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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.

 

 

 

 

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

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J Appl Genet. 2015 Aug;56(3):393-401. doi: 10.1007/s13353-014-0258-1. Epub 2015 Jan 21.

First insights into the microbial diversity in the omasum and reticulum of bovine using Illumina sequencing.

Peng S1, Yin J, Liu X, Jia B, Chang Z, Lu H, Jiang N, Chen Q.

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Abstract

The digestive systems of mammals harbor a complex gut microbiome, comprising bacteria and other microorganisms that confer metabolic and immunological benefits to the host. Ruminants that digest plant-based foods have a four-compartment stomach consisting of the rumen, reticulum, omasum, and abomasum. The microorganisms in the stomach are essential for providing the host with critical nutrients. However, the majority of these microorganisms are unknown species. The microbiome of the stomach is diverse, and the majority of these organisms cannot be cultured. Next-generation sequencing (NGS) combined with bioinformatic analysis tools have allowed the dissection of the composition of the microbiome in samples collected from a specific environment. In this study, for the first time, the bacterial composition in two compartments, the reticulum and the omasum, of bovine were analyzed using a metagenomic approach and compared to the bacterial composition of the rumen. These data will assist in understanding the biology of ruminants and benefit the agricultural industry. The diversity and composition of the bacterial community in samples collected from the rumen, reticulum, and omasum of bovines in the Changchun Region of Northeast China were analyzed by sequencing the V3 region of the 16S rRNA gene using a barcoded Illumina paired-end sequencing technique, and the primary composition of the microbiome in the rumen, reticulum, and omasum of the bovines was determined. These microbiomes contained 17 phyla and 107 genera in all three samples. Five phyla, Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes, and Lentisphaerae, were the most abundant taxonomic groups. Additionally, the different stomach compartments harbored different compositions of the microorganisms.

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PMID: 25604266 PMCID: PMC4543427 DOI: 10.1007/s13353-014-0258-1

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Select item 25599982

33.

Clin Exp Allergy. 2015 Mar;45(3):632-43. doi: 10.1111/cea.12487.

Infant gut microbiota and food sensitization: associations in the first year of life.

Azad MB1, Konya T, Guttman DS, Field CJ, Sears MR, HayGlass KT, Mandhane PJ, Turvey SE, Subbarao P, Becker AB, Scott JA, Kozyrskyj AL; CHILD Study Investigators.

Collaborators (33)

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Abstract

BACKGROUND:

The gut microbiota is established during infancy and plays a fundamental role in shaping host immunity. Colonization patterns may influence the development of atopic disease, but existing evidence is limited and conflicting.

OBJECTIVE:

To explore associations of infant gut microbiota and food sensitization.

METHODS:

Food sensitization at 1 year was determined by skin prick testing in 166 infants from the population-based Canadian Healthy Infant Longitudinal Development (CHILD) study. Faecal samples were collected at 3 and 12 months, and microbiota was characterized by Illumina 16S rRNA sequencing.

RESULTS:

Twelve infants (7.2%) were sensitized to ≥ 1 common food allergen at 1 year. Enterobacteriaceae were overrepresented and Bacteroidaceae were underrepresented in the gut microbiota of food-sensitized infants at 3 months and 1 year, whereas lower microbiota richness was evident only at 3 months. Each quartile increase in richness at 3 months was associated with a 55% reduction in risk for food sensitization by 1 year (adjusted odds ratio 0.45, 95% confidence interval 0.23-0.87). Independently, each quartile increase in Enterobacteriaceae/Bacteroidaceae ratio was associated with a twofold increase in risk (2.02, 1.07-3.80). These associations were upheld in a sensitivity analysis among infants who were vaginally delivered, exclusively breastfed and unexposed to antibiotics. At 1 year, the Enterobacteriaceae/Bacteroidaceae ratio remained elevated among sensitized infants, who also tended to have decreased abundance of Ruminococcaceae.

