- 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
- Christian Riesenfeld 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?
http://www.mobio.com/ has what you need and should be consideredFollowing
- 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.
- Abdelrahman Mahmoud 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.Really, i need to thank you about that clear and great answers
Deep thanks to you :)Following
- 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:Which one is proper for the human gut microbial community: microbiome or microbiota?.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