An Overview of Next Generation Sequencing

Agnes Caruso, Product Manager at Canon BioMedical on August 13, 2018

What is Next Generation Sequencing?

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.

NGS sample preparation

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. 

NGS steps

There are several commercially available methods for NGS. Most all of them have the same basic steps in the procedure.  These steps are:

  1. Library construction. This step involves, with some exceptions:
    1.  Fragmentation of the DNA into the optimal size range for the procedure.  When investigating targeted panels the fragmentation step is not needed.
    2. End repair.
    3. Phosphorylation of 5’ ends.
    4. Ligation of adapters. Adapters are small DNA fragments ligated to the ends that are critical to the sequencing step.
    5. PCR enrichment of the library performed on an end-point PCR thermocycler.
    6. Library QC and if needed size selection.
  2. Sequencing of the DNA. There are several methods for this step. Some examples are:  
    1. Sequencing by synthesis - using cluster PCR and fluorescently-labeled nucleotides.
    2. Semiconductor sequencing - using sequencing by synthesis process with emulsion PCR and hydrogen ions as means of signal detection using semiconductors.
    3. Single molecule sequencing - using long, single strands of nucleic acids.
  3. Alignment and data analysis. The use of bioinformatics to rebuild the entire sequence from the sequenced fragments and compare to known sequence is what finally delivers the sequencing report with all the identified sequence variants.

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.