Sanger Sequencing: the beginning
For 40 years Sanger Sequencing was the most widely used sequencing method. Sanger Sequencing is developed by Frederick Sanger and his colleagues in 1977 and for many years this method was seen as the golden standard. The process of Sanger Sequencing is based on the detection of labeled chain-terminating nucleotides that are incorporated by a DNA polymerase during the replication of a template.
There are three steps to Sanger Sequencing:
- Generating DNA fragments of varying lengths, each terminated with a fluorescent labeled dideoxynucleotide. To enable this, double stranded DNA needs to be denaturated. A primer is attached to the single stranded DNA and elongated with a mixture of nucleotides and and a small quantity of chain-terminating dideoxynucleotides. No nucleotide can be added to the DNA chain once a dideoxynucleotide has been incorporated.
- Separate DNA sequences to length with capillary gel electrophoresis. The shorter fragments move faster and will therefor are fed first into the third step.
- A laser excites the label on the dideoxynucleotide at the end of each sequence and this is translated into a ‘peak’. Each type of dideoxynucleotide is tagged with a different label, which is detected by a light sensor.
The future of Sanger Sequencing
Sanger Sequencing will remain useful in many labs in the future. It is still the most robust and accurate technique to sequence your DNA. Sanger Sequencing comes in handy in small-scale sequencing applications like checking genotypes, or to fill in the ‘gaps’ other techniques may have in their data. But with the introduction of Next-Generation Sequencing (NGS), the amount of Sanger sequencing analysis is decreasing rapidly. Still, the Sanger sequencing is frequently used for smaller projects and for sequencing long DNA sequences.
And then there was NGS
The amount of data generated with Next-Generation Sequencing is massive and have drastically increased the sequencing throughput and reduced the cost of sequencing. Next-generation sequencing involves three steps:
- Library preparation. In this first step DNA is fragmented either enzymatically or by sonication to create smaller strands. After fragmentation, adapters are ligated to these fragments with the help of DNA ligase. The adaptors enable the sequence to become bound to a complementary counterpart.
- In the second step of the Next-Generation Sequencing process libraries are loaded onto a flow cell and placed on the sequencer. The clusters of DNA fragments are amplified in a process called cluster generation, resulting in millions of copies of single-stranded DNA. The exact method of sequencing can vary between sequencers.
- Data analysis. Since Next-Generation Sequencing generates large volume of data, software is needed to analyse all this data. After sequencing, the software of the instrument identifies nucleotides. This process is called base calling. The software also predicts the accuracy of those base calls.
The future of NGS
With Next-Generation sequencing hundreds to thousands of genes or gene regions can be sequenced simultaneously. In one single experiment the mutation status of relevant genes is determined, using less DNA compared to the Sanger sequencing method. Like with every technique, there are also some downsides for Next-generation sequencing. The amount of data is not only an advantage, analyzing all this data can be challenging. Also it is important to consider the ethics. All data generated with Next-generation sequencing contains loads of genetic information about an individual. This genetic information can predict illnesses that may or may not be expressed in the future. It is therefore truly important to handle this data confidential.