Vance McCracken added an answer:Do you know if PowerLyzer PowerSoil DNA isolation Kit has been used for bacterial DNA extraction from colonic mucosa in the HumanMicrobiomeProject?
bacterial DNA extracted will be used for metagenomic analysis
As Muther Mansoor pointed out above, the Human Genome Project uses the PowerSoil kits (or a modification of them) as the primary method for DNA isolation not just from feces, but from many parts of the body. Although there are pros and cons to every method and/or kit (also pointed out in Muther's article), and tons of others, MoBio was on the scene fairly early and seems to perform consistently well as far as kits go.
Though often used for fecal isolation, these kits are often used for mucosal swabs, scrapings, or luminal contents also. The HMP protocol can be found here: http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/GetPdf.cgi?id=phd003190.2Following
Muhammad Farooq added an answer:How to isolate and cultivate bacteria from body fluid?
how to isolate and cultivate bacteria from body fluid.
you can isolate bacteria by making the dilutions of fluids and then streak them on solid media ( mostly nutrient agar)Following
Ibrahim Shnawa added an answer:Why is our normal flora so important from a immunological point of view?
Normal flora is so hard first defense line against attacking microbes and this is due to different mechanisms including nitches.
Microbiome is a biological barrier of natural immunity oragainst host pathogenic invaders .Actually it reduces the their population sizes through secretion of bacteriocins or through other probiotic secretion.Recently ,microbiome gains intense investigations its immunologic roles.Following
Vancheswaran Gopalakrishnan added an answer:Does the buccal mucosa sufficiently capture the oral microbiome?
Will a buccal swab be sufficiently representative of the oral microbiome, or do I need to take swabs from other regions of the oral cavity as well ?
Thank you for replying. Did you use the Catch-All swab or did you use a separate swab for each of the regions that you mentioned?
Meriem Fassatoui added an answer:Has anyone tried this kit innuPREP Stool DNA Kit Analyticjena ?
I plan to use this kit innuPREP Stool DNA Kit to extract microbial DNA from human feces, however it has not been quoted in articles or publications, the most cited kit to perform this task is Qiamp DNA STOOL MINI KIT (Qiagen). Who can tell me the name of an item at which was cited including information on its efficiency?
thank you for your helpFollowing
Diana H Taft added an answer:Has anyone used the QIAamp DNA microbiome kit?
Has anyone used the QIAamp DNA microbiome kit for human microbiome study? Is it good? Would it be challenging if the amount of starting material is low? Would it unspecifically remove bacterial DNA? Thanks.
Hi Yang, I'd worry less about removing bacteria and more about adding it. DNA extraction protocol tends to add contaminant DNA (see this article: http://www.biomedcentral.com/1741-7007/12/87#sec5) Since you expect a low amount of DNA from your samples, this could be a major problem. I would make sure you extract a blank sample to control for contamination.Following
Jawwad Ahmad added an answer:Does anyone have suggestions for optimizing a PCR using DMSO and Betaine for a metagenomic study?
Hi everyone....I am doing targeted sequencing of the 16S rRNA bacterial gene. I have been struggling to optimize my PCR with broad range 16S primers of the v1-v3 region (I have used the ones from the human microbiome study). I have a few non-specific bands that show up on the gel. I have been using PCR additives ( DMSO and Betaine) combination but as the non-specifics decrease my primer dimers increase in yield. I have decreased the primer concentration and it has helped a bit but not enough. Ive tried all sorts of concentrations for both additives and Im having a hard time finding a balance. Anyone have an idea how to help? Also is the use of these additives ok in a metagenomic study. I am targeting an amplicon size of ~550 bp and since I do not want to extract the band from the gel and would rather just do a post-pcr cleanup I would like to remove as much non-specific bands as possible. Any help?
David Robert Clark added an answer:Which microbial ecology studies have both 16s amplicons and WGS metagenomes of same samples?
I am conducting some bioinformatics experiments which require comparisons between 16s amplicons and whole genome shotguns. I therefore need sequence data from samples that have been sequenced using both strategies. I am aware of the PAMPA datasets, as well as the Human microbiome project and the studies cited in the PiCRUST paper, but any other suggestions would be welcomed. Thank you in advance.
Thanks Thomas, I'll add that to my list!
Jane Ludvigsen added an answer:How do I separate a mixed sample of Human and microbial DNA in a Microbiome study ?
Are there anyway to increase the microbial DNA concentration in a Mixed sample of Human and microbial DNA ?
