Second- and third-generation NGS platforms are separated by their need for DNA amplification prior to sequencing. Second-generation sequencing technologies are all dependent on the amplification of DNA before analysis because a large enough number of template DNA is required for good signal strength of each base addition, whereas third generation platforms do not require this step. The three different types of amplification used are emulsion PCR, bridge amplification and DNA nanoball generation.
PCR components, such as template DNA, polymerase, primers, nucleotides and minerals are soluble in water but not oil. This forms the basis for the concept behind emulsion PCR, which is a technique that amplifies DNA molecules in physically separated water-in-oil droplets. Both Ion Torrent and GenapSys sequencing platforms utilize the approach.
Find below a step-by-step guide for Emulsion PCR:
- Step #1: The DNA is fragmented into small pieces and purified, and the ends are repaired with oligonucleotides.
- Step #2: The aqueous solution (now containing the library of DNA fragments, a large number of prepared beads and the necessary PCR reagents) is emulsified in oil from very small droplets.
- Step #3: The droplets contain a bead and a single DNA fragment. The DNA is replicated by DNA polymerase after one of the ends of the fragment anneals to its complementary oligonucleotides covalently attached to the bead.
- Step #4: The entire emulsion is heated to denature the original DNA fragment from the newly copied one, which remains covalently bonded to the bead. There are now two fragments in the droplet – the original DNA floating free and the other attached to the bead.
- Step #5: The original DNA fragment then hybridizes again to an oligonucleotide on the bead, and both fragments are replicated by DNA polymerase.
- Step #6: The emulsion is heated again – now there are two fragments covalently bonded linked to the bead and two fragments in the solution.
- Step #7: Once the desired amount of DNA has been reached, the beads with amplified fragments on them are purified from any beads that do not have any DNA attached by density centrifugation.
A diagram showing how emulsion PCR works. Image credit: Sequencing Buyer’s Guide, 2021
This process is repeated, with the number of DNA fragments covalently linked to the bead doubling at each step. The amount of amplification is entirely dependent on the diameter of the beads. For example, the original 454 sequencing platform used beads that were 28 microns in diameter, and these were capable of generating up to 1 million copies of the starting DNA fragment. In contrast, the Ion Torrent sequencing platform uses beads with a 5-micron diameter, and so a much smaller surface area. Therefore, the technology is only capable of amplifying the original DNA fragment into around 50,000 copies.
The main disadvantage of the technique is that PCR errors can occur during some of the early stages, which are then magnified during later amplification steps. Also, the multistep process is largely inconvenient, especially as it involves the intricate use of oil and water emulsions. The increase of automation will reduce this issue though – Thermo Fisher Scientific markets a machine, called the Ion Chef System, that automates the entire process!
Illumina sequencing is based on a technique called bridge amplification, whereby DNA molecules are repeatedly replicated on a glass flow cell containing complementary oligonucleotides.
Find below a step-by-step guide to bridge amplification:
- Step #1: The glass flow cell is coated with two types of oligonucleotides.
- Step #2: The DNA is fragmented and adapters are added to each end of the strand. Each of these adapters is complementary to one of the oligonucleotides on the flow cell.
- Step #3: DNA fragments are added to the flow cell and hybridize with one of the oligonucleotides on the flow cell surface.
- Step #4: A DNA polymerase moves along the strand, replicating it.
- Step #5: The double-stranded fragment is denatured and the original strand is washed away.
- Stpe #6: The new remaining strand folds over and its adapter attaches to the second type of oligonucleotide on the flow cell.
- Step #7: DNA polymerase replicates this fragment forming a double-stranded bridge.
- Step #8: The bridge is denatured, resulting in two single stranded copies of the DNA anchored to the flow cell.
- Step #9: This process is repeated until the desired amount of DNA has been reached.
A diagram showing the step-by-step process of bridge amplification. Image credit: atdbio
Bridge amplification is not a very efficient method for clonal amplification – 35 cycles yield just around 1,000 copies of the original molecule. Furthermore, there is a high chance that the template strands hybridize which each other, rather than annealing to a new primer site on the flow cell. Also, there are strict size requirements for the length of the template DNA and some polymerases have shown bias towards specific DNA regions.
Nevertheless, the bridge amplification approach that Illumina employs yields a high number of clusters – total reads generated can be up to 180 million using the HiSeq 2000. If 2 flow cells are sequenced in parallel, 600 gigabases of data can be achieved.
DNA nanoball generation
DNA nanoball generation was developed by Complete Genomics, which was purchase by BGI in 2012. This technology does not use PCR amplification, but instead clones a library of DNA fragments into a retroviral vector to produce circles.
Find below a step-by-step guide for DNA nanoball generation:
- Step #1: The DNA is fragmented and adapter sequences are attached.
- Step #2: A splint oligonucleotide hybridizes to the ends of the fragments, causing the DNA to form a circle.
- Step #3: The remaining linear DNA strands are removed, leaving only completed circular single-stranded DNA templates.
- Step #4: The circular DNA is replicated by phi29 DNA polymerase and the newly synthesized strand is released from the template.
- Step #5: The resulting long single-stranded DNA strand contains several copies of the circular template and self-assembles into a tight ball, called a nanoball. These remain separated due to repelling negative charges.
- Step #6: The DNA nanoballs are deposited onto patterned flow cells for sequencing.
A diagram to show the process of DNA nanoballing. Phi29 DNA polymerase copies the circular DNA and displaces the new strand, which forms a ball due to the adapter sequences. Image credit: Wikimedia, 2011
The number of copies made is lower than from emulsion PCR or bridge amplification, with only 300 copies being made from each original fragment. However, an advantage of DNA nanoball generation is that it uses phi29 DNA polymerase, which enables accurate amplification of the circular template. Therefore, it does not make errors that may be present in PCR-based amplification and does not suffer from barcode hopping. Also, all of the copies are compacted into a small area, resulting in an intense signal strength.
The Sequencing Buyer’s Guide report includes in-depth information about how different NGS platforms utilize various DNA amplification techniques. It contains David I Smith’s advice for reducing the cost of NGS workflows and explains the current patent landscape for sequencing. Check it out here:
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