The PCR products are pooled together into an Eppendorf tube and is called a library. In a typical experiment that takes place in our lab, there can be more than 400 samples which constitutes at least five libraries. Generally, a set of 96 barcode primers are used, hence a library can have a maximum of 96 samples. All the reads from one sample has a particular barcode in that library and this is crucial when calling sequences (explained later) after sequencing is done.
Each library is run on the Bioanalyzer to assess the concentration and the size of the PCR products. It is then purified on the Pipinprep to purify each library so that it only contains the PCR products of the desirable size while PCR products shorter or longer than desirable size, primers, enzymes etc. are removed. The purified library is again diluted to get around 22 – 26 pM which is adequate concentration for sequencing. The PCR products are denatures into single strands and then subjected to emulsion PCR, where each strand or read binds to one bead via the adapter sequence. The adapter on the bead is complement to the adapter primer so that it attaches firmly. This is where the concentration is crucial so that only one strand binds to one bead. In other words, one bead carries single strand. During emulsion PCR, again PCR is done on the bead, so that the whole bead is covered with the same sequence.
These beads are then washed and subjected to ion-torrent sequencing. The ion-torrent sequencer machine has several input ports. The main input is the microchip consisting of minute wells onto which the beads are loaded. It is taken care that one bead enters each well, sometimes more than a bead may enter a well. The other inputs are bottles each filled with four dNTPS separately – one bottle each for dATP, dGTP, dCTP, dTTP. Once the necessary metadata (chip number, sample labels, date, user, barcodes) are entered to the software in the machine, the sequencing begins. At a time, one of the four NTP is flooded onto the chip from the bottle, for example dATP is used. The beads with T as their first nucleotide in the sequence would take up the dATP. Those wells will release an ion (H+) which changes the pH of the solution. An ion-sensitive layer below the well measures the change in pH and converts into to voltage. The voltage change is recorded and thus a base is called, so when each dNTP is passed through the well, it records a voltage when it is attached, or if it is not the required base then no voltage change is recorded. So, each voltage difference from each well is recorded, and converted into digital information. Each base is flooded every 15 seconds. A typical run is finished in 3 – 4 hours.
Once the sequences are obtained it can be viewed from the ion torrent server software and can get details of the quality of the run as well. The barcode primer sequences can be input into the software so that it splits the reads according to the barcodes, so that it is easy for the user to identify which sequences come from which sample. Once the sequences are obtained it may be downloaded and then OTU tables are generated to find taxonomy of the microbes. The data can be then utilized to interpret statistical information with appropriate software and can also predict functions from databases. Hence, we decipher microbiome data from a plant.
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