Knowing that acquiring the samples was very difficult because of strict inclusion and exclusion criteria, so I can not re-sample !!!!:)
You can try the NEBNext® Microbiome DNA Enrichment Kit from New England Biolabs. This kit enhances microbial DNA in a mixed sample- a bit expensive!Following
Kathleen Bailey added an answer:Are there any food scientists/microbiologists willing to test kombucha and kefir SCOBYs for pathology/probiota and reactions with resident microbiota?
Or has anyone DONE studies already and can point me toward them? I am not a scientist (Ha! I hate that they used this phrase to make ME look stupid now), but I am a researcher and health advocate, nurse, future Dr. of something and really, really want to get in the weeds on how fermented foods/lactobacillus interact with the human microbiome to improve or not improve health.
I am just developing the vocabulary to talk around these issues, so bear with me...
Thank you so much! I am having fun going down the rabbit hole of the bibliographies on all the reviews and studies I have found thus far and you have helped me fill in some more pieces!
Thank you for you work and your help!!!Following
Ami Rameshbhai Patel added an answer:What is the ratio bacteria/yeasts in the human microbiome?
We hear a lot about bacteria from the human microbiome. But what about the fungi? How many, and who are part of our microbiome?
Thank you for sharing new information!Following
M. Atif Khan added an answer:What is the cost of sequencing 16s rDNA amplicon which is taken from a stool sample?I need to know the types and diversity of the bacterial community in stool samples using next generation sequencing but i don't know the cost per sample.
I am from Science Exchange (a marketplace for scientific experiments). Since you've already got your answers, I would just like you to check this platform, through which you can access several labs and core facilities who can render the sequencing services for you. Please check using the following link.
Suheir Nassar added an answer:Which kits are best to use for isolating microbial DNA from stool samples?
We are planning on doing microbiome analyses with next generation sequencing and would like to know what kits we should use for collection and extraction of microbial DNA?
concerning washing the stool sample with PBS , would this work with frozen stool samples as well? Knowing that the QIAamp kit notes that stool samples should not thaw before adding the ASL buffer , which i found practically very difficult to do.what do you think according to your experience?Following
Jay Siddharth added an answer:Where can I find the barcodes of study SRP000913?
I am trying to help a friend to compare his 454 datasets with published ones.
I looked into this record but didn't find any barcodes. As I recall, the barcodes are usually in the design description, yet there is no barcode sequence under this record. The external link pointed to VAMPS. So the author wanted us to use VAMPS to compare the datasets from different researches?
Thanks in advance.
The samples are usually submitted post demultiplexing.
So if you download each sample set, you should be able to have all sample sequences even though there are no barcodes.
Or you can go here and put the metadata together for yourself and proceed
derived from here
Abdelrahman Mahmoud added an answer:How many clones need to be sequenced for 16s rRNA genes from human stool sample?
I will work on 40 human stool samples to identify the bacterial community of each sample by creating cloning library and sequencing their 16s rRNA genes. My question is what is the number of clones needed to be sequenced for each sample and from the point of view of the cost, what will be the lower cost (cloning library or Next generation sequencing)?
Thanks Fang :)Following
Khaled Saad added an answer:How far has recent research come in the use of probiotica as prophylactic treatment to NEC (necrotising enterocolitis)?I have not been able to find newer publications on whether studies of the use of probiotica as prophylactic treatment to NEC have been concluded, terminated or if they are still ongoing.Since the preterm gut demonstrates delayed commensal colonization and low bacterial diversity, it may be particularly amenable to therapeutic manipulation by probiotic administration. In keeping with this idea, a number of clinical studies have demonstrated the benefit of probiotic administration in reducing the incidence and severity of NEC. Most of these trials have used strains of probiotics from the Genus Lactobacillus or Bifidobacteria, although the treatment regimen, including dose and duration of therapy vary widely . In addition, concerns regarding the risks of therapy, optimal species or combination of species, duration of therapy and dosing remain unanswered.
(Therapeutic Use of Prebiotics, Probiotics, and Postbiotics to Prevent Necrotizing Enterocolitis: What is the Current Evidence? Ravi Mangal Patel et al; Clin Perinatol. Mar 2013; 40(1): 11–25.)Following
Imran Khan added an answer:Why are only some bacteria able to colonize our gut? Any molecular mechanisms?Human guts are inhabited by trillions of bacteria. However this is not a random selection. Why is it that only some bacteria are able to stably colonize our gut while others cannot? What is the molecular mechanism for this selection?Few paragraphs from my paper that is accepted, I have attached a figure that will help to understand the mechanism.
GM is proven to induce repair of damaged intestinal epithelium , and to use this barrier to interact with the immune system . GM follows several pathways in its interaction with the immune system; some bacterial species penetrate directly into the mucus and contact the epithelial cell layer  via germline encoded pattern recognition receptors, such as the toll-like receptors, cytosolic RIG-I-like receptors, Nodlike receptors and HIN-200 family members . However, other bacterial species are presented to the immune system through dendritic cells (DC), which phagocytize antigens from the intestinal lumen, and by M cells overlaying the Peyers Patches (PP), forming a dome like structure below the epithelium (Fig. 1) .
The GM-immune system interaction is of a mutual understanding nature. GM sensitizes the immune system by presenting bacterial components such as lipopolysaccharide, flagellin and muramyl dipeptide, inducing acquire local and systemic responses ; in reward, GM gets shelter and nutrition by the Gastrointestinal tract. Beside symbiotic association, the immune system keeps a check on GM density and composition, controlled by the Regenerating islet-derived protein 3γ (REG3γ), an antibacterial lectin that is expressed in epithelial cells . This lectin regulates the number of bacteria that contact the epithelial surface . Immunoglobulin A (IgA) is secreted into the intestinal lumen, contributing to microbiota composition; also, dependent changes in GM community was related to α-defensin , Fig1. The hosts can sense a shift in GM composition by detecting microbial byproducts such as the SCFAs, including butyrate and propionate . The dysbiosis has substantial effects on host body, leading to poor epithelial architecture , deficient antimicrobial defense by IgA and CD8αβ , and immature development of the mucosal-associated lymphoid tissue associated with reduced IgA levels and drop in T and B cell populations . The absence of GM caused limited generation of the TH17 cells in the gut and spleen; these cells are known to action mucosal linings by recruiting monocytes and neutrophils in response to infection .
GM contributes to systemic autoimmune and allergy conditions . The GF mice show increased susceptibility to invariant natural killer T cell–mediated oxazolone-induced colitis and ovalbumin-induced asthma . The altered expression of NF-κB, a transcription factor involved in inflammatory and immune responses of the gut, are linked to pathologies of chronic inflammatory bowel disease or obesity . It is expected that future GM research could find solutions to intestinal immune pathologies and systemic immune diseasesFollowing
Grant Gallagher added an answer:Using PCR to obtain 16s rRNA amplicons from thermally injured skin?I am attempting to use 16s rRNA primers to amplify bacterial DNA for sequencing. Does anyone have experience using pcr to obtain amplicons? I am worried my thermocycler settings are not optimized...Bonnie - relax - this is done all the time everywhere. Take a wander down the corridor to your local friendly micro lab and you'll get the answer.
Watch out for cross-contamination once you start though.
Afshan Malik asked a question:How to distinguish between bacterial and host DNA in circulation?I am interested in the composition of cell free DNA found in human blood. Where has it come from. Is it waste product from dying or stressed cells in the body? I know it contains human nuclear and mitochondrial DNA but if we find unmethylated DNA here could this have come from bacteria in the gut? Is there a way to distinguish between bacterial, mitochondrial and human ? Any views welcomeFollowing
Boe Hlabano asked a question:Are there bacteria that have a positive effects on the nervous system?Are there bacteria that have a positive effects on the nervous system?Following
Paul I Costea added an answer:Which level of identification can be acheived by using 454 pyrosequencing for gut microbiome?Level of bacterial identification by 454 pyrosequencer for 16S rRNA sequencing.The simple answer is: it's complicated.
Level of resolution will depend on a lot of variables, but i think the most important are going to be the choice of region and the length of the reads.
Here's something to read that should make the point quite nicely:
Now even assuming you go for the 97% OTU identity (which some will argue is a species level resolution) you might find that it's not very easy to transform those otu's into a relevant taxonomy. It's doable, but it isn't easy.
Bottom line, you could get species resolution. Using some fancy sequencing you might even be able to get strain:
Bhawna Vyas added an answer:xxyy.Microbiota is the appropriate word.Following
Carlos Wolfgang Nossa added an answer:Whats the best software for 16S data analysis?Ive been using RDP for my 16S rRNA analysis since I started in this field 4 years ago. But now I see people using different tools.
Plus I have reviewers demanding that I denoise my data, which I had never done before.
Should I switch to QIIME or stay with RDP? Any other options that are good (and user friendly for the computationally impaired?)Thanks, Ive started to use QIIME. The Genboree online toolkit looks promising, when it is completly up and running.Following