CONCLUSIONS AND CLINICAL RELEVANCE:

Low gut microbiota richness and an elevated Enterobacteriaceae/Bacteroidaceae ratio in early infancy are associated with subsequent food sensitization, suggesting that early gut colonization may contribute to the development of atopic disease, including food allergy.

TRIAL REGISTRATION:

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© 2015 John Wiley & Sons Ltd.

PMID: 25599982 DOI: 10.1111/cea.12487

[PubMed - indexed for MEDLINE]

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Select item 25557481

34.

J Microbiol. 2015 Jan;53(1):60-9. doi: 10.1007/s12275-015-4601-y. Epub 2015 Jan 4.

Improved pipeline for reducing erroneous identification by 16S rRNA sequences using the Illumina MiSeq platform.

Jeon YS1, Park SC, Lim J, Chun J, Kim BS.

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Abstract

The cost of DNA sequencing has decreased due to advancements in Next Generation Sequencing. The number of sequences obtained from the Illumina platform is large, use of this platform can reduce costs more than the 454 pyrosequencer. However, the Illumina platform has other challenges, including bioinformatics analysis of large numbers of sequences and the need to reduce erroneous nucleotides generated at the 3'-ends of the sequences. These erroneous sequences can lead to errors in analysis of microbial communities. Therefore, correction of these erroneous sequences is necessary for accurate taxonomic identification. Several studies that have used the Illumina platform to perform metagenomic analyses proposed curating pipelines to increase accuracy. In this study, we evaluated the likelihood of obtaining an erroneous microbial composition using the MiSeq 250 bp paired sequence platform and improved the pipeline to reduce erroneous identifications. We compared different sequencing conditions by varying the percentage of control phiX added, the concentration of the sequencing library, and the 16S rRNA gene target region using a mock community sample composed of known sequences. Our recommended method corrected erroneous nucleotides and improved identification accuracy. Overall, 99.5% of the total reads shared 95% similarity with the corresponding template sequences and 93.6% of the total reads shared over 97% similarity. This indicated that the MiSeq platform can be used to analyze microbial communities at the genus level with high accuracy. The improved analysis method recommended in this study can be applied to amplicon studies in various environments using high-throughput reads generated on the MiSeq platform.

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PMID: 25557481 DOI: 10.1007/s12275-015-4601-y

[PubMed - indexed for MEDLINE]

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Select item 25538865

35.

PeerJ. 2014 Dec 9;2:e659. doi: 10.7717/peerj.659. eCollection 2014.

The microbes we eat: abundance and taxonomy of microbes consumed in a day's worth of meals for three diet types.

Lang JM1, Eisen JA2, Zivkovic AM3.

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Abstract

Far more attention has been paid to the microbes in our feces than the microbes in our food. Research efforts dedicated to the microbes that we eat have historically been focused on a fairly narrow range of species, namely those which cause disease and those which are thought to confer some "probiotic" health benefit. Little is known about the effects of ingested microbial communities that are present in typical American diets, and even the basic questions of which microbes, how many of them, and how much they vary from diet to diet and meal to meal, have not been answered. We characterized the microbiota of three different dietary patterns in order to estimate: the average total amount of daily microbes ingested via food and beverages, and their composition in three daily meal plans representing three different dietary patterns. The three dietary patterns analyzed were: (1) the Average American (AMERICAN): focused on convenience foods, (2) USDA recommended (USDA): emphasizing fruits and vegetables, lean meat, dairy, and whole grains, and (3) Vegan (VEGAN): excluding all animal products. Meals were prepared in a home kitchen or purchased at restaurants and blended, followed by microbial analysis including aerobic, anaerobic, yeast and mold plate counts as well as 16S rRNA PCR survey analysis. Based on plate counts, the USDA meal plan had the highest total amount of microbes at 1.3 × 10(9) CFU per day, followed by the VEGAN meal plan and the AMERICAN meal plan at 6 × 10(6) and 1.4 × 10(6) CFU per day respectively. There was no significant difference in diversity among the three dietary patterns. Individual meals clustered based on taxonomic composition independent of dietary pattern. For example, meals that were abundant in Lactic Acid Bacteria were from all three dietary patterns. Some taxonomic groups were correlated with the nutritional content of the meals. Predictive metagenome analysis using PICRUSt indicated differences in some functional KEGG categories across the three dietary patterns and for meals clustered based on whether they were raw or cooked. Further studies are needed to determine the impact of ingested microbes on the intestinal microbiota, the extent of variation across foods, meals and diets, and the extent to which dietary microbes may impact human health. The answers to these questions will reveal whether dietary microbes, beyond probiotics taken as supplements-i.e., ingested with food-are important contributors to the composition, inter-individual variation, and function of our gut microbiota.

