Nutr. 2016 Feb;62(2):292-303.

 

DOI: 10.1097/MPG.0000000000001068

PMID: 26655942  [PubMed - indexed for MEDLINE]

 

 

112. Cell Metab. 2014 Nov 4;20(5):731-41. doi: 10.1016/j.cmet.2014.10.003. Epub 2014

Nov 4.

 

Determining microbial products and identifying molecular targets in the human

microbiome.

 

Joice R(1), Yasuda K(1), Shafquat A(1), Morgan XC(2), Huttenhower C(3).

 

Author information:

(1)Department of Biostatistics, Harvard School of Public Health, Boston, MA

02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

(2)Department of Biostatistics, Harvard School of Public Health, Boston, MA

02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

Electronic address: xmorgan@hsph.harvard.edu. (3)Department of Biostatistics,

Harvard School of Public Health, Boston, MA 02115, USA; Broad Institute of MIT

and Harvard, Cambridge, MA 02142, USA. Electronic address:

chuttenh@hsph.harvard.edu.

 

Human-associated microbes are the source of many bioactive microbial products

(proteins and metabolites) that play key functions both in human host pathways

and in microbe-microbe interactions. Culture-independent studies now provide an

accelerated means of exploring novel bioactives in the human microbiome; however,

intriguingly, a substantial fraction of the microbial metagenome cannot be mapped

to annotated genes or isolate genomes and is thus of unknown function. Meta'omic

approaches, including metagenomic sequencing, metatranscriptomics, metabolomics,

and integration of multiple assay types, represent an opportunity to efficiently

explore this large pool of potential therapeutics. In combination with

appropriate follow-up validation, high-throughput culture-independent assays can

be combined with computational approaches to identify and characterize novel and

biologically interesting microbial products. Here we briefly review the state of

microbial product identification and characterization and discuss possible next

steps to catalog and leverage the large uncharted fraction of the microbial

metagenome.

 

Copyright © 2014 Elsevier Inc. All rights reserved.

 

DOI: 10.1016/j.cmet.2014.10.003

PMCID: PMC4254638

PMID: 25440055  [PubMed - indexed for MEDLINE]

 

 

113. Curr Opin Rheumatol. 2014 Jul;26(4):410-5. doi: 10.1097/BOR.0000000000000075.

 

Microbiome and probiotics: link to arthritis.

 

Bedaiwi MK(1), Inman RD.

 

Author information:

(1)Division of Rheumatology, UHN - Toronto Western Hospital, Toronto, Ontario,

Canada.

 

PURPOSE OF REVIEW: The gut microbiome plays an integral role in the development

and maintenance of the host immune system. Expanding knowledge about this

microbial microenvironment has raised the possibility of new treatments based on

this knowledge. In this review, we describe the recent evidence of the impact of

the gut microbiome on arthritis and possible novel therapeutic approaches to

alter the gut flora.

RECENT FINDINGS: Recent studies support the growing evidence of microbiome as a

causative agent underlying certain rheumatic diseases like ankylosing spondylitis

and rheumatoid arthritis. There is intriguing yet still inconclusive evidence to

support the use of probiotics as a treatment for these diseases.

SUMMARY: There is recently a new level of understanding how the microbiome

interacts with the immune system. Gene-environment interaction is another

important element that sets the stage for initiation of autoimmune disease, which

calls for further investigation. Probiotics could be an appealing therapeutic

strategy, but further interventional studies exploring the dynamic interaction of

microbiome and probiotics are still needed.

 

DOI: 10.1097/BOR.0000000000000075

PMID: 24841227  [PubMed - indexed for MEDLINE]

 

 

114. JAMA. 2015 Sep 15;314(11):1127-8. doi: 10.1001/jama.2015.10700.

 

The Human Microbiome and the Future Practice of Medicine.

 

Relman DA(1).

 

Author information:

(1)Departments of Medicine and of Microbiology and Immunology, Stanford

University, Stanford, California2Veterans Affairs Palo Alto Health Care System,

Palo Alto, California.

 

DOI: 10.1001/jama.2015.10700

PMID: 26372576  [PubMed - indexed for MEDLINE]

 

 

115. Biol Blood Marrow Transplant. 2014 May;20(5):640-5. doi:

10.1016/j.bbmt.2014.01.030. Epub 2014 Jan 31.

