It has been over 30 years since the first generation of DNA sequencing technology was developed in 1977. Since then, sequencing technology has made considerable progress – from first generation to third generation NGS. Every transformation of sequencing technology leads to a huge shift towards enabling genome research, clinical disease research and drug development.
First generation NGS
All first-generation machines are capable of reading short fragments, between 300 and 500 base pairs in length. The technology is based on the chain-termination method developed by Sanger and Coulson in 1975, or the chain-degradation method invented by Maxam and Gulbert in 1977.
The Sanger method has evolved considerably but the core principles remain unchanged – specific dideoxynucleotides are used to disrupt the DNA synthesis reaction, each with a base-specific radioactive isotope label. This means that after gel electrophoresis, the DNA sequences of the sample can be determined according to the position of the electrophoretic band.
The main advantages of first-generation NGS technologies are that they have a good overall sequence output, and that they have a high accuracy of 99.99%. Also, preparing nucleic acids of the ideal size for first-generation NGS is relatively easy for many different types of samples, apart from formalin-fixed paraffin-embedded (FFPE) specimens. However, this form of sequencing is considered high cost and low throughput, both of which have a significant impact on large-scale applications.
The ABI PRISM range of sequencers were produced by Applied Biosystems in 2008, which allowed the simultaneous sequencing of hundreds of samples and were used to help draft the human genome as part of the Human Genome Project. Thermo Fisher Scientific acquired the company in 2014 and currently markets Applied Biosystems genetic analysis systems for Sanger sequencing and fragment analysis by capillary electrophoresis.
Second generation NGS
Researchers developed a luminescent method for measuring pyrophosphate synthesis – a process that converts pyrophosphate into ATP. In this reaction, light is produced proportionately to pyrophosphate. It was discovered that this approach can be used to infer DNA sequence by measuring the pyrophosphate produced by each nucleotide. This pyrosequencing technique was pioneered by Pal Nyren and was later licensed to 454 Life Sciences. These machines greatly increased the amount of DNA that could be sequenced at once.
Second-generation NGS machines were born and immediately began to drive the ‘genomics revolution’. They increased the yield of sequencing efforts massively and allowed researchers to completely sequence an entire genome far more quickly and cheaply than Sanger sequencing could. Whilst the initial machines were only capable of producing very short reads, the accuracy and read length of second-generation DNA platforms has grown rapidly. In recent years, Illumina sequencers have essentially monopolized the market and could be considered to have made the greatest contribution to second-generation NGS
Here are some examples of a variety of second-generation sequencing platforms:
- SOLiD: Sequencing by Oligonucleotide Ligation and Detection was developed by Life Technologies in 2006. It has a read length of 50 base pairs per minute and it takes 14 days to complete each run. The platform costs around $350,000 and has an extremely low error rate of 0.01%.
- 454 GS FLX+: Produced by Roche in 2011, the 454 GS FLX+ can read 700 base pairs per run in between 24 and 48 hours. It uses pyrosequencing chemistry and has a 1% raw error rate. The machine costs around $500,000.
- NextSeq 550Dx: In 2017, Illumina released the NextSeq 550Dx instrument. It can read up to 300 million base pairs per run in a total time of less than 35 hours. The overall accuracy is around 99%.
- Revolocity Supersequencer: This nanoball DNA sequencer was refined by the Beijing Genomics Institute after the company purchased Complete Genomics. It can read 50 base pairs in 14 days, with a 0.1% error rate. It uses nanoball technology.
Third generation NGS
There is some confusion as to what defines third-generation DNA sequencing. The simplest conclusion is probably that third-generation technologies are capable of sequencing single molecules without the requirement for DNA amplification, as this separates them from all other previous technologies.
Single molecule sequencing was commercialized by Helicos Biosciences around 2009. Whilst it was relatively slow, expensive and produced short reads, it was the first machine to allow sequencing of non-amplified DNA. This avoided all the associated biases and errors. Helicos Biosciences became bankrupt in 2012, allowing other companies to take over the marketing of third-generation technologies.
Single molecule sequencing shows huge promise for the future of genomic medicine. This is mainly because these technologies offer extremely long read lengths, at high accuracies, in regions of the genome that are mostly inaccessible by short-read platforms. Extreme GC content regions and complex gene loci can now be explored, opening up new clinical avenues that can be applied to a variety of research areas, such as pharmacogenomics, cancer and neurological disorders.
Examples of third-generation NGS platforms include:
- SMRT: Single-molecule real-time sequencing was commercialized by Pacific Biosciences in 2011. It can read more than 10,000 base pairs in under two hours. The machine costs around $750,000. The error rate is relatively high at 12%, but the errors are random and without bias, so the deviation can be corrected through multiple sequencing.
- ONT: Oxford Nanopore Technologies created a portable platform in 2014, that uses nanopore sequencing to read over 5,000 base pairs in between 48 and 72 hours. The platform only costs around $1,000. Notably, a quarter of all the world’s SARS-CoV-2 virus genomes haven been sequenced with nanopore devices.
The importance of DNA sequencing has led to researchers around the world investing a great deal of time and resources into developing and improving NGS platforms. Innovation over the years has led to increased technical capabilities of sequencing, whilst decreasing the costs to allow the reading of thousands of DNA base pairs in a matter of hours. It is clear that NGS has a rich history and understanding this history can provide some insights into what future sequencing endeavours may be still to come.
For more information on sequencing technologies, check out our Sequencing Buyer’s Guide.
Image credit: LabManager
