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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.





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301. BMC Bioinformatics. 2013 Mar 5;14:79. doi: 10.1186/1471-2105-14-79.


Comparative analysis of microbiome measurement platforms using latent variable

structural equation modeling.


Wu X(1), Berkow K, Frank DN, Li E, Gulati AS, Zhu W.


Author information:

(1)Department of Applied Mathematics and Statistics, Stony Brook University,

Stony Brook, NY, USA.


BACKGROUND: Culture-independent phylogenetic analysis of 16S ribosomal RNA (rRNA)

gene sequences has emerged as an incisive method of profiling bacteria present in

a specimen. Currently, multiple techniques are available to enumerate the

abundance of bacterial taxa in specimens, including the Sanger sequencing, the

'next generation' pyrosequencing, microarrays, quantitative PCR, and the rapidly

emerging, third generation sequencing, and fourth generation sequencing methods.

An efficient statistical tool is in urgent need for the followings tasks: (1) to

compare the agreement between these measurement platforms, (2) to select the most

reliable platform(s), and (3) to combine different platforms of complementary

strengths, for a unified analysis.

RESULTS: We present the latent variable structural equation modeling (SEM) as a

novel statistical application for the comparative analysis of measurement

platforms. The latent variable SEM model treats the true (unknown) relative

frequency of a given bacterial taxon in a specimen as the latent (unobserved)

variable and estimates the reliabilities of, and similarities between, different

measurement platforms, and subsequently weighs those measurements optimally for a

unified analysis of the microbiome composition. The latent variable SEM contains

the repeated measures ANOVA (both the univariate and the multivariate models) as

special cases and, as a more general and realistic modeling approach, yields

superior goodness-of-fit and more reliable analysis results, as demonstrated by a

microbiome study of the human inflammatory bowel diseases.

CONCLUSIONS: Given the rapid evolution of modern biotechnologies, the measurement

platform comparison, selection and combination tasks are here to stay and to

grow--and the latent variable SEM method is readily applicable to any other

biological settings, aside from the microbiome study presented here.


DOI: 10.1186/1471-2105-14-79

PMCID: PMC3608994

PMID: 23497007  [PubMed - indexed for MEDLINE]



302. PLoS One. 2010 Nov 29;5(11):e15046. doi: 10.1371/journal.pone.0015046.


Resistant starches types 2 and 4 have differential effects on the composition of

the fecal microbiota in human subjects.


Martínez I(1), Kim J, Duffy PR, Schlegel VL, Walter J.


Author information:

(1)Department of Food Science and Technology, University of Nebraska, Lincoln,

Nebraska, United States of America.


BACKGROUND: To systematically develop dietary strategies based on resistant

starch (RS) that modulate the human gut microbiome, detailed in vivo studies that

evaluate the effects of different forms of RS on the community structure and

population dynamics of the gut microbiota are necessary. The aim of the present

study was to gain a community wide perspective of the effects of RS types 2 (RS2)

and 4 (RS4) on the fecal microbiota in human individuals.

METHODS AND FINDINGS: Ten human subjects consumed crackers for three weeks each

containing either RS2, RS4, or native starch in a double-blind, crossover design.

Multiplex sequencing of 16S rRNA tags revealed that both types of RS induced

several significant compositional alterations in the fecal microbial populations,

with differential effects on community structure. RS4 but not RS2 induced

phylum-level changes, significantly increasing Actinobacteria and Bacteroidetes

while decreasing Firmicutes. At the species level, the changes evoked by RS4 were

increases in Bifidobacterium adolescentis and Parabacteroides distasonis, while

RS2 significantly raised the proportions of Ruminococcus bromii and Eubacterium

rectale when compared to RS4. The population shifts caused by RS4 were

numerically substantial for several taxa, leading for example, to a ten-fold

increase in bifidobacteria in three of the subjects, enriching them to 18-30% of

the fecal microbial community. The responses to RS and their magnitudes varied

between individuals, and they were reversible and tightly associated with the

consumption of RS.

CONCLUSION: Our results demonstrate that RS2 and RS4 show functional differences

in their effect on human fecal microbiota composition, indicating that the

chemical structure of RS determines its accessibility by groups of colonic

bacteria. The findings imply that specific bacterial populations could be

selectively targeted by well designed functional carbohydrates, but the

inter-subject variations in the response to RS indicates that such strategies

might benefit from more personalized approaches.