 

Metagenomic analysis of the stool microbiome in patients receiving allogeneic

stem cell transplantation: loss of diversity is associated with use of systemic

antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

 

Holler E(1), Butzhammer P(2), Schmid K(3), Hundsrucker C(2), Koestler J(4), Peter

K(3), Zhu W(2), Sporrer D(3), Hehlgans T(5), Kreutz M(3), Holler B(3), Wolff

D(3), Edinger M(3), Andreesen R(3), Levine JE(6), Ferrara JL(6), Gessner A(4),

Spang R(2), Oefner PJ(2).

 

Author information:

(1)Department of Hematology/Oncology, University Medical Center, Regensburg,

Germany. Electronic address: ernst.holler@ukr.de. (2)Institute of Functional

Genomics, University of Regensburg, Regensburg, Germany. (3)Department of

Hematology/Oncology, University Medical Center, Regensburg, Germany. (4)Institute

of Microbiology, University Medical Center, Regensburg, Germany. (5)Institute of

Immunology, University of Regensburg, Regensburg, Germany. (6)University of

Michigan Medical Center, Blood and Marrow Transplantation Program, Ann Arbor,

Michigan.

 

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene

isolated from serial stool specimens collected from 31 patients receiving

allogeneic stem cell transplantation (SCT) was performed to elucidate variations

in the composition of the intestinal microbiome in the course of allogeneic SCT.

Metagenomic analysis was complemented by strain-specific enterococcal PCR and

indirect assessment of bacterial load by liquid chromatography-tandem mass

spectrometry of urinary indoxyl sulfate. At the time of admission, patients

showed a predominance of commensal bacteria. After transplantation, a relative

shift toward enterococci was observed, which was more pronounced under antibiotic

prophylaxis and treatment of neutropenic infections. The shift was particularly

prominent in patients that developed subsequently or suffered from active

gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of

enterococci in post-transplant stool specimens was 21% in patients who did not

develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD

and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of

Enterococcus faecium or both E. faecium and Enterococcus faecalis in these

specimens. As a consequence of the loss of bacterial diversity, mean urinary

indoxyl sulfate levels dropped from 42.5 ± 11 μmol/L to 11.8 ± 2.8 μmol/L in all

post-transplant samples and to 3.5 ± 3 μmol/L in samples from patients with

active GVHD. Our study reveals major microbiome shifts in the course of

allogeneic SCT that occur in the period of antibiotic treatment but are more

prominent in association with GI GVHD. Our data indicate early microbiome shifts

and a loss of diversity of the intestinal microbiome that may affect intestinal

inflammation in the setting of allogeneic SCT.

 

Copyright © 2014 American Society for Blood and Marrow Transplantation. Published

by Elsevier Inc. All rights reserved.

 

DOI: 10.1016/j.bbmt.2014.01.030

PMCID: PMC4973578

PMID: 24492144  [PubMed - indexed for MEDLINE]

 

 

116. Appl Environ Microbiol. 2015 Nov;81(22):7893-904. doi: 10.1128/AEM.02294-15. Epub

2015 Sep 4.

 

Coexistence of Lactic Acid Bacteria and Potential Spoilage Microbiota in a Dairy

Processing Environment.

 

Stellato G(1), De Filippis F(1), La Storia A(1), Ercolini D(2).

 

Author information:

(1)Department of Agricultural Sciences, Division of Microbiology, University of

Naples Federico II, Portici, Italy. (2)Department of Agricultural Sciences,

Division of Microbiology, University of Naples Federico II, Portici, Italy

ercolini@unina.it.

 

Microbial contamination in food processing plants can play a fundamental role in

food quality and safety. In this study, the microbiota in a dairy plant was

studied by both 16S rRNA- and 26S rRNA-based culture-independent high-throughput

amplicon sequencing. Environmental samples from surfaces and tools were studied

along with the different types of cheese produced in the same plant. The

microbiota of environmental swabs was very complex, including more than 200

operational taxonomic units with extremely variable relative abundances (0.01 to

99%) depending on the species and sample. A core microbiota shared by 70% of the

samples indicated a coexistence of lactic acid bacteria with a remarkable level

of Streptococcus thermophilus and possible spoilage-associated bacteria,

including Pseudomonas, Acinetobacter, and Psychrobacter, with a relative

abundance above 50%. The most abundant yeasts were Kluyveromyces marxianus,

Yamadazyma triangularis, Trichosporon faecale, and Debaryomyces hansenii.

Beta-diversity analyses showed a clear separation of environmental and cheese

samples based on both yeast and bacterial community structure. In addition,

predicted metagenomes also indicated differential distribution of metabolic

pathways between the two categories of samples. Cooccurrence and coexclusion

pattern analyses indicated that the occurrence of potential spoilers was excluded

by lactic acid bacteria. In addition, their persistence in the environment can be

helpful to counter the development of potential spoilers that may contaminate the

cheeses, with possible negative effects on their microbiological quality.