TRIAL REGISTRATION:

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KEYWORDS:

16S; Bioinformatics; Food microbiology; Illumina amplicon sequencing; Microbial communities; Microbial ecology; Microbiome; Microbiota; PICRUSt; QIIME

PMID: 25538865 PMCID: PMC4266855 DOI: 10.7717/peerj.659

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Select item 25392361

36.

J Clin Microbiol. 2015 Jan;53(1):237-47. doi: 10.1128/JCM.02555-14. Epub 2014 Nov 12.

Analysis of changes in diversity and abundance of the microbial community in a cystic fibrosis patient over a multiyear period.

Stokell JR1, Gharaibeh RZ2, Hamp TJ3, Zapata MJ3, Fodor AA3, Steck TR4.

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Abstract

The evolution of pulmonary disease in cystic fibrosis (CF) usually begins when bacteria get trapped in mucus in the lungs and become established as a chronic infection. While most CF patients experience periods of stability, pulmonary exacerbations (PEs) can occur multiple times per year and result in permanent damage to the lungs. Little is known of the shift from a period of stability to a PE, but this shift is likely to be attributed to changes in the bacterial community. Here, we identified changes in the lung microbiota to determine if they reflect patient health, indicate the onset of exacerbations, or are related to antibiotic treatment. In contrast to most bacterial studies on CF, we collected weekly samples from an adult CF patient over a period of 3 years and performed quantitative PCR (qPCR) and Illumina sequencing on those samples. While many DNA-based studies have shown the CF microbiota to be relatively stable, we observed an increase in the total bacterial abundance over time (P < 0.001), while the number of different taxa (bacterial richness) and the number of different taxa and their abundances (diversity) significantly decreased over time (P < 0.03), which was likely due to repeated antibiotic exposure. Using genus-specific primers with qPCR, we observed an increase in the abundance of Burkholderia multivorans, a CF-associated pathogen, prior to the occurrence of a PE (P = 0.006). Combining these DNA-based techniques with frequent sampling identified a potential initiator for exacerbations and described a response of the CF microbiota to time and antibiotic treatment not observed in previous CF microbiota studies.

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

PMID: 25392361 PMCID: PMC4290929 DOI: 10.1128/JCM.02555-14

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37.

PLoS One. 2014 Sep 24;9(9):e106491. doi: 10.1371/journal.pone.0106491. eCollection 2014.

Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods.

Snelling TJ1, Genç B2, McKain N1, Watson M3, Waters SM4, Creevey CJ5, Wallace RJ1.

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Abstract

Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥ 91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6-V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to 16S rRNA gene references produced taxonomic identification to Order level including 2-3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19-35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10-18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6-V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.

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PMID: 25250654 PMCID: PMC4175461 DOI: 10.1371/journal.pone.0106491

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Select item 25208077

38.

PLoS One. 2014 Sep 10;9(9):e106707. doi: 10.1371/journal.pone.0106707. eCollection 2014.

A comparative metagenome survey of the fecal microbiota of a breast- and a plant-fed Asian elephant reveals an unexpectedly high diversity of glycoside hydrolase family enzymes.

