- Mohsen Sohrabi added an answer:7Which primer do you recomend for saliva microbiome profiling? Which one can give me more data on bacterial species level?
I will sequence my samples on illumina Illumina MiSeq (paired-end).
The candidate primers are:
My concern is to have more bacterial diversity and have a better assembling data.
Dear Dr. Nazar
Thanks for your answer. Is there a significant difference between V1-V3 and V3-V4 primer in terms of species identification? I can see lots of publications used V3-V4 for MiSeq. I am a bit confuse why people are stil doing sequencing while they are not able to identify at the species level? I guess genera classification cannot give use significant data for using bacteria as biomarkers.Following
- Alain J Benitez added an answer:6Does 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 ?
I used a micro brush to collect sample from all areas. I agree with Alexander, it would be great to do all areas separately allowing for individual and combined analysis.Following
- 6Has 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?
take a look at the new PureLink Microbiome DNA Purification Kit
- Lahmadi Mohamed added an answer:5Is there a method for extracting DNA from sputum?
Is there a method to extract DNA from sputum, considering sputum's microbiome?
Thank you Alexander
that what I am looking for ...Following
- 14Which 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?
the new PureLink Microbiome DNA Purification Kit works really well for stool, and has protocols for additional microbiome sample types.
- 7Has 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.
you can also try the new PureLink Microbiome DNA Purification Kit – that has protocols for a wide variety of sample types, including challenging samples such as stool and soil - in addition to urine, saliva, misc swabs, transport media, growth media etc.
- Jean Philippe Blankert added an answer:7Does anyone have any information on the gram-positive bacterial genus Streptococcus in the human gut -- where it is normally found, what it lives on?
Streptococcus is a genus of bacteria that is normally found in the human microbiome. From what I can establish a range of species are found in the mouth and upper GIT but I am finding it hard to establish much more about it's ecology.
Is anyone aware of any papers on the genus? Are species faculative anaerobes and if so what do they ferment? What do they do in the GIT? What part of the GIT do the species occupy -- the fluid, the mucus, the lumen?
The reason I ask is that the genus is flagged as potentially being an over-abundant gram-positive anaerobes in the colon. The over abundance results in an increase in D-lactic Acid in the bowels and production of by-products that are suspected as causal agents in chronic fatigue syndrome, irritable bowel syndrome and more.
I am trying to grapple with why these seemly upper GIT species would grow and become over-abundant in the colon of people eating normal amounts of food? I am aware that this syndrome has been documented in cattle fed with corn (that they are not able to digest) and documented in post-operative patients following small bowel syndrome (food unable to be completely digested before moving on to the colon) but don't understand the processes that would lead to a sustained population in the large intestine of a normal human.
Hence the desire to collate what I can on one group of species involved in this syndrome to better understand what might be going on.
So, does anyone have any information on Streptococcus in the human gut -- where it is normally found, what it lives on, anything? Actually any information on the genus would be good; even if it is purely biochemical.
Yes, very interested to cooperate with you...see http://blankert.bio/immuunsysteem-stress-burnout-chronisch-vermoeidheids-syndroom-Borreliosis-Lyme.html
I go on holiday now, back around August 25.Following
- 12Any thoughts on uncultured microbe isolation?
With the involvement of NGS, most of the researchers have moved to desk than to bench. At bench we understand the biological entity with chemicals and pipettes by monitoring them at specific laboratory conditions day over night, at desk we hypothesize of a biological relevance using the arsenal of computational tools of biology. I myself do microbial ecology but I wonder, are we unnoticing and losing those so called uncultured entities who are definitely of significance.
What would be the push towards making the uncultivable cultivable ??
Rajal- great point. i am one from the crowd who is just isolating total microbial DNA, and analyze by qPCR and NGS. dont have any expertise with culturing. the only thing i know- there is a huge number of microbes that cant be cultured at the moment, and its obvioulsy not trivial to address at the moment. but should be done one way or anotherFollowing
- Paul I Costea added an answer:3Oral to gut microbial transmission?
What is the expectation when in comes to microbial transmission from the oral cavity to the gut?
I would think not much gets through because of the stomach barrier. Is that a common expectation? I'm finding it hard to get literature on this.
Thanks Mark. I realize that in infancy, the transmission may be considerable, as the oral community is "seeding" the gut. However, given that the overlap in the mature human (between oral and stool) in terms of species is minor, i expect this seeding to not mean that much.
Generally, i don't expect a lot of the oral microbes to be viable gut colonizers. I'm just trying to make sure this is a valid assumption.Following
- Paul Rutland added an answer:3Is there a reduction in the sensitivity of PCR when using universal primers with degenerate sequences?
I am using a pair of primers each oligo has a degeneracy of 4, so an overall degeneracy of 8 when they are used together.
The primers are for the amplification of 16S rDNA so are being used to identify the microbiota of particular tissues.
In my mind I feel there may be a a loss of sensitivity when each reaction is relying on one out of 8 primers to anneal to its target. This, in my mind, will have a greater impact when bacterial DNA present in my samples is particularly low.
Can someone please let me know one way or another, if I am just fussing over nothing, or better still if there is a study where LOD was investigated for primers sets with varying levels of degeneracy, that would be amazing!!
Thanks in advance
ok the degeneracy in the middle is good. specific primers must be better as they will produce only one product so amplification is efficient bbut if you have many products then some will amplify better depending on whether the product melts easily and the primer sticks well. Too many products will also lead to reagent depletion earlier in some cases.
You may want to consider that in the making of oligos the g base phosphoramidite ads slightly less efficiently than other bases so you will get slightly less of the g containing oligo in a redundant mixture. Tech help at the company that made them can advise how much difference this makes on long oligos but probably not great. you may need to check sequences of the non amplifying species for high cg content so poor melting of the product
- Tewodros Debebe added an answer:3How to isolate and cultivate bacteria from body fluid?
how to isolate and cultivate bacteria from body fluid.
you can also use Brucella Blood Agar for the isolation and quantification of anaerobic bacteria. In this case you need to have anaerobic jar or plastic to make the culture/plate anaerobic environment. As it has been mentioned by someone else, it is advisable to make a serial dilution as your fluid might have high number of microbes.Following
- 6Do 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
try the PureLink Microbiome DNA Purification Kit – that enables fast purification of high-quality microbial (and host, where applicable) DNA from a wide variety of sample types, including challenging samples such as stool and soil - in addition to urine, saliva, misc swabs, transport media, growth media. http://www.thermofisher.com/order/catalog/product/A29790?ICID=search-product. yield is substantially higher vs MoBio kit or QiagenFollowing
- Ibrahim Shnawa added an answer:6Why 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
- Jawwad Ahmad added an answer:5Does 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:7Which 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:5How 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:2Are 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:5What 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:5What 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.
- Jay Siddharth added an answer:1Where 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:11How 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:2How 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:7Why 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:2Using 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.
- 1How 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 welcome
there is a new MagMax kit for isolation of circulating/cell free DNA from blood. and then using corresponding TaqMan assays or sequencing you can figure which DNA is there- definitely mostly human, but some microbial will be present as wellFollowing
- Boe Hlabano asked a question:OpenAre 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:4Which 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:3xxyy.Microbiota is the appropriate word.Following