The evolution of next-generation sequencing technologies

Abstract

The genetic information that dictates the structure and function of all life forms is encoded in the DNA. In 1953, Watson and Crick first presented the double helical structure of a DNA molecule. Their findings unearthed the desire to elucidate the exact composition and sequence of DNA molecules. Discoveries and the subsequent development and optimization of techniques that allowed for deciphering the DNA sequence has opened new doors in research, biotech, and healthcare. The application of high-throughput sequencing technologies in these industries has positively impacted and will continue to contribute to the betterment of humanity and the global economy. Improvements, such as the use of radioactive molecules for DNA sequencing to the use of florescent dyes and the implementation of polymerase chain reaction (PCR) for amplification, led to sequencing a few hundred base pairs in days, to automation, where sequencing of thousands of base pairs in hours became possible. Significant advances have been made, but there is still room for improvement. Here, we look at the history and the technology of the currently available high-through put sequencing platforms and the possible applications of such technologies to biomedical research and beyond.

Keywords: DNA-seq; Next-generation sequencing; RNA-seq; high-throughput sequencing; single-molecule sequencing.

Figure 1.

Sanger sequencing workflow. The reverse strand of linear DNA is synthesized using DNA polymerase and four fluorescently labeled dideoxyribonucleotides (ddNTPs), which when incorporated will terminate chain elongation and emit a fluorescence signal unique to each ddNTP. The resulting fragments are analyzed via capillary gel electrophoresis. Created with BioRender.com.

Figure 2.

454/Roche and Ion Torrent sequencing. DNA is fragmented and attached to a bead via ligated adapters sequences. Amplification of the fragment attached to the beads occurs by emulsion PCR (emPCR). Beads (clones) are collected in wells of picotiter plates followed by the addition of DNA polymerase and dNTPs. For 454/Roche sequencing, the sequence is determined by measuring the light signal, which is unique to each dNTP, emitted using a charged couple device (CCD) camera, following pyrophosphate (PP_i) release during base incorporation and extension. Ion Torrent sequencing follows a similar workflow, but sequence determination is made by measuring H⁺ ion release during incorporation by a pH sensor. Created with BioRender.com.

Figure 3.

Illumina sequencing platform. DNA is fragmented, linearized, and ligated to adapters that will bind surface-tethered oligos via complementarity on a flow cell. The DNA fragments bend over to form bridges, followed by bridge amplification and cluster formation. Sequences are determined following incorporation of reversible dye ddNTPs. The fragments in the library are then assembled using various bioinformatic pipelines. Created with BioRender.com.

Figure 4.

Oxford Nanopore sequencing. A ssDNA fragment is pushed through a membrane nanopore composed of a motor enzyme. Each nucleotide will alter the current through the pore by a different magnitude as it moves through the pore, allowing for sequence determination. Created with BioRender.com.