Next-Generation Sequencing (NGS)- Definition, Types


Next-Generation Sequencing (NGS) is a term that refers to a group of technologies that enable rapid and high-throughput sequencing of DNA or RNA molecules. NGS can be used for various applications, such as gene expression profiling, variant/mutation detection, epigenetic changes, and molecular analysis. NGS has revolutionized genomic research and personalized medicine by allowing labs to study biological systems at a level never before possible.

NGS is different from the previous Sanger sequencing technology, which was slower and less scalable. Sanger sequencing relies on the chain-termination method, which uses dideoxynucleotides to terminate the synthesis of DNA strands and produces fragments of different lengths that are separated by electrophoresis and detected by fluorescence. Sanger sequencing is best for analyzing small numbers of gene targets and samples and can be accomplished in a single day. It is also considered the gold-standard sequencing technology, so NGS results are often verified using Sanger sequencing.

NGS, on the other hand, uses a massively parallel approach, which means that millions of DNA or RNA fragments are sequenced simultaneously in a single run. NGS technologies vary in the way they prepare, amplify, and sequence the DNA or RNA molecules, but they all share some common steps: library preparation, sequencing, and data analysis.

  • Library preparation involves fragmenting the DNA or RNA sample and attaching adapter sequences that are specific to each technology. These adapters may also have unique molecular barcodes that allow for multiplexing, which means that multiple samples can be pooled and sequenced together in the same run.
  • Sequencing involves adding fluorescently labeled nucleotides to the DNA or RNA fragments and detecting the incorporation of each nucleotide by a camera or a sensor. Sequencing chemistry can be based on synthesis (adding one nucleotide at a time), ligation (adding short oligonucleotides at a time), or hybridization (probing with complementary oligonucleotides) methods.
  • Data analysis involves processing the raw images or signals into sequences of nucleotides (called reads) and aligning them to a reference genome or assembling them into contigs. The data analysis can also include quality control, variant calling, annotation, and interpretation steps, depending on the application.

NGS offers several advantages over Sanger sequencing, such as lower cost per base, higher throughput per run, higher accuracy, lower sample input requirements, and the ability to detect variants at lower allele frequencies. NGS also enables new applications that were not possible with Sanger sequencing, such as whole-genome sequencing, transcriptome sequencing, metagenomics sequencing, epigenomics sequencing, and single-cell sequencing. NGS has transformed the fields of genomics and clinical research, reproductive health, and environmental, agricultural, and forensic science.

In this article, we will provide an overview of the generations of sequencing technologies and describe some of the most common types of NGS platforms available today. We will also discuss their advantages and disadvantages, as well as their applications and challenges.