Ilmberger N1, Güllert S1, Dannenberg J1, Rabausch U1, Torres J1, Wemheuer B2, Alawi M3, Poehlein A2, Chow J1, Turaev D4, Rattei T4, Schmeisser C1, Salomon J5, Olsen PB5, Daniel R2, Grundhoff A6, Borchert MS5, Streit WR1.

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Abstract

A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant) was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs) were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH) genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs), which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.

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PMID: 25208077 PMCID: PMC4160196 DOI: 10.1371/journal.pone.0106707

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39.

BMC Microbiol. 2014 Sep 7;14:225. doi: 10.1186/s12866-014-0225-3.

The presence of nitrate dramatically changed the predominant microbial community in perchlorate degrading cultures under saline conditions.

Stepanov VG, Xiao Y, Tran Q, Rojas M, Willson RC, Fofanov Y, Fox GE, Roberts DJ.

Abstract

BACKGROUND:

Perchlorate contamination has been detected in both ground water and drinking water. An attractive treatment option is the use of ion-exchange to remove and concentrate perchlorate in brine. Biological treatment can subsequently remove the perchlorate from the brine. When nitrate is present, it will also be concentrated in the brine and must also be removed by biological treatment. The primary objective was to obtain an in-depth characterization of the microbial populations of two salt-tolerant cultures each of which is capable of metabolizing perchlorate. The cultures were derived from a single ancestral culture and have been maintained in the laboratory for more than 10 years. One culture was fed perchlorate only, while the other was fed both perchlorate and nitrate.

RESULTS:

A metagenomic characterization was performed using Illumina DNA sequencing technology, and the 16S rDNA of several pure strains isolated from the mixed cultures were sequenced. In the absence of nitrate, members of the Rhodobacteraceae constituted the prevailing taxonomic group. Second in abundance were the Rhodocyclaceae. In the nitrate fed culture, the Rhodobacteraceae are essentially absent. They are replaced by a major expansion of the Rhodocyclaceae and the emergence of the Alteromonadaceae as a significant community member. Gene sequences exhibiting significant homology to known perchlorate and nitrate reduction enzymes were found in both cultures.

CONCLUSIONS:

The structure of the two microbial ecosystems of interest has been established and some representative strains obtained in pure culture. The results illustrate that under favorable conditions a group of organisms can readily dominate an ecosystem and yet be effectively eliminated when their advantage is lost. Almost all known perchlorate-reducing organisms can also effectively reduce nitrate. This is certainly not the case for the Rhodobacteraceae that were found to dominate in the absence of nitrate, but effectively disappeared in its presence. This study is significant in that it reveals the existence of a novel group of organisms that play a role in the reduction of perchlorate under saline conditions. These Rhodobacteraceae especially, as well as other organisms present in these communities may be a promising source of unique salt-tolerant enzymes for perchlorate reduction.

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PMID: 25194715 PMCID: PMC4174249 DOI: 10.1186/s12866-014-0225-3

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40.

Mol Ecol. 2014 Sep;23(18):4498-510. doi: 10.1111/mec.12885. Epub 2014 Sep 8.

The Sphagnum microbiome supports bog ecosystem functioning under extreme conditions.

Bragina A1, Oberauner-Wappis L, Zachow C, Halwachs B, Thallinger GG, Müller H, Berg G.

