What are the steps involved in ion torrent sequencing?

The four main steps involved in ion torrent sequencing include: DNA fragmentation, addition of adapter sequences, size selection, and library quantification. 

1. During DNA fragmentation, to obtain the necessary DNA for subsequent processes like ligation or amplification with adaptor sequences, isolated DNA undergoes fragmentation using physical or enzymatic techniques. The resulting libraries from this process are known as fragment libraries. 
2. Following DNA fragmentation, specific DNA adapter sequences are attached to both the 5' and 3' ends of the fragmented DNA. These DNA adapters (typically 20 to 40 base pairs long) possess sequences that complement those found on beads. Typically, two distinct adapter sequences can bind to the DNA fragments, oriented either at the 5' or 3' end. One adapter sequence includes the primer annealing site for the sequencing primer, and the second adapter sequence is commonly utilized to secure the DNA fragment to a bead's surface. This is done through Watson & Crick base pairing with the complementary sequence on the bead's surface. 
3. The third step involves choosing the sizes of library fragments required for the sequencing run. This selection can be achieved through either bead-based size selection method or gel electrophoresis. In the gel-based method, the adapted library fragments are run on a gel to separate them by size and the bands corresponding to the desired size are gathered. In the bead-based method, magnetic beads are used with different buffer concentrations to isolate DNA fragments of the desired sizes. It is crucial for the final library fragment size to be consistent to ensure efficient DNA sequencing in subsequent steps. 
4. The last step involves library quantification and quality control (QC). Two main methods are commonly used for library quantification. The first method involves analysis using the Bioanalyzer™ system, which provides information on fragment size and library concentration. The second method is qPCR, which gives the most precise library quantification by measuring only amplifiable library fragments. It does not provide information on library size however.