Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

The overall goal of this procedure is to sequence 16 S-R-R-N-A. Genes amplified from environmental samples using a benchtop sequencing machine. This is accomplished by first amplifying the 16 S-R-R-N-A gene using modified primers.

The second step is to purify the amplification products, quantify them, and pool them. Next, the amplicon pool is attached to sequencing spheres amplified clonally during emulsion PCR and sequenced on a benchtop sequencer. The final step is to analyze the resulting sequence data sets.

Ultimately 16 S-R-R-N-A. Gene amplicon sequencing is used to determine the composition and diversity of the microbial community in an environmental sample. The main advantage of this technique over existing methods like DGG or cloning sequencing, is that it gives a deeper and clearer perspective into microbial community composition and diversity.

This method can help you answer one of the key questions in microbial ecology, who's there To begin the forward and reverse primers? The two X hot start tac plus master mix and the samples, the sequence of the forward and reverse primers fit the template shown. The sequence contains ion torrent specific adapters.

A sequencing key for calibrating the signal intensity at the beginning of the sequencing. Run a template specific primer and a 10 base pair barcode. Then mix the primers and samples thoroughly before use.

Prepare A PCR reaction. Mix 20 microliters per reaction containing 0.5 micromolar of the reverse primer, 0.4 milligrams per milliliter of BSA and one X hot start tac plus master mix. Next pipette 18.5 microliters of the master mix into each PCR tube add 0.5 microliters of a 20 millimolar forward primer stock solution containing a different barcode to each of the samples that will be sequenced together.

Then outside the PCR hood, add one microliter of a sample at a concentration of one to 10 nanograms per microliter to the PCR tube. Set a program with an initial denaturation of 95 degrees Celsius for five minutes. Then 25 cycles of 95 degrees Celsius for 30 seconds.

55 degrees Celsius for 30 seconds and 72 degrees Celsius for 45 seconds. The final elongation should be at 72 degrees Celsius for 10 minutes. Place the tubes in a PCR machine and start the program.

The final PCR products can be placed at four degrees Celsius or negative 20 degrees Celsius until used. Prepare a 2%aros gel using the largest combs available. Then mix five microliters of six x gel loading dye with each PCR reaction and load the samples on the gel separating each sample by an empty.

Well run the gel at 70 volts for 50 minutes. After the gel is run, take a picture of the gel and cut the bands using a scalpel. Each band is expected to be around 250 base pairs, which contains a 190 base pair product with 63 base pairs that make up the adapters and barcodes.

Next, weigh the excised bands and proceed with the gel extraction and purification of the PCR products. Using a kit such as the kayak quick gel extraction kit, then quantify each PCR product with pico green and make dilutions to obtain a stock solution at five times 10 to the ninth molecules per microliter, which is approximately one nanogram per microliter. If some samples show lower amplification, adjust the concentration as necessary, but do not go below 1.57 times 10 to the seventh molecules per microliter.

Pool 10 microliters of each diluted PCR product to obtain an equal molar pool of samples, prepare one separate pool for each of the planned sequencing runs. The pooled PCR products can be kept in the freezer until used to perform emulsion. PCR first, dilute the pooled PCR products to 26 picaMolar in a volume of 25 microliters and haw the reagents from an ion PGM template kit.

Then work in a PCR hood and prepare the emulsion PCR. Mix the pooled PCR products and the sphere particles can be added when working on the bench. Mix the solution thoroughly and then insert the mixture in a filter cartridge and carefully top with emulsion oil.

Next, slowly invert the filter cartridge and load the cartridge onto the automated emulsion PCR apparatus. Then select the appropriate program and start the procedure. After the automated emulsion PCR is complete, remove the supernatant from the collection tubes and retrieve the sphere particles at the bottom of the tubes.Resus.

Suspend the spheres in 100 microliters of wash solution and take a two microliter aliquot for library quantification. To sequence the PCR products, prepare a sequencing run plan that specifies the details of the sequencing run. Then open the instrument, click on initialize and when prompted, install wash solution number two, wash solution number one, and the auto pH buffer solution into the sequencing instrument.

Next, continue the initialization procedure and when prompted, add the D-A-T-P-D-G-T-P-D-C-T-P and DTTP nucleotides into their respective 50 milliliter tubes. Then screw the tubes onto the sequencing machine and continue the initialization procedure. Once the initialization procedure is complete, immediately start the sample loading procedure to prepare the samples for sequencing.

Centrifuge the enriched spheres from the emulsion PCR at 15, 500 times G for 1.5 minutes to collect the spheres at the bottom of the tube. Then remove the S natin and leave exactly three microliters in the tube. Next, add three microliters of the sequencing primer to the tube and pipette up and down to resuspend the particles and mix the solution thoroughly.

Incubate the tube for two minutes at 95 degrees Celsius, and then for two minutes at 37 degrees Celsius. When the incubation is complete, add one microliter of polymerase and mix well then incubate the mixture at room temperature for five minutes. While the mixture is incubating, load the sequencing chip onto the sequencer and perform a chip quality check.

Then remove the liquid from the chip with a quick centrifugation and a micro centrifuge. Load the sample in the chip by slowly pipetting down seven microliters of the enzyme sphere mix and avoiding introducing bubbles in the chip. Once the sample is loaded in the chip, place the chip into the instrument and start the sequencing run.

To begin analysis of the data, connect to the sequencing data server and download the fast Q file. Then create a tab, delimited oligos file containing the primer and barcode information. Next, download the Green Genes reference files from the mother website, launch mother, and perform the analysis.

Convert the FASTQ files to a fast a quality file using the command shown on the bottom of the screen. Determine the group membership of each sequence and trim the sequences. Using this command, then classify the sequences in the Green Genes taxonomy.

Using this command, the average output when sequencing using a three 14 ship on an ion torrent PGM is around 0.3 to 0.5 million. Good quality reads after the results are filtered in mother. Here is a typical breakdown of the number of reads after each step of this next generation sequencing procedure shown here is the average community composition at the phylum class level of all 36 multiplexed environmental samples that were amplified with primers targeting the V three to four region of the 16 s ribosome and analyzed using mother One master.

This technique can be done in two days if performed properly. After watching this video, you should have a good understanding of how to sequence 16 s ribosomal RNA applicants using a benchtop sequencing machine.