Major platforms of Next generation sequencing

   Illumina sequencing

Illumina platform was first introduced by Solexa in 2006 later re-branded by Illumina. Two of the major platforms of illumine are HiSeq, MiSeq. This technology utilizes sequencing by synthesis approach where adapter ligated template molecules flow into flow cells made glass hollow from inside. Template hybridizes to primer on the flow cell surface and gets copied onto the flow cell. Basically, bridge amplification occurs which results in a generation of reverse complimentary copies of the template. Each cluster holds approximately 1 million copies of the original fragment. Each nucleotide added contains a base specific fluorescently labelled probe with chemically blocked OH group. The fluorescently labelled probe is probe is helpful in detection of nucleotide s being incorporated. These incorporated nucleotide s are then imaged using CCD camera and then DNA Polymerase removes 3’ block chemically to prepare each strand for next incorporation.

   454 Sequencing/Roche

Roche technology became commercialized in the year 2005 which uses bead based em-PCR (Emulsion PCR) approach to amplify the copies of template of DNA molecule. Later these beads are sequenced by pyrosequencing in parallel fashion. Pyrosequencing involves four different nucleotides flowed in sequential manner through solid surface wells, known as picotiter plates into which single bead is incorporated. Pyrosequencing is based on principle of generation of light signal through release of pyrophosphate (PPi) on each nucleotide incorporation. The signal intensity per nucleotide is recorded for each bead and later analyzed by CCD camera attached to computer system to detect the exact sequence.

    ABI SOLiD

Sequencing by Oligonucleotide Ligation and Detection is developed by life technologies and has been commercially available in the year 2006. This Technology is similar to Roche technology but the only difference is that the beads in SOLiD are smaller as compared to Roche sequencers. A library of DNA fragments is used to prepare clonal bead populations. Only one fragment with universal adapter is present on each bead so that the starting sequence of every fragment is both known and identical. The resulting PCR products attached to the beads are then covalently bound to a glass slide. Primers hybridize to the adapter sequence within the library template. Later a set of four fluorescently labelled probes of 16 dinucleotide groupings compete for ligation for the sequencing primer. The nucleotides are recognized by analyzing the color light emitted as a result of two consecutive ligation reactions. One of the drawback of this system is the issues with sequencing palindromic sequences.

     Ion Torrent

Ion semiconductor sequencing is based on the detection of hydrogen ions that are released during polymerization of DNA by using sequencing by synthesis approach. Ion torrent detects the nucleotide's and then converts them into digital format 1 and 0 on a semiconductor chip. When a nucleotide is incorporated in the strand of DNA, H+ ion is released which is detected by semiconductor chip. The charge from the ion will change the PH, of solution which can be detected in the form of peaks by ion sensor.