Skip to 0 minutes and 2 seconds In this method of whole genome sequencing, specially designed plastic slides called flow cells are used. Attached to the surface of the flow cell are short sequences of DNA that match the adapters attached to each fragment in the DNA library. Before the DNA library can be sequenced, each DNA fragment needs to be separated into single strands. This allows the fragments of the DNA library to stick to the complementary DNA on the flow cell. Each fragment is then copied thousands of times, producing many clusters of fragments along the flow cell. To explain how the DNA fragments are sequenced, we must first revisit the concept of DNA replication from Week 1.
Skip to 0 minutes and 45 seconds The complementary base pairing rule of DNA is the basis for replication, and it is this rule that is used during DNA sequencing. During DNA replication, using an existing DNA strand as a template, enzymes called polymerases build two new complementary strands of DNA. Whole genome sequencing uses these principles of DNA replication - but with notable differences. One of the fundamental differences is the use of specially modified DNA bases to build the new strand of DNA. These modified bases have specific colours attached to them that help the sequencer identify the base. For instance, the DNA base T is the colour green. The template DNA is provided by the fragments of DNA in each cluster on the flow cell.
Skip to 1 minute and 34 seconds As the sequencing begins, polymerases add the coloured bases to the template strand. When a base is added, the reaction stops temporarily. The sequencer then takes a snapshot of the base that has been added. The machine software then ‘converts’ the colour it has recorded to the corresponding letter. This process continues until the entire sequence of the fragment has been read by the sequencer.
Skip to 1 minute and 58 seconds This process occurs simultaneously with every fragment in every cluster across the whole flow cell, building up multiple reads for each cluster which the sequencer software then combines into one single read. By recording multiple reads of the same fragment the confidence that the correct order of bases has been recorded is increased. Therefore, each cluster on the flow cell produces a single read for each of the fragments from the genome being sequenced. One single run on a next generation sequencer can produce up to 4 billion single reads. The single reads now need to be put back together and the genome analysed.
What happens inside the sequencer?
Once DNA has been extracted from the sample and prepared for sequencing, it will be entered into a next generation sequencing machine. But what happens inside the ‘black box’? How does the machine ‘read’ the genome sequence and convert it into something that scientists can understand?
This animation will take you ‘inside the sequencer’ to highlight how it produces a read of every single base of many multiple fragments of DNA simultaneously, using one particular type of technology as an example. The animation highlights how the process of DNA replication - in which the guanine base (G) is always paired with the complementary cytosine base (C), and the adenine base (A) is always paired with the complementary thymine base (T) - is highjacked by the sequencing process. During the sequencing process new strands of DNA are built using an existing strand as a template and as each new base is added to the strand, the sequencer can ‘take a snapshot’ of the new base and so produce a read of the DNA sequence. The animation visualises, and should help you to understand, the complex processes that take place inside the machine.