Lecture 26 - Next Generation Sequencing Flashcards
What are NGS technologies
Using so-called ‘Next Generation Sequencing’ (NGS) technologies, today an entire human genome can be sequenced within an hour at a cost of just a few hundred dollars
What are 1st generation NGS techniques
The major hindrance in the Sanger sequencing method is the requirement to perform a DNA size determination step, as this hugely constrains the number of DNA clones that can be sequenced in a single sequencing run
Thus instead of sequence determination hinging on size- and light-detection, the aim in the early days of NGS development was to come up with a new method that relied essentially only on light-detection
What are the two key features of the first wave of NGS methods
Clonal amplification of DNA fragments using ‘massively parallel’ PCR-based techniques
Sequencing of all the produced DNA clones in parallel using a light detection-only method
What is Pyrosequencing
‘Emulsion-PCR’ used for massively parallel generation of millions of clones from a fragmented genomic sample
‘Pyrosequencing’ chemistry used to determine DNA sequence of the DNA clones
How does pyrosequencing take place
Fragment genome into tiny pieces
Ligate bits of DNA (adaptors) of known sequence to the ends of each fragment
Create DNA:water/oil emulsion
Allow PCRs to proceed from each DNA fragment in contained water-droplet-compartments
Spread individual bead/droplet PCR reactions onto ‘pico-titer plates’: typically couple of million wells per plate
Perform pyrosequencing in wells, again using primers complementary to the adaptors
(slide 6)
What is the main pro of Pyrosequencing
Cheaper and quicker than Sanger when applied to genome-scale projects
What are the cons to pyrosequencing
However, not quite well-suited for sequencing of larger (i.e. mammalian) genomes
High error-rate compared to Sanger sequencing
What is illumina sequencing
Localised ‘bridge amplification’ on a glass slide to yield clonal clusters of DNA
Reversible dye terminator chemistry to determine DNA sequence of the clonal clusters
What are the pros of illumina sequencing
A human genome can be sequenced in just an hour or so for well under a thousand pounds using the latest ‘NovaSeq 6000’ model
Accuracy comparable to sanger (i.e. >99.9%)
Currently, by far the most widely used NGS platform in both biomedical research and medicine
What are some newer NGS techniques
These allow DNA to be sequenced without any PCR amplification step
Sequence data is obtained in real-time
Enable direct detection of non-canonical bases
The two best examples are SMRT and Nanopore sequencing
What is nano pore sequencing
Has all the practical capabilities/advantages offered by SMRT sequencing
Also has higher throughput than SMRT and generates longer read lengths
Commercially released recently by Oxford Nanopore Technologies (ONT)
