Written by Andres Melendez, Jake Pearson, Katie Ceraso, Arijit Nukala
An Overview of Sequencing
DNA sequencing has been a topic of interest for decades and, in current times, has evolved into an invaluable tool in understanding biology and in creating technologies that have revolutionized healthcare. On a broad scale, DNA sequencing involves determining the order of nucleotides in DNA so scientists can access genetic information. With a DNA sequence of an organism, scientists can discover genetic diseases and how to treat them, augment organisms using genetic engineering, treat diseases such as cancer, and more. Understanding DNA at this level opens an infinite range of possibilities in medicine, biology, and a plethora of other fields.
Ever since the structure of DNA was first captured by Watson, Crick, and Franklin, scientists have pursued sequencing technology. Milestones in the 1960s and early 1970s shaped DNA sequencing. Robert Holley’s use of RNase enzymes to produce the first whole nucleic acid sequence in 1965 was an initial effort to sequence tRNA which is significantly shorter than DNA. These experiments eventually evolved into DNA sequencing like that done by Maxam and Gilbert in 1977. Maxim and Gilbert used radiolabeled DNA treated with chemicals which broke the DNA chain at specific bases. Running a polyacrylamide gel allowed the length of the created fragments to be determined and from that, the sequence could be inferred. This was the first DNA sequencing technique that was widely adopted.
The real breakthrough in sequencing that would shape the field for decades to come was Sanger sequencing. In 1977, Frederic Sanger sequenced the genome of a bacteriophage, marking the first ever genome to be sequenced. Sanger used a technique called shotgun sequencing which involves breaking DNA into small fragments and sequencing each piece individually. The sequences found from these pieces are then stitched together using a computer program until the complete sequence of the original DNA is generated. This process was revolutionary and as the technology was refined, larger scale projects such as the Human Genome Project were made possible.
Sanger sequencing was once the gold standard of sequencing but newer, faster, and cheaper methods (Next Generation Sequencing or NGS) will make sequencing more accessible and applicable than ever before. Despite the emergence of next-gen sequencing, Sanger sequencing still has its place in the market due to several advantages it holds over NGS. Though NGS is less expensive than Sanger on a large scale, Sanger sequencing is cheaper for smaller sample sizes and areas. Additionally, the capillary sequencer instruments used in Sanger sequencing are significantly cheaper than those used in Next Generation Sequencing. Another major benefit of Sanger sequencing is the relative simplicity compared to NGS.
However, Sanger sequencing does have its drawbacks as well. Sanger sequencing is only able to process short pieces of DNA, which makes the process timely. Additionally, Sanger can only process up to 384 DNA fragments at a time, while Sanger can sequence millions simultaneously. Finally, Sanger sequencing is limited by quality declining after 700 to 900 bases of DNA. Thus, Sanger sequencing still has its place in the market, but it is overshadowed by NGS sequencing in many regards.
Future of Microorganism Sequencing
As previously mentioned, microorganisms are best identified using Sanger sequencing. However, there has been major advancement in the field of sequencing such as the use of next generation sequencing (NGS) & single-molecule sequencing (SMS).
NGS is used to sequence complete genomes and is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. NGS platforms perform sequencing of millions of small fragments of DNA in parallel and Bioinformatics analyses are used to piece together these fragments by mapping the individual reads to the human reference genome. NGS can be used to sequence entire genomes or constrained to specific areas of interest. NGS can be roughly divided into the process elements of sample pre-processing, library preations, sequencing itself and bioinformatics. Yet despite all the modern advancements, modern sequencing technologies require dedicated sample preparation to yield the sequencing library loaded onto the instrument. Sequencing libraries consist of DNA fragments of a defined length distribution with oligomer adapters at the 5′ and 3′ end for barcoding, as well as the actual sequencing process.
Single-molecule sequencing(SMS) platforms have become available to researchers and are currently being tested for clinical applications. SMS offers long read lengths, high consensus accuracies, direct identification of base modifications, direct RNA sequencing, portability, and more democratized access to sequencing platforms. SMS offers direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA.
In short, we’ve highlighted the impact of DNA sequencing, and how this incredible technology has influenced the healthcare industry. Even more important than the current impact is the potential of sequencing technology in cancer research, understanding genetic diseases, and potentially changing the healthcare landscape. The first initial breakthrough in Gene sequencing for commercial use was Sanger Sequencing. Sanger Sequencing has long been considered the gold standard of sequencing. Yet Next Generation Sequencing and Single Molecule Sequencing proved an in-depth understanding of the genome and can become powerful diagnostic tools in the years to come. The potential of sequencing technology is immense, and will greatly affect the healthcare industry and diagnostic care in years to come.
- Heather, James M, and Benjamin Chain. “The sequence of sequencers: The history of sequencing DNA.” Genomics vol. 107,1 (2016): 1–8. doi:10.1016/j.ygeno.2015.11.003
- Kozińska, A., Seweryn, P. & Sitkiewicz, I. A crash course in sequencing for a microbiologist. J Appl Genetics vol. 60, 103–111 (2019). https://doi.org/10.1007/s13353-019-00482-2
- “Shotgun Sequencing.” Genome.gov, www.genome.gov/genetics-glossary/Shotgun-Sequencing#:~:text=Shotgun%20sequencing%20is%20a%20laboratory,fragments%20that%20are%20sequenced%20individually.
- Shaffer, Dr. Catherine. “Challenges with Sanger Sequencing.” News, News Medical, 26 Feb. 2019, www.news-medical.net/life-sciences/Challenges-with-Sanger-Sequencing.aspx.
- J.F. Hess, T.A. Kohl, M. Kotrová, K. Rönsch, T. Paprotka, V. Mohr, T. Hutzenlaub, M. Brüggemann, R. Zengerle, S. Niemann, N. Paust. “Library preparation for next generation sequencing: A review of automation strategies.” Biotechnology Advances vol. 41, 2020. https://doi.org/10.1016/j.biotechadv.2020.107537.
- Adam Ameur, Wigard P. Kloosterman, Matthew S. Hestand. “Single-Molecule Sequencing: Towards Clinical Applications” Trends in Biotechnology vol. 37, 1 (2019). https://doi.org/10.1016/j.tibtech.2018.07.013.