Next Generation Sequencing (NGS) is also known as high-throughput sequencing. It allows for rapid sequencing of whole genomes or specific PCR amplicons of interest. NGS can be used for sequencing DNA or RNA from various sources. In some ways, it is not that different from some older methods, such as capillary sequencing. Just like capillary sequencing, NGS provides the DNA sequence, however, it uses different instrumentation and provides more information in a shorter period of time. The initial sequencing of the human genome took 13 years to complete and now it is possible to get the same sequence information in just days or in medical emergencies even within 24 hours.
The starting material for any type of sequencing is typically blood or saliva. However, in order for the sample to be tested by NGS it must first be converted into a library. A library consists of fragments of DNA from the original sample with adapters added that are necessary for sequencing. The library of DNA fragments is then sequenced and aligned to create a continuous DNA sequence. Methods for preparation of the library vary by input sample type (DNA or RNA) and the sequencing method used. While more PCR-free methods are being developed, the use of PCR in NGS sample preparation allows the user to start with very small nucleic acid amounts and is of great value when sequencing from a limited input. There are two main areas where the use of a thermocycler for PCR is necessary. The first is in whole genome sequencing where PCR is required for library enrichment. The second is, if sequencing only part of the genome, PCR is required for the creation of the amplicons that are to be sequenced.
There are several commercially available methods for NGS. Most all of them have the same basic steps in the procedure. These steps are:
NGS is dramatically faster than Sanger sequencing by virtue of how it sequences multiple fragments at once. However, the sample preparation for NGS is time-consuming. New developments to every step of NGS are aimed at reducing the turn-around time from initial sample to a final clinical report. There is a need to improve sample quality, sample volumes, and overall sequencing costs in order to provide faster, cost-effective and more accurate diagnostic tools.