DOI: 10.1371/journal.pone.0015046

PMCID: PMC2993935

PMID: 21151493  [PubMed - indexed for MEDLINE]



303. Gut. 2013 Jan;62(1):159-76. doi: 10.1136/gutjnl-2012-302167. Epub 2012 Jun 22.


Intestinal microbiota in functional bowel disorders: a Rome foundation report.


Simrén M(1), Barbara G, Flint HJ, Spiegel BM, Spiller RC, Vanner S, Verdu EF,

Whorwell PJ, Zoetendal EG; Rome Foundation Committee.


Author information:

(1)Department of Internal Medicine, Institute of Medicine, Sahlgrenska Academy,

University of Gothenburg, Gothenburg S-41345, Sweden.


It is increasingly perceived that gut host-microbial interactions are important

elements in the pathogenesis of functional gastrointestinal disorders (FGID). The

most convincing evidence to date is the finding that functional dyspepsia and

irritable bowel syndrome (IBS) may develop in predisposed individuals following a

bout of infectious gastroenteritis. There has been a great deal of interest in

the potential clinical and therapeutic implications of small intestinal bacterial

overgrowth in IBS. However, this theory has generated much debate because the

evidence is largely based on breath tests which have not been validated. The

introduction of culture-independent molecular techniques provides a major

advancement in our understanding of the microbial community in FGID. Results from

16S rRNA-based microbiota profiling approaches demonstrate both quantitative and

qualitative changes of mucosal and faecal gut microbiota, particularly in IBS.

Investigators are also starting to measure host-microbial interactions in IBS.

The current working hypothesis is that abnormal microbiota activate mucosal

innate immune responses which increase epithelial permeability, activate

nociceptive sensory pathways and dysregulate the enteric nervous system. While we

await important insights in this field, the microbiota is already a therapeutic

target. Existing controlled trials of dietary manipulation, prebiotics,

probiotics, synbiotics and non-absorbable antibiotics are promising, although

most are limited by suboptimal design and small sample size. In this article, the

authors provide a critical review of current hypotheses regarding the

pathogenetic involvement of microbiota in FGID and evaluate the results of

microbiota-directed interventions. The authors also provide clinical guidance on

modulation of gut microbiota in IBS.


DOI: 10.1136/gutjnl-2012-302167

PMCID: PMC3551212

PMID: 22730468  [PubMed - indexed for MEDLINE]



304. ISME J. 2012 Aug;6(8):1469-79. doi: 10.1038/ismej.2011.211. Epub 2012 Jan 26.


Architectural design influences the diversity and structure of the built

environment microbiome.


Kembel SW(1), Jones E, Kline J, Northcutt D, Stenson J, Womack AM, Bohannan BJ,

Brown GZ, Green JL.


Author information:

(1)Biology and the Built Environment Center, Institute of Ecology and Evolution,

Department of Biology, University of Oregon, Eugene, OR 97405, USA.


Buildings are complex ecosystems that house trillions of microorganisms

interacting with each other, with humans and with their environment.

Understanding the ecological and evolutionary processes that determine the

diversity and composition of the built environment microbiome--the community of

microorganisms that live indoors--is important for understanding the relationship

between building design, biodiversity and human health. In this study, we used

high-throughput sequencing of the bacterial 16S rRNA gene to quantify

relationships between building attributes and airborne bacterial communities at a

health-care facility. We quantified airborne bacterial community structure and

environmental conditions in patient rooms exposed to mechanical or window

ventilation and in outdoor air. The phylogenetic diversity of airborne bacterial

communities was lower indoors than outdoors, and mechanically ventilated rooms

contained less diverse microbial communities than did window-ventilated rooms.

Bacterial communities in indoor environments contained many taxa that are absent

or rare outdoors, including taxa closely related to potential human pathogens.

Building attributes, specifically the source of ventilation air, airflow rates,

relative humidity and temperature, were correlated with the diversity and

composition of indoor bacterial communities. The relative abundance of bacteria

closely related to human pathogens was higher indoors than outdoors, and higher

in rooms with lower airflow rates and lower relative humidity. The observed

relationship between building design and airborne bacterial diversity suggests

that we can manage indoor environments, altering through building design and

operation the community of microbial species that potentially colonize the human

microbiome during our time indoors.


DOI: 10.1038/ismej.2011.211

PMCID: PMC3400407

PMID: 22278670  [PubMed - indexed for MEDLINE]



305. PLoS One. 2011;6(5):e19709. doi: 10.1371/journal.pone.0019709. Epub 2011 May 13.


The microbial communities in male first catch urine are highly similar to those

in paired urethral swab specimens.


Dong Q(1), Nelson DE, Toh E, Diao L, Gao X, Fortenberry JD, Van der Pol B.


Author information:

(1)Department of Biology, University of North Texas, Denton, Texas, United States

of America.


Urine is the CDC-recommended specimen for STI testing. It was unknown if the

bacterial communities (microbiomes) in urine reflected those in the distal male

urethra. We compared microbiomes of 32 paired urine and urethral swab specimens

obtained from adult men attending an STD clinic, by 16S rRNA PCR and deep

pyrosequencing. Microbiomes of urine and swabs were remarkably similar,

regardless of STI status of the subjects. Thus, urine can be used to characterize

urethral microbiomes when swabs are undesirable, such as in population-based

studies of the urethral microbiome or where multiple sampling of participants is



DOI: 10.1371/journal.pone.0019709

PMCID: PMC3094389

PMID: 21603636  [PubMed - indexed for MEDLINE]



306. Gut Microbes. 2013 May-Jun;4(3):193-200. doi: 10.4161/gmic.23867. Epub 2013 Apr



A pig model of the human gastrointestinal tract.


Zhang Q(1), Widmer G, Tzipori S.


Author information:

(1)Tufts Cummings School of Veterinary Medicine, Division of Infectious Diseases,

North Grafton, MA, USA.


Easy access to next generation sequencing has enabled the rapid analysis of

complex microbial populations. To take full advantage of these technologies,

animal models enabling the manipulation of human microbiomes and the study of the

impact of such perturbations on the host are needed. To this aim we are

developing experimentally tractable and clinically relevant pig models of the

human adult and infant gastro-intestinal tract. The intestine of germ-free

piglets was populated with human adult or infant fecal microbial populations, and

the piglets were maintained on solid or milk diet, respectively. Amplicons of 16S

rRNA V6 region were deep-sequenced to monitor to what extent the transplanted

human microbiomes changed in the pig. Within 24 h of transfer of human fecal

microbiome to pigs, bacterial microbiomes rich in Proteobacteria emerged. These

populations evolved toward a more diverse composition rich in Bacteroidetes and

Firmicutes. In the experiment where infant microbiome was used, the phylogenetic

composition of the transplanted bacterial population converged toward that of the

human inoculum. A majority of sequences belonged to a relatively small number of

operational taxonomic units, whereas at the other end of the abundance spectrum,

a large number of rare and transient OTUs were detected. Analysis of fecal and

colonic microbiomes originating from the same animal indicate that feces closely

replicate the colonic microbiome. We conclude that the pig intestine can be

colonized with human fecal microbiomes to generate a realistic model of the human

GI tract.


DOI: 10.4161/gmic.23867

PMCID: PMC3669164

PMID: 23549377  [PubMed - indexed for MEDLINE]



307. Nat Rev Microbiol. 2008 Oct;6(10):776-88. doi: 10.1038/nrmicro1978.


Worlds within worlds: evolution of the vertebrate gut microbiota.


Ley RE(1), Lozupone CA, Hamady M, Knight R, Gordon JI.


Author information:

(1)Center for Genome Sciences, Washington University School of Medicine, St

Louis, Missouri 63108, USA.


In this Analysis we use published 16S ribosomal RNA gene sequences to compare the

bacterial assemblages that are associated with humans and other mammals, metazoa

and free-living microbial communities that span a range of environments. The

composition of the vertebrate gut microbiota is influenced by diet, host

morphology and phylogeny, and in this respect the human gut bacterial community

is typical of an omnivorous primate. However, the vertebrate gut microbiota is

different from free-living communities that are not associated with animal body

habitats. We propose that the recently initiated international Human Microbiome

Project should strive to include a broad representation of humans, as well as

other mammalian and environmental samples, as comparative analyses of microbiotas

and their microbiomes are a powerful way to explore the evolutionary history of

the biosphere.


DOI: 10.1038/nrmicro1978

PMCID: PMC2664199

PMID: 18794915  [PubMed - indexed for MEDLINE]



308. PLoS Comput Biol. 2016 Jan 28;12(1):e1004658. doi: 10.1371/journal.pcbi.1004658.

eCollection 2016.


NINJA-OPS: Fast Accurate Marker Gene Alignment Using Concatenated Ribosomes.


Al-Ghalith GA(1), Montassier E(2,)(3), Ward HN(4), Knights D(1,)(3).


Author information:

(1)Biomedical Informatics and Computational Biology, University of Minnesota,

Minneapolis, Minnesota, United States of America. (2)University of Nantes,

Nantes, France. (3)Department of Computer Science and Engineering, University of

Minnesota, Minneapolis, Minnesota, United States of America. (4)Lawrence

University, Appleton, Wisconsin, United States of America.


The explosion of bioinformatics technologies in the form of next generation

sequencing (NGS) has facilitated a massive influx of genomics data in the form of

short reads. Short read mapping is therefore a fundamental component of next

generation sequencing pipelines which routinely match these short reads against

reference genomes for contig assembly. However, such techniques have seldom been

applied to microbial marker gene sequencing studies, which have mostly relied on

novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking

Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method

enabling reference-based marker gene matching (picking Operational Taxonomic

Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment

using an artificial reference chromosome composed of concatenated reference

sequences, the "concatesome," as the BW input. Other features include automatic

support for paired-end reads with arbitrary insert sizes. NINJA is also free and

open source and implements several pre-filtering methods that elicit substantial

speedup when coupled with existing tools. We applied NINJA to several published

microbiome studies, obtaining accuracy similar to or better than previous

reference-based OTU-picking methods while achieving an order of magnitude or more

speedup and using a fraction of the memory footprint. NINJA is a complete

pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted

taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S

genes in under 10 minutes on a dual-core laptop.


DOI: 10.1371/journal.pcbi.1004658

PMCID: PMC4731464

PMID: 26820746  [PubMed - indexed for MEDLINE]



309. Int J Food Microbiol. 2013 May 15;163(2-3):171-9. doi:

10.1016/j.ijfoodmicro.2013.02.022. Epub 2013 Mar 6.


Metatranscriptomic analysis of lactic acid bacterial gene expression during

kimchi fermentation.


Jung JY(1), Lee SH, Jin HM, Hahn Y, Madsen EL, Jeon CO.


Author information:

(1)Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of



Barcode-based 16S rRNA gene pyrosequencing showed that the kimchi microbiome was

dominated by six lactic acid bacteria (LAB), Leuconostoc (Lc.) mesenteroides,

Lactobacillus (Lb.) sakei, Weissella (W.) koreensis, Lc. gelidum, Lc. carnosum,

and Lc. gasicomitatum. Therefore, we used completed genome sequences of

representatives of these bacteria to investigate metatranscriptomic

gene-expression profiles during kimchi fermentation. Total mRNA was extracted

from kimchi samples taken at five time points during a 29 day-fermentation.

Nearly all (97.7%) of the metagenome sequences that were recruited on all LAB

genomes of GenBank mapped onto the six LAB strains; this high coverage rate

indicated that this approach for assessing processes carried out by the kimchi

microbiome was valid. Expressed mRNA sequences (as cDNA) were determined using

Illumina GA IIx. Assignment of mRNA sequences to metabolic genes using MG-RAST

revealed the prevalence of carbohydrate metabolism and lactic acid fermentation.

The mRNA sequencing reads were mapped onto genomes of the six LAB strains, which

showed that Lc. mesenteroides was most active during the early-stage

fermentation, whereas gene expression by Lb. sakei and W. koreensis was high

during later stages. However, gene expression by Lb. sakei decreased rapidly at

25 days of fermentation, which was possibly caused by bacteriophage infection of

the Lactobacillus species. Many genes related to carbohydrate transport and

hydrolysis and lactate fermentation were actively expressed, which indicated

typical heterolactic acid fermentation. Mannitol dehydrogenase-encoding genes

(mdh) were identified from all Leuconostoc species and especially Lc.

mesenteroides, which harbored three copies (two copies on chromosome and one copy

on plasmid) of mdh with different expression patterns. These results contribute

to knowledge of the active populations and gene expression in the LAB community

responsible for an important fermentation process.


Copyright © 2013 Elsevier B.V. All rights reserved.


DOI: 10.1016/j.ijfoodmicro.2013.02.022

PMID: 23558201  [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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