 

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

 

DOI: 10.1128/AEM.02294-15

PMCID: PMC4616952

PMID: 26341209  [PubMed - indexed for MEDLINE]

 

 

 

108. BMC Microbiol. 2013 Dec 28;13:303. doi: 10.1186/1471-2180-13-303.

 

The murine lung microbiome in relation to the intestinal and vaginal bacterial

communities.

 

Barfod KK(1), Roggenbuck M, Hansen LH, Schjørring S, Larsen ST, Sørensen SJ,

Krogfelt KA.

 

Author information:

(1)Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.

kkb@nrcwe.dk.

 

BACKGROUND: This work provides the first description of the bacterial population

of the lung microbiota in mice. The aim of this study was to examine the lung

microbiome in mice, the most used animal model for inflammatory lung diseases

such as COPD, cystic fibrosis and asthma.Bacterial communities from

broncho-alveolar lavage fluids and lung tissue were compared to samples taken

from fecal matter (caecum) and vaginal lavage fluid from female BALB/cJ mice.

RESULTS: Using a customized 16S rRNA sequencing protocol amplifying the V3-V4

region our study shows that the mice have a lung microbiome that cluster

separately from mouse intestinal microbiome (caecum). The mouse lung microbiome

is dominated by Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and

Cyanobacteria overlapping the vaginal microbiome. We also show that removal of

host tissue or cells from lung fluid during the DNA extraction step has an impact

on the resulting bacterial community profile. Sample preparation needs to be

considered when choosing an extraction method and interpreting data.

CONCLUSIONS: We have consistently amplified bacterial DNA from mouse lungs that

is distinct from the intestinal microbiome in these mice. The gut microbiome has

been extensively studied for its links to development of disease. Here we suggest

that also the lung microbiome could be important in relation to inflammatory lung

diseases. Further research is needed to understand the contribution of the lung

microbiome and the gut-lung axis to the development of lung diseases such as COPD

and asthma.

 

DOI: 10.1186/1471-2180-13-303

PMCID: PMC3878784

PMID: 24373613  [PubMed - indexed for MEDLINE]

 

 

109. PLoS One. 2014 Jan 15;9(1):e84963. doi: 10.1371/journal.pone.0084963. eCollection

2014.

 

Recruiting human microbiome shotgun data to site-specific reference genomes.

 

Xie G(1), Lo CC(1), Scholz M(1), Chain PS(1).

 

Author information:

(1)Genome Science Group, Los Alamos National Laboratory, Los Alamos, New Mexico,

United States of America ; Microbial and Metagenome Program, Joint Genome

Institute, Walnut Creek, California, United States of America.

 

The human body consists of innumerable multifaceted environments that predispose

colonization by a number of distinct microbial communities, which play

fundamental roles in human health and disease. In addition to community surveys

and shotgun metagenomes that seek to explore the composition and diversity of

these microbiomes, there are significant efforts to sequence reference microbial

genomes from many body sites of healthy adults. To illustrate the utility of

reference genomes when studying more complex metagenomes, we present a

reference-based analysis of sequence reads generated from 55 shotgun metagenomes,

selected from 5 major body sites, including 16 sub-sites. Interestingly, between

13% and 92% (62.3% average) of these shotgun reads were aligned to a

then-complete list of 2780 reference genomes, including 1583 references for the

human microbiome. However, no reference genome was universally found in all body

sites. For any given metagenome, the body site-specific reference genomes,

derived from the same body site as the sample, accounted for an average of 58.8%

of the mapped reads. While different body sites did differ in abundant genera,

proximal or symmetrical body sites were found to be most similar to one another.

The extent of variation observed, both between individuals sampled within the

same microenvironment, or at the same site within the same individual over time,

calls into question comparative studies across individuals even if sampled at the

same body site. This study illustrates the high utility of reference genomes and

the need for further site-specific reference microbial genome sequencing, even

within the already well-sampled human microbiome.

 

DOI: 10.1371/journal.pone.0084963

PMCID: PMC3893169

PMID: 24454771  [PubMed - indexed for MEDLINE]

 

 

 

 

 

16s rRNA Sequencing with MR DNA

16S ribosomal  (rRNA) sequencing using next generation sequencing is a method used to identify and compare bacteria and archaea present within almost any type of sample. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study.

 

 

 

 

16s sequencing illumina or PGM low cost prices with MR DNA

MR DNA is a next generation sequencing provider with low cost 16s sequencing services.

 

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