The overall goal of this high-throughput 16S rRNA-Amplicon Sequencing is to characterize the global microbiome composition starting from fecal samples. This method can help answer key questions about global microbial composition associated with many field of interests such as metabolism, digestion, and the gut and systemic inflammation. The main advantage of this technique is that it provides a census of aerobic and anaerobic microbial communities present in the fecal sample including those bacteria that are difficult to culture.
This method can simultaneously handle large numbers of fecal samples thanks to rapid colon-free processing pump line and it resulted in robust and reproducible 16S rRNA-Amplicon Sequencing. For this protocol, have frozen feces samples that are roughly the size of a pencil eraser. The samples should be stored in two milliliter tubes with the trimmed off tip of the collection swab.
Include negative controls consisting of swabs with no feces and internal duplicates controls and have sample duplicates from the sample original stool to serve as an internal control. Begin with thawing the extraction and dilution solutions at room temperature along with the fecal samples and control samples. Once thawed, add 250 microliters of extraction solution to each sample tube and mix the feces into solution using a vortex.
Introduce to buffer-only negative controls at this point. Next, heat the samples for 10 minutes in a boiling water bath. After the incubation in boiling water, add 250 microliters of dilution solution to each sample and vortex the samples to mix.
Then store the sample tubes at four degrees Celsius until PCR can be performed. Set up the PCRs at a pre-PCR workstation that has been cleansed of potential DNA contaminants like templates and Amplicons. Label the primers according to their bar code and dilute them in double distilled water to a concentration of 50 micromolar.
Store the stocks of diluted primer at minus 20 degrees Celsius and thaw them completely before use. For each sample, prepare a 96-well plate for 32 reactions in triplicate. After warming to room temperature, dilute aliquots of the forward primer and the 32 reverse primers to five micromolar for the PCR.
Then prepare enough base PCR mix for 100 reactions. Combine 100 microliters of five micromolar forward primer with one milliliter of 2X PCR master mix and 400 microliters of double distilled water. Vortex this mixture and then load 15 microliters of this mixture into each of the 96 wells.
Next, load sets of three wells each with one microliter of five micromolar reverse primer. Thus, load all 32 different reverse index into the plate. Then to every plate well, add four microliters of the extracted DNA sample.
This is done on a clean bench. Now run the PCR with a three-minute initial denaturation followed by 35 cycles with one minute of denaturation and kneeling at 55 degrees Celsius and one minute of extension. Finish with a 10-minute final extension.
After the reaction is completed, work in a post-PCR dedicated workbench. There, combine each set of triplicate PCR products into a single tube for 60 microliters per sample. Then run four microliters of each sample on an ethidium bromide stained 1%agarose gel.
The positive Amplicons will appear between 375 and 425 base pairs. The negative controls are shown and are processed along with the study positive Amplicons. Quantify the DNA concentrations of the positive reaction products according to manufacturer instructions.
Then combine 500 nanograms of DNA from each of the products into a single tube. Vortex this mixture and then run out 200 microliters on a 1%gel as before. Now extract the bands from the gel between 375 and 425 base pairs.
Next, prepare the library for sequencing. Measure the concentration using a highly sensitive double-stranded DNA detecting kit according to manufacturer instructions followed by use of a highly sensitive separation and analysis kit. Then dilute the library to seven picomolar and combine it four to one with a control library.
Now that the sample is ready for sequencing, transfer it to the sequencing machine. The described method for a library preparation from stool samples was used to analyze the microbiome of hospitalized patients with suspected infectious diarrhea. In parallel, stool samples were also subjected to traditional culturing in a microbiology lab.
Stool samples from healthy adults were sequenced for comparison. Several negative control samples were included, PCR without template, PCR on clean and sterile swabs in extraction and dilution solution and PCR on mixed extraction and dilution solutions. Each of these had less than 118 total reads which were mainly Mycoplasm taxa.
Standard samples averaged greater than 10, 000 reads with no identification of Mycoplasma after quality control. Samples consistency and variability was measured using 16 stool samples prepared with two different bar coded primers to hence construct two different libraries from each sample. Half of the samples were positive for Campylobacter salmonella or Shigella in traditional culturing technique.
Using the 16S sequencing results, an area plot at the phylo level shows consistency between libraries made from the same samples independent of the traditional culturing results. From this pool of 16 paired samples, principal coordinate analysis revealed that the sample pairs are closely aligned and that the positive culture samples had a significantly different PC1 value from the negative culture samples. Further analysis of the samples revealed that high levels of Proteobacteria were only found in the hospitalized patients.
After watching this video, you should have a good understanding of how to simultaneously process a large number of fecal samples using a colon-free DNA extraction and PCR to generate bar coded sequencing libraries of the microbial variable V4 region. While attempting this procedure, it's important to remember to work in an organized and clean environment especially when preparing the libraries. Don't forget that working with human stool samples is hazardous and personal protection equipment should always be worn while performing this procedure.
