The overall goal of this experiment is to understand the bacterial community that is present within a silage sample, although these procedures can be applied to a range of different sample types. This method can help answer key questions when identifying the procaryotic communities found within complex microbial systems, such as in the case presented here, silage for animal feed. The main advantage of this technique is that shotgun sequencing is employed rather than the more conventional 16S amplicon sequencing, enabling a much greater accuracy when classifying the microbiome.
This method can be applied to a wide range of other samples where complex microcosms may form, such as in the human gut, on the surface of the skin, in water samples, or even around the office coffee machine. A commercial kit is used to extract DNA from the silage sample. Begin this procedure by adding 100 to 400 milligrams of the sample to 978 microliters of sodium phosphate buffer and 122 microliters of soil lysis buffer in the supplied lysis tube.
Include a negative control that has no silage sample. Homogenize the samples by placing the lysis tubes into the homogenizer for 40 seconds at a speed of 6.0 meters per second. Centrifuge the lysates for 15 minutes.
Transfer the supernatant to a clean microcentrifuge tube containing 250 microliters of Protein Precipitate Solution and mix the solution by inverting 10 times. Then centrifuge for five minutes. Transfer the supernatant to a clean 15 milliliter centrifuge tube that contains one milliliter of DNA binding matrix.
Mix the solution by inverting the tube constantly for three minutes. Allow the mixture to settle for three minutes, then discard 500 microliters of the supernatant and mix the remaining supernatant by pipetting. Transfer 600 microliters of the suspension to a spin filter and centrifuge at 14, 000 times G for one minute.
Discard the filtrate and repeat the process with the remaining suspension. After discarding the filtrate, add 500 microliters of wash buffer to the DNA binding matrix within the spin filter, mix by pipetting, and centrifuge at 14, 000 times G for one minute. Discard the filtrate and centrifuge the spin filter for two minutes to ensure all wash buffer is removed.
Dry the spin filter at 23 degrees Celsius for five minutes. Add 100 microliters of pre-warmed DNase-free water to the DNA binding matrix and transfer the spin filter to a clean 1.5 milliliter microcentrifuge tube. Centrifuge at 14, 000 times G for one minute to elute the DNA.
Store the DNA at minus 20 degrees Celsius until needed for further analysis. The DNA extracted from silage samples will be purified using DNA purification beads. Remove the beads from the refrigerator and leave them at 23 degrees Celsius at least 30 minutes before use.
Add two volumes of beads to each DNA sample and incubate the solution at 23 degrees Celsius for five minutes. Place the samples onto a separation magnet for five minutes and then discard the supernatant. Wash the beads twice with 200 microliters of fresh 80%ethanol.
Air-dry the beads for 10 minutes The beads need to be sufficiently dry, but not over dried, so it is crucial to visually inspect the beads before continuing. Remove the samples from the separation magnet, add 50 microliters of elution buffer, and mix by pipetting. Incubate the suspension at 23 degrees Celsius for five minutes, and then place the samples back onto the separation magnet for three minutes.
Transfer the supernatant which contains the DNA to a clean tube and discard the beads. To begin this procedure, prepare a working solution using a 199 to one ratio of buffer to reagent. Add 10 microliters of each DNA standard to 190 microliters of working solution.
Add 10 microliters of purified DNA to 190 microliters of working solution. Incubate the standard and DNA samples at 23 degrees Celsius for two minutes. Subsequently, analyze the standards before the DNA samples on the fluorometer following the onscreen instructions.
It is essential that the purified DNA be of good quality and accurately quantified, so if there is any doubt regarding the quality and the concentration of the DNA, the DNA extraction must be repeated. The shotgun sequencing library is prepared using a commercial library preparation kit. Dilute the DNA samples to 0.2 nanograms per microliter using elution buffer.
Mix five microliters of the purified DNA with 10 microliters of tagmentation buffer and five microliters of tagmentation enzyme mix. Incubate the samples at 55 degrees Celsius for five minutes. Add five microliters of neutralizing buffer to each sample and incubate the solution at 23 degrees Celsius for five minutes.
Next, add five microliters of each of the sample-specific sequencing indices and 15 microliters of the PCR mastermix. Place the samples into a thermocycler and perform PCR. Purify the prepared DNA using the bead purification method as demonstrated earlier, but with a final elution of 30 microliters of elution buffer.
Subsequently, the DNA is sequenced and a range of bioinformatics tools are used to analyze the sequence data as described in the text protocol. Taxonomic classification of the bacterial microbiome was performed with Kracken software and then visualized with CHROMA software. The majority of the bacterial species present in the silage metagenome are found within four procaryotic fila, firmicutes at 34%actinobacteria at 28%proteobacteria at 27%and bactoroitis at 7%The distribution of classes present within these fila is also indicated.
Functional annotation performed on assembled reads resulted in 6, 357 predicted proteins annotated as a putative carbohydrate active enzyme and one of the five enzyme classes that make up the carbohydrate active enzymes database. This venn diagram shows the distribution of protein annotations, including those containing more than one enzyme class annotation. Of these, 3, 591 were predicted to have glycoside hydrolase activity.
Once mastered, the preparation of DNA sequencing libraries can be completed in a single day if it is performed properly. While attempting this procedure, it's important to ensure there is no cross-contamination between samples and to always use suitable controls such as a blank. Following this procedure, other biofomatic analyzes can be performed in order to validate the classifications made by Kracken.
These metagenomic techniques enable researchers to explore the microbiome in many complex systems. After watching this video, you should have a good understanding of how to prepare metagenomic DNA sequencing libraries and perform bioinformatic analyses on the DNA sequence reads obtained. Don't forget that working with DNA binding reagents can be extremely hazardous, and precautions such as gloves and lab coats should always be worn while performing this procedure.
