The PacBio (Pacific Biosciences) sequencing technique is the most widely used third generation sequencing method, mainly due to the fact that the reads obtained with it are very long, even greater than 20.000 bp. Do you think that with this technique it is possible, with a single sequencing run, to sequence an entire bacterial genome.

Again it is necessary to build a library before proceeding with the actual sequencing. For the construction of the library it is necessary to fragment the extracted DNA to be sequenced, generally by sonication and, subsequently, to add circular adapters to the ends of the DNA fragments obtained. These are called SMRTbell DNA.

Fig. 1 PacBio SMRTbell Template Preparation Workflow for PacBio RS II system. PacBio SMRTbell Template Preparation Workflow for PacBio RS II system. This workflow is used to prepare libraries from fragmented and concentrated DNA using Covaris g-TUBE and concentrated using the AMPure magnetic beads before following PacBio SMRTbell 10 kb Library Preparation procedures.
Fonte: Kong N, Ng W, Thao K, Agulto R, Weis A, Kim KS, Korlach J, Hickey L, Kelly L, Lappin S, Weimer BC. Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing. Stand Genomic Sci. 2017 Mar 23;12:27. doi: 10.1186/s40793-017-0239-1. PMID: 28344744; PMCID: PMC5363030.

After building the library we proceed directly with the sequencing of these without going through an amplification or cloning phase as we have seen instead in the second and first generation sequencing techniques. In particular, for their sequencing, the fragments are loaded on a support which is called SMRT cell. This comes with thousands of defined nano wells ZMW (Zero-Mode Waveguide), which have a diameter of about 70 nm and a depth of 100 nm and the detection volume is about 20 zeptoliters (2 ∙ 10-20% L). It is important to note that there will be a single library fragment in each well. In fact, on the bottom of each well there is a DNA polymerase molecule to which the fragment that will be sequenced is bound.

Fig 2. Fonte: https://www.pacb.com/

The sequencing process begins when the different types of nucleotides (dsNTPs) are added into the SMRT cell. Since the nucleotides are labeled with different fluorescent molecules it is possible to distinguish which nucleotide is added for complementarity by the DNA polymerase. In fact, the DNA polymerase enzyme, starting from a sequencing primer that matches the fragment adapter, adds a specific type of nucleotide in the case of complementarity with the fragments being sequenced. When a nucleotide is about to be added, a light signal from the well will be recorded relating to the fluorescence emitted by the fluorochrome linked to the phosphate group of the nucleotide that is about to be added for the construction of the sequence. Then when the nucleotide is added the phosphate group is removed together with the fluorochrome, with consequent decay of the relative fluorescence. The sequencing process of the fragment present in each well of the SMRT cell occurs several times thanks to the “kicking” activity of the DNA polymerase. This allows for more reads to be produced from each individual fragment despite the absence of the amplification process.

Fig. 3 A schematic illustration representing the highly parallel optic system used in Single Molecule Real-Time (SMRT) DNA sequencing technology (Pacific Biosciences). This method uses SMRT chips that contain thousands of zero-mode waveguides (ZMWs). The presence of a fluorescent dye within the detection volume (within the ZMWs) indicates nucleotide incorporation. This leads to a light flash that is separated into a spatial array, from which the identity of the incorporated base can be determined. Fonte: Pushpendra K. Gupta,
Single-molecule DNA sequencing technologies for future genomics research, Trends in Biotechnology, Volume 26, Issue 11, 2008, Pages 602-611, ISSN 0167-7799, https://doi.org/10.1016/j.tibtech.2008.07.003. (http://www.sciencedirect.com/science/article/pii/S0167779908002047)

The light signal coming from the wells of the SMRT cell is recorded by an extremely powerful chamber, positioned on the bottom of this support, and for each well a trace is produced relating to the fluorescence peaks recorded in it at the addition of each nucleotide (therefore for each nucleotide we have a peak recorded). It should also be noted that despite having inside the well all the types of nucleotides with the different fluorescent molecules linked to them, in reality the camera can only detect the fluorescence of the nucleotide that is about to be added by the DNA polymerase thanks to the sensitivity of the chamber and due to the fact that the detection volume is an extremely small volume that allows only the light signal of the nucleotide to be recorded near the active site of the enzyme.

I know, as usual, explaining such an articulated process in detail is rather difficult for me but below I have reported some videos that will help you in understanding, taken directly from the Pacific Biosciences (PacBio) official youtube channel.

And here we are at the end of this article. If you like it, write a comment or leave a “like”.

Bye-bye and see you soon.