First generation DNA sequencing technology was an elaborate multistep procedure involving separation of chromosomes using Pulse Field Gel Electrophoresis (PFGE), restriction digestion, and ligation to high-capacity cloning vectors like BAC, YAC, and PAC. The DNA segments were physically mapped and sub-cloned into sequencing plasmids. This method was time-consuming due to extensive cloning and sub-cloning steps, and was prohibitively expensive.

Next generation DNA sequencing technology overcomes these drawbacks by using a massively parallel approach where millions of sequencing reactions occur simultaneously in one machine. This technology eliminates the need for time-consuming cloning and sub-cloning steps, thereby significantly reducing the cost of genome sequencing projects.

The methodology involves advance computing algorithms that process millions of short sequence outputs (75 to 300 base long) and assemble them into large contigs representing DNA sequences of large genomes. Unlike the first generation's sequencing by chain termination method using fluorescence-tagged terminating bases, next generation methods provide simple workflow and high data accuracy due to high depth of coverage.

Next generation sequencing offers a broad range of applications including whole genome sequencing (WGS), targeted resequencing, clinical exome sequencing, Chipseq, and RNAseq.