PACBIO SEQUENCING - PRINCIPLE, TYPES, APPLICATION, ADVANTAGE AND DISADVANTAGE

PacBio’s SMRT (single molecule real time) sequencing is one of the most commonly used third-generation sequencing technologies.
Compared with the previous two generations, PacBio long-read sequencing enabled by SMRT Sequencing technology requires no PCR amplification
The read length is 100 times longer than that of NGS.

1. PAC BIO PLATFORMS BY KAVIYA PRIYA A I MSc BIOTECHNOLOGY

2. PAC BIO Pacific Biosciences of California, Inc . (aka PacBio ) is an American biotechnology company founded in 2004 that develops and manufactures systems for gene sequencing and some novel real time biological observation. PacBio has two principal sequencing platforms: single-molecule real-time sequencing (SMRT), based on the properties of zero-mode waveguides and sequencing by binding (SBB) chemistry, which uses native nucleotides and scarless incorporation for DNA binding and extension.

3. PACBIO COMPANY: The company was founded based on research done at  Cornell University  that combined semiconductor processing and  photonics  with biotechnology research. The company’s first commercial product, the PacBio RS (The PacBio RS is the only microbial sequencing platform that finishes genomes, determines structure and resolves strains) was sold to a limited set of customers in 2010 and was commercially released in early 2011. A subsequent version of the sequencer called the PacBio RS II ( sequencer uses single molecule, real time (SMRT) sequencing technology) was released in April 2013.

4. Pac Bio SEQUENCING PacBio’s SMRT (single molecule real time) sequencing is one of the most commonly used third-generation sequencing technologies. Compared with the previous two generations, PacBio long-read sequencing enabled by SMRT Sequencing technology requires no PCR amplification The read length is 100 times longer than that of NGS.

5. PRINCIPLE Zero-mode waveguides (ZMWs), subwavelength optical nanostructures fabricated in a thin metallic film. These are powerful analytical tools that are capable of confining an excitation volume to the range of attoliters . A llows individual molecules to be isolated for optical analysis at physiologically relevant concentrations of fluorescently labeled biomolecules. Arrays of such nanostructures can also be engineered into systems for real-time analysis of a mass of single-molecule reactions or binding events, which is the principle of PacBio SMRT sequencing.

6. ZMW A  zero-mode waveguide  is an  optical waveguide  that guides light energy into a volume that is small in all dimensions compared to the  wavelength  of the light. SMRT CELL A single SMRT Cell. Each SMRT Cell contains 150,000 ZMWs. Approximately 35,000-75,000 of these wells produce a read in a run lasting 0.5-4 h, resulting in 0.5-1 Gb of sequence.

7. LIBRARY CONSTRUCTION The workflow for library construction involves the following steps: Determine the quality of genomic DNA ( gDNA )
Shear gDNA using a g-TUBE ( Covaris )
Select size and adjust concentration
Repair DNA damage and ends of fragmented DNA
Conduct DNA purification
Blunt-end ligation using blunt adapters
Purify template for submission to a sequencer The template, called a SMRTbell , is a closed single-stranded circular DNA, which is created by ligating hairpin adapters to both ends of target double-stranded DNA (dsDNA) molecules.

8. G TUBE

9. PacBio Single Molecule Real-Time (SMRT) Sequencing System A SMRTbell (grey) diffuses into a ZMW, and the adaptor binds to a polymerase immobilized at the bottom. Four types of nucleotides are labeled with a different fluorescent dye (indicated in red , yellow , green , and blue , respectively for G, C, T, and A ) so that they have distinct emission spectrums. As a nucleotide is held in the detection volume by the polymerase , a light pulse that identifies the base is produced.

10. STEPS A fluorescently- labeled nucleotide binds to the template in the active site of the polymerase. The fluorescence output of the color corresponding to the incorporated base is elevated. The dye linker-pyrophosphate product is cleaved from the nucleotide and diffuses out of the ZMW to end the fluorescence pulse. The polymerase is translocate to the next position. The next nucleotide binds to the template in the active site of the polymerase and initiates the next fluorescence pulse, which corresponds to base.

11. Highly accurate HiFi sequencing for more complete views of genomes, epigenomes , and transcriptomes. Throughput to run up to 1,300 human HiFi genomes per year.
Smart consumables for simple handling and less plastic waste. Powerful compute with Google Health Deep Consensus on-board. SEQUENCING SYSTEMS REVIO SYSTEM
Long-read sequencing

12. SEQUEL IIe SYSTEM Long-read sequencing Long, accurate HiFi reads with DNA methylation direct from the instrument in every run. Supports a wide range of applications – targeted sequencing, RNA sequencing, and whole-genome sequencing Throughput match for microbial genome sequencing, AAV vector sequencing, Iso-Seq method, and more

13. ONSO SYSTEM
Short-read sequencing Up to 15x higher accuracy than other benchtop sequencers Reduced requirement for sequencing coverage depth versus SBS sequencer.
Resolve homopolymer and difficult to sequence regions Low duplication rate , negligible index hopping
Rapid conversion of existing P5/P7 libraries for sequencing on the Onso system

14. APPLICATION OF SMRT SEQUENCING Whole genome sequencing : for humans, plants, animals and microbes. RNA Sequencing : Analysis of cDNA sequences across entire transcriptomes or targeted genes. Population studies: Understanding variants among bacterial, viral and cancer cells in complex populations. Targeted sequencing : of relevant genome targets in regions of interest. Epigenetics : To detect DNA modifications in samples through epigenetic studies.

15. ADVANTAGE OF PACBIO SMRT SEQUENCING LONG READS HIGH ACCURACY UNIFORM COVERAGE IN SEQUENCING De Novo Genome Assembly REAL-TIME ACQUISITION OF SIGNAL SINGLE MOLECULE RESOLUTION

16. Low throughput Time taken for accuracy Sequencing Error rate Size and cost of the machine DISADVANTAGES OF SMRT SEQUENCING

17. https://youtu.be/_lD8JyAbwEo?si=5qBYRwEAWM7wT7Ix