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Abstract

Sphagnum-dominated bogs represent a unique yet widely distributed type of terrestrial ecosystem and strongly contribute to global biosphere functioning. Sphagnum is colonized by highly diverse microbial communities, but less is known about their function. We identified a high functional diversity within the Sphagnum microbiome applying an Illumina-based metagenomic approach followed by de novo assembly and MG-RAST annotation. An interenvironmental comparison revealed that the Sphagnum microbiome harbours specific genetic features that distinguish it significantly from microbiomes of higher plants and peat soils. The differential traits especially support ecosystem functioning by a symbiotic lifestyle under poikilohydric and ombrotrophic conditions. To realise a plasticity-stability balance, we found abundant subsystems responsible to cope with oxidative and drought stresses, to exchange (mobile) genetic elements, and genes that encode for resistance to detrimental environmental factors, repair and self-controlling mechanisms. Multiple microbe-microbe and plant-microbe interactions were also found to play a crucial role as indicated by diverse genes necessary for biofilm formation, interaction via quorum sensing and nutrient exchange. A high proportion of genes involved in nitrogen cycle and recycling of organic material supported the role of bacteria for nutrient supply. 16S rDNA analysis indicated a higher structural diversity than that which had been previously detected using PCR-dependent techniques. Altogether, the diverse Sphagnum microbiome has the ability to support the life of the host plant and the entire ecosystem under changing environmental conditions. Beyond this, the moss microbiome presents a promising bio-resource for environmental biotechnology - with respect to novel enzymes or stress-protecting bacteria.

TRIAL REGISTRATION:

ClinicalTrials.gov .

© 2014 John Wiley & Sons Ltd.

KEYWORDS:

FISH-CLSM; Sphagnum moss; bog ecosystem; illumina-based metagenomics; plant microbiome

PMID: 25113243 DOI: 10.1111/mec.12885

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Microbiome. 2015 Oct 27;3:50. doi: 10.1186/s40168-015-0116-3.

Microbiomes of the dust particles collected from the International Space Station and Spacecraft Assembly Facilities.

Checinska A1, Probst AJ2, Vaishampayan P1, White JR3, Kumar D4, Stepanov VG4, Fox GE4, Nilsson HR5, Pierson DL6, Perry J7, Venkateswaran K8.

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Abstract

BACKGROUND:

The International Space Station (ISS) is a unique built environment due to the effects of microgravity, space radiation, elevated carbon dioxide levels, and especially continuous human habitation. Understanding the composition of the ISS microbial community will facilitate further development of safety and maintenance practices. The primary goal of this study was to characterize the viable microbiome of the ISS-built environment. A second objective was to determine if the built environments of Earth-based cleanrooms associated with space exploration are an appropriate model of the ISS environment.

RESULTS:

Samples collected from the ISS and two cleanrooms at the Jet Propulsion Laboratory (JPL, Pasadena, CA) were analyzed by traditional cultivation, adenosine triphosphate (ATP), and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assays to estimate viable microbial populations. The 16S rRNA gene Illumina iTag sequencing was used to elucidate microbial diversity and explore differences between ISS and cleanroom microbiomes. Statistical analyses showed that members of the phyla Actinobacteria, Firmicutes, and Proteobacteria were dominant in the samples examined but varied in abundance. Actinobacteria were predominant in the ISS samples whereas Proteobacteria, least abundant in the ISS, dominated in the cleanroom samples. The viable bacterial populations seen by PMA treatment were greatly decreased. However, the treatment did not appear to have an effect on the bacterial composition (diversity) associated with each sampling site.

CONCLUSIONS:

The results of this study provide strong evidence that specific human skin-associated microorganisms make a substantial contribution to the ISS microbiome, which is not the case in Earth-based cleanrooms. For example, Corynebacterium and Propionibacterium (Actinobacteria) but not Staphylococcus (Firmicutes) species are dominant on the ISS in terms of viable and total bacterial community composition. The results obtained will facilitate future studies to determine how stable the ISS environment is over time. The present results also demonstrate the value of measuring viable cell diversity and population size at any sampling site. This information can be used to identify sites that can be targeted for more stringent cleaning. Finally, the results will allow comparisons with other built sites and facilitate future improvements on the ISS that will ensure astronaut health.

PMID: 26502721 PMCID: PMC4624184 DOI: 10.1186/s40168-015-0116-3

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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.

 

 

 

 

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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