Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

Profiling of microbiome is the first step toward defining the role of microbiome in health and disease.Here, we are describing a simple procedure for isolating high-quality bacterial DNA from fecal sample preparing library, performing 16SR RNA and a meta genomic sequencing and microbiome data analysis.This protocol will help researchers new to microbiome field, as well as researchers working in microbiome field in establishing the microbiome pipeline in their lab.This protocol can be extended for profiling of microbiome from other bio specimens such as nasal, oral, or skin samples of animal and human subjects.Bead picking is the most important steps as all downstream steps are depend on high quality DNA.Proper sealing of preserve plate is important as incomplete sealing might lead to the oppressing of amplified product resulting in low quality sequencing data.To begin, sterilize the divider boxes with 70%ethanol.Place the mice in sterilized divider boxes and allow them to defecate normally for up to one hour.Use sterile forceps to collect the fecal pellets in an empty pre-labeled 1.5 milliliter micro centrifuge tube.Enclose the tube securely.Sterilize the forceps with 70%ethanol, followed by a germicidal disposable wipe, after collecting fecal from each mouse.Weigh 200 milligrams of fecal pellets into a 1.5 milliliter micro centrifuge tube, with 0.1 milliliter glass beads, and place the bead tube into a bead beating homogenizer and homogenize the samples for 45 seconds at room temperature, at a speed of 4.5 meters per second.Extract DNA as per instructions of the kit manufacturer and elute DNA in a 1.5 milliliter micro centrifuge tube.After isolating DNA, quantify the isolated DNA by loading 1 microliter of the DNA on a fluorometer.Setup 16S ribosomal RNA gene amplification PCR1 in a 96-Well PCR plate, using a 25 microliter reaction volume.Using a multi-channel pipette, add 40 nano grams of DNA, 13.5 micro liters of 2X high-fidelity polymerase enzyme mix, containing buffer, plus DNTPs in forward and reverse primer.Seal the PCR plate properly from the top to the bottom in along all four edges.And centrifuge at 1000 times G at 20