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?
We use PSP stool kit (Wang et al. 2010) which provides better quality and yield and also equivalent to traditional hot phenol extraction method.
Hi Lesley, what yields of DNA do you get on average per sample using these kits?? Using the QIAamp DNA Stool mini kit gives yields of approximately 50-200ng/ul. I have found that DNA yields are usually low from stool samples, and a kit that removes inhibitors for downstream processing us essential
Thanks so much for all your valuable inputs. Lesley, this might be a stupid question, but what do you mean by negative ctrl with regards to the extraction - do you mean to perform the whole extraction process, but without adding any stool sample, so only using the different buffers etc? We will be comparing 2 sample groups to each other. so I thought that kind of thing would be sorted out, since it would be similar for both groups so it would not lead to any other differential results? I might be misunderstanding something.
Also, I just want to make sure about your initial steps, how much PBS do you add to your stool sample? And do you wash it by centrifugation and adding PBS again?
We have a trial kit from Zymo which I am also going to give a go, has anyone perhaps used that before and do you know if it works well?
I have been researching DNA extraction kits and methods recently myself. You should have a look at: Yu Z, Morrison M (2004) Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques 36: 808–812.
Hello previous speakers, I have problem with extraction of DNA from stool samples too. I use broiler chickens stool and after DNA extraction I send samples to NGS (Ilumina) analysis, but results are not reliable. I use E.Z.N.A stool DNA kit D4015 (http://omegabiotek.com/store/product/stool-dna-kit/ from ). I have information about good results of swine stool preparation, but specific poultry material is more demanding. Do you have experience with animal stool DNA extraction in focus on poultry (broiler chickens) ?
http://www.mobio.com/ has what you need and should be considered
Hi Stefanie there are several kits for isolation of bacterial DNA from stool- MoBio, Qiagen, Norgen, Invitrogen/Ambion. You can choose between columns for low throughput and magnetic beads for high throughput isolation. Beat beating is crucial to "open" all bacteria uniformly. However in the near some efficient straight lysis- based technologies will become available, simplifying the isolation workflows (so that you wont need bead beating)
Hi Stefanie, I agree with Alexander and you can more details from a recently published article of Henderson et al. 2014 (Plos One) who discussed the details of each kit and compared them with traditional DNA extraction method. However, we are using bead-beating and PSP kit for DNA extraction from rumen and fecal contents and getting good coverage of bacterial sequences including Firmicutes.
Hi Lesely!
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?
the new PureLink Microbiome DNA Purification Kit works really well for stool, and has protocols for additional microbiome sample types.
http://www.thermofisher.com/order/catalog/product/A29790?ICID=search-product
With stool samples it’s also important to ensure effective removal of humic acids and other inhibitors for PCR, NGS and so on. Here are a few papers that used kits to isolate microbial DNA from rats and human feces for example.
Fecal microbiota in rats:
(http://www.ncbi.nlm.nih.gov/pubmed/26898931)
Detection of Campylobacter in human feces: (http://ojs.wpro.who.int/ojs/index.php/wpsar/article/view/262/431)
Something else to keep in mind is collection of stool samples for reliable preservation of microbiota profiles. For example please see this related poster presented at the Plant & Animal Genomics (PAG) Meeting
http://bit.ly/1MvSokb
Hope this information is helpful!
Does anyone know any article or protocol independent of a kit for Microbial DNA extraction from fecal samples of mice? Since I´m going to do a quick experiment it won´t be interesting for me to buy a kit...
I found one based on the use of Lysozyme and proteinase K to treat the sample and DNA extraction through the classical organic method, but the article lacks some details (i.e. the quantity of sample used.
Regardless of what kit you decide on, you can increase your recovery by as much as 50% by adding an enzymatic pre-treatment step. Simple suspend your sample in PBS 7.5 pH and add metapolyzyme at 5ul/100ul of sample. Incubate 1-4 hours. Metapolyzyme is a turbo charged lysozyme geared to Gram +, -, Yeasts, and fungi. Available from Sigma. Developed by Me and the ABRF Metagenomics Research Group.
Hey @Scott Tighe , do you have any publications that described the used of the method you described? I would be very interested in read more about the method.
Does anyone try the Quick-DNA Fecal/Soil Microbe Miniprep Kit (D6010) for human fecal samples? I have problems with tough samples because the first step isn`t enough to homogenize all the samples, and the I get variable performances. Someone washed frozen stool samples with PBS previous to purification? thanks.
Thomas H A Haverkamp . Info about metapolyzyme https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/mac4lpis